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2015
Complementary Immune Roles for Infected and Uninfected Cells During Legionella Pneumophila infection
Alan M. Copenhaver University of Pennsylvania, [email protected]
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Part of the Allergy and Immunology Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Microbiology Commons
Recommended Citation Copenhaver, Alan M., "Complementary Immune Roles for Infected and Uninfected Cells During Legionella Pneumophila infection" (2015). Publicly Accessible Penn Dissertations. 1032. https://repository.upenn.edu/edissertations/1032
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1032 For more information, please contact [email protected]. Complementary Immune Roles for Infected and Uninfected Cells During Legionella Pneumophila infection
Abstract The innate immune system responds to virulent pathogens, yet many pathogens manipulate host- signaling pathways, which should limit immune activation. The intracellular bacterium Legionella pneumophila is the cause of the severe pneumonia Legionnaire's disease. L. pneumophila encodes a type IV secretion system (T4SS) to translocate bacterial proteins into the cytosol of infected host cells. Several of these bacterial effectors (Lgt1, Lgt2, Lgt3, SidI, SidL, Pkn5, and Lpg1489) inactivate host cell elongation factors involved in protein translation. Despite the ability of L. pneumophila to block host protein translation, inflammatory cytokines are still made during infection both in vivo and in vitro. It is unclear how infected cells can mount a cytokine response when host protein synthesis is blocked. By creating a fluorescence resonance energy transfer-based system to track the activity of the T4SS in infected host cells, this study investigates how innate immune cells produce cytokines during L. pneumophila infection. In vitro, cells targeted by the T4SS of L. pneumophila are poor producers of cytokines critical for control of infection, such as TNF, IL-6, IL-12, and do not express CD86 in response to infection. Instead, uninfected, bystander cells produce these cytokines. Infected host cells do produce IL-1alpha; and IL-1beta; de novo and transcribe many proinflammatory genes. During pulmonary infection, alveolar macrophages and neutrophils are targeted by the T4SS for translocation and contain viable L. pneumophila. These cells provide a niche for bacterial replication during infection, but also secrete IL-1 in response to virulent bacteria. Uninfected alveolar macrophages, neutrophils, as well as inflammatory monocytes and dendritic cells produce TNF during L. pneumophila infection in vivo. Inflammatory monocytes and dendritic cells also increase CD86 expression during infection. Importantly, this bystander TNF production and CD86 expression requires IL-1 signaling, as mice deficient for the IL-1R have diminished levels of TNF and CD86 expression during infection. These data suggest that infected cells have mechanisms to overcome protein synthesis inhibition to produce IL-1 and that uninfected bystander cells are important contributors to the immune response during infection with L. pneumophila. This mechanism of immune activation has broad significance as many other bacterial pathogens manipulate host cell processes, including immune cell signaling.
Degree Type Dissertation
Degree Name Doctor of Philosophy (PhD)
Graduate Group Immunology
First Advisor Sunny Shin
Keywords IL-1, IL-1R, Legionella pneumophila, Type IV secretion system
Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology | Microbiology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1032 COMPLEMENTARY IMMUNE ROLES FOR INFECTED AND UNINFECTED CELLS DURING LEGIONELLA PNEUMOPHILA INFECTION
Alan M. Copenhaver
A DISSERTATION
in
Immunology
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
2015
Supervisor of Dissertation
______Sunny Shin Assistant Professor of Microbiology
Graduate Group Chairperson
______David Michael Allman Associate Professor of Pathology and Laboratory Medicine
Dissertation Committee:
Michael S. Marks, Ph.D., Professor of Pathology and Laboratory Medicine, Chair George Scott Worthen, M.D., Professor of Pediatrics Carolina B. López, Ph.D., Assistant Professor of Pathobiology Igor E. Brodsky, Ph.D., Assistant Professor of Microbiology COMPLEMENTARY IMMUNE ROLES FOR INFECTED AND UNINFECTED CELLS DURING LEGIONELLA PNEUMOPHILA INFECTION
COPYRIGHT
2015
Alan M. Copenhaver
ACKNOWLEDGEMENTS
I would like to thank my mentor Sunny Shin for applying many hours of time and effort into my development both as a researcher as well as a member of the scientific community. My writing, my thinking, and my experiments have forever been altered by her tutelage. I thank my committee for their input and advice and for showing me that no matter how hard an experiment or an assay is, getting four professors to appear in the same room at the same time is far more difficult. I would specifically like to thank Mickey Marks for his consistent enthusiasm for my development. I thank Carolina Lopez for showing me what unbridled excitement for scientific discussion looks like. I thank Jeff Weiser for his dry sense of humor, which is always welcome in the midst of a long meeting. I thank Igor for the countless hours he spent editing my writing, thinking about my project, and for his excellent skills at mixing margaritas. I thank Chris Hunter for his combative presence, his air of confidence, as well as for the time he bought me tea. I would also like to thank Sarah Hughes for keeping Chris on schedule. Last, I thank George Scott Worthen for his tough questions and his insight into pulmonary immunology.
There are a myriad of people who have physically helped make this work possible. First and foremost, I thank Cierra Casson for being a constant companion in all experiments. I thank Cierra for her vigilance, her attention to detail, her criticism, her work ethic, and her commitment to helping others, including myself. I thank Hieu Nguyen for always agreeing to help, although begrudgingly, with tissue harvests and for bringing fresh life and art to a very scientific space. I thank Janet Yu for performing tasks before I even knew I needed them done. I apologize to Liam Bradley for all the years he had to share a bay with me.
I thank all my friends for dealing with my unpleasant demeanor and my acerbic nature. I thank Irene Chernova for showing me that no matter how much I achieve someone is doing more. I also thank Irene for being a constant driver of excellence. I thank Martin Naradikian for demonstrating perseverance, determination, and naïveté. I thank Leigh Kinczewski for being a pinnacle of hard work, blue-collar life, and for being a hero of labor. I also thank Irene, Martin, and Leigh for dragging me to cultural events. I thank James Barnes and Phillip Hains for all the years of friendship, cooking, and discussion.
Lastly, I thank my mother, Laurie Klingenberg, for sacrificing so much and striving for so much better than we had. I thank all of my family, including my step-father, my brother, my grandparents, my aunt, and my uncle, for living with me and molding me throughout my youth into what I am today.
iii ABSTRACT
COMPLEMENTARY IMMUNE ROLES FOR INFECTED AND UNINFECTED CELLS DURING LEGIONELLA PNEUMOPHILA INFECTION
Alan M. Copenhaver Sunny Shin
The innate immune system responds to virulent pathogens, yet many pathogens manipulate host-signaling pathways, which should limit immune activation. The intracellular bacterium Legionella pneumophila is the cause of the severe pneumonia Legionnaire’s disease. L. pneumophila encodes a type IV secretion system (T4SS) to translocate bacterial proteins into the cytosol of infected host cells. Several of these bacterial effectors (Lgt1, Lgt2, Lgt3, SidI, SidL, Pkn5, and Lpg1489) inactivate host cell elongation factors involved in protein translation. Despite the ability of L. pneumophila to block host protein translation, inflammatory cytokines are still made during infection both in vivo and in vitro. It is unclear how infected cells can mount a cytokine response when host protein synthesis is blocked. By creating a fluorescence resonance energy transfer- based system to track the activity of the T4SS in infected host cells, this study investigates how innate immune cells produce cytokines during L. pneumophila infection. In vitro, cells targeted by the T4SS of L. pneumophila are poor producers of cytokines critical for control of infection, such as TNF, IL-6, IL-12, and do not express CD86 in response to infection. Instead, uninfected, bystander cells produce these cytokines. Infected host cells do produce IL-1α and IL-1β de novo and transcribe many proinflammatory genes. During pulmonary infection, alveolar macrophages and neutrophils are targeted by the T4SS for translocation and contain viable L. pneumophila. These cells provide a niche for bacterial replication during infection, but also secrete IL-1 in response to virulent bacteria. Uninfected alveolar macrophages, neutrophils, as well as inflammatory monocytes and dendritic cells produce TNF during L. pneumophila infection in vivo. Inflammatory monocytes and dendritic cells also increase CD86 expression during infection. Importantly, this bystander TNF production and CD86 expression requires IL-1 signaling, as mice deficient for the IL-1R have diminished levels of TNF and CD86 expression during infection. These data suggest that infected cells have mechanisms to overcome protein synthesis inhibition to produce IL-1 and that uninfected bystander cells are important contributors to the immune response during infection with L. pneumophila. This mechanism of immune activation has broad significance as many other bacterial pathogens manipulate host cell processes, including immune cell signaling.
iv TABLE OF CONTENTS
CONTENT PAGE
Copyright Notice……………………………………………………………………….……...... ii Acknowledgements……………………………….………………………………………….....iii Abstract…………………………………………………………………………………………...iv Table of Contents………………………….……………………………………………………..v List of Abbreviations…………………….………………………………...... …………...…….vii List of Illustrations..…………………………………………………………………………….viii
CHAPTER 1: INTRODUCTION….……………..…………………………………….………..1
A. Pathogenic manipulation of host cells……………………………….……………………..3 B. Immune responses during pathogenic manipulation………………………………….….4 C. Legionella pneumophila ecology and infection………………………………………...….5 D. Legionella pneumophila type IV secretion system…………………………………….….7 E. Type IV secretion system effectors…………………………………………………..…….9 F. The immune response to Legionella pneumophila……………………………………...12 G. Inflammasome activation during Legionella pneumophila infection…………………...15 H. The cytokine response to Legionella pneumophila…………………………………...... 18 I. The role of IL-1α and IL-1β during infection……………………………………………….20 J. Pulmonary immune responses………………………………………………………..……22 K. Dissertation aims……………………………………………………………………………25
CHAPTER 2: MATERIALS AND METHODS………………………………………………..29
CHAPTER 3: ALVEOLAR MACROPHAGES AND NEUTROPHILS ARE THE PRIMARY RESERVOIRS FOR LEGIONELLA PNEUMOPHILA AND MEDIATE CYTOSOLIC SURVEILLANCE OF TYPE IV SECRETION………………………………………………..36
Abstract………………………………………………………………………………………….37 Introduction……………………………………………………………………………………...37 Results…………………………………………………………………………………………..41 Conclusion…………………………………………………………….………………………...51 Acknowledgements………………………………………………………………………….....51
CHAPTER 4: IL-1 INDUCES INNATE BYSTANDER CYTOKINE PRODUCTION TO BYPASS BACTERIAL BLOCKADE OF HOST PROTEIN SYNTHESIS…………………63 Abstract……………………………………………………………………………………….…64 Introduction……………………………………………………………………………………...65 Results…………………………………………………………………………………………..67 Conclusion……………………………………………………………………………………....74 Acknowledgements…………………………………………………………………………….75
v CHAPTER 5: DISCUSSION………………………………………………………………..…93
A. Alveolar macrophages and neutrophils are targeted by the type IV secretion system of L. pneumophila………………………………………………………………………….…..93 B. Non-phagocytic cells are not targeted by L. pneumophila for injection…………….....95 C. Alveolar macrophages and neutrophils produce cytokines in response to virulent L. pneumophila infection…………………………………………………………………...….98 D. Bystander cytokine production by uninjected cells………………………………….....100 E. Injected cells produce IL-1α and IL-1β during protein translations inhibition………..102 F. IL-1 induces TNF production and CD86 expression in dendritic cells and alveolar macrophages……………………………………………………………………………….104 G. Bystander signaling occurs in other models of infection………………………………109 H. Final Conclusions……………………………………………………………………….…111
BIBLIOGRAPHY………………………………………………………………………………116
vi LIST OF ABBREVIATIONS
AEC: Alveolar epithelial cell AMΦ: Alveolar macrophage BFA: Brefeldin-A BlaM: β-lactamase BMDC: Bone marrow-derived dendritic cell BMDM: Bone marrow-derived macrophage CFU: Colony forming unit cDC: Conventional dendritic cell CYE: Charcoal yeast extract DAP Diaminopimelic acid DC: Dendritic cell ELISA: Enzyme-linked immuno assay ER: Endoplasmic reticulum EΦ: Eosinophil FAK: Focal adhesion kinase FBS: Fetal bovine serum FRET: Fluorescent resonance energy transfer GEF: Guanine nucleotide exchange factor iMC: Inflammatory monocyte JNK: c-Jun N-terminal kinase LCV: Legionella containing vacuole LPS: Lipopolysaccharide MAPK: Mitogen-activated protein kinase MDP: Muramyl dipeptide MKK: MAPK kinase MyD88: Myeloid differentiation primary response 88 NF-κB: Nuclear factor kappa-light-chain enhancer of activated B cells NK: Natural killer NLR: Nod-like receptor or nucleotide-binding oligerimization domain receptor NΦ: Neutrophil PAMP: Pathogen-associated molecular pattern PBS: Phosphate buffered saline PRR: Pattern recognition receptor RIP2: Receptor-interacting protein 2 TLR: Toll-like receptor T3SS: Type III secretion system T4SS: Type IV secretion system Unfx: Uninfected Untx: Untreated UPR: Unfolded protein response WT: Wild Type
vii LIST OF ILLUSTRATIONS
FIGURE 1-1. Bacterial pathogens inhibit host cell signaling and protein translation during infection………………………………………………………………………………………….27
FIGURE 1-2. Legionella pneumophila activates multiple immune pathways, but inhibits protein synthesis in infected cells…………………………………………………………….28
FIGURE 3-1. β-lactamase translocation into cells requires fusion to RalF and a functional type IV secretion system…………………………………………………..………53
FIGURE 3-2. Viable Legionella pneumophila are predominantly associated with macrophages positive for T4SS-dependent translocation…………………………………54
FIGURE 3-3. Gating strategies for fluorescence-based imaging flow cytometry and conventional flow cytometry…………………………………………………………………...55
FIGURE 3-4. The BlaM-RalF expression plasmid is stably maintained during both in vitro and in vivo infection…………………………………………………………………………….56
FIGURE 3-5. Legionella pneumophila T4SS-dependent translocation is detected in dendritic cells and alveolar epithelial cells during in vitro infection………………………..57
FIGURE 3-6. Bacterial motility increases the frequency of T4SS-injected macrophages, but not T4SS-injected alveolar epithelial cells………………………………………………58
FIGURE 3-7. Alveolar macrophages and neutrophils in the airway space are injected by the Legionella pneumophila T4SS……………………………………………………………59
FIGURE 3-8. Infection with ΔflaA Legionella pneumophila recruits immune cells to the lungs of infected mice………………………………………………………………………….60
FIGURE 3-9. The Legionella pneumophila T4SS injects alveolar macrophages and neutrophils in the lung………………………………………………………………………….61
FIGURE 3-10. Alveolar macrophages and neutrophils from infected mice contain viable Legionella pneumophila and secrete cytokines……………………………………………..62
FIGURE 4-1. T4SS-injected and uninjected macrophages produce different cytokines in vitro………………………………………………………………………………………………77
FIGURE 4-2. IL-12p40 and CD86 are produced by bystander cells during infection…...78
FIGURE 4-3. Type IV secretion system effectors that block protein translation inhibit early TNF production in injected cells………………………………………………………..79
FIGURE 4-4. Bystander alveolar macrophages and neutrophils produce TNF during L. pneumophila infection………………………………………………………………………….80
viii FIGURE 4-5. T4SS-injected alveolar macrophages produce IL-1 during pulmonary infection………………………………………………………………………………………….81
FIGURE 4-6. Bystander alveolar macrophages and neutrophils from the lung produce TNF during infection……………………………………………………………………………82
FIGURE 4-7. Bystander inflammatory monocytes and conventional dendritic cells produce TNF and express CD86 during infection…………………………………………..83
FIGURE 4-8. Alveolar macrophages and neutrophils do not express CD86 during infection………………………………………………………………………………………….84
FIGURE 4-9. IL-1 signaling induces cytokine production by bystander cells……………85
FIGURE 4-10. IL-1 signaling leads to the expression of CD86 and TNF by bystander cells………………………………………………………………………………………………86
FIGURE 4-11. Lack of IL-1 signaling does not alter the production of TNF by avirulent L. pneumophila infection………………………………………………………………………….87
FIGURE 4-12. Both IL-1α and IL-1β contribute to cytokine production during L. pneumophila infection………………………………………………………………………….88
FIGURE 4-13. Bone marrow-derived macrophages are unresponsive to IL-1 treatment………………………………………………………………………………………...89
FIGURE 4-14. Bone marrow-derived dendritic cells produce cytokines and express CD86 in response to IL-1 treatment………………………………………………………….90
FIGURE 4-15. Alveolar macrophages produce TNF in response to IL-1 treatment…….91
FIGURE 4-16. Inflammatory monocytes do not respond to IL-1 treatment………………92
FIGURE 5-1. Alveolar macrophages and neutrophils are targeted by the T4SS of L. pneumophila and contain viable bacteria during pulmonary infection…………………..113
FIGURE 5-2. T4SS effectors block the production of cytokines in infected cells, yet IL-1α and IL-1β are still translated during infection………………………………………………114
FIGURE 5-3. Direct and indirect IL-1 signaling induces bystander cytokine production during L. pneumophila infection from uninfected innate host cells………………………115
ix CHAPTER 1
INTRODUCTION
Mammals provide large and diverse niches for microbial species. To date, every mammalian surface, including the skin, gastrointestinal tract, airways, and oral cavities are known to contain a multitude of living microbes including bacteria, archaea, viruses, and fungi (Bäckhed et al., 2005; Beck et al., 2012; Delwart, 2013; Dewhirst et al., 2010;
Fontana et al., 2012; Grice and Segre, 2011; Hoffmann et al., 2013; Somerville, 1969).
The vast majority of known microorganisms do not infect their hosts and induce disease.
These organisms are referred to as non-pathogenic organisms or commensals. Some of these non-pathogenic organisms benefit their hosts by aiding digestion and nutrient uptake or by preventing colonization and infection of the host with pathogenic organisms
(Casadevall and Pirofski, 2003; Case et al., 2009; Kamada et al., 2013a; 2013b; Kau et al., 2011). Pathogenic organisms are known to cause infection and disease, and thus their presence is detrimental to host organisms (Casadevall and Pirofski, 2003; Kamada et al., 2013a; 2013b; Kau et al., 2011; Mouchtouri et al., 2010; Nguyen et al., 2006). To detect pathogenic organisms, hosts encode and express pattern recognition receptors
(PRR) (Janeway and Medzhitov, 2002). These PRRs detect conserved pathogen- associated molecular patterns (PAMP) expressed by pathogens (Vance et al., 2009).
These PAMPs include lipopolysaccharide (LPS), flagellin, foreign peptidoglycans, and foreign genetic material. However, commensal organisms express many of these
PAMPs (Vance et al., 2009). For instance, LPS is a component of the Gram-negative cell wall, and thus is ubiquitously expressed by all Gram-negative organisms, including non-pathogenic microbes (Rietschel et al., 1994). Similarly, flagellin is a component of the Gram-negative flagellum, which is used by these organisms for locomotion and is not
1 specifically associated with pathogenic organisms (Bardy et al., 2003; Neville et al.,
2013). Thus, PRRs alone cannot distinguish between non-pathogenic and pathogenic organisms.
One distinction between commensal bacteria and their virulent counterparts is that many pathogens access the cytosol of host cells either by using specialized secretion systems to translocate bacterial products across host cell membranes or by using pore-forming proteins to disrupt host cell membranes and gain direct access to the cytosol (Krachler et al., 2011; Roy and Mocarski, 2007; Vance et al., 2009). Pathogens, such as Yersinia and Salmonella spp., encode type III secretion systems (T3SS) that form syringe-like structures to translocate bacterial effectors proteins across the plasma or phagosomal membranes (Cornelis, 2006; Viboud and Bliska, 2005). Burkholderia spp. also encode a
T3SS, but unlike Yersinia and Salmonella spp, these bacteria utilize their T3SS to break out of the phagosome allowing for bacterial replication within the cytosol of host cells
(Cornelis, 2006; Galyov et al., 2010). Other bacterial pathogens, such as certain Vibrio spp. and Listeria monocytogenes, utilize pore-forming proteins to either translocate effectors into the cell or disrupt phagosomal and other membranes allowing for access to the cytosol (De Haan and Hirst, 2004; Hamon et al., 2012). These secretion systems and pore-forming proteins often translocate bacterial effector proteins into the cytosol of host cells to manipulate host cell processes such as cytoskeletal rearrangement, vesicular trafficking, host cell signaling, and protein translation (Kahn et al., 2002; Roy and Mocarski, 2007). These mechanisms benefit the replication, and spread of the pathogens within hosts and are essential for their survival.
2 A. Pathogenic manipulation of host cells
A variety of bacterial pathogens encode effectors that modulate the activity of host cell kinases, ultimately altering host cell activation (Figure 1-1) (Krachler et al., 2011). Vibrio parahaemolyticus translocates the acetyltransferase VopA/P into host cells, which inactivates a number of MAP kinase kinases (MKK), ultimately inhibiting host cell growth
(Selyunin et al., 2011; Trosky et al., 2007). Shigella flexneri encodes two effectors, OspF and OspG that inhibit extracellular signal-regulated kinases (ERK) and nuclear factor kappa-light-chain-enhancer of B cells NF-κB activation respectively, preventing the production of proinflammatory immune responses against the pathogen (Arbibe et al.,
2007; Kim et al., 2005; Kramer et al., 2007; Li et al., 2007). Yersinia spp. encode three effectors that alter host cell kinase activity. YopJ and its more pathogenic homolog YopP are acetyltransferases that inactivate both MAPK and NF-κB signaling in host cells via an unknown mechanism (Mukherjee et al., 2006; Palmer et al., 1998; Schesser et al.,
1998). Yersinia spp. encodes YopH that inhibits the protein kinases Fyn and focal adhesion kinase (FAK) signaling, preventing phagocytosis of the bacteria (Black and
Bliska, 1997; Yuan et al., 2005). Finally, Yersinia spp. encode invasin that inhibits FAK signaling and prevents phagocytosis (Uliczka et al., 2009).
Beyond inhibiting host cell signaling, a handful of pathogenic bacterial species encode toxins or effectors that inhibit protein translation by host cells (Fontana and Vance,
2011). This host protein translation inhibition is thought to limit the production of many proteins involved in immune responses, including cytokines, antimicrobial agents, and lysosomal enzymes. This inability to produce proteins that are important for immune responses promotes infection (Fontana and Vance, 2011). Shigella spp. encode shiga toxin, a protein translation inhibitor that acts by cleaving the 28S RNA of the host ribosome (Sandvig and van Deurs, 1996). Corynebacterium diphtheriae express
3 diphtheria toxin that ribosylates elongation factor 2 in host cells, thus inhibiting protein translation (Wilson and Collier, 1992). Pseudomonas aeruginosa exotoxin ribosylates elongation factor 2 and therefore inhibits protein synthesis in a similar fashion (Wilson and Collier, 1992). Legionella pneumophila encodes seven known effectors that inhibit host protein synthesis via inhibition of host elongation factors or via currently unknown mechanisms (Barry et al., 2013; Belyi et al., 2006; 2008; Fontana et al., 2011; Shen et al., 2009).
B. Immune responses during pathogenic manipulation
Although bacterial pathogens manipulate host cells processes and therefore limit the immune response against these pathogens, successful immune responses are still mounted against many bacterial pathogens. Importantly, these immune responses are required for protection against infection. Several immune pathways are important for controlling Yersinia spp. infections, including the inflammasome, cytokine production, and T cell responses (Bohn and Autenrieth, 1996; Brodsky et al., 2010). Shigella spp. activate IFNγ production via IL-12 and IL-18 signaling that are critical for the survival of infected mice (Pore et al., 2012; Sansonetti et al., 2000; Way et al., 1998). P. aeruginosa infection induces inflammasome activation and neutrophil recruitment, both of which limit bacterial replication and dissemination (Hirche et al., 2008; Sutterwala et al., 2007). Like
P. aeruginosa infection, Legionella pneumophila infection is controlled by a number of cytokines, the activation of the inflammasome, and neutrophil recruitment (Brieland et al., 1998; Ren et al., 2006; Tateda et al., 2001b).
Thus, many pathogens express toxins, secretion systems, or effector proteins to block and manipulate host cell process, including the immune response, responses are still mounted against these pathogens and are critical for pathogen control and clearance.
4 Little work, however, has uncovered how a successful immune response is mounted during pathogenic manipulation of host cells. Some pathways have been elucidated.
Manipulation of MAPK and NF-κB signaling during Y. pseudotuberculosis infection results in a pro-inflammatory form of cell death known as necroptosis that has been shown to activate the immune response in infected mice (Philip et al., 2014). Likewise, signaling by pattern recognition receptors on host cells during protein translation inhibition induce or increase induction of a number of proinflammatory genes (Dunbar et al., 2012; Fontana and Vance, 2011; Fontana et al., 2012; McEwan et al., 2012). Yet, these phenomena have not been shown to be important in all bacterial infections and other pathways may exist to combat pathogenic manipulation. To study this phenomenon, we have utilized the pathogenic bacterium L. pneumophila.
C. Legionella pneumophila ecology and infection
Legionella pneumophila is a species of Gram-negative bacteria of the genus Legionella, which is found ubiquitously in freshwater environments including ponds, lakes, and streams (Fields, 1996). It parasitizes and replicates within various species of freshwater amoeba, including Hartmannella vermiformis and Acanthamoeba castellani. L. pneumophila can form planktonic biofilms on surfaces in freshwater environments (Piao et al., 2006). In its natural environment, L. pneumophila is generally harmless to mammalian hosts and no documented disease was caused by L. pneumophila until the
20th century. Modern technology has allowed for the collection and aerosolization of vast quantities of fresh water for use in devices such as air conditioners, cooling towers, and misters. If the source of water used for aerosolization is contaminated with L. pneumophila, the bacteria become aerosolized where mammalian hosts can inhale them. This can lead to a severe form of pneumonia, known as Legionnaires’ disease, or cause a milder, flu-like illness known as Pontiac fever (Chandler et al., 1977; Fraser et
5 al., 1977; McDade et al., 1977; Winn et al., 1978). L. pneumophila was first discovered in
Philadelphia in 1976 when 200 people attending the convention of the American Legion fell ill with a mysterious pneumonia that killed 34 individuals (Fraser et al., 1977;
McDade et al., 1977). The source of infection was determined to be a contaminated air conditioner at the Bellevue hotel where many of the convention-goers were housed.
Since then, many efforts have been made to prevent contamination of aerosolizing machines, although L. pneumophila infection still occurs. Cooling towers have been implicated as having the greatest ability to spread infection, as contaminated aerosols can travel up to 6 kilometers and infect individuals over a wide radius (Mouchtouri et al.,
2010; Nguyen et al., 2006; Vance et al., 2009). L. pneumophila is one of the leading causes of community-acquired pneumonia (Musher and Thorner, 2014; Rietschel et al.,
1994). There is no immunization for L. pneumophila; however, treatment with antibiotics resolves most infections (Bardy et al., 2003; Garau et al., 2010; Neville et al., 2013; Yu et al., 2004).
Once in the lung of an individual, L. pneumophila encounters specialized macrophages of the airway space, known as alveolar macrophages (Chandler et al., 1977; Krachler et al., 2011; Roy and Mocarski, 2007; Vance et al., 2009). Infection of a single cell by L. pneumophila lasts approximately 16-24 hours, allowing for approximately five rounds of intracellular replication (Abu Kwaik et al., 1993; Cornelis, 2006; Horwitz, 1983; Horwitz and Silverstein, 1980; Viboud and Bliska, 2005). After this time, L. pneumophila is released from host cells via an unknown mechanism, resulting in death of the host cell.
The bacteria are then able to disseminate to new cells. It is thought that some individuals have been exposed to L. pneumophila multiple times, as evidenced by anti-L. pneumophila serum titers of antibodies in the human population (Borella et al., 2008;
Boshuizen et al., 2006; Cornelis, 2006; Galyov et al., 2010; Nagelkerke et al., 2003).
6 This indicates that most people control infection without subsequent disease. In those that cannot control infection, however, L. pneumophila disseminates throughout the body and bacteria can be found in the kidneys and heart (De Haan and Hirst, 2004; Hamon et al., 2012; Lowry and Tompkins, 1993). Patients who succumb to L. pneumophila infection often die of multi-organ failure (Fraser et al., 1977; Kahn et al., 2002; Roy and
Mocarski, 2007). Smokers, individuals with chronic lung disease, recent transplant recipients, and immunocompromised individuals are at the greatest risk of acquiring L. pneumophila (Boer et al., 2006; Kool et al., 1998; Singh et al., 1993; Yu et al., 2004).
The risk of L. pneumophila infection increases with age, but infection can occur in both children and young adults (Centers for Disease Control and Prevention (CDC), 2011). In
2009, there were 3,522 reported cases of legionellosis in the US. Etiological studies estimate that only a third of all infections with Legionella spp. are reported and estimate that there are approximately 8,000 to 18,000 cases of legionellosis per year (Centers for
Disease Control and Prevention (CDC), 2011). The prevalence of legionellosis has increased over the past decade, highlighting a need for better preventative measures, including proper water treatment (Parr et al., 2014).
D. Legionella pneumophila type IV secretion system
Legionella pneumophila expresses a specialized type IVb secretion system (T4SS) that is encoded by dot/icm genes and is essential for virulence as well as survival of L. pneumophila inside of host cells (Berger and Isberg, 1993; Hubber and Roy, 2010;
Marra et al., 1992). This secretion system is similar, but genetically distinct from the type
IVa secretion system expressed by P. aeruginosa and other Gram-negative bacteria
(Vincent et al., 2006). Many of the components of this T4SS share genetic sequence and protein structure homology with the type IVa/VirB secretion system of such bacteria as Brucella spp. and Agrobacterium tumefaciens (Juhas et al., 2008). Despite this
7 homology, many aspects of the secretion system are unique and distinct from the type
IVa secretion system (Nagai and Kubori, 2011). L. pneumophila lacking key components of the T4SS is still phagocytosed by amoebae and macrophages, but once inside host cells are unable to establish a replicative niche and are quickly shuttled to a degradative lysosome (Berger and Isberg, 1993; Marra et al., 1992). About 30 genes encode the
T4SS of L. pneumophila (Vincent et al., 2006). Deletion of some of these genes, such as dotA, dotB, icmC, and icmD render the T4SS completely inactive, whereas mutations in other genes such as icmS, dotF, icmF, and dotU allow for some T4SS activity to still occur, but for intracellular growth to be partially impaired (Nagai and Kubori, 2011; Vogel et al., 1996). Beyond replication and translocation, the dot/icm protein, IcmT, has a demonstrated role in allowing for L. pneumophila egress from host cells at the end of infection, but the exact mechanism for this escape is unknown (Molmeret et al., 2002).
Research has resolved some of the base structure of the T4SS, mainly those components that are embedded in bacterial membranes or are localized to the bacterial cytosol (Sutherland et al., 2013). Unlike the type III secretion system and the type IVa secretion system, no syringe-like structure has been visualized for the T4SS of L. pneumophila and no pore-formation into host-derived membranes has been directly assessed (Nagai and Kubori, 2011). Therefore, the exact mechanism of T4SS-mediated translocation into the cytosol of host cells has not been described.
For pathogenesis, L. pneumophila uses its T4SS to translocate bacterial effector proteins from the cytosol of the bacterium, through the phagosomal membrane into the cytosol of the host cell (Ensminger and Isberg, 2009; Ninio and Roy, 2007). To date, over 300 different effector proteins have been identified via screening, but the vast majority of these effectors have not been characterized (Burstein et al., 2009). The overall activity of the T4SS on host cells, however, has been described (Roy and
8 Mocarski, 2007). Translocation by the T4SS occurs in as little as five minutes post phagocytosis and translocation of effectors is thought to occur throughout the infectious cycle (Nagai et al., 2005; Ninio and Roy, 2007). Not all effectors are translocated at all times, however, and many effector proteins have opposing activities, suggesting that translocation is temporally orchestrated (Bardill et al., 2005; Kubori et al., 2010). Unlike with other secretion system, the cues for this temporal structure as well as the overall signal to induce translocation by the T4SS of L. pneumophila are not known (Qiu and
Luo, 2013). Likewise, no definitive motif that targets bacterial proteins to the T4SS to be translocated has been identified, although many effectors have “disorganized” C-termini
(Nagai et al., 2005). Other effectors, however, do not contain this disordered C-terminus and attachment of the disordered C-terminus alone to reporter constructs does not confer translocation (Nagai et al., 2005). Many of the T4SS effectors contain eukaryotic protein domains, such as ankyrin repeats and serine/threonine kinase domains (Ge et al., 2009; Pan et al., 2008). Likewise, the genes encoding these effectors have a lower
GC content than other L. pneumophila genes on the bacterial chromosome, indicating that many effectors have been acquired via horizontal gene transfer, although this has not been directly demonstrated (Juhas et al., 2008).
E. Type IV secretion system effectors
The T4SS and many of the effectors are involved in preventing phagosomal maturation, as evidenced by a lack of maturation markers on the Legionella containing vacuole
(LCV) (Roy et al., 1998). The T4SS promotes the assembly of the LCV, which is comprised of ER-derived vesicles and resembles an ER-like compartment by electron and fluorescent microscopy (Nash et al., 1984; Tilney et al., 2001). Much of vesicular transport in eukaryotic cells is controlled by the activity of small, Ras-like GTPases such as Arf1 (Goody and Itzen, 2013). Interestingly, many of the characterized T4SS effectors
9 alter the activity of GTPases, often by mimicking the activity of guanine nucleotide exchange factors (GEF) or GTPase activating proteins (GAP) and therefore can recruit or exclude various Rabs to the LCV (Goody and Itzen, 2013). RalF, one of best- characterized effectors, recruits the host GTPase, Arf1, to the LCV, although deletion of
RalF does not alter infection in mammalian macrophages (Amor, 2004; Nagai et al.,
2005). The effectors DrrA, SidD, LepB, LidA, AnkX, Lem3, and VipD have been demonstrated to interact with various host Rabs that are crucial regulators of ER-Golgi trafficking (Goody and Itzen, 2013). Similar to RalF, deletion of a single effector does not greatly alter infection in host macrophages, although minor effects on the LCV are evidenced. In fact, deletion of over 70 of the known effectors in combination does not alter L. pneumophila growth in murine macrophages, suggesting a large amount of redundancy in the activity of various effectors (O'Connor et al., 2011). Indeed, many effectors have overlapping activities or similar outcomes to pathogenesis. This redundancy appears to be required for a large host range, as deletion of various groups of effectors alters L. pneumophila replication in certain species of amoeba, but not in others (Ensminger et al., 2012; O'Connor et al., 2011). Currently, only one T4SS effector has a demonstrated, non-redundant role: SdhA (Creasey and Isberg, 2012). This effector is involved in preventing PlaA-induced lysis of the LCV during infection, and deletion of this effector releases L. pneumophila into the cytosol where it is degraded by autophagosomes, thus limiting growth of SdhA-deficient L. pneumophila in vitro
(Creasey and Isberg, 2012).
Although many of the effectors described have roles in preventing degradation of L. pneumophila and the creation and maintenance of the LCV, other T4SS effectors have different activities. The effector RomA is a methyltransferase that enters the nucleus of host cells and directly trimethylates histone H3 at the K14 residue, thereby globally
10 altering the chromatin landscape of infected host cells (Rolando et al., 2013). Autophagy of L. pneumophila can be blocked by the activity of the effector RavZ, which inactivates the host protein Atg8 (Choy et al., 2012). The effector LegK1 directly phosphorylates
IκBα on the serine 32 and 36 residues, ultimately leading the activation of NF-κB (Ge et al., 2009). Like the effectors that alter ER-Golgi trafficking, however, deletion of these effectors either singly or in combination does not alter bacterial replication in murine macrophages.
Many groups have observed a greater than 90% reduction in host protein synthesis during L. pneumophila infection that depends on the presence of a functional T4SS
(Barry et al., 2013; Belyi et al., 2006; McCusker et al., 1991). Currently, seven effectors have been described to inhibit host protein translation (Figure 1-2). The effectors Lgt1,
Lgt2, and Lgt3 are all glycosyltransferases that modify the host factor eEF1A, allowing for translational initiation but blocking elongation (Belyi et al., 2006; 2008). SidI binds and inactivates eEF1A, but can also bind the host factor eEF1Bγ (Shen et al., 2009).
SidL, Pkn5, and Lpg1489 block host protein translation via unknown mechanisms (Barry et al., 2013; Belyi et al., 2013). Of note, these effectors are toxic to mammalian cells as demonstrated in ectopic expression assays (Fontana et al., 2011). Deletion of five or all seven of the known effectors, however, does not restore translational activity in infected cells and does not alter bacterial replication in vitro (Barry et al., 2013; Fontana et al.,
2011). These data suggest that other effectors may block host protein translation.
Alternatively, macrophages treated with rapamycin and infected with avirulent L. pneumophila translate less protein than control-infected cells, and cells infected with virulent L. pneumophila exhibit suppressed mTOR signaling, suggesting that host mTOR signaling may also diminish translational activity in infected cells (Ivanov and Roy, 2013).
11 F. The immune response to Legionella pneumophila
Despite the ability of the T4SS of L. pneumophila to alter many of the aspects of host cell physiology, including chromatin accessibility, ER-Golgi trafficking, authophagy, and protein translation, mammalian host cells still mount an immune response to virulent L. pneumophila (Friedman et al., 2002; Spörri et al., 2008). This immune response includes the production of many proinflammatory cytokines, the recruitment of innate and adaptive immune cells to the site of infection, and even the induction of a form of proinflammatory cell death (Casson and Shin, 2013; Neild and Roy, 2004; Tateda et al.,
2001a). The immune response to L. pneumophila is critical both in vitro and in vivo in the control and eventual clearance of bacteria from the host, as deletion or manipulation of many key immune pathways leads to subsequent increases in bacterial burden, increased bacterial growth in host phagocytes, and, in severe cases, the death of infected animals (Archer and Roy, 2006; Brieland et al., 1998; 1995; Derré and Isberg,
2004; Hawn et al., 2006; Spörri et al., 2006).
Many of the downstream signaling pathways activated by L. pneumophila in host cells have been characterized during in vitro and in vivo infection. The production of the majority of cytokines in vitro depends heavily on the activity of the adaptor protein myeloid differentiation primary response 88 (MyD88), and macrophages deficient for
MyD88 have markedly reduced or absent production of many proinflammatory cytokines
(Archer and Roy, 2006; Shin et al., 2008). MyD88-deficient mice are the most susceptible to L. pneumophila infection in vivo and die around 14 days post infection
(Archer et al., 2010). The adaptor protein TIR-domain-containing adaptor-inducing interferon-beta (TRIF) plays a minor role in vitro in the induction of type I interferon and other cytokines, though the role of TRIF in vivo has never been investigated (Shin et al.,
2008; Stetson and Medzhitov, 2006). The kinase receptor-interacting serine/threonine-
12 protein kinase 2 (Rip2) contributes mildly to IL-6 production in vitro and helps clear bacterial CFUs as well as induce production of IL-6, KC, and G-CSF in vivo (Archer et al., 2010; Frutuoso et al., 2010; Shin et al., 2008). IRF3 is induced by L. pneumophila infection and leads to the production of type I interferon (Lippmann et al., 2011; Stetson and Medzhitov, 2006). In vivo, this signaling pathway has little to no effect on bacterial burden.
Many of the aforementioned signaling pathways converge upon classical immune signaling pathways. MyD88, TRIF, and Rip2 all lead to the induction of NF-κB signaling as measured by IκB degradation (Losick and Isberg, 2006; Shin et al., 2008).
Interestingly, in the absence of these signaling adaptors, IκB degradation still occurs during WT L. pneumophila infection, but not T4SS-deficient bacteria, suggesting that additional signaling pathways lead to NF-κB induction (Shin et al., 2008). MyD88, TRIF, and Rip2 lead to the induction of MAPK signaling, including ERK1/2 and the kinase c-
Jun N-terminal kinase (JNK), which like NF-κB are also activated by other, unknown
T4SS-dependent signals as well as in response to T4SS-dependent protein translation inhibition (Fontana et al., 2012; Shin et al., 2008).
Toll-like receptor (TLR) signaling during L. pneumophila infection has been extensively studied both in vivo and in vitro. Although TLR4 is known to recognize bacterial LPS,
LPS isolated from L. pneumophila is not a strong activator of TLR4 signaling (Archer and
Roy, 2006; Lettinga et al., 2002). TLR2 is activated by outer wall components of L. pneumophila and TLR2 signaling leads to the production of many of the cytokines seen in response to L. pneumophila in vitro (Archer and Roy, 2006; Hawn et al., 2006; Shin et al., 2008). During L. pneumophila infection in vitro, TLR9 is stimulated upon recognition of CpG DNA motifs enriched in bacterial genomes (Archer et al., 2010; Newton et al.,
13 2007). Although L. pneumophila expresses flagellin, determining a role for TLR5 signaling in vitro is complicated by the absence of TLR5 on bone marrow-derived macrophages (Chandler et al., 1977; Hayashi et al., 2001; Means et al., 2003). In vivo, the absence of TLR5 does not alter the course of pulmonary infection (Archer et al.,
2010). In fact, mice deficient for either TLR2, TLR4, TLR5, or TLR9 demonstrate no defect or only a slight defect in controlling pulmonary L. pneumophila infection (Archer et al., 2010; Hawn et al., 2007). Even mice triply deficient in TLR2, 4, and 9 show a minimal defect in control. This is in stark contrast to the dramatic phenotype observed in MyD88- deficient mice during infection, which are deficient in TLR signaling as well as IL-1 family signaling. No role has been determined for TLRs 3, 7, and 8 that recognize synthetic viral motifs. Likewise, no role has been investigated for TLRs 11, 12, or 13. In humans, certain mutations in TLR5 are associated with an increased prevalence of legionellosis
(Hawn et al., 2003). This is in contrast to the phenotype of TLR5-deficient mice, indicating that the role of various immune pathways may differ between mouse models of L. pneumophila infection and clinical manifestations of disease (Hawn et al., 2007).
Various intracellular PRRs are activated during WT L. pneumophila infection, but not with bacteria lacking a T4SS (Berrington et al., 2010; Neild and Roy, 2004; Shin and
Roy, 2008). This T4SS-dependent intracellular activation is thought to be important for the distinction between virulent and avirulent L. pneumophila infection (Shin et al., 2008).
Nod1 and Nod2, which signal via Rip2, are activated in response to the cell well components diaminopimelic acid (DAP) and muramyl dipeptide (MDP), respectively, in the cytosol of host cells (Girardin et al., 2003a; 2003b; Inohara et al., 2005). Deletion of
Nod1 and Nod2 in vivo leads to different effects on pathogenesis; deletion of Nod1 enhances disease, while deletion of Nod2 limits infection (Berrington et al., 2010;
Frutuoso et al., 2010). RIG-I and MDA5 are activated by WT L. pneumophila infection
14 (Monroe et al., 2009). Both RIG-I and MDA5 signal via MAVS to induce the production of
IFNβ.
Additionally, intracellular pathways may sense the effects of the T4SS on host cell processes. Deletion of five of the effectors that inhibit protein translation alters the proinflammatory genes expressed in response to infection, notably the transcription of
Gmcsf and Il23 (Fontana et al., 2011). Perturbations in mTOR signaling may partially drive the response to protein translation inhibition (Ivanov and Roy, 2013). Interestingly, deletion and manipulation of the known intracellular signaling pathways does not fully explain the difference in immune responses between virulent L. pneumophila and bacteria lacking a functional T4SS (Shin et al., 2008). Whether the host directly senses other, specific components of the T4SS or if other effects on the host cell, such as manipulation of ER-Golgi trafficking, activate the immune response is not known.
G. Inflammasome activation during Legionella pneumophila infection
The inflammasome is a large, multi-protein complex that forms in the cytosol of cells upon induction by various infectious or stress-induced signals. Many different inflammasomes have been characterized, but all inflammasome activation leads to the same downstream consequences: release of IL-1 family members and an inflammatory form of cell-death known as pyroptosis (Lamkanfi and Dixit, 2009; Moltke et al., 2013).
Activation of the inflammasome is a two-step process. First, NF-κB induction leads to the translation of many of the components of the inflammasome, including the upstream
NLRs and downstream caspases and IL-1 family members (Rathinam et al., 2012).
NLRs are thought to be sensors that either directly or indirectly recognize stress or infectious components. Some of these NLRs, such as NLRC4, can then directly oligomerize and recruit caspase-1 (Broz et al., 2010). Others, such as NLRP3, require
15 the adaptor protein ASC to bridge the interaction between NLR and caspase
(Mariathasan et al., 2006; Martinon et al., 2002). Both caspase-1 and caspase-11 are involved in inflammasome activation during L. pneumophila infection (Akhter et al., 2012;
Case et al., 2009; Casson et al., 2013; Ren et al., 2006; Zamboni et al., 2006). Various
NLRs are activated by infection and, importantly, inflammasome activation is exquisitely dependent on the T4SS (Casson et al., 2013). L. pneumophila lacking a functional T4SS fail to activate the inflammasome and no release of IL-1 family members or cell death is observed. In vivo, mice lacking caspase-1 and caspase-11 have increased bacterial burdens compared to the WT counterparts, but IL-1 family members can still be detected during infection, suggesting that other enzymes or pathways can mimic inflammasome activation in vivo (Barry et al., 2013; Casson et al., 2013).
The earliest characterized inflammasome important for L. pneumophila infection is the
Naip5/NLRC4 inflammasome (Derré and Isberg, 2004; Zamboni et al., 2006). Naip5, in conjunction with NLRC4, binds monomers of L. pneumophila flagellin (Kofoed and
Vance, 2011). In C57BL/6 mice, this inflammasome is restrictive to growth and, thus, bacteria expressing flagellin are rapidly cleared in vivo and replicate poorly in macrophages derived from these mice in vitro (Molofsky et al., 2006; Ren et al., 2006).
The discovery of Naip5 as an intracellular flagellin sensor occurred because although B6 mice are restrictive, A/J mice and their macrophages are permissive for replication of
WT L. pneumophila. A/J mice have a polymorphism in the Naip5 gene, resulting in a hypomorphic allele (Beckers et al., 1995; Dietrich et al., 1995; Diez et al., 2003; Wright et al., 2003). Either deletion of Naip5 from mice or deletion of the L. pneumophila flagellin gene flaA, results in restored replication in mice and macrophages (Coers et al.,
2007; Molofsky et al., 2006; Ren et al., 2006). As activation of this inflammasome
16 requires the T4SS, it is assumed the flagellin monomers are translocated into the cytosol of host cells, although this has never been fully demonstrated.
In the absence of the Naip5/NLRC4/flagellin axis, inflammasome activation still occurs in a less robust and less rapid fashion (Case et al., 2009; 2013; Casson et al., 2013). Part of this remaining response is controlled by NLRP3, an inflammasome that requires the adaptor ASC (Case et al., 2013; Casson et al., 2013). What exactly NLRP3 senses during infection has not been described, although a variety of other signals, such as intracellular reactive oxygen species, uric acid crystal, extracellular ATP, and other cellular stressors lead to NLRP3 activation (Leemans et al., 2011).
The interferon-inducible protein AIM2 recognizes cytosolic dsDNA, but is not normally activated by infection with L. pneumophila (Fernandes-Alnemri et al., 2009; Hornung et al., 2009; Roberts et al., 2009). The AIM2 inflammasome is induced, however, during infection with the SdhA mutant of L. pneumophila (Aachoui et al., 2013; Creasey and
Isberg, 2012). It is not known how AIM2 ligands enter the cytosol, but it is assumed that cytosolic degradation of bacteria leads to the release of genetic content.
More recently, caspase-11 has been demonstrated to be activated by L. pneumophila infection (Akhter et al., 2012; Barry et al., 2013; Case et al., 2013; Casson et al., 2013).
The only known activator of caspase-11 is LPS as it can bind directly to caspase-11 in the absence of an NLR (Hagar et al., 2013; Kayagaki et al., 2013; Shi et al., 2014).
Caspase-11 activation during L. pneumophila infection leads to the release of IL-1α and death of the host cell (Case et al., 2013; Casson et al., 2013). Interestingly, deletion of caspase-11 in murine macrophages ameliorates not only IL-1α release and cell death, but also diminishes IL-1β release, suggesting that caspase-11 activation feeds into
17 caspase-1-mediated responses (Case et al., 2013; Casson et al., 2013). It has not been directly shown that L. pneumophila LPS is the signal for caspase-11 activation.
H. The cytokine response to Legionella pneumophila
As a consequence of the various signaling cascades and PRRs activated by L. pneumophila, a variety of cytokines are produced during infection and many of these cytokines are critical for controlling and clearing infection. TNF, which binds the TNF receptor, is produced both in vitro and in vivo downstream of MyD88 and NF-κB signaling and is critical for limiting pulmonary infection (Brieland et al., 1998). In vitro,
TNF restricts L. pneumophila growth in macrophages via an unknown mechanism
(Coers et al., 2007). Importantly, human patients on TNF therapeutics that inhibit or limit
TNF signaling are more susceptible to L. pneumophila infection (Aringer et al., 2009;
Beigel et al., 2009; Jinno et al., 2009).
IL-12 is produced in response to L. pneumophila infection and, like TNF, is protective
(Brieland et al., 1998; 2000). IL-12 leads to the production of IFNγ (Hsieh et al., 1993).
Mice deficient for IFNγ contain high bacterial burdens, although whether IFNγ deficiency is fatal in mice has not been reported (Heath et al., 1996; Spörri et al., 2006). IL-18 can lead to the production of IFNγ from natural killer (NK) cells during L. pneumophila infection; however, blocking IL-18 has no impact on bacterial burdens (Archer et al.,
2009; Brieland et al., 2000; Spörri et al., 2008). Blocking both IL-12 and IL-18 during infection leads to greater bacterial burdens than blocking either alone, suggesting synergy between these two cytokines (Brieland et al., 2000).
Type I interferon and IL-6 are produced in response to L. pneumophila infection. In vitro, type I interferon restricts bacterial replication in macrophages; however, the effect of
18 deleting the type I interferon receptor, IFNAR, has a mild to absent phenotype during L. pneumophila infection (Ang et al., 2010; Plumlee et al., 2009). Type I interferon signaling may be more important in the absence of IFNγ signaling (Lippmann et al., 2011). A role for IL-6 has not been demonstrated during L. pneumophila infection, but is protective in other models of bacterial pneumonia (Jones et al., 2006; Sutherland et al., 2008; van der
Poll et al., 1997).
Beyond cytokines, chemokines are produced and play a role in controlling L. pneumophila infection. The chemokine KC is mildly protective during infection as it recruits neutrophils to the site of infection (Tateda et al., 2001b). Blockade of CXCR2, the receptor for KC, has a greater impact on infection than blocking KC alone (Tateda et al., 2001b). These data indicate a role for other chemokine ligands of CXCR2 during infection. Whether other chemokine receptors are critical for control of L. pneumophila infection, such as CCR2 or CXCR4 which recruit other immune cell types like monocytes, has not been investigated (Hasenberg et al., 2013).
Both IL-1α and IL-1β are produced during L. pneumophila infection in vitro and in vivo.
IL-1 signaling in vivo rapidly recruits neutrophils to the airway space during infection
(Barry et al., 2013; Casson et al., 2013; LeibundGut-Landmann et al., 2011). Deletion of the IL-1R or IL-1α or antibody-mediated neutralization of IL-1 is deleterious to the host as evidenced by increased bacterial burdens and delayed clearance (Barry et al., 2013;
Casson et al., 2013). Of note, there is a case study of a patient taking Anakinra, an IL-1 antagonist, who became infected with L. pneumophila during therapy, suggesting a role for IL-1 in human as well as murine L. pneumophila infection (Scholtze et al., 2011).
19 I. The role of IL-1α and IL-1β during infection
IL-1α and β are part of larger family of cytokines, known as the IL-1 family (Dinarello,
1996). IL-1β and IL-1α are the founding members of the IL-1 family and were first described in the 1980s (Auron et al., 1984; March et al., 1985). Unlike other conventional cytokines, IL-1 family members are translated by cytosolic ribosomes and do not traffic through the ER-Golgi network (Stevenson et al., 1992). The transcription and translation of IL-1α and β, like many cytokines, require the induction of NF-κB signaling. Once translated, these cytokines exist in a cytosolic, inactive pool in their pro-forms.
Translation of IL-1β and IL-1α alone does not lead to their activation. Instead, cleavage and activation of IL-1α and β require the activity of the inflammasome (Lamkanfi and
Dixit, 2009). Cleavage is absolutely required for the release of IL-1β and its ability to bind the IL-1R (Black et al., 1988). IL-1α does not require cleavage for release and can bind and activate the IL-1R in either its full pro-form or mature form (Chen et al., 2007;
Howard et al., 1991). The binding affinity to IL-1R is higher for pro- and mature IL-1α than for IL-1β (Symons et al., 1995).
The IL-1R is expressed on a variety of cell types, including epithelial cells, endothelial cells, astrocytes, and T cells (Dinarello, 2013). The IL-1R itself is comprised of two chains. IL-1R1 physically binds the ligands IL-1α and IL-1β, as well as a non-signaling competitive antagonist IL-1Ra (Greenfeder et al., 1995). The second chain in the receptor is the IL-1Racp - a common chain that, in part, also forms the receptors for IL-
33 and IL-36. Signaling by the IL-1R requires MyD88, which is important for many of the
TLRs (Weber et al., 2010). Induction of IL-1R signaling leads to the activation of NF-κB and MAPK pathways, ultimately leading to transcription of pro-inflammatory genes
(Dinarello, 1996). IL-1R signaling on epithelial and endothelial cells leads to the production of IL-6 and various chemokines, including KC (Kirnbauer et al., 1989;
20 Nakamura et al., 1991; Schröder et al., 1990; Sironi et al., 1989). For immune cells, IL-1 signaling on DCs leads to CD86 and CCR7 expression and IL-1 on T cells leads to the activation of T cells as well as Th17 skewing (Lichtman et al., 1988; Pang et al., 2013;
Veldhoen et al., 2006). In vivo, injection of IL-1 leads to the rapid recruitment of neutrophils to the site of injection as well as the production of IL-6 and certain chemokines (Dinarello, 1996). IL-6 production induced by IL-1 leads to the induction of fever in humans and other mammals. Beyond immune responses, injection of IL-1β can diminish sexual receptivity in female, but not in male rats, highlighting the broad and systemic responses that IL-1 imparts on host organisms (Yirmiya et al., 1995).
IL-1 and the IL-1R signaling have been shown to be protective in a variety of infectious models, including Salmonella enterica Typhimurium, Yersinia pseudotuberculosis,
Staphylococcus aureus, and Mycobacterium tuberculosis, as well as others (Moltke et al., 2013). Interestingly, although IL-1α and IL-1β bind the same receptor, deletion of either IL-1α or IL-1β alone reduces resistance to M. tuberculosis in mice, suggesting a non-redundant role for the two cytokines (Mayer-Barber et al., 2011). In contrast, IL-1 has been shown to be deleterious to the host in a number of infectious models as well as models of autoinflammatory disease (Dinarello, 1996). For example, IL-1β increases bacterial burden during Burkholderia pseduomallei infection (Ceballos-Olvera et al.,
2011). For autoinflammatory disorders, a role for IL-1 or inflammasome-associated genes has been demonstrated for cryopyrin-associated periodic syndrome, familial cold autoinflammatory syndrome, familial Mediterranean fever, rheumatoid arthritis, and others (de Jesus et al., 2015; Jesus and Goldbach-Mansky, 2014; Ting et al., 2006).
Therapy for many of these syndromes and diseases relies on IL-1 antagonists, such as anakinra (Dinarello and van der Meer, 2013). Thus, IL-1 signaling is highly proinflammatory in a variety of settings. This inflammation can limit infection, but can
21 have pathological consequences Therefore, activation of the inflammasome and production of IL-1 family members is tightly regulated.
J. Pulmonary immune responses
The mammalian lung is a highly vascularized structure containing a variety of cell types.
Alveolar macrophages are tissue resident macrophages of the lung. Many alveolar macrophages exist outside the body in the alveolar sacs (Hussell and Bell, 2014). During homeostasis they clear debris and surfactant from the airway space, facilitating respiration. Alveolar macrophages require GM-CSF for both their presence and function in the lung (Stanley et al., 1994). Their absence impairs lung function and in humans leads to a disease known as pulmonary alveolar proteinosis (Stanley et al., 1994).
Although thought to be free of association with other cells, a subset of alveolar macrophages were recently shown to have intimate contact with the airway epithelium and were termed sessile alveolar macrophages (Westphalen et al., 2014). These macrophages are defined by their localization and no markers currently distinguish between these two types of alveolar macrophages. Alveolar macrophages express TLRs and are known to make cytokines such as TNF (Hussell and Bell, 2014). During
Legionnaire’s disease, alveolar macrophages harbor L. pneumophila as evidenced by detection of Gram-negative bacteria by electron microscopy in human patients as well as by detection of GFP+ L. pneumophila in a murine model of infection (Horwitz and
Silverstein, 1981; LeibundGut-Landmann et al., 2011). Alveolar macrophages, then, are thought to be the primary reservoir of L. pneumophila during pulmonary infection.
Whether or not other cell types harbor L. pneumophila, however, has not been fully investigated.
22 There are two main types of epithelial cells in the mammalian lung, termed airway epithelial cells (AEC) type I and type II. AECIs physically perform the act of respiration by governing gas exchange (Borok et al., 2002; Johnson et al., 2002). AECIIs produce surfactant to lower surface tension facilitating gas exchange (Fleming et al., 1994). AECI and AECII cells are often connected via gap junctions, and signaling cascades initiated in one cell can migrate via these junctions to adjacent cells (Guo et al., 1999). In vitro, epithelial cells can be infected with L. pneumophila and the bacteria can adhere to the outside of epithelial cells, but no evidence for epithelial cell infection has been demonstrated in vivo (Maruta et al., 1998; Mody et al., 1993; Prashar et al., 2012).
Epithelial cells produce chemokines, such as KC, during L. pneumophila infection, which are important to recruit immune cells to the site of infection (Tateda et al., 2001b).
Neutrophils are not generally present in the lung tissue itself, but due to the vascularized nature of the tissue these cells are found in vessels throughout the tissue. Neutrophils are considered highly inflammatory cells in most tissues, including the lung (Mizgerd,
2002). Neutrophils are often associated with tissue damage, as they are present at sites of damage to the epithelial barrier (Puljic et al., 2007). Neutrophils are recruited into the lung tissue and subsequently into the airway space itself by chemokines produced by epithelial cells during inflammation (Tateda et al., 2001a; 2001b). Neutrophils are highly bactericidal and L. pneumophila products can be detected in neutrophils during infection in vivo (LeibundGut-Landmann et al., 2011). Depletion of neutrophils with the antibody
RB68C5 leads to a reduction in IL-18 and a subsequent decrease in IFNγ during L. pneumophila infection, ultimately leading to an increase in bacterial burdens (Spörri et al., 2008). Depletion with the RB68C5 clone, however, perturbs inflammatory monocytes, though the role of monocytes during infection has not been investigated on
23 its own (Daley et al., 2008). Thus, neutrophils are important for controlling infection, both for their ability to destroy live bacteria and their contribution to the cytokine milieu.
Inflammatory monocytes and natural killer (NK) cells are recruited to the lung tissue during infection (LeibundGut-Landmann et al., 2011; Spörri et al., 2008). NK cells respond to IL-12 and IL-18 produced during infection and rapidly produce IFNγ that can be detected as early as two days post infection in mice (Archer et al., 2010). This IFNγ production is critical for control of infection. The role of inflammatory monocytes during L. pneumophila infection has not been investigated; however, inflammatory monocytes are important in other models of bacterial infection (Serbina et al., 2008). Inflammatory monocytes are capable of producing a wide array of proinflammatory cytokines, including TNF, IL-6, and IL-12 (Serbina et al., 2008). These cells are capable of differentiating into other cells types, including dendritic cells (DC) (León et al., 2004;
Yang et al., 2014). Inflammatory monocytes have recently been demonstrated to be bactericidal in certain models of infection (Narni-Mancinelli et al., 2011). The recruitment of inflammatory monocytes during other infections, and in other tissues, is often dependent on the expression of CCR2 on the monocytes and production of CCR2 ligands, such as CCL2, CCL7, and CCL12 in the infected site (Serbina et al., 2008).
However, not all monocytes express CCR2; thus, certain subsets of monocytes are recruited independently of this pathway.
A variety of cells exist in the mammalian lung, but their roles in controlling L. pneumophila infection have not been investigated. Conventional DCs (cDC) exist in the lung during both homeostasis and infection (Ang et al., 2010). In vitro, cDCs rapidly undergo apoptosis when infected with virulent L. pneumophila in a manner that is independent of the inflammasome, suggesting that they may not be able to harbor L.
24 pneumophila in vivo (Nogueira et al., 2009). Eosinophils, although well characterized for their ability to exacerbate the symptoms of asthma in the lung, have no demonstrated role in either controlling or worsening L. pneumophila infection (Busse and Sedgwick,
1992; Stevens et al., 2007). B and T cells are recruited to the lung during infection and in other infectious models tertiary lymphoid structures (BALT) are formed weeks into pulmonary infection, which contain a large number of these cells (Sminia et al., 1989;
Trunk and Oxenius, 2012). Antibody, and therefore B cell, responses are induced during
L. pneumophila infection and passive transfer of immune sera protects mice from a low- dose challenge model of infection (Weber et al., 2012). T cells have been demonstrated to produce both IFNγ and IL-17 during L. pneumophila infection, but the contribution of either of these responses has not been fully investigated (Trunk and Oxenius, 2012).
K. Dissertation Aims
It is evident, then, that rapid and robust immune responses are mounted in response to
L. pneumophila infection both in vitro and in vivo. In vitro, this response is diminished, but still present, in response to L. pneumophila lacking a functional T4SS. In vivo, the immune response to T4SS-deficient L. pneumophila is nearly undetectable and these bacteria are rapidly cleared from the host. This leads to two conclusions. First, the proinflammatory immune response to L. pneumophila both in vitro and in vivo is greatly enhanced by the presence of the T4SS. And second, although the protein translation inhibition mediated by effectors of the T4SS should limit the immune response against L. pneumophila, an immune response is still mounted and is vital for control and ultimate clearance of bacteria from the host. This paradoxical T4SS-dependent immune response to L. pneumophila leads to two questions. Which cells are targeted by the
T4SS of L. pneumophila during infection? And how does the immune system overcome the T4SS-dependent block in protein translation to produce cytokines?
25
In chapter 3 of this dissertation, I investigate the cell types targeted by the T4SS of L. pneumophila by using a fluorescence-based translocation assay. I find that alveolar macrophages and neutrophils are the primary reservoir for L. pneumophila during pulmonary infection as they are both targeted by the T4SS and contain viable L. pneumophila. Although the T4SS can actively inject dendritic cells in vitro, I see no injection into dendritic cells in vivo. Likewise, I see no injection into alveolar epithelial cells. Surprisingly, although only a small percentage of neutrophils harbor viable L. pneumophila, neutrophils are the largest reservoir of viable bacteria in vivo.
In chapter 4, I investigate the ability of injected cells and uninjected, bystander cells to produce proinflammatory cytokines in response to infection by combining our β- lactamase (BlaM)-based assay with intracellular cytokine staining. Injected cells are able to produce IL-1α and β, but are poor producers of the cytokines TNF, IL-6, IL-12p40, and do not increase expression of CD86. Instead, cells not targeted by the T4SS during infection, including non-targeted alveolar macrophages and neutrophils, produce TNF in response to L. pneumophila infection in vivo and increase expression of CD86.
Importantly, this TNF production and CD86 expression is partially dependent on IL-1R signaling, suggesting that the IL-1 produced by alveolar macrophages and neutrophils during infection is critical for the response of uninfected, bystander cells. Together, these data suggest a coordinated effort between cells that harbor viable L. pneumophila during infection and uninfected, bystander cells to produce a successful immune response in spite of pathogenic manipulation of host cell processes. Part of the communication between infected and uninfected cells depends on the production of IL-1α and β. This signaling pathway may be more broadly applicable to other infections, as IL-1 is produced in response to variety of bacterial pathogens and aids in bacterial clearance.
26
Bacterium or bacterial product! ! ! PRR PRR ! !
E. coli! Adaptor Adaptor
Y. pestis! S. flexneri! V. parahaemolyticus! B. antracis! NF-κB! MAPK! S. dysenteriae! P. aeruginosa! C. diphtheriae! L. pneumophila!
Nucleus! Ribosome!
Proinflammatory Infected cell! cytokines!
Figure 1-1. Bacterial pathogens inhibit host cell signaling and protein translation during infection. A number of bacterial pathogens encode virulence factors that inhibit host cells processes. NF-κB signaling is inhibited by virulence factors from Escherichia coli, Yersinia spp., and Shigella flexneri. MAPK signaling is also inhibited by proteins from Yersinia spp. and S. flexneri as well as Vibro parahaemolyticus and Bacillis anthracis. Downstream of receptor signaling Shigella dysenteriae, Pseudomonas aeruginosa, Corynebacterium diphtheria, and Legionella pneumophila encode virulence factors that inhibit host protein synthesis. Despite this, immune responses are still mounted against these pathogens as evidenced by proinflammatory cytokine production, indicating that hosts have mechanisms to bypass pathogenic manipulation.
27 TLR2/5/9*! L. pneumophila! !
MyD88/ TRIF!
NF-κB! MAPK!
T4SS effectors!
Naip5, NLRP3, ! Flagellin!
NLRC4! & others!
!
ASC!
! NOD1/2! Caspase 1/11! RIP2!
Lgt 1/2/3! ! Pro-IL-1! SidI/L, Pkn5! Lpg1489! !
Nucleus! Ribosome! !
IL-1! Cell death!
TNF, IL-6, IL-12, & others! !
Figure 1-2. Legionella pneumophila activates multiple immune pathways, but inhibits protein synthesis in infected cells. Infection with Legionella pneumophila activates multiple signaling pathways, leading to the induction of NF-κB and MAPK signaling. These pathways induce the transcription of proinflammatory genes. The T4SS effectors Lgt1, Lgt2, Lgt3, SidI, SidL, Pkn5, and Lpg1489 block the translation of these proteins; yet, TNF, IL-6, and IL-12 are produced during L. pneumophila infection. In tandem, bacterial products in the cytosol of infected cells activates the inflammasome, leading to the processing of mature IL-1 and death of the host cell. The coordination of all these events is poorly characterized.
28 CHAPTER 2
MATERIALS AND METHODS
Ethics statement
All experiments performed in this study were done so in accordance with the Animal
Welfare Act (AWA), the recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The Institutional Animal Care and
Use Committee of the University of Pennsylvania approved all procedures (protocols
#803465, #803459, and #804928).
Bacterial strains and plasmids
All experiments used Legionella pneumophila serogroup 1 strains. For in vitro studies, macrophages, dendritic cells, and epithelial cells were infected with Lp02 (rpsL, hsdR, thyA), a thymidine auxotroph derived from strain Lp01, or ΔdotA or ΔflaA isogenic mutant strains (Berger and Isberg, 1993). Certain experiments also used strains of L. pneumophila with deletions in Lgt1, Lgt2, Lgt3, SidI, and SidL on the Lp02 background
(WTΔ5) or L. pneumophila with deletions in Lgt, Lgt2, Lgt3, SidI, SidL, Pkn5, and
Lpg1489 on the ΔflaA background (Δ7ΔflaA) (Barry et al., 2013; Fontana et al., 2011).
For in vivo studies, mice were infected with JR32-derived (rpsL, hsdR) ΔdotA or ΔflaA isogenic mutant strains (Marra and Shuman, 1989; Ren et al., 2006). For in vivo experiments requiring cell sorting, the aforementioned Lp02 strains were used. For in vitro and in vivo studies L. pneumophila was cultured on charcoal yeast extract agar containing 6.25µg/mL chloramphenicol for 48 hours at 37°C prior to infection (Feeley et al., 1979; Neild et al., 2005). For studies requiring motile L. pneumophila, 48 hour cultures grown on CYE agar were grown overnight in AYE broth containing chloramphenicol with shaking at 37°C until >50% of the bacteria were observed to be
29 motile by light microscopy. Plasmids encoding M45 tagged β-lactamase-RalF fusion protein or M45-tagged β-lactamase were generated as follows. Briefly, the pJB1806 plasmid (RSF1010 ori, tdΔI, Ampr, Cmr) was first modified by cloning the icmR promoter and M45 epitope tag into the EcoRI and BamHI sites (Bardill et al., 2005). The mature
TEM-1 β-lactamase gene (BlaM) was then amplified from a Y. pseudotuberculosis
YopE-BlaM-encoding plasmid using primers that introduced a 5’ BglII site (5’-
AATAAGATCTTGCACCCAGAAACGCTGGTG-3’) and 3’ BamHI site (5’-
GCCTCACTGATTAAGCATTGGGGGATCCAATA-3’
) (Brodsky and Medzhitov, 2008). The resulting PCR product was digested with BglII and
BamHI and cloned into the BamHI site of the pJB1806 PicmR:M45 plasmid to create a plasmid encoding M45-tagged β-lactamase. To generate the plasmid encoding a translational fusion of M45-β-lactamase-RalF, RalF was amplified from Lp01 genomic
DNA using primers that introduced BamHI sites at the 5’ and 3’ ends (5’-
AATAGGATCCGGCATCCAGAAATTGAAAAAGCCC-3’) and (5’-
GAAAAAGGTAGACAATTAAAATTTTAAGGATCCAATA-3’). The resulting PCR product was digested with BamHI and cloned into the BamHI site downstream of gene encoding
M45-BlaM. The resulting plasmids were then electroporated into L. pneumophila and transformed colonies were selected for with chloramphenicol (Marra et al., 1992).
Mice
All mice were purchased from Jackson Laboratories. Mice were maintained in accordance with the guidelines of the University of Pennsylvania Institutional Animal Use and Care Committee. For infections, 8-12 week old male and female mice were anesthetized by intraperitoneal injection of a ketamine/xylazine/PBS solution at a dose of
100mg/kg ketamine and 10mg/kg xylazine. Mice were then infected intranasally with
40µl of a bacterial suspension containing 1-5x106 CFU L. pneumophila or PBS vehicle
30 control. At the indicated time points after infection, mice were sacrificed. To isolate lung airway cells, bronchoalveolar lavage was performed 3-5 times with 1mL of cold PBS each time. Lungs were then excised and digested for 30 minutes at 37°C with occasional shaking in 5mL of PBS containing 5% FBS, 250U/mL of collagenase IV (Worthington
Biochem), and 20U/mL DNAse I (Roche). Lungs were then mechanically homogenized and a single-cell suspension was obtained. To determine bacterial burden, lungs were mechanically homogenized in sterile, distilled H2O and a portion of the lysate was spread onto CYE plates containing either chloramphenicol or streptomycin.
Antibody-mediated neutralizations and depletions
All antibodies were administered by intraperitoneal injection in 500µl PBS. For IL-1 antibody neutralizations 16 hours prior to infection, mice were given 200µg of either isotype antibody (hamster IgG), antibody against IL-1α (α-IL-1α) (ALF-161), IL-1β (α-IL-
1β) (B122), or both (BioXcell).
Cell culture
For macrophages, C57BL/6 mouse bone marrow cells were differentiated in RPMI containing 30% L929 cell supernatant and 20% FBS at 37°C, 5% CO2 in a humidified incubator. The macrophages were replated in RPMI containing 15% L929 cell supernatant and 10% FBS (Casson et al., 2013). For dendritic cells, bone marrow cells were differentiated in RPMI containing 10% FBS, 50µM β-mercaptoethanol, 2mM L- glutamine, and 20ng/mL GM-CSF (Peprotech) (Inaba et al., 1992). Semi-adherent dendritic cells were then isolated and replated in medium lacking GM-CSF. A549 cells and NIH/3T3 cells (ATCC) were cultured in DMEM containing 10% FBS
(Wickremasinghe et al., 1999). For infections, cells were treated with 10-100ng/mL LPS
31 from E. coli strain 055:B5 (Sigma), 0.4µg/mL PAM3CSK4, 10µl of bacterial suspension, or 10µL of PBS vehicle control.
Isolation of inflammatory monocytes and alveolar macrophages
Alveolar macrophages were harvested from the airways of mice by bronchoalveolar lavage in 5 x 1mL cold PBS. For intracellular cytokine staining, cells were centrifuged, counted, and resuspended at 10,000 cells per well in 150µl DMEM + 10% FBS.
Inflammatory monocytes were isolated from the bone marrow of mice using a magnetic negative selection kit (Miltenyi). Inflammatory monocytes were assayed for purity post selection and were consistently 90-95% CD45+, Ly6Chi, Ly6G-, CD11bint to CD11bhi. For intracellular cytokine staining, inflammatory monocytes were resupsended at 30,000 cells per well in 150µl DMEM with 10% FBS.
Flow cytometry, fluorescence-based imaging flow cytometry, and cell sorting
For in vitro experiments, infected cells were lifted and loaded with CCF4-AM (Invitrogen) per the manufacturer’s instructions. Cells were then washed and treated with Live/Dead
Fixable Dead Cell Stain (Invitrogen). Bone marrow-derived dendritic cells were stained with antibodies specific for CD11c and MHCII (eBioscience). To stain for intracellular L. pneumophila, cells were fixed with BD Cytofix, permeabilized with BD Phosflow Perm
Buffer III (BD Biosciences), and then stained with a rabbit polyclonal antibody against L. pneumophila followed by a rabbit-specific secondary antibody tagged to a fluorophore
(Invitrogen). For in vivo studies, lung and airway cells were loaded with CCF4-AM and treated with the Live/Dead stain. Cells were then stained with antibodies specific for the cell surface antigens CD45, CD11c, Ly6G, Ly6C, NK1.1 (BioLegend), MHCII, CD19,
CD3ε, CD31, CD326 (eBioscience), Siglec F, CD11b, and Ter119 (BD Biosciences).
Data were collected on a LSR II flow cytometer (BD Biosciences) and post-collection
32 data were analyzed using FlowJo (Treestar). For fluorescent imaging experiments, data and images were collected on an Amnis ImageStreamX Mark II and data were analyzed using IDEAS software (EMD Millipore), Cells were gated on live singlets that had retained the CCF4-AM dye. Cell sorting experiments were performed on a FACSAria II flow cytometer (BD Biosciences).
Intracellular staining
For in vitro experiments, infected cells were treated with 10µg/mL brefeldin A with or without 2µM monensin (Sigma) three hours prior to harvest. To stain for intracellular cytokines cells were fixed with BD Cytofix/Cytoperm (BD Biosciences), and then stained with antibodies specific for IL-6 (BioLegend), TNF, IL-1α and IL-1β (eBioscience). For in vivo studies, lung and airway cells were resuspended in RPMI or DMEM containing 10%
FBS and 10µg/mL brefeldin A for 3 hours. Intracellular staining was performed as described above. Data were collected on a LSR II flow cytometer (BD Biosciences) and post-collection data were analyzed using FlowJo (Treestar). Cells were gated on live singlets that had retained the CCF4-AM dye. Cell sorting experiments were performed on a FACSAria II flow cytometer (BD Biosciences).
IL-1 Stimulation
For treatment of cells with IL-1, mature recombinant IL-1α, IL-1β, or IL-1α and IL-1β
(eBioscience) combined were added to cells at various concentrations. For intracellular staining of BMDCs, inflammatory monocytes, and alveolar macrophages, IL-1 was added at 100ng/mL for 6 hours with BFA added 3 hours prior to harvest. BMDCs were stained for CD11c, MHCII, and CD86. BMDCs were then fixed and stained for TNF.
Inflammatory monocytes were stained for CD45, Ly6C, Ly6G, CD11b, Ter119, CD86
33 and then fixed and stained for TNF. Alveolar macrophages were fixed and then stained for TNF.
ELISA
Harvested supernatants from cultured cells or BAL were assayed using capture and detection antibodies specific for IL-1α, IL-6, TNF, IL-12p70 (BioLegend), KC (R&D system), and IL-12p40 (BD Biosciences).
RT-PCR
Sorted bone marrow-derived macrophages were centrifuged and resuspended in RLT lysis buffer and RNA was isolated using an RNeasy kit as per kit’s protocol (Qiagen).
RNA was then reverse transcribed into cDNA using Superscript II reverse transcriptase
(Invitrogen). Quantitative PCR was then performed using the prepared cDNA with primers for Il1b (5’-GCA ACT GTT CCT GAA CTA AAC T-3’, 5’-ATC TTT TGG GGT
CCG TCA ACT-3’), Il1a (5’-GCA CCT TAC ACC TAC CAG AGT-3’, 5’-TGC AGG TCA
TTT AAC CAA GTG G-3’), Il6 (5’-GAC TTC CAT CCA GTT GCC TTC TTG G-3’, 5’-CCA
GTT TGG TAG CAT CCA TCA TTT CT-3’), Tnf (5’-GAC GTG GAA CTG GCA GAA
GAG-3’, 5’-TTG GTG GTT TGT GAG TGT GAG-3’), or the housekeeping gene Hprt (5’-
GTT GGA TAC AGG CCA GAC TTT GTT G-3’,5’-GAG GGT AGG CTG GCC TAT AGG
CT-3’). Data were analyzed using by comparing the CT values obtained for each reaction and then comparing them with the appropriate samples’ housekeeping gene to determine relative expression.
Immunoblot analysis
Legionella pneumophila expressing the appropriate reporter plasmids were harvested from a two-day heavy patch and lysed. Lysates were then subjected to SDS-PAGE,
34 transferred to PVDF membrane, and probed with an anti-M45 epitope monoclonal antibody (Obert et al., 1994).
Statistical analysis
Plotting of data and statistical analysis were performed using Graphpad Prism software.
Statistical significance was determined using the unpaired, two-tailed Student’s t test or one-way ANOVA with Tukey post-test. Differences were considered significant if the p value was <0.05.
35 CHAPTER 3
ALVEOLAR MACROPHAGES AND NEUTROPHILS ARE THE PRIMARY RESERVOIRS FOR LEGIONELLA PNEUMOPHILA AND MEDIATE CYTOSOLIC SURVEILLANCE OF TYPE IV SECRETION
This chapter appeared as a published peer-reviewed article titled “Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion” by Alan M. Copenhaver, Cierra N. Casson, Hieu T. Nguyen, Thomas C Fung, Matthew M. Duda, Craig R. Roy, and Sunny Shin. Infection and Immunity, 2014.
36 Abstract
Legionella pneumophila, an intracellular pathogen responsible for the severe pneumonia
Legionnaires’ disease, uses its dot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol, L. pneumophila also activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate control of infection. Thus, the pulmonary cell types that L. pneumophila infects may act not only as an intracellular niche that facilitates its pathogenesis, but also contribute to the immune response against L. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain of L. pneumophila producing a fusion protein consisting of β- lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils are both the primary recipients of T4SS-translocated effectors and harbor viable L. pneumophila during pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced TNF and IL-1α in response to T4SS- sufficient, but not T4SS-deficient, L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir for L. pneumophila and a source of proinflammatory cytokines that contribute to the host immune response against L. pneumophila during pulmonary infection.
Introduction
Legionella pneumophila is a Gram-negative bacterium found ubiquitously in fresh water environments, where it is often found in association with its natural host, protozoan amoebae (Fields, 1996). L. pneumophila has recently become a human pathogen due to modern technologies, such as cooling towers and air conditioners, which can aerosolize
37 fresh water contaminated with L. pneumophila (Fraser et al., 1977; McDade et al., 1977;
Phin et al., 2014). Humans can then inhale these contaminated droplets, thus allowing L. pneumophila to gain access to the pulmonary airway. L. pneumophila infection can lead to a severe bacterial pneumonia known as Legionnaires’ disease (Fraser et al., 1977), with mortality rates approaching 30% (Domínguez et al., 2009).
Once in the lung, L. pneumophila encounters a specialized subset of pulmonary phagocytes called alveolar macrophages (Chandler et al., 1977). Following phagocytosis, the Legionella-containing phagosome avoids endocytic maturation and bacterial degradation and is converted into an ER-derived vacuole that supports bacterial replication (Shin and Roy, 2008). To establish infection, L. pneumophila utilizes its type IV secretion system (T4SS), encoded by the dot/icm genes, to translocate approximately 300 effector proteins into the host cell cytosol (Berger and Isberg, 1993;
Ensminger and Isberg, 2009; Hubber and Roy, 2010; Marra et al., 1992; Nagai, 2002;
Roy et al., 1998; Segal et al., 1998; Vogel et al., 1998). Many of these effector proteins are thought to be involved in recruiting ER-derived vacuoles to the Legionella-containing vacuole or preventing endocytic maturation (Hubber and Roy, 2010). Other effector proteins modulate host cell processes such as autophagy or host protein synthesis
(Barry et al., 2013; Belyi et al., 2006; 2008; Choy et al., 2012; Shen et al., 2009). These virulence activities ultimately prevent destruction of L. pneumophila and allow for its replication within host cells. The T4SS is essential for the ability of L. pneumophila to survive and replicate within host cells, as L. pneumophila mutants lacking a functional
T4SS do not replicate and reside in phagosomes that mature along a canonical endocytic pathway (Berger and Isberg, 1993; Roy et al., 1998).
38 While the Dot/Icm T4SS is essential for L. pneumophila to survive intracellularly and to cause disease, cytosolic immune surveillance systems activate host defense responses to T4SS activity that are critical for the control of L. pneumophila infection (Shin, 2012).
For example, the NAIP5/NLRC4 inflammasome detects T4SS-dependent delivery of flagellin, leading to the caspase-1-dependent secretion of IL-1 family cytokines and pyroptotic cell death (Lightfield et al., 2008; Molofsky et al., 2006; Ren et al., 2006).
Cytosolic detection of T4SS activity is also required for the robust secretion of inflammasome-independent cytokines, such as TNF (Fontana et al., 2011; Shin et al.,
2008; Spörri et al., 2006). The IL-1 of family cytokines and TNF are critical for host defense against L. pneumophila (Barry et al., 2013; Brieland et al., 1995; Casson et al.,
2013; LeibundGut-Landmann et al., 2011). Thus, the cells that interact with L. pneumophila in the lung and receive T4SS-translocated effectors may have a dual role during in vivo infection, in that they can enable intracellular survival of the pathogen and also contribute directly to the immune response by detecting T4SS-translocated products. However, the identities of the pulmonary cell types that interact with L. pneumophila and receive T4SS-translocated effectors are poorly understood.
Alveolar macrophages are thought to be the primary cell type infected by L. pneumophila and to support bacterial replication in vivo (Nash et al., 1984). However, it is unknown whether other immune phagocytes in the lung, such as neutrophils, inflammatory monocytes, or dendritic cells, may also receive T4SS-translocated effectors and contribute to the immune response or support L. pneumophila survival. Previous studies have demonstrated that in addition to alveolar macrophages, L. pneumophila can be detected in neutrophils during pulmonary infection (LeibundGut-Landmann et al., 2011).
Neutrophils are thought to be highly bactericidal, and their presence in the lung and airway space during pulmonary L. pneumophila infection correlates with lower bacterial
39 burden (Barry et al., 2013; Casson et al., 2013; Nauseef, 2007; Tateda et al., 2001a;
2001b). Whether L. pneumophila can survive within neutrophils and translocate T4SS effectors into these cells during pulmonary infection is unknown. L. pneumophila can be taken up by a wide variety of cell types in vitro, such as neutrophils, bone marrow- derived dendritic cells, type I and type II alveolar epithelial cells, endothelial cells, and plasmacytoid dendritic cells (Ang et al., 2010; Horwitz and Silverstein, 1981; Maruta et al., 1998; Mody et al., 1993; Neild and Roy, 2003). However, the efficiency of L. pneumophila replication within these cell types varies greatly, and whether these cell types are injected by the T4SS or productively infected in vivo is unknown. We thus decided to investigate which cell types receive T4SS-translocated effectors and therefore may support L. pneumophila survival and contribute to cytosolic immunosurveillance during pulmonary infection.
Using a FRET-based reporter of T4SS translocation, we were able to detect effector translocation into macrophages, dendritic cells, and airway epithelial cells in vitro. We also demonstrate that only T4SS-injected cells contain viable L. pneumophila, whereas infected cells that have not received T4SS effectors do not contain viable bacteria. In vivo, alveolar macrophages and neutrophils in the airway space and lung tissue were the primary recipients of T4SS-translocated effectors and harbored viable bacteria.
Consistent with the critical role of immune sensing of T4SS activity in triggering host cytokine production, alveolar macrophages and neutrophils from mice infected with
T4SS-competent L. pneumophila, but not T4SS-deficient bacteria, secreted the cytokines TNF and IL-1α, which are known to be important for immune-mediated clearance of infection (Brieland et al., 1995; Casson et al., 2013; LeibundGut-Landmann et al., 2011). We did not observe T4SS-mediated injection into other lung cell populations, including airway epithelial cells and dendritic cells, suggesting that these
40 cells are neither a primary intracellular niche for L. pneumophila, nor do they directly participate in cytosolic immunosurveillance of T4SS activity during lung infection.
Collectively, our data indicate that alveolar macrophages and neutrophils play a dual role as both an intracellular niche and immune mediator during pulmonary L. pneumophila infection.
Results
A reporter system tracks translocation of type IV secretion system effectors by
Legionella pneumophila into mammalian cells
L. pneumophila uses its type IV secretion system (T4SS) to translocate effector proteins into the cytosol of host cells. To track this translocation, we constructed a plasmid in which the well-characterized L. pneumophila icmR promoter drives transcription of a gene encoding a translational fusion of the mature TEM-1 BlaM and the well- characterized T4SS effector protein RalF and introduced this plasmid into L. pneumophila (Figure 3-1A & B) (Gal-Mor et al., 2002; Knapp et al., 2003; Zlokarnik et al.,
1998). We chose RalF because it is translocated into the cytosol of infected cells immediately following intimate interaction of L. pneumophila with host cells (Nagai et al.,
2005; Ninio and Roy, 2007). Following infection of host cells by bacterial strains expressing the BlaM-RalF fusion protein, the enzymatic activity of translocated BlaM-
RalF was detected in host cells by means of the membrane-permeable BlaM substrate
CCF4-AM (Zlokarnik et al., 1998). CCF4-AM consists of coumarin joined to fluorescein by a β-lactam ring. When excited at 409nm, fluorescence resonance energy transfer
(FRET) between coumarin and fluorescein results in green fluorescence emission at
518nm. T4SS-injected BlaM will cleave the CCF4-AM substrate in the host cytosol and eliminate FRET, thus resulting in blue fluorescence emission at 447nm.
41 We generated L. pneumophila strains expressing either BlaM or BlaM-RalF and infected
C57BL/6 bone marrow-derived macrophages (BMDM) with these strains for 8 hours.
Following infection, the cells were loaded with CCF4-AM and analyzed by flow cytometry to determine whether blue fluorescence emitted by cleaved CCF4-AM was detected
(Figure 3-1C). Approximately 20-25% of macrophages infected with BlaM-RalF- expressing WT L. pneumophila and L. pneumophila lacking flagellin (ΔflaA), which evade NAIP5 inflammasome responses, were positive for blue fluorescence resulting from cleaved CCF4-AM, but not following infection with strains lacking a functional T4SS
(ΔdotA). This indicates that CCF4-AM is efficiently cleaved only by BlaM-RalF translocated by T4SS-sufficient bacteria and that BlaM-RalF remaining within bacteria does not generate a detectable signal in this assay. The frequencies of injected cells in
WT and ΔflaA L. pneumophila infections were comparable, although the frequency of injection was consistently lower in WT infections (Figures 3-1C, 3-2A, & 3-5A). The robust detection of injection by WT L. pneumophila is surprising considering that a higher percentage of vacuoles containing WT L. pneumophila fail to avoid rapid endocytic maturation and that flagellin induces NAIP5-dependent cell death in C57BL/6 macrophages (Amer and Swanson, 2005; Amer et al., 2006; Byrne et al., 2013; Molofsky et al., 2006; Ren et al., 2006). Following infection with L. pneumophila strains expressing
BlaM alone, we found that a much lower percentage of macrophages became positive for blue CCF4-AM fluorescence compared to macrophages infected with L. pneumophila expressing BlaM-RalF (Figure 3-1C). Importantly, this small percentage of CCF4-AM positive cells was still dependent on infection with T4SS-sufficient bacteria, suggesting that BlaM lacking a canonical T4SS signal sequence may be inefficiently delivered into the host cytosol by the T4SS.
42 T4SS-injected host cells contain viable Legionella pneumophila.
The T4SS is essential for the survival of L. pneumophila within host cells. To determine whether cells injected with BlaM-RalF contain L. pneumophila, we infected BMDMs with these reporter strains and loaded the cells with CCF4-AM. After loading, the macrophages were fixed, permeabilized, and stained with an antibody specific for L. pneumophila (Figure 3-2A). Infection with all three strains (WT, ΔdotA, ΔflaA) of L. pneumophila resulted in macrophages staining positive for the presence of bacteria. 90-
100% of cells that were positive for BlaM-RalF injection were also positive for L. pneumophila staining in both the WT and ΔflaA strains. With both strains, we detected a subset of cells that was positive for L. pneumophila but translocation of BlaM-RalF was not within a detectible range, revealing heterogeneity in BlaM-RalF translocation at the single cell level. The percentage of cells positive for L. pneumophila but negative for
BlaM-RalF translocation could result from bacteria that failed to successfully translocate
T4SS effectors into the host cell, either because they were non-viable, were not in the transmissive phase, or failed to efficiently evade rapid phagosomal maturation.
To determine whether the L. pneumophila associated with injected or uninjected macrophages were intact or degraded, we infected macropohages with our reporter strains of L. pneumophila and analyzed these macrophages with fluorescence-based imaging flow cytometry (Figure 3-2B and Figures 3-3A). The majority of macrophages infected with the ΔdotA strain showed dim L. pneumophila staining, with multiple small puncta present per cell (Figure 3-2C). Because the ΔdotA mutants are unable to evade endocytic maturation due to their lack of a functional T4SS, punctate staining could result from bacteria that were degraded. Alternatively, punctate staining could represent uninfected cells that had phagocytosed bacterial debris. When we infected macrophages with ΔflaA L. pneumophila encoding a functional T4SS, we again could identify T4SS-
43 injected and uninjected cells. Many of the uninjected cells stained positive for intracellular L. pneumophila (Figure 3-3B) as determined by analysis of the images obtained of infected cells using fluorescent-based imaging flow cytometry, but the majority of these cells exhibited dim, punctate staining similar to the staining seen for
ΔdotA-infected macrophages (Figure 3-2B & C). This may represent cells containing bacteria that had not successfully evaded endocytic maturation or uninfected cells that had phagocytosed bacterial debris. In contrast, the majority of injected cells showed a single bright punctum of L. pneumophila staining, indicating the presence of an intact bacterium that had not been transported to a hydrolytic compartment.
To test whether injected macrophages contain viable L. pneumophila, we sorted infected macrophages that were either positive or negative for the cleaved CCF4-AM signal, lysed the macrophages, and enumerated bacterial CFUs in these distinct cell populations (Figure 3-2D). T4SS-injected cells recovered from a ΔflaA L. pneumophila infection contained the vast majority (nearly 6 bacteria for every injected BMDM) of viable L. pneumophila as determined by CFU count. Uninjected cells from the same infection contained a minimal number of viable L. pneumophila (less than 1 bacterium for each uninjected BMDM), comparable to the number of viable bacteria recovered from a
ΔdotA infection. To exclude the possibility that the uninjected cells contained viable L. pneumophila that lost the BlaM-RalF reporter plasmid encoding chloramphenicol resistance, we also plated cell lysates in the presence or absence of chloramphenicol
(Figure 3-4A). The CFUs obtained on plates with and without chloramphenicol were indistinguishable, suggesting that the plasmid is stably maintained during in vitro infection in the absence of antibiotics. Collectively, our data indicate that viable bacteria are associated primarily with cells that have received translocated BlaM-Ralf, whereas uninjected cells are either not infected or contain non-viable bacteria.
44
Translocation by the type IV secretion system can be detected in dendritic cells and alveolar epithelial cells in vitro
L. pneumophila can infect a variety of cell types in vitro, including dendritic cells and airway epithelial cells (Maruta et al., 1998; Mody et al., 1993; Neild and Roy, 2003). We thus examined whether T4SS-mediated translocation into these cell types could be detected using the β-lactamase reporter system. At a given MOI, as compared to infected BMDMs, we detected a much lower frequency of T4SS-mediated injection into bone marrow-derived dendritic cells (BMDCs) infected with WT or ΔflaA L. pneumophila
(Figure 3-5A & B). We also infected A549 cells, an alveolar epithelial cell line, and detected a low frequency of injection into these cells (Figure 3-5C).
Other researchers have noted an increase in bacterial uptake by host cells when L. pneumophila is grown under conditions that promote bacterial motility (Ren et al., 2006).
Indeed, infection of macrophages with motile L. pneumophila resulted in a large increase in the frequency of injected macrophages, as the percentage of injected cells increased from 10% to more than 80% (Figure 3-6B). In contrast, in A549 cells, we did not observe an increase in injection regardless of bacterial motility in that 1.3% of cells infected with non-motile or motile L. pneumophila were injected (Figure 3-6A). For all cell types, the percentage of cells injected by the T4SS of L. pneumophila increased over time (Figure
3-5). In all instances, cleaved CCF4-AM signal required expression of a functional T4SS, suggesting that the β-lactamase reporter operates in a T4SS-dependent manner in a variety of cell types. As we observed more robust injection into macrophages and dendritic cells than into non-phagocytic alveolar epithelial cells, these data suggest that both increased cell contact and efficient uptake by professional phagocytes contribute to the ability of L. pneumophila to efficiently translocate effector proteins.
45
Legionella pneumophila translocates bacterial effectors into alveolar macrophages and neutrophils during pulmonary infection
Our data suggest that the L. pneumophila T4SS can translocate effectors into alveolar epithelial cells, dendritic cells, and macrophages during in vitro infection. During pulmonary infection, replicating L. pneumophila can be detected in alveolar macrophages (Nash et al., 1984), indicating that alveolar macrophages receive T4SS- translocated effectors. However, whether alveolar epithelial cells, dendritic cells, and other cell types receive T4SS-translocated effectors in vivo has not been investigated.
To identify the cells that receive translocated effectors during a permissive model of in vivo infection, we intranasally infected C57BL/6 mice with ΔflaA L. pneumophila expressing BlaM-RalF, as WT L. pneumophila do not establish a productive infection in mice that encode a functional NAIP5 allele as CFUs do not increase and are cleared from C567BL/6 mice (Figure 3-4D) (Molofsky et al., 2006; Ren et al., 2006). In this model, similar to WT L. pneumophila infection of A/J mice expressing a hypomorphic
NAIP5 allele, the lungs of C57BL/6 mice exhibit approximately a one-log increase in
ΔflaA L. pneumophila CFUs by 24-48 hours post-infection (Figure 3-4C & D) (Case et al., 2009). The mice are subsequently able to control infection, with minimal bacterial
CFUs detected in the lungs by 5 days post-infection (Figure 3-4C & D) (Molofsky et al.,
2006). Expression of the plasmid containing BlaM-RalF did not affect the replication of
ΔflaA L. pneumophila in vivo (Figure 3-4C). After intranasal inoculation with L. pneumophila, we performed bronchoalveolar lavage to isolate cells from the airway space at various time points and loaded them with CCF4-AM to detect T4SS-mediated injection of BlaM-RalF. At four hours post-inoculation, we detected T4SS-mediated translocation of β-lactamase activity in nearly 50% of cells recovered from the airway of mice infected with ΔflaA L. pneumophila (Figure 3-7A). Greater than 95% of the T4SS-
46 injected cells were alveolar macrophages, as indicated by their expression of CD11c and
Siglec F (Stevens et al., 2007; Sung et al., 2006). Similar results were obtained with WT
L. pneumophila at this time point (data not shown). Consistent with our in vitro data, we did not observe injection of BlaM-RalF in mice infected with the ΔdotA strain, which is unable to translocate effectors into host cells and cannot establish a productive infection in vivo.
At later times post-infection, we detected recruitment of a large population of neutrophils to the airway space of ΔflaA L. pneumophila-infected mice that did not occur in mice infected with ΔdotA L. pneumophila (Figures 3-8A & B), consistent with previous studies indicating that neutrophil recruitment is T4SS-dependent (Barry et al., 2013; Casson et al., 2013; Frutuoso et al., 2010; LeibundGut-Landmann et al., 2011). When we identified cells injected by L. pneumophila in the airway space at 24 hours post-infection, we again identified alveolar macrophages as being positive for T4SS-mediated injection, but we could also identify injected cells that expressed high levels of Ly6G and were negative for MHC class II (Figure 3-7B). We determined these injected Ly6G+ cells to be neutrophils, as they expressed low levels of Ly6C, a cell surface marker highly expressed on inflammatory monocytes (Figure 3-3C) (Fleming et al., 1993; Sunderkötter et al., 2004). The frequency of injected neutrophils was much lower than that of injected alveolar macrophages (Figure 3-7C). However, due to the large influx of neutrophils, the total number of injected neutrophils was comparable to or greater than the total number of injected alveolar macrophages at 24, 48, and 72 hours post-infection (Figure 3-7D).
As we could detect robust T4SS-mediated injection of BlaM-RalF into cells of the airway space, we wanted to determine whether cells within the lung interstitium were injected by
L. pneumophila as well. Notably, we again observed T4SS-mediated injection into
47 alveolar macrophages and neutrophils within lung homogenates (Figure 3-9A). As in the airway space, we detected a large influx of neutrophils into the lung tissue of ΔflaA- infected mice, but not in mice infected with the ΔdotA strain of L. pneumophila (Figures
3-8C & D). Though in vitro we observed T4SS-mediated injection into bone marrow- derived dendritic cells as well as A549 alveolar epithelial cells (Figure 3-5B & C), we did not detect injection into lung dendritic cells or CD326+ airway epithelial cells, suggesting that L. pneumophila does not efficiently infect or translocate effectors into these cell types during a permissive mouse model of infection (Figure 3-9A). We also did not observe injection into inflammatory monocytes, plasmacytoid dendritic cells, eosinophils,
B cells, T cells, NK cells, or endothelial cells within the lung tissue at any time assayed post infection (Figure 3-3C & D). The frequency of T4SS-injected neutrophils in the lung tissue was much lower than that seen in alveolar macrophages, similar to what we observed in the airway space (Figure 3-9B). At four hours post-infection, the majority of cells receiving T4SS-translocated effectors in the lung tissue were alveolar macrophages, but at later times, many of the T4SS-injected cells were neutrophils
(Figure 3-9C). Importantly, L. pneumophila recovered at 48 and 72 hours post-infection retained the reporter plasmid, indicating that the plasmid is stably maintained during in vivo infection even in the absence of antibiotic selection (Figure 3-4B). A previous study examining a non-permissive model of C57BL/6 mice infected with WT L. pneumophila also found that CD45-negative cells or lung epithelial cells did not appear to have taken up L. pneumophila, with alveolar macrophages appearing to be the primary cells infected at early time points post-infection, followed by infection of recruited neutrophils at one day post-infection (LeibundGut-Landmann et al., 2011), suggesting that in both permissive and non-permissive mouse models, similar lung cell types are infected.
48 Neutrophils and alveolar macrophages in the lungs of infected mice harbor viable
Legionella pneumophila bacteria and produce cytokines
Alveolar macrophages are thought to be the primary cell type that is infected by L. pneumophila and supports bacterial replication (Nash et al., 1984). A previous study using a non-permissive model of C57BL/6 mice infected with WT L. pneumophila also found that that recruited neutrophils take up L. pneumophila in the lung, but whether L. pneumophila could translocate effectors into neutrophils or survive within these cells was not examined (LeibundGut-Landmann et al., 2011). As we observed that both alveolar macrophages and neutrophils in L. pneumophila–infected lungs were injected by the
T4SS, we sought to determine whether in addition to alveolar macrophages, neutrophils also contained viable bacteria. We therefore sorted total alveolar macrophages and neutrophils from the lungs of mice infected intranasally with L. pneumophila and enumerated bacteria from lysed cells. As a comparison, we also sorted total inflammatory monocytes, a population of cells negative for T4SS injection, from these infected mice as well. As expected, alveolar macrophages isolated twenty-four hours after infection contained viable bacteria (Figure 3-10A). In contrast, inflammatory monocytes contained very few viable L. pneumophila, consistent with the lack of observed T4SS-dependent translocation into these cells (Figure 3-10A). Interestingly, neutrophils from ΔflaA-infected mice contained viable bacteria at a frequency consistent with the extent of injection, suggesting that injected neutrophils harbor viable bacteria in the airway and lung tissue. Although the absolute frequency of viable L. pneumophila in alveolar macrophages was greater than within neutrophils (nearly 10 bacteria per 100 alveolar macrophages vs. 1 bacterium per 100 neutrophils), the higher absolute numbers of neutrophils present during infection results in the unexpected finding that neutrophils actually contain nearly twice as many viable bacteria as alveolar macrophages (Figure 3-10B). To examine whether this was also the case at the peak of
49 pulmonary bacterial load, we sorted alveolar macrophages and neutrophils from mice 48 hours PI (Figure 5C &D). As with 24 hours PI, although a higher frequency of alveolar macrophages contained viable L. pneumophila, in total there were more L. pneumophila in neutrophils than alveolar macrophages.
The presence of neutrophils in the airway space during infection correlates with lower bacterial burden, thought to be in part due to their potent bactericidal activity (Barry et al., 2013; Casson et al., 2013; LeibundGut-Landmann et al., 2011). However, as our data suggest that L. pneumophila inject and survive within neutrophils, thus potentially activating cytosolic immunosurveillance pathways within these cells, we next examined whether or not infected neutrophils may also contribute to the T4SS-dependent production of pro-inflammatory cytokines important for bacterial clearance. To test this, following intranasal infection with either ΔdotA or ΔflaA L. pneumophila, we measured cytokines secreted by alveolar macrophages isolated at four hours post-infection or neutrophils isolated at twenty-four hours post-infection. Alveolar macrophages from
ΔflaA-infected mice secreted TNF and IL-1α, whereas macrophages from ΔdotA– infected mice did not. Interestingly, neutrophils from mice infected with ΔflaA L. pneumophila also secreted substantial amounts of TNF and IL-1α, whereas neutrophils from mice infected with ΔdotA L. pneumophila did not secrete detectable levels of IL-1α and secreted significantly less TNF, which correlates with the lack of detectable cytokine production observed during pulmonary infection with ΔdotA L. pneumophila (Fig 3-10E &
F). Intriguingly, these data demonstrate that in addition to alveolar macrophages, neutrophils also produce proinflammatory cytokines in the context of T4SS-competent L. pneumophila infection. This indicates that in addition to their potent bactericidal activity, neutrophils may contribute to the control of infection by other immune effector mechanisms such as cytokine production.
50 Conclusion
Here, I describe the creation of a sensitive and faithful fluorescent reporter to track the activity of the T4SS of L. pneumophila within in host cells. Using this system, I am able to detect injection by the T4SS into cells both in vitro and in vivo. In vitro, injected harbor viable L. pneumophila, demonstrating that this fluorescent detection system correlates with active infection in host cells. Cells not injected by the T4SS do not contain viable L. pneumophila. Injection is detectable in macrophages, dendritic cells, and alveolar epithelial cells in vitro. These data indicate that injection can occur into a variety of cell types. In a pulmonary model of L. pneumophila infection in vivo, however, injection is only detected in alveolar macrophages and neutrophils at multiple times PI. Importantly, viable CFUs are obtained from these cell types, indicating that L. pneumophila are able to survive and potentially replicate within these cells. Beyond alveolar macrophages and neutrophils being a niche for L. pneumophila in vivo, the cells also secrete IL-1α and
TNF in response to infection with virulent, but not avirulent infection. These data indicate a dual role for phagocytic cells during pulmonary L. pneumophila infection as both intracellular niche and important activators of subsequent immune responses.
Acknowledgements
We thank Andree Hubber for generating the pJB908 plasmid containing the icmR promoter and M45 epitope tag, the laboratory of Michael Betts, specifically Morgan
Reuter, for the use of their imaging flow cytometer and the help required to operate it,
Sara Cherry and members of the Brodsky laboratory for helpful discussions, and Igor
Brodsky and Liam Bradley for critical review of the manuscript. This work was supported in part by National Institutes of Health grants K99/R00AI087963 (to S.S.),
R01AI0487700 (to C.R.R.), T32GM007229 (to A.M.C.), American Lung Association grant RG-268528-N (to S.S.), the University of Pennsylvania University Research
51 Foundation (to S.S.), American Heart Association grant 13BGIA14780070 (to S.S.), and the Roy and Diana Vagelos Scholars Program in Molecular Life Sciences (to M.M.D.).
This material is based upon work supported by the National Science Foundation under
Grant No. DGE-0822 (C.N.C., graduate research fellowship). Cierra N, Casson aided in in vivo experiments as well as all experiments involving cell sorting and was instrumental in the writing and formatting of this manuscript. Hieu T. Nguyen aided in all in vivo experiments. Thomas C. Fung provided data and protocols for assays involving dendritic cells. Matthew M. Duda helped in enumerating L. pneumophila CFUs.
52
Figure 3-1. β-lactamase translocation into cells requires fusion to RalF and a functional type IV secretion system. (A) Schematic of the BlaM-RalF expression plasmid. β- lactamase (BlaM) is tagged with the M45 peptide and fused to the N-terminus of the effector protein RalF. The icmR promoter drives the expression of this fusion protein. (B) Lysates from WT, ΔdotA, and ΔflaA L. pneumophila expressing either β-lactamase (BlaM) alone or Blam-RalF fusion reporter plasmids were blotted with an α-M45 antibody to detect reporter protein expression. (C) Bone marrow-derived macrophages (BMDMs) were treated with PBS (Unfx) or infected with WT, ΔdotA, and ΔflaA strains of Legionella pneumophila (Lp) expressing BlaM or BlaM-RalF for 8 hours with an MOI=5. Cells were then loaded with CCF4-AM and analyzed by flow cytometry. Cells positive for cleaved CCF4-AM (blue) fluorescence are gated and the frequency of injected cells for each condition is denoted within the gate. Bar graph shows mean ± standard error of the mean (SEM) of triplicate wells. Representative of three independent experiments.
53 A Unfx WT ΔdotA ΔflaA 0.227 4.53e-3 2.33 12.1 0.15 0.859 18.6 100 0.2 0.0 2.4 12.1 0.1 0.0354 0.1 0.9 18.6 80 +
Lp 60 40 (%)
Injection (blue) 20 99.799.7 0.10.0227 60.060 25.525.5 70.470.4 29.429.4 29.529.5 51.051 Legionella Unfx dotA Δ injected injected uninjected uninjected WT ΔflaA B C D Uncleaved Cleaved Legionellag Mergeg 100 800 p<0.05 Unfx
75 s 600 Φ ΔdotA 50 400 ΔflaA
uncleaved 25 CFU/ 100 M 200 (%) multiple puncta ns ΔflaA cleaved dotA dotA Δ Δ injected injected uninjected uninjected ΔflaA ΔflaA
Figure 3-2. Viable Legionella pneumophila are predominantly associated with macrophages positive for T4SS-dependent translocation. (A) BMDMs were left untreated (Unfx) or infected with WT, ΔdotA, or ΔflaA Lp for 8 hours with an MOI=5. Cells were then loaded with CCF4-AM, fixed, permeabilized, and stained with a polyclonal antibody against Lp. Cells were analyzed by flow cytometry. The percentages of cells positive for Lp as determined by staining and cells positive for T4SS injection are denoted within the gates. (B & C) Cells were treated similarly to (A), but infected with an MOI=10 for 4 hours and analyzed using an Amnis ImageStream imaging flow cytometer. (D) BMDMs were left untreated, treated with LPS, or infected with ΔdotA or ΔflaA Lp for 8 hours with an MOI=5 in the presence of exogenous thymidine (100µg/mL) and loaded with CCF4-AM. Samples infected with ΔdotA were sorted in bulk for loaded cells. Cells infected with ΔflaA were then sorted based on uncleaved or cleaved CCF4-AM signal, lysed, and plated on CYE plates. CFUs were then enumerated. Bar graphs show mean only or mean ± SEM of triplicate samples. Representative of 2 independent experiments. ns=not significant.
54
Figure 3-3. Gating strategies for fluorescence-based imaging flow cytometry and conventional flow cytometry. (A & B) BMDMs were left untreated or infected with ΔdotA or ΔflaA Lp for 8 hours with an MOI=10. Cells were then loaded with CCF4-AM, fixed, permeabilized, and stained with an antibody against Lp. Cells were then analyzed using the Amnis ImageStream imaging flow cytometer. Focused singlets that took up CCF4- AM were selected as shown in (A) and the frequency of injected (cleaved) cells was deteremined by gating as well as the amount of staining for Lp in either uninjected (uncleaved) or injected cells. The frequency of injection and Lp+ cells was then determined as shown in (B). (C & D) Murine lung was homogenized and single cell suspensions were stained with extracellular markers to identify various populations of immune cells including alveolar macrophages (AMΦ) (CD45+, Ly6G-, Ly6C-, CD11c+, Siglec F+), neutrophils (NΦ) (CD45+, Ly6G+, Ly6Clow), inflammatory monocytes (iMC) (CD45+, Ly6G-, Ly6Chi), conventional DCs (cDC) (CD45+, Ly6G-, Ly6C-, CD11c+, Siglec F-, MHCII+) T cells (CD45+, CD3ε+, CD19-), B cells (CD45+, CD3ε-, CD19+) and NK cells (CD45+, CD3ε-, CD19-, NK1.1+). Airway epithelial cells (AEC) (CD45-, CD31+, CD326+) and lung endothelial cells (LEC) (CD45-, CD31+, CD326-) were also identified. Gates shown are representative of the gates used throughout this study.
55
Figure 3-4. The BlaM-RalF expression plasmid is stably maintained during both in vitro and in vivo infection. (A) BMDMs were infected with ΔdotA or ΔflaA Lp for 8 hours with an MOI=5. Cells were then sorted based on cleaved CCF4-AM signal, lysed, and plated on CYE agar plates containing either streptomycin or chloramphenicol. CFUs were then enumerated. Bar graph shows mean ± SEM. N=3. (B) Mice were infected intransally with ΔdotA or ΔflaA Lp for 48 or 72 hours. The lungs of the infected mice were then homogenized and the cells were lysed and plated on CYE plates as in (A). Points represent individual mice with the mean denoted as a line. N=3-4 mice per group. (C) Mice were infected with infected intranasally with 1x106 JR32 ΔflaA, 5x106 JR32 ΔflaA, or 5x106 JR32 ΔflaA expressing the BlaM-RalF plasmid. CFUs were enumerated on CYE plates containing streptomycin. N=3 mice per group. (D) Mice were infected with 5x106 CFUs of BlaM-RalF-expressing strains of JR32 WT, ΔdotA, or ΔflaA L. pneumophila and bacterial burdens were enumerated. N= 2-4 mice per group.
56
A Unfx WT ΔdotA ΔflaA 30 Unfx WT ΔdotA 20 ΔflaA 0.1460.146 11.611.7 0.2020.202 18.318.3 10 (%) Injected CCF4-AM (green) Injection (blue) 4 8 12 Hours PI B Unfx WT ΔdotA ΔflaA 10 8 6
0.1360.136 2.472.47 0.1180.118 4.014.01 4
(%) Injected 2 CCF4-AM (green) Injection (blue) 4 8 12 Hours PI C Unfx WT ΔdotA ΔflaA 6
4
0.522 1.24 0.551 2.83 0.522 1.24 0.551 2.83 2 (%) Injected (%) CCF4-AM (green) Injection (blue) 4 8 12 Hours PI
Figure 3-5. Legionella pneumophila T4SS-dependent translocation is detected in dendritic cells and alveolar epithelial cells during in vitro infection. BMDMs (A), bone marrow-derived dendritic cells (BMDCs) (B), and A549 cells (C) were left untreated or infected with WT, ΔdotA, or ΔflaA Lp for 4, 8, or 12 hours with an MOI=5. Cells were then loaded with CCF4-AM and analyzed for injection by flow cytometry. Representative plots show injection 8 hours post infection. Graphs show mean ± SEM of triplicate wells. Representative of 2 independent experiments, N=3.
57 1.28 0.309 1.33
0.433 1.25 0.266
10.3 0.315 15.1
0.284 83.4 0.256
Figure 3-6. Bacterial motility increases the frequency of T4SS-injected macrophages, but not T4SS-injected alveolar epithelial cells. A549 cells (A) and BMDMs (B) were left untreated, treated with LPS, or infected with WT, ΔdotA, or ΔflaA Lp at MOI=5 for 4 hours that were either non-motile or grown under conditions that induce motility. Cells were then loaded with CCF4-AM and analyzed for injection by flow cytometry. Bar graphs are mean ± SEM. Representative of 2 independent experiments, N=3.
58 A Unfx ΔdotA ΔflaA
99 CD11c 99.0 0.6510.651 1.291.29 47.847.8 CCF4-AM (green) Injection (blue) Siglec F B Unfx ΔdotA ΔflaA
47.247.2 49.049 Ly6G CD11c 0.6050.606 1.001 4.494.49 CCF4-AM (green) Injection (blue) Siglec F MHCII CD 50 Alveolar MΦs Neutrophils 40 10x104
30
20 5x104 (%) Injected (%)
10 Total injected cells
4 24 48 72 4 24 48 72 Hours PI Hours PI
Figure 3-7. Alveolar macrophages and neutrophils in the airway space are injected by the Legionella pneumophila T4SS. C57BL/6J mice were infected intranasally with PBS vehicle control, ΔdotA Lp, or ΔflaA Lp. At 4 (A) or 24 (B) hours post infection, cells in the airway space were isolated, enumerated, loaded with CCF4-AM, and stained for cell surface markers. Cells were then analyzed for injection by flow cytometry and cell surface marker expression. Representative of 2 independent experiments, N=4 mice per group. (C & D) Alveolar macrophages (MΦ) and neutrophils from the airway space of mice infected with ΔflaA Lp were isolated at 4, 24, 48, or 72 hours post infection and loaded with CCF4-AM. The percentage (C) and total number (D) of cells injected by Lp in each population was quantified. Graphs show mean ± SEM. N=3-4 mice per group.
59
Figure 3-8. Infection with ΔflaA Legionella pneumophila recruits immune cells to the lungs of infected mice. Mice were infected intranasally with ΔflaA (A & C) or ΔdotA (B & D) Lp. At various times post infection, airway space cells (A & B) and lung cells (C & D) were separately isolated, enumerated, and stained for extracellular markers. The frequency and number of alveolar macrophages, neutrophils, monocytes, conventional DCs, T cells, B cells, and NK cells was calculated. Graphs show mean ± SEM. N=3-4 mice per group.
60 A B ΔdotA ΔflaA Alveolar MΦs 20 Alveolar MΦs Neutrophils 15
0.6380.638 7.637.63 CD11c 10 (%) Injected (%) 5 Neutrophils
4 24 48 72 Hours PI 0.7200.72 1.911.91
Ly6G C 20x105
5 cDCs 15x10
10x105
0.45 0.494 5x105 Total injected cells CD11c 0.450 0.494
4 24 48 72 Airway epithelial cells Hours PI
CD326 0.3730.372 0.7250.723
Injection (blue)
Figure 3-9. The Legionella pneumophila T4SS injects alveolar macrophages and neutrophils in the lung. (A) Mice were infected intranasally with ΔdotA or ΔflaA L. pneumophila for 24 hours. Lung cells were then isolated, loaded with CCF4-AM, and stained. Flow plots are pre-gated on the denoted cell populations. Representative of 3 independent experiments, N=4 mice per group. (B & C) Mice were infected with ΔflaA Lp and lung cells were isolated at various times post infection. The percentage (B) and total number (C) of cells injected by Lp in each population was quantified. Graphs show mean ± SEM. N=3-4 mice per group. cDC=conventional dendritic cell.
61 24 Hours PI
48 Hours PI 2.0 0.5 0.4 1.5 0.3 1.0 0.2 0.5 0.1
EF Alveolar MΦs 4 Hours PI Neutrophils 24 Hours PI
Figure 3-10. Alveolar macrophages and neutrophils from infected mice contain viable Legionella pneumophila and secrete cytokines. (A+C) Mice were intransally infected with ΔdotA or ΔflaA Lp. At 24 (A) or 48 (B) hours post-infection, the lungs and airway space were homogenized and single cell suspensions were stained for cell surface markers to identify alveolar macrophages, neutrophils and inflammatory monocytes. These cell populations were sorted using a flow cytometer, lysed, and plated on CYE agar to ennumerate L. pneumophila CFUs. (B) The frequency of viable bacteria per cell type in (A) was then multiplied by the frequency of the appropriate cell type found in a total of 106 lung cells. (D) Similarly, the frequency of viable bacteria per cell type in (C) was multiplied by the frequency of the appropriate cell type found in a total of 106 lung cells. (E) Alveolar macrophages were isolated by bronchoalveolar lavage 4 hours after intranasal infection with ΔdotA or ΔflaA Lp and cultured overnight. Supernatants were collected and TNF and IL-1α concentrations were determined by ELISA. (F) Neutrophils were isolated and sorted at 24 hours PI from mice treated as in (A) and then cultured overnight as in (C). Graphs show mean ± SEM. *p<0.05, ***p<0.0005
62 CHAPTER 4
IL-1 INDUCES INNATE BYSTANDER CYTOKINE PRODUCTION TO BYPASS
BACTERIAL BLOCKADE OF HOST PROTEIN SYNTHESIS
This chapter has been submitted and reviewed as an article entitled “IL-1 induces innate bystander cytokine production to bypass bacterial blockade of host protein synthesis” by Alan M. Copenhaver, Cierra N. Casson, Hieu T. Nguyen, Matthew M. Duda, and Sunny Shin. Proceedings of the National Academy of Sciences, 2015.
63 Abstract
The innate immune system is critical for host defense against microbial pathogens, yet many pathogens utilize virulence factors to impair immune cell function. Here, we employed the intracellular bacterium Legionella pneumophila to understand how the immune system overcomes pathogenic activities. L. pneumophila utilizes a type IV secretion system (T4SS) to translocate bacterial effectors that potently block host translation in infected host cells. Despite this translational block, virulent L. pneumophila elicits a robust inflammatory response, but the basis for this is poorly understood. Single cell analysis of cytokine production by infected and uninfected cells revealed that infected macrophages produce IL-1α and IL-1β but poorly synthesize the cytokines IL-6,
TNF, and IL-12, which are critical for controlling L. pneumophila infection. Intriguingly, uninfected bystander immune cells produced IL-6, TNF, and IL-12, and IL-1 receptor (IL-
1R) signaling was required for this cytokine production. Thus, our data demonstrate functional heterogeneity in production of critical protective cytokines in the response to bacterial infection. These data suggest collaboration between infected and uninfected cells that enables the immune system to bypass protein synthesis inhibition and generate a robust immune response against L. pneumophila.
Significance Statement
Pathogens use virulence factors to inhibit key immune cell functions and would be expected to impair immune responses to infection. However, immune responses are still activated during infection, suggesting that the immune system has evolved mechanisms for overcoming pathogenic activity. Here, we demonstrate that cells infected with L. pneumophila produce IL-1 despite a pathogen-imposed host translational block, but are unable to robustly produce other critical cytokines. IL-1R signaling allowed uninfected bystander cells to produce protective cytokines. Our data thus demonstrate a key role for
64 communication between infected and uninfected bystander cells in overcoming pathogenic activities. This mechanism of immune activation has broad significance for our understanding of how successful immune responses are generated against pathogens.
Introduction
Initiation of innate immune responses to microbial pathogens is thought to involve the direct recognition of pathogen-associated molecular patterns (PAMPs) by membrane- bound and cytosolic pattern recognition receptors (PRRs) on infected cells (Janeway and Medzhitov, 2002; Vance et al., 2009). However, many pathogens utilize virulence factors that interfere with essential immune signaling processes, including NF-κB and
MAPK signaling and host protein synthesis (Krachler et al., 2011; Roy and Mocarski,
2007; Viboud and Bliska, 2005). Such virulence factors limit cell-intrinsic immune activation of infected cells. The mechanisms that enable the host to circumvent pathogen manipulation of host cell signaling processes remain poorly understood.
The Gram-negative bacterium Legionella pneumophila encodes a specialized Dot/Icm type IV secretion system (T4SS) that injects bacterial effector proteins into host cells to facilitate bacterial intracellular survival and replication (Berger and Isberg, 1993; Hubber and Roy, 2010; Marra et al., 1992). A subset of effector proteins, Lgt1, Lgt2, Lgt3, SidI,
SidL, Pkn5, and Lpg1489, block host protein synthesis, in part by targeting translational elongation factors (Barry et al., 2013; Belyi et al., 2006; 2008; Shen et al., 2009).
Furthermore, translational initiation is inhibited during infection due to diminished mTOR signaling (Ivanov and Roy, 2013). These activities result in a greater than 90% block in host translation in infected host cells (Fontana et al., 2011; McCusker et al., 1991).
Nevertheless, infection by L. pneumophila leads to the robust production of pro-
65 inflammatory cytokines, such as TNF, IL-6, IL-1α, IL-1β, and IL-12 (Barry et al., 2013;
Casson et al., 2013; LeibundGut-Landmann et al., 2011; Shin et al., 2008; Spörri et al.,
2006). Moreover, the presence of the T4SS paradoxically enhances levels of cytokine production in response to L. pneumophila, suggesting that much of the cytokine response against L. pneumophila is mediated by cytosolic immune sensing of bacterial ligands and virulence activities (Fontana et al., 2011; 2012; Krachler et al., 2011; Roy et al., 1998; Shin et al., 2008; Spörri et al., 2008).
It is unclear, then, how the host is able to mount a pro-inflammatory cytokine response when L. pneumophila infection potently blocks host protein synthesis. At the population level, decreased host protein synthesis leads to preferential translation of the most abundant cytokine transcripts (Ivanov and Roy, 2013). At the single cell level, directly infected cells overcome the translational block and selectively synthesize IL-1α and IL-
1β through a mechanism involving MyD88-dependent transcriptional expression of IL-1α and IL-1β (Asrat et al., 2014). However, whether mechanisms that enable selective translation of IL-1 also apply more broadly to other key cytokines and immune effector proteins is unclear. Alternatively, as uninfected bystander cells are present both in vitro and in vivo during infection, we considered the possibility that these bystander cells might somehow respond to infection and produce cytokines instead (Copenhaver et al.,
2014). Here, by tracking immune responses in L. pneumophila-infected cells at the single cell level, we demonstrate that although infected cells that have received T4SS effectors are able to synthesize IL-1α and IL-1β, they are poor producers of other key cytokines. Instead, bystander cells that have not received T4SS effectors are the primary producers of TNF, IL-6, IL-12, and the costimulatory molecule CD86 during both in vitro and in vivo infection. Importantly, loss of IL-1R signaling leads to reduced bystander cytokine production and increased bacterial burden in vivo, suggesting that the IL-1
66 released from infected cells mediates production of pro-inflammatory cytokines by bystander cells. Overall, our data suggest that release of IL-1 by infected cells signals the presence of virulent infection, enabling the host to generate a robust innate immune response despite a pathogen-imposed translational block.
Results
Cells that have not received bacterial effectors are the primary producers of pro- inflammatory cytokines during in vitro infection
Infection with virulent L. pneumophila expressing a type IV secretion system (T4SS) leads to an enhanced cytokine response despite bacterial inhibition of host translation.
How this enhanced T4SS-dependent cytokine response is generated remains unclear.
We considered the possibility that directly infected macrophages possess cell-intrinsic mechanisms for overcoming the translational block to selectively synthesize cytokines.
Alternatively, cytokines may be produced by bystander cells that are uninfected or have taken up bacteria that failed to translocate effector proteins (Copenhaver et al., 2014).
To determine whether T4SS-injected cells or uninfected bystander cells produce cytokines during infection, we utilized a fluorescence-based system that detects a translocated L. pneumophila effector protein (RalF) fused to β-lactamase (BlaM)
(Copenhaver et al., 2014; Knapp et al., 2003). In the absence of BlaM activity, 409nm excitation of the cell-permeable BlaM fluorescent substrate CCF4-AM results emission of green fluorescence at 518 nm. However, T4SS-translocated BlaM-RalF results in cleavage of CCF4-AM and a shift in emission to blue fluorescence at 447 nm. This system enables robust discrimination of productively infected and uninfected cells within tissues in vivo or in cultured cells in vitro (Copenhaver et al., 2014).
67 We infected bone marrow-derived macrophages with L. pneumophila encoding the
BlaM-RalF reporter. As flagellin delivered by the T4SS into the host cell cytosol induces rapid cell death via NAIP5 detection and inflammasome activation, we infected macrophages with the virulent ΔflaA strain lacking flagellin (Molofsky et al., 2006; Ren et al., 2006; Zamboni et al., 2006). Following infection, macrophages were loaded with
CCF4-AM and intracellularly stained for various cytokines at multiple time points post- infection. (Figure 4-1 & 4-2). Macrophages infected with avirulent L. pneumophila lacking the Dot/Icm T4SS (ΔdotA) demonstrated no BlaM-RalF activity as expected. In the virulent ΔflaA infection, we found that TNF is rapidly produced in response to infection, but that T4SS-injected cells were poor producers of TNF at all time points assayed
(Figure 4-1A). Instead, the majority of cells robustly producing TNF were cells that had not received T4SS effectors. Likewise, T4SS-injected cells poorly produced IL-6.
Instead, IL-6 was primarily produced by uninjected cells (Figure 4-1B). Similar findings were obtained for IL-12 and CD86 (Figure 4-2). In contrast, IL-1α and IL-1β were robustly produced by both T4SS-injected cells and uninjected cells (Figure 4-1C &D), indicating that T4SS-injected cells produce IL-1α and IL-1β despite the translational block imposed by L. pneumophila effector proteins, consistent with recent findings (Asrat et al., 2014).
To determine whether the decreased cytokine production in T4SS-injected cells is due to decreased translation or transcription, we performed qRT-PCR on sorted T4SS-injected and uninjected cells (Figure 4-1E). Both injected and uninjected cells displayed marked increases in transcript levels for the cytokines IL-1α, IL-1β, TNF, and IL-6 relative to uninfected cells, with injected cells consistently exhibiting a greater increase in cytokine transcript levels, consistent with recent findings (Asrat et al., 2014). Thus, the decreased production of immune proteins by injected cells is not due to a lack of transcriptional
68 activation and most likely is a direct consequence of the translational block. Taken together, these data suggest that although productively infected cells can bypass the translational block to produce IL-1α and IL-1β, they poorly translate TNF, IL-6, IL-12, and
CD86. Instead, bystander cells preferentially produce these proteins.
There are currently seven identified effectors in virulent L. pneumophila that inhibit protein translation in host cells (Barry et al., 2013; Belyi et al., 2013). To determine if these effectors prevent cytokine production in injected cells, we infected BMDMs with strains of L. pneumophila that lack either five of the known effectors (Lgt1, Lgt2, Lgt3,
SidI, SidL)(Δ5) or all seven (Δ5 and Pkn5 and Lpg1489) (Δ7ΔflaA) of the known effectors. At four hours PI, cells injected by the Δ5 or Δ7ΔflaA strains produced more
TNF than cells injected by their parental WT counterparts (Figure 4-3A &D). This increase occurred both in the frequency of TNF-producing injected cells as well as the gMFI of injected cells producing TNF. At 16 hours PI, however, there was no difference in the frequency of TNF-producing injected cells in either the Δ5 or Δ7ΔflaA strains and gMFI was only different in the Δ7ΔflaA strain compared to the parent strain (Figure 4-3B
&E). IL-6 production by injected cells was also unaffected by the presence or absence of these effectors (Figure 4-3C &F). These data indicate that T4SS effectors that block protein translation effect early, but not later cytokine production in infected cells.
Bystander alveolar macrophages and neutrophils are predominant producers of
TNF during L. pneumophila infection
Our in vitro findings indicate that many key protective cytokines are primarily generated by bystander macrophages during infection by pathogens that interfere with cell intrinsic immune defense. In vivo, multiple cell populations exist, and distinct immune populations may possess alternative mechanisms for overcoming pathogen-induced translational
69 blockade. We therefore intranasally infected WT mice with L. pneumophila strains expressing BlaM-RalF and examined TNF production in airway-resident alveolar macrophages and recruited neutrophils, as they are the primary cell types that receive L. pneumophila T4SS effectors (Copenhaver et al., 2014). Following infection with ΔflaA L. pneumophila, we observed a significant increase in both the percentage and total numbers of TNF-producing airway alveolar macrophages and neutrophils (Figure 4-4), consistent with previous findings showing that cytosolic immune sensing of T4SS activity is required for maximal TNF production (Shin et al., 2008; Spörri et al., 2006). We observed that T4SS-injected alveolar macrophages and neutrophils did not produce
TNF. Instead, TNF was produced almost exclusively by uninjected alveolar macrophages and neutrophils (Figure 4-4). These data, along with our data in macrophages in vitro, indicate that infected cells are poor producers of proinflammatory cytokines and that uninfected cells are the major TNF-producers.
In contrast, both injected and uninjected alveolar macrophages produced IL-1α and IL-
1β, in agreement with our in vitro findings (Figure 4-5). PBS vehicle control and avirulent
ΔdotA infection yielded no significant increase in TNF-positive alveolar macrophages or neutrophils (Figure 4-4), corroborating with previous findings that TNF is undetectable in the bronchoalveolar lavage fluid or serum isolated from similarly treated mice (Shin et al., 2008; Spörri et al., 2006). Consistent results were also obtained from alveolar macrophages and neutrophils isolated from lung tissue (Figure 4-6). These data indicate that although cytosolic immune sensing of T4SS-translocated bacterial products is critical to elicit TNF production, TNF is produced by uninjected bystander alveolar macrophages and neutrophils that have not received T4SS effectors.
70 Bystander inflammatory monocytes and dendritic cells produce TNF and CD86 during L. pneumophila infection
Inflammatory monocytes and conventional dendritic cells are also recruited to the lung during L. pneumophila infection. In contrast to alveolar macrophages and neutrophils, these cell populations are not productively infected by L. pneumophila and do not receive T4SS effectors (Copenhaver et al., 2014; LeibundGut-Landmann et al., 2011).
Even so, inflammatory monocytes and dendritic cells produced significant amounts of
TNF and were the primary cell types in the lung responsible for producing TNF during
ΔflaA L. pneumophila infection (Figures 4-7A & B). In contrast, inflammatory monocytes and dendritic cells from ΔdotA-infected mice did not exhibit an increase in TNF production compared to cells from PBS-infected mice. Inflammatory monocytes and dendritic cells also substantially increased expression of CD86 during infection with virulent ΔflaA L. pneumophila compared to PBS or ΔdotA infection, and were the primary cell types expressing CD86 (Figure 4-7C & D). Alveolar macrophages and neutrophils did not increase expression of CD86 during ΔflaA infection (Figure 4-8). These data indicate that although inflammatory monocytes and dendritic cells are not productively infected and do not receive T4SS-translocated products, they are the primary cell types that express TNF and CD86 during virulent L. pneumophila infection as measure by intracellular cytokine staining.
IL-1 signaling is critical for activating bystander immune cells to produce cytokines and express CD86
Maximal immune responses to L. pneumophila require cytosolic sensing of T4SS activity
(Shin et al., 2008), yet our data indicate that the majority of cytokine- and CD86- producing cells are bystander innate immune cells that have not received T4SS effectors. Given that T4SS-injected cells are capable of producing IL-1α and IL-1β, we
71 considered the possibility that IL-1α and IL-1β released by infected cells might play a role in instructing bystander cells to produce other key inflammatory cytokines. Notably,
IL-1α and IL-1β can elicit expression of other cytokines (Dinarello, 1996; Kohase et al.,
1987), and IL-1R signaling is critical for innate immune control of L. pneumophila infection (Barry et al., 2013; Casson et al., 2013; LeibundGut-Landmann et al., 2011;
Mascarenhas et al., 2015). The crucial role of IL-1 in early host defense has been primarily attributed to IL-1-dependent expression of neutrophil-attracting chemokines by epithelial cells and subsequent neutrophil recruitment to the site of infection (Barry et al.,
2013; Casson et al., 2013; LeibundGut-Landmann et al., 2011; Mascarenhas et al.,
2015). We hypothesized that IL-1R signaling would also be critical for instructing bystander innate immune cells to produce cytokines. We therefore intranasally infected
WT or Il1r1-/- (IL-1R-/-) mice with ΔflaA L. pneumophila and assayed TNF production 24 hours later. Critically, IL-1R-/- mice exhibited significant reductions in both the percentages and total numbers of TNF-producing alveolar macrophages, neutrophils, dendritic cells and inflammatory monocytes compared to WT mice, in which large numbers of the aforementioned cells produced TNF (Figure 4-9 & 4-10). Although a percentage of IL-1R-/- dendritic cells and inflammatory monocytes remained positive for
TNF, it was only minimally higher than the basal percentage of TNF-positive cells observed in WT or IL-1R-/- mice infected with avirulent ΔdotA L. pneumophila (Figure 4-
11). Consistent with the decreased numbers of TNF-producing cells in IL-1R-/- mice, TNF levels in the bronchoalveolar lavage of IL-1R-/- mice were significantly reduced compared to WT mice (Figure 4-11E). IL-12p40 levels in IL-1R-/- mice were also significantly reduced (Figure 4-9E). Furthermore, IL-1R-/- mice also exhibited a significant decrease in the percentages and total numbers of CD86-expressing inflammatory monocytes and dendritic cells compared to WT controls (Figure 4-10). In contrast, IL-6 levels were unaffected, indicating that IL-1 signaling is not required for production of all responses by
72 bystander cells during L. pneumophila infection (Figure 4-9E). Finally, injection of neutralizing antibodies against IL-1α and IL-1β resulted in a significant decrease in the levels of TNF and IL-12p40, but not IL-6, with neutralization of IL-1α and IL-1β together having the greatest effect (Figure 4-12). Overall, these data indicate a crucial role for IL-
1 signaling in directing the optimal production of the pro-inflammatory cytokines TNF and
IL-12p40 as well as the costimulatory molecule CD86 by bystander innate immune cells during pulmonary L. pneumophila infection.
BMDMs are reported to not express the IL-1R and should therefore not respond to IL-1 stimulation in vitro (Demuth et al., 1996). To see, however, whether IL-1R signaling occurs during L. pneumophila infection in vitro, we infected with WT or IL-1R-/- BMDMs with L. pneumophila and assayed for cytokine production by flow as well as be ELISA
(Figure 4-13A &B). An equal frequency of IL-1R-/- bystander BMDMs produced TNF in response to L. pneumophila as compared to WT cells (Figure 4-13A) and IL-6 production in response to infection was also equivalent between the two genotypes of BMDMs
(Figure 4-13B).Indeed, treating BMDMs with varying concentrations of recombinant IL-
1α, IL-1β, or both combined did not elicit TNF or IL-6 production from these cells (Figure
4-13C). Treatment of NIH/3T3 cells, an epithelial cell line known to express the IL-1R, elicited IL-6 production, demonstrating that the recombinant IL-1 is competent to induce signaling (Figure 4-13D). These data, along with the decreased, but present cytokine response to L. pneumophila infection from IL-1R-/- mice in vivo, suggest that additional signals may drive bystander cytokine responses during infection.
To determine if IL-1 signaling is sufficient to drive TNF production on other innate cell types, we treated alveolar macrophages, bone marrow-derived dendritic cells (BMDCs) or inflammatory monocytes isolated from mouse bone marrow and treated with IL-1α, IL-
73 1β, or both in combination. A small, but consistent frequency of BMDCs stained for TNF production in response to IL-1 treatment (Figure 4-14A). These BMDCs also increased expression of CD86 in response to IL-1 treatment (Figures 4-14B). BMDCs also produced a low level of TNF and IL-6 in response to IL-1 treatment as measured by
ELISA (Figure 4-14D). Alveolar macrophages also produced TNF in response to IL-1 treatment (Figure 4-15A). Importantly, this response was dependent on expression of the IL-1R, as alveolar macrophages from IL-1R-/- mice did not produce TNF when treated with IL-1 (Figure 4-15B). Inflammatory monocytes were unresponsive to IL-1 treatment as measured by TNF production and CD86 expression (Figure 4-16). We were also unable to see increased CD86 expression in response to LPS on inflammatory monocytes, suggesting that inflammatory monocytes may not express CD86 ex vivo
(Figure 4-16A). Together, these data indicate that some innate cell populations, such as dendritic cells and alveolar macrophages, may directly respond to IL-1 signaling during
L. pneumophila infection in a cell-intrinsic manner. Other cells types, however, do not respond to IL-1 and may require either additional signals to produce cytokines or require a intermediate cell type to respond to IL-1.
Conclusion
Cells targeted by the T4SS of L. pneumophila were poor producers of the cytokines
TNF, IL-6, IL-12p40, and fail to increase expression of CD86 upon infection. Instead, uninjected, bystander cells produced the majority of these cytokines in vitro and in vivo.
Injected cells were still able to produce IL-1α and IL-1β during infection and IL-1 signaling was important in vivo for the production of cytokines by bystander cells including uninjected alveolar macrophages, neutrophils, inflammatory monocytes, and dendritic cells. The 7 currently known T4SS effectors that block protein translation were important for preventing TNF production in injected cells early, but not later during
74 infection in vitro. Deleting these effectors did not affect the production of IL-6 by injected cells, suggesting that other effectors or a host-driven response prevent IL-6 and late
TNF production by injected cells. BMDMs did not respond to IL-1 stimulation in vitro and do not require IL-1R signaling to produce bystander cytokines. IL-1R-/- mice in vivo produced less TNF and IL-12 during L. pneumophila infection, but still produced a small amount of cytokine. Together, these data suggest that other mechanisms besides IL-1 signaling drive bystander cytokine production. Both alveolar macrophages and BMDCs were able to produce TNF in response to IL-1 stimulation and BMDCs also increased expression of CD86. Inflammatory monocytes, however, did not respond to IL-1. Thus,
IL-1 signaling is sufficient in some, but not all cell types shown to produce bystander cytokines in vivo. Together, these data detail a mechanism by which cells infected with a pathogen that blocks host protein synthesis are able to communicate to uninfected cells to initiate a protective immune response.
Acknowledgements
I thank Russell Vance for his generous gift of the WTΔ5 and Δ7ΔflaA L. pneumophila strains. I thank Mike Askenase is the lab of Yasmine Belkaid for giving us protocols to isolate and stimulate inflammatory monocytes. I thank members of the Brodsky laboratory for helpful discussions and Igor Brodsky for critical reading of the manuscript.
This work was supported in part by National Institutes of Health grants
K99/R00AI087963 (to S.S.), T32GM007229 (to A.M.C.), American Lung Association grant RG-268528-N (to S.S.), the University of Pennsylvania University Research
Foundation (to S.S.), American Heart Association grant 13BGIA14780070 (to S.S.), and the Roy and Diana Vagelos Scholars Program in Molecular Life Sciences (to M.M.D.).
This material is based upon work supported by the National Science Foundation under
Grant No. DGE-0822 (C.N.C., graduate research fellowship). Cierra N. Casson was
75 instrumental in the design and execution of the in vivo and sorting experiments as well as in editing the manuscript and maintaining the Il1r1-/- colony. Hieu T. Nguyen aided in harvesting tissues and counting cells. Matthew M. Duda aided in the performance of in vitro experiments.
76 IL-6 TNF
30 60 Uninjected UninjectedUninjected ! A! TNF Unfx!Injected ΔdotA! ΔflaA! InjectedInjected ! 0.497 0.0166 0.673 0.0392 19.8 3.58 60
! 60! 20 0.5! Total0.1! 0.7! 40 0.1! 19.8! 3.6! TotalTotal ! Uninjected
! Injected 4040! Total % IL-6+ 10 % TNF+ 20
% TNF+ 20 % TNF + % 20!
0 Injection(blue) 0 99.199.2 ! 0.30.315! 85.885.7! 13.513.5! 62.862.7! 13.913.9! 4 8 4 8 0 12 16 20 12 16 20 4 8 TNF! 4! 8! 1212 ! 1616 ! Hours PI Hours PI HoursHours PI PI! B! Unfx! ΔdotA! ΔflaA! IL-6 0.456 0.0331 0.634 0.0561 18.7 4.21
! 30 0.5! 0.1! 0.6! 0.1! 18.7! 4.2! 30! Uninjected IL-1alpha IL-1beta ! Injected 80 100 2020! Total Uninjected Uninjected Injected Injected 80 % IL-6+ 10 60 %IL-6+ Total 10! Total 60 Injection(blue) 99.399.2! 0.20.219! 94.894.7! 4.64.55! 59 18.218.2! 40 58.9! 0 4 8 IL-640! 4! 8! 1212 ! 1616 ! % IL-Beta+
% IL-alpha+ HoursHours PI PI! 20 C! 20 Unfx! ΔdotA! ΔflaA! IL-1alpha 1.03 0.0956 0.611 0 0.244 17.3 25.9 0 ! 1.0! 0.1! 0.6! 0.2! 17.3! 25.9! 8080! 4 8 Uninjected 4 8 12 16 20 ! 12 16 20 Injected Hours PI + 6060!
Hours PI α Total 4040! % IL-alpha+ %IL-1 2020! Injection(blue) 98.798.8! 0.10.147! 8585.1! 14.114.1 ! 1717.1! 39.739.7! 0 4 8 IL-1α! 4! 8! 1212 ! 1616 ! HoursHours PI PI! D! Unfx! ΔdotA! ΔflaA! IL-1beta 1.07 0.126 0.237 0.388 8.63 32.1 100 ! 1.1! 0.1! 0.2! 0.4! 8.6! 32.1! 100! Uninjected ! 8080! Injected + Total β 6060! 4040! % IL-Beta+ %IL-1 2020! Injection(blue) 98.598.5! 0.30.341! 26.526.5! 72.872.8! 2.983.0! 56.356.3! 0 4 8 IL-1β! 4! 8! 1212 ! 1616 ! HoursHours PI PI! E! Tnf over HPRT * new TNF Il1a over HPRT Il1b over HPRT
! IL-6 over HPRT Tnf! Il6! 3 Il1a! Il1b! 101022! 101022! 10103! 101033! Unfx bulk! ***! ***! ***! 2 ***! 1011! 1022! 102! 101 10 10 10 ΔflaA bulk!
! 1! ) 10 1000! 1011! 10 1011! 10
10Hprt 0 -1 0 ΔflaA uninjected! 100! 1010-1! / 100!
Hprt 10 100 10
Il6 / Hprt Il6 0! Tnf / Hprt Tnf Il1a Il1b / Hprt Il1b ( 10-2-2! 10 10-1-1! ΔflaA injected! -1 10 10 10-1! -1 10 10-1! -2 10-3-3! 10 1010-2! 10 ND! 10-2-2! 10-4-4! 10-2-2! 10-3-3!
RelativeExpression 10 10 10 10 Unfx Unfx Unfx Unfx flaA bulk flaA bulk flaA bulk flaA bulk flaA injected flaA injected flaA injected flaA injected flaA uninjected flaA uninjected flaA uninjected flaA uninjected Figure 4-1. T4SS-injected and uninjected macrophages produce different cytokines in vitro. (A-D) Bone marrow-derived macrophages (BMDMs) were infected with Lp02 strains of L. pneumophila for 4, 8, 12, or 16 hours. BMDMs were treated with brefeldin A and monensin starting at 3 hours prior to harvest. Cells were then loaded with CCF4- AM, fixed, and then stained with antibodies against TNF (A), IL-6 (B), IL-1α (C), and IL- 1β (D). Plots show cytokine production at 16 hours post infection (PI). Line graphs show ΔflaA-infected macrophages where total frequency of cytokine producing cells as well as the contribution of injected and uninjected cells to cytokines are shown. N=3 wells per condition per timepoint. Graphs show mean ± SEM. Representative of 3 independent experiments. (E) BMDMs were infected with Lp02 ΔflaA L. pneumophila for 8 hours. Cells were then loaded with CCF4-AM and sorted based upon cleavage of the dye, or were collected after going through the sorter without separating the cells based on dye cleavage (bulk). Sorted cells were then lysed and relative abundance of transcripts for proinflammatory cytokines were assayed using RT-PCR. Bar graphs show mean relative abundance of 2 separate wells. Statistics represent the results of Tukey’s post test between ΔflaA uninjected and ΔflaA injected columns. Representative of 3 independent experiments. ***p<0.0005
77 A! IL-6 PBS! ΔdotA! TNF ΔflaA! IL-12p40 0.416 0.0121 0.519 0.0467 22 1.24 30 0.4! 0.1! 0.5! 600.1! 22.1! 1.2! 88! ! ! Uninjected UninjectedUninjected ! Uninjected Injected Injected 6 InjectedInjected ! 6! Total 20 Total 40 TotalTotal ! 44! % IL-6+ % TNF+
10 20 % IL-12p40 + %IL-12p40 + 22! Injection(blue)
99.599.4! 0.10.0484! 93.6 ! 5.85.78! 72.1! 4.6! 72.1 4.65 0 0 0 IL-12p40! 44! 88 ! 12! 16! 4 8 4 8 12 16 12 16 20 12 16 20 HoursHours PI PI! Hours PI Hours PI B! PBS! ΔdotA! ΔflaA! CD86 0.52 0.101 1.2 0.092 23 0.686
! 0.5! 0.1! 1.2! 0.1! 23! 0.7! 1010! Uninjected! IL-1alpha IL-1beta Uninjected
80 100 ! 88! Injected! Injected Uninjected Uninjected Total! Total Injected 80 66! Injected 60 Total Total 60 44! % CD86 + % CD86 +
40 Injection(blue) 40 22! % IL-Beta+ % IL-alpha+ 97.5 ! 1.91.92! 93.9 ! 4.84.81! 68.8 ! 7.57.49! 20 20 0 CD86! 88! HoursHours PI PI! 0 0 4 8 4 8 Figure12 16 420-2. IL-12p40 and CD86 are expressed by12 bystander16 20 cells during infection. (A) Hours PI HoursBone PI marrow-derived macrophages (BMDMs) were infected with Lp02 strains of L. pneumophila at an MOI = 5 for 4, 8, 12, or 16 hours. BMDMs were treated with brefeldin A and monensin starting at 3 hours prior to harvest. Cells were then loaded with CCF4- AM, fixed, and then stained with antibodies against IL-12p40. Plots show cytokine production at 16 hours post infection (PI). Line graph shows ΔflaA-infected macrophages where total = the total frequency of cells positive for the given cytokine. Injected and uninjected = the relative contribution of each subset of cells to the total frequency of cytokine positive cells. N=3 wells per condition per timepoint. Line graphs show mean ± SEM (B) Cells infected as in (A) were harvested 8 hours PI, loaded with CCF4-AM and stained for CD86. Bar graph shows mean ± SEM.
78
A! LPS! WT! ΔdotA! ΔflaA! WTΔ5! ΔflaAΔ7! 0.01860.1! 0.10.137! 10.1 ! 10.510.5! 0.1420.1! 0.40.431! 9.7 ! 9.39.28! 2.112.1! 13.113.1 ! 2.832.8! 14.114.1 ! ! Injection(blue)
4.324.3! 95.595.5! 17.8 ! 61.661.6! 3232.0! 67.567.5! 27.9 ! 53.153.1! 9.08.98 ! 75.875.8! 15.7 ! 67.467.4! TNF! B! LPS! WT! ΔdotA! ΔflaA! WTΔ5! ΔflaAΔ7! 0.1470.1! 0.20.196! 32.532.5! 5.55.53! 0.1390.1! 0.10.125! 3232.0! 8.38.25! 29.229.2! 5.45.43! 39.1 ! 6.36.26! ! Injection(blue)
88.6 ! 11.111.1! 41.5 ! 20.520.5! 83.7 ! 16.116! 44.3 ! 15.415.4! 38.2 ! 27.227.2! 29.8 ! 24.824.8! TNF! C! LPS! WT! ΔdotA! ΔflaA! WTΔ5! ΔflaAΔ7! 0.3760.4! 0.08180.1! 2929.0! 4.74.72! 0.3760.4! 0.10.0139! 34.634.6! 5.65.62! 30.6 ! 6.76.66! 29.4 ! 3.83.79! ! Injection(blue)
94.5 ! 5.05.03! 50.5 ! 15.815.8! 95.6! 4.04.03! 5151.0! 8.88.77! 38.138.0! 24.724.7! 53.153.0! 13.813.8 ! IL-6! D! E! F!
TNF 4 hours ! MFI 4 hour TNF TNF 16 hours gMFI 16 hour TNF IL-6 16 hours 4 hour PI! 4 hour PI! 16 hour PI! ! 16 hour PI! 16 hour PI! ! ! ! 100100! 8,0008000! *** *** 3030! 4000 2525! ns ns ******! ***! ***! ***! nsns! nsns! 8,000! nsns! **! ns! ns! 9090! 2020! 6,0006000! 8080! 2020! 3,5003500! 1515! 7070! 4,0004000! 1010! 6060! 1010! 3,0003000! 2,0002000! TNF (injected TNF cells) 55! 5050! (injected TNF cells) % IL-6+ (of injected cells) % TNF+ (of injected cells) % TNF+ (of injected cells) gMFI of TNF+ injected cells gMFI of TNF+ injected cells
%IL-6+(injected cells) 0
2500 ! ! ! %TNF+ (injected%TNF+ cells) (injected%TNF+ cells) ! 0 0 !
2,500! ! !
40 ! ! ! ! ! ! ! ! ! ! ! ! !
40! gMFI gMFI 7 7 5 7 7 7 5 5 D5 5 D7 D5 5 D7 WT D5 D7 WT flaA D5 D7 D5 D7 flaA Δ Δ
WT WT Δ Δ Δ Δ WT flaA flaA flaA Δ WT Δ Δ Δ WT WT WT WT flaA flaA flaA flaA flaA Δ Δ Δ Δ Δ WT WT flaA flaA WT WT WT flaA flaA flaA Δ Δ Δ Δ Δ
Figure 4-3. Type IV secretion system effectors that block protein translation inhibit early TNF production in injected cells. BMDMs were treated with LPS or infected with Lp02 WT, ΔdotA, ΔflaA, Δ5, or Δ7ΔflaA strains of L. pneumophila at an MOI = 5, infected for the indicated amount of time, loaded with CCF4-AM, and stained for ICS. (A & B) Representative flow plots showing TNF production 4 (A) or 16 (B) hours PI. The frequency of TNF-producing cells and the geometric MFI (gMFI) of TNF-producing- injected cells are quantified in (D) for 4 hours and (E) for 16 hours. (C) Plots showing IL- 6 production at 16 hours PI. The frequency of IL-6-producing injected cells is quantified in (F). Representative of 2 independent experiments. Graphs show individual wells and mean. ANOVA was performed and statistics show significance between Δ5 and WT or Δ7ΔflaA and ΔflaA as determined by Tukey post test. *p<0.05, **p<0.005, ***p<0.0005, ns=not significant.
79 A! B! Alveolar Macrophages! ΔflaA! BALF Alveolar Macs TNFBALF + Alveolar Macs TNF injection ! PBS! ΔdotA! ΔflaA! ) ! ! 0.0689 0.0345 0.0663 0.116 16.3 1.37 150,0004 0.0026 3030 ! 0.1! 0.1! 0.1! 0.1! 16.3! 1.4! 15 **! ***! ! 100,00010! 2020! TNF + TNF % TNF+ 50,0005! 1010! % TNF + % Injection(blue) 97.497.3! 2.52.53! 97.197.0! 2.82.75! 71.271.1! 11.211.2! Total TNF + (x10 + TNF Total ! ! ! ! 0 ! 0 ! TNF! - B6 flaA B6 PBS B6 dotA flaA Injected PBS dotA Uninjected Δ Unin jected Δ C! D! Injected Neutrophils! ΔflaA! BALF Neuts TNF + neuts BALF TNF injection !
PBS! ΔdotA! ΔflaA! ) ! ! 0.195 0.195 0.253 0 3.15 0.536 600,0005 60 ! 0.2! 0.2! 0.3! 0! 3.2! 0.5! 6 0.0031 **! 60 **! ! 400,0004! 4040! TNF + TNF 200,000! % TNF+ 20!
2 TNF + % 20 Injection(blue) 98.198.0! 1.61.56! 97.697.5! 2.22.15! 8787.0! 9.39.29! Total TNF + (x10 + TNF Total ! ! ! !
0 ! 0 ! TNF! - B6 flaA B6 PBS B6 dotA flaA Injected PBS dotA Uninjected Δ Unin jected Δ Injected
Figure 4-4. Bystander alveolar macrophages and neutrophils produce TNF during L. pneumophila infection. Mice were infected and cells were harvested from the bronchoalveolar lavage and lung tissue, treated with BFA, loaded with CCF4-AM, stained, fixed and stained with an antibody against TNF. (A-D) Representative plots from alveolar macrophages (A) and neutrophils (C) collected from the BAL 24 hours PI and quantified in (B & D). Graphs show individual mice and mean from 3 pooled independent experiments. Graphs on far right show the relative contribution of uninjected and injected cells to TNF production from ΔflaA-infected mice. N=2-3 mice per group per experiment. Student’s T-tests were performed for total TNF producing cells between ΔdotA and ΔflaA infected mice. **p<0.005, ***p<0.0005.
80 A! Alveolar Macrophages! PBS! ΔdotA! ΔflaA! 0.2160.2! 0.10.0345! 0.1540.2! 0!0 2.492.5! 5.75.7! ! Injection(blue)
97.397.3! 2.42.42! 95.395.2! 4.64.57! 73.973.9! 17.917.9! IL-1α! B! Alveolar Macrophages! PBS! ΔdotA! ΔflaA! 0.0977 0.0391 0.181 0.0345 3.56 4.49
! 0.1! 0.1! 0.2! 0.1! 3.6! 4.5! Injection(blue)
9897.9! 1.91.87! 95.595.4! 4.34.25! 66.566.4! 25.525.5! IL-1β!
Figure 4-5. T4SS-injected alveolar macrophages produce IL-1 during pulmonary infection. Mice were infected with 5x106 CFU of JR32 ΔdotA or ΔflaA L. pneumophila intranasally or given PBS. 24 hours PI, cells were harvested from the lung tissue, treated with BFA, loaded with CCF4-AM, stained, fixed and stained with an antibody against IL- 1α (A) and IL-1β (B). Representative plots are shown. N=2-3 mice per group. Representative of 2 independent experiments.
81 A! B! Alveolar Macrophages! Lung Alveolar Macs TNF injection PBS! ΔdotA! ΔflaA! Lung Alveolar Macs TNF + 0.0413 0.062 0.0498 0.0249 4.5 0.183 ! 1,500,000! ! 6060 0.1! 0.1! 0.1! 4.5! 0.2! ) 15 ns! **!
0.1! 5 0.1677 ! ! 1,000,00010! 4040! TNF + TNF ! % TNF+ 20! 500,0005 TNF + % 20 Injection(blue) Total TNF + (x10 + TNF Total 0
0 ! ! ! 90.390.4 ! 9.52 ! 9.5! 86.987 ! 12.912.9! 59.1 ! 36.236.2 ! ! ! TNF! - B6 PBS B6 flaA flaA PBS B6 dotA dotA Injected Δ
UninjectedUnin jected Δ
C! D! Injected Neutrophils! Lung neuts TNF injection PBS! ΔdotA! ΔflaA! Lung Neuts TNF +
0.202 0.239 0.128 0.348 1.11 1.01 ! 07 40! 0.2! 0.2! 0.1! 0.3! 1.1! 1.0! 1.0×) 10 ! 40 6 10 **! ***! ! 0.0020 ! ! 8,000,0008 3030! 6,000,0006! 2020! TNF + TNF ! TNF + % 4,000,0004 % TNF+ ! 1010!
Injection(blue) 2,000,0002 Total TNF + (x10 + TNF Total
97.497.5! 2.12.11! 95.395.4! 4.24.22! 6767.0! 30.930.9! !
0 ! ! !
! 0 ! TNF! -
B6 flaA flaA
B6 PBS PBS B6 dotA dotA Injected Δ Unin jected
Δ Uninjected Injected
Figure 4-6. Bystander alveolar macrophages and neutrophils from the lung produce TNF during infection. Alveolar macrophages (A) and neutrophils (C) were harvested from the lung tissue of infected mice and stained for TNF production. Representative flow plots are shown. Graphs show individual mice and mean from 3 pooled independent experiments. (B & D) Graphs on far right show the relative contribution of uninjected and injected cells to TNF production from ΔflaA-infected mice. N=2-3 mice per group per experiment. Student’s T-tests were performed for total TNF producing cells between ΔdotA and ΔflaA infected mice. ns=not significant, **p<0.005, ***p<0.0005.
82 A! Inflammatory monocytes! Lung iMCs TNF +
PBS! ΔdotA! ΔflaA! ! 0.0179 0.116 0.0222 0.189 0.0292 0.608 )
6 07 ! ! 0.1! 0.1! 0.1! 0.1! 0.1! 0.6! 1.5×1510 0.0014 **!
1.0×1007! TNF + TNF 5,000,0005! Injection(blue) 78.178.0! 21.821.8! 79.279.2! 20.620.6! 20.520.4! 78.978.9! ! ! Total TNF + (x10 + TNF Total 0 TNF! ! B6 flaA
B6 PBS B6 dotA flaA PBS dotA Δ Δ B! Conventional dendritic cells! Lung cDCs TNF +
PBS! ΔdotA! ΔflaA! ! 0.134 0.0448 0.299 0.239 0.284 0.0568 )
5 ! ! 1,500,000 0.1! 0.1! 0.1! 0.1! 0.1! 0.1! 15 0.0200 **!
1,000,00010! TNF + TNF 500,0005! Injection(blue) 89.589.6! 10.210.2! 87.788.0! 11.811.8! 74.975.1! 24.724.7! Total TNF + (x10 + TNF Total ! !
0 ! TNF! B6 flaA B6 PBS B6 dotA flaA PBS dotA Δ Δ C! Inflammatory monocytes!
! Lung iMCs CD86 + )
PBS! ΔdotA! ΔflaA! 6 0.0894 0.0179 0.156 0.0222 0.248 0.224 6,000,000! 0.0038 ! 0.1! 0.1! 0.2! 0.1! 0.2! 0.2! 6 **!
4,000,0004! CD86 + 2,000,0002!
Injection(blue) 96.996.8! 3.02.95! 95.495.3! 4.44.41! 66.466.5! 33.133.1 ! ! !
0 ! CD86! CD86 hi (x10 Total
B6 PBS B6 dotA B6 flaA flaA PBS dotA Δ Δ D! Conventional dendritic cells!
! Lung cDCs CD86 + PBS! ΔdotA! ΔflaA! ) 5 0.112 0 0.149 0.0186 0.216 0.0341 ! 0.0007 ! 0.1! 0! 0.1! 0.1! 0.2! 0.1! 500,0005 ***! 400,0004! 300,0003! ! CD86 + 200,0002 100,0001! Injection(blue) 96.9! 3.0! 96.3! 3.5! 88.5! 11.3! 97 3.05 96.1 3.48 88.4 11.3 0 ! ! ! CD86! CD86 hi (x10 Total B6 flaA
B6 PBS B6 dotA flaA PBS dotA Δ Δ
Figure 4-7. Bystander inflammatory monocytes and conventional dendritic cells produce TNF and express CD86 during infection. Cells were harvested from the lung tissue of mice, treated with BFA, loaded with CCF4-AM, stained, fixed and stained with an antibody against TNF (A & B) or CD86 (C & D). Representative plots are shown. Graphs show individual mice and mean from 3 pooled independent experiments. Student’s T- test were performed between ΔdotA and ΔflaA infected mice. **p<0.005, ***p<0.0005.
83 A! Alveolar Macrophages! PBS! ΔdotA! ΔflaA! 0.6130.2! 0.10! 0.3830.2! 0.01780! 14.814.8! 5.10.0642! ! Injection(blue)
98.197.3! 2.41.31! 97.197.2! 2.62.58! 81.596.5! 3.63.63! CD86! B! Neutrophils! PBS! ΔdotA! ΔflaA! 0.499 0.0441 0.894 0.0789 2.18 0.0473 ! 0.5! 0.1! 0.9! 0.1! 2.2! 0.1! Injection(blue)
98.798.6! 0.80.801! 98.198.0! 1.00.973! 97.197.0! 0.70.684! CD86!
Figure 4-8. Alveolar macrophages and neutrophils do not express CD86 during infection. 24 hours PI, cells were harvested from the lung tissue, treated with BFA, loaded with CCF4-AM, stained with an antibody against CD86, and fixed. Representative plots for alveolar macrophages (A) and neutrophils (B) are shown. N=2-3 mice per group. Representative of 3 independent experiments.
84 A! B! Alveolar Macrophages! -/-! Lung Alveolar Macs % TNF Lung+ Alveolar Macs TNF +
WT! IL-1R ! 0.0143
0.0016 ) 14.2 0.225 11 0 1,500,000! 5 ! 14.2! 0.2! 11.0! 0! 6060! **! 15 *! !
! 4040! 1,000,00010 TNF + TNF
% TNF+ ! % TNF + % 2020! 500,0005
Injection(blue) 70.9! 14.7! 87.6! 1.4! 70.8 14.7 87.6 1.4 0 0 TNF! WT! IL-1R-/-! (x10+ TNF Total WT! IL-1R-/-! C! D! B6 flaA B6 flaA Conventional dendritic cells! IL-1R-/- flaA IL-1R-/- flaA -/-! Lung cDCs % TNF + Lung cDCs TNF + WT! IL-1R !
< 0.0001 ) 0.268 0.107 0.184 0.023 0.0016 30 1,500,0005 ! ! 0.3! 0.1! 0.2! 0.1! 30! ***! 15 **! !
2020! 1,000,00010! TNF + TNF % TNF+ 10 500,000! % TNF + % 10! 5 Injection(blue) 80.580.5! 19.119.1 ! 94.594.5! 5.35.25! 0 (x10+ TNF Total 0 TNF! WT! IL-1R-/-! WT! IL-1R-/-! IL-6 IL-12p40 TNF B6 flaA B6 flaA
E! ! ! ! ! IL-1R-/- flaA IL-1R-/- flaA
! 500 1500 1000 500 < 0.0001***! ! 1.5 0.0994ns! 1,000 < 0.0001***!
400400! /mL) 800800! /mL) /mL) ! 1000 pg 300! 1.0 600! pg 300 ng 600
200! 400! 200 500! 400 IL-6 (pg/mL) IL-6 TNF (pg/mL) TNF 0.5 IL-6( TNF ( 100100! (pg/mL) IL-12p40 200200!
0 0 IL-12p40( 0 WT! IL-1R-/-! WT! IL-1R-/-! WT! IL-1R-/-! B6 flaA B6 flaA B6 flaA Figure 4-9. IL-1R-/-IL -flaA1 signaling inducesIL-1R-/- flaA cytokine productionIL-1R-/- flaA by bystander cells. Cells were harvested from the lungs of infected WT or Il1r1-/- (IL-1R-/-) mice, treated with BFA, loaded with CCF4-AM, stained, fixed and then stained with an antibody against TNF. Representative plots from alveolar macrophages (A & B) and conventional dendritic cells (C & D) are shown. Graphs show individual mice and mean pooled from 4 independent experiments. (E) BAL fluid was collected from mice and cytokine levels were measured by ELISA. Graphs show individual mice and mean pooled from 3 independent experiments. N=2-4 mice per group. Student’s T-test were performed between pooled groups. ns=not significant, *p<0.05, **p<0.005, ***p<0.0005.
85 A! Neutrophils! WT! IL-1R-/-! Lung Neuts % TNF + Lung Neuts TNF +
1.06 0.262 0.15 0.208 < 0.0001 ! < 0.0001 40 1.5) ×1007!
! 1.1! 0.3! 0.2! 0.2! ***! 40! ***! 6 15 ! 3030! 1.0×1007! 2020! TNF + TNF % TNF+
% TNF + % 5,000,0005! 1010! Injection(blue) Total TNF + (x10+ TNF Total 86.9! 11.7! 98.7! 0.9! 0 0 87 11.7 98.8 0.86 WT! IL-1R-/-! WT! IL-1R-/-! TNF! B6 flaA B6 flaA
B! IL-1R-/- flaA IL-1R-/- flaA Inflammatory Monocytes! WT! IL-1R-/-! Lung iMCs % TNF + Lung iMCs TNF + 0.0628 0.431 0.0633 0.274 < 0.0001 ! 07 0.0496 100 1.5) ×10 ! 0.1! 0.4! 0.1! 0.3! 100! ***! 6 15 *! ! ! 8080! 1.0×1007! 6060! TNF + TNF % TNF+ % TNF + % 4040! 5,000,0005! 2020! Injection(blue) Total TNF + (x10+ TNF Total 52.5! 47.0! 80.3! 19.3! 0 0 52.5 47 80.4 19.3 WT! IL-1R-/-! WT! IL-1R-/-! TNF! B6 flaA B6 flaA C! IL-1R-/- flaA IL-1R-/- flaA Inflammatory Monocytes! -/-! Lung iMCs % CD86 + Lung iMCs CD86 + !
WT! IL-1R ) 0.0002 0.0194 0.2180.2! 0.10.127! 0.1270.1! 0.10.0667! 4040! ***! 6,000,0006 6! *! ! ! 3030! 4,000,0004! 2020! CD86 + % CD86 + 2,000,0002! % CD86 hi 1010! Injection(blue) 71.0! 28.7! 84.8! 15.0! 0 CD86 hi (x10 Total 0 71 28.7 84.8 15 WT! IL-1R-/-! WT! IL-1R-/-! CD86! B6 flaA B6 flaA D! IL-1R-/- flaA IL-1R-/- flaA Conventional dendritic cells! -/-! Lung cDCs % CD86 + Lung cDCs CD86 +
WT! IL-1R ! 0.361 0.0623 0.521 0.104 0.0211 ) 0.0054 0.4! 0.1! 0.5! 0.1! 2525! *! 500,0005 5! **! ! ! 2020! 400,0004! 1515! 300,0003! ! 1010 CD86 + 200,0002! % CD86 + % CD86 hi 55! 100,0001! Injection(blue)
89.1! 92.5! 6.9! 0 CD86 hi (x10 Total 0 89.2 10.410.4! 92.3 6.92 WT! IL-1R-/-! WT! IL-1R-/-! CD86! B6 flaA B6 flaA IL-1R-/- flaA IL-1R-/- flaA Figure 4-10. IL-1 signaling leads to the expression of CD86 and TNF by bystander cells. Cells were harvested from the lungs of infected WT or Il1r1-/- (IL-1R-/-) mice, treated with BFA, loaded with CCF4-AM, stained with an antibody against TNF (A & B) or CD86 (C & D). Representative flow plots from various cell populations are shown. Graphs show individual mice and mean pooled from 4 independent experiments. N=2-4 mice per group. Student’s T-test were performed between pooled groups. *p<0.05, **p<0.005, ***p<0.0005.
86 A! Inflammatory monocytes! WT! IL-1R-/-! ΔdotA! ΔflaA! ΔdotA! ΔflaA! TNF + iMCs B6 vs. IL1R 0.0508 0.554 0.0377 0.199 6.14e-3 0.203 0.0773 0.211 0.1! 0.6! 0.1! 0.2! 0! 0.2! 0.1! 0.2! 100100! !
! 8080!
6060!
40 % TNF + % TNF + % 40!
Injection(blue) 2020!
85.285.1! 14.214.2! 57.557.4! 42.342.3! 82.2 ! 17.617.6! 81.981.9! 17.817.8! ! ! ! 0 ! TNF! flaA B6 flaA flaA B6 dotA dotA dotA Δ Δ Δ Δ IL-1R-/- dotAIL-1R-/- flaA WT! IL-1R-/-! B! Conventional dendritic cells! WT! IL-1R-/-! ΔdotA! ΔflaA! ΔdotA! ΔflaA! % TNF + cDCs B6 vs. IL1R 0.403 0.127 0.19 0.0632 0.0447 0.0223 0.172 0.115 0.4! 0.1! 0.2! 0.1! 0.1! 0.1! 0.2! 0.1! 3030! ! ! 2020!
% TNF + 10
% TNF + % 10! Injection(blue)
93.193.1! 6.46.38! 80.280.3! 19.419.4! 94.894.3! 5.25.18! 9393.1! 6.76.7! ! !
0 ! ! TNF! flaA B6 flaA flaA B6 dotA dotA dotA Δ Δ Δ IL-1R-/- dotAIL-1R-/-Δ flaA -/-! WT! IL-1R Figure 4-11. Lack of IL-1 signaling does not alter the production of TNF by avirulent L. pneumophila infection. Cells were harvested from the lungs of infected WT or Il1r1-/- (IL- 1R) mice, treated with BFA, loaded with CCF4-AM, stained, fixed and stained with an antibody against TNF. Representative flow plots showing TNF production from inflammatory monocytes (A) or conventional dendritic cells (B) from the lung. Bar graphs on right show individual mice and pooled mean from 3 independent experiments.
87 6 6 6 6 1x10 JR32 ΔflaA inoculum, 24 hours1x10 PI JR32 ΔflaA inoculum, 24 hours1x10 PI JR32 ΔflaA inoculum, 24 hours1x10 PI JR32 ΔflaA inoculum, 24 hours PI ! ! ! ! ! ! ! ! ! ! ! ! 7! ns ns ns ! * ** *** ! ns ns ns ! ns * ** 1010 300300 2,0002.0 ! 1,5001.5 ! ! ! 1066! 1,5001.5! /mL) 10 /mL) ! ! 200200 ng 1,0001.0 /mL) pg 55! 1,000! 1010 ng 1.0 ! 500! TNF ( 100 IL-6 (pg/mL) IL-6 CFU/g Lung TNF (pg/mL) TNF 100 0.5 CFU/g lung 44! 500!
10 IL-6( 10 0.5 (pg/mL) IL-12p40 IL-12p40(
3 0 0 ! ! 0 ! ! ! ! ! ! 10 ! ! ! ! α! α! α! α! α β α β α β β! β! β! α β β β β! α β β α β β α β β α 1 1 1 1 1 1 1 1 IL- IL- IL- -IL-1 -IL- /IL- -IL-1 -IL- / -IL-1 -IL- / -IL-1 -IL- / Isotype α Isotype α α Isotype α α Isotype α α α α α 1 α 1 α 1 1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL-1 -IL- -IL- -IL-
-IL- isotype α IL-1 + isotype α IL-1 + isotype α IL-1 + isotype α IL-1 + α α α α α α α α α α α α Figure 4-12. Both IL-1α and IL-1β contribute to cytokine production during L. pneumophila infection. Mice were then infected with JR32 ΔflaA L. pneumophila intranasally. 24 hours PI, BAL and lungs were harvested and CFUs were plated for viable L. pneumophila from lung homogenates. Graphs show individual mice and mean. Statistics are one-way ANOVA with the stats representing the significance of the Tukey post-test between isotype mice and the groups of interest. ns=not significant, **p<0.05, **p<0.005, ***p<0.0005.
88 A! B! -/- 24 hours PI, MOI 10 Unfx WT MΦ" ΔflaA + WT MΦ" ΔflaA + IL-1R MΦ" ΔdotA! 1.051.1" 0.10.122 " 8.348.3" 4.54.62 " 10.2 " 3.13.14" 25,00025" "
" ΔflaA! 20,00020" dotA flaA 15,000/mL) 15" ng 10,00010" IL-6 pg/mL IL-6 IL-6( 5,000 Injection(blue) 5" 97.7" 1.1" 36.4" 50.6" 38.2" 48.5" 97.7 1.11 36.4 50.6 38.2 48.5 0 " -/- TNF" WTB6 MΦ IL-1R MΦ"
IL-1R-/- C! 24 hours post24 treatment hours post treatment 24 hours post treatment D! 24 hours Post treatment 22,000 4,0004,000" 22,00022,000" IL-1alpha" 600600" 20,000 IL-1alpha IL-1α IL-1alpha 3,000 20,000 " 3,000" " " " 20,000" IL-1beta 18,000 IL-1beta IL-1β IL-1beta 2,000 18,000 50 IL-1IL-1alpha/IL-1betaα+β" 50 IL-1alpha/IL-1beta50 IL-1alpha/IL-1beta400 /mL) 50" 50" /mL) /mL) " 400" 40 LPSLPS 40 LPS 40 LPS pg pg 40" pg 40" 30 3030" 3030" IL-6 (pg/mL) IL-6 20
IL-6 (pg/mL) IL-6 200 IL-6 (pg/mL) IL-6 TNF (pg/mL) TNF 2020" 200" IL-6(
20 IL-6( TNF ( 20" 10 1010" 1010" 0 0 " 0 2 3 4 5 0 " 2 3 10 4 10 5 10 10 2 3 4 5 β" β" β" β" α" 2" 3" 4" 5" 2" 3" 4" 5" α" 1010 1010 1010 1010 1010 1010 1010 10 α α β β /β /β + α α + [pg/mL] PBS 1 1 α [pg/mL] [pg/mL] α [pg/mL]" [pg/mL]" PBS IL- IL- 100ng LPS10ng IL-1 10ng IL-1 ng ng 100ng IL-1 100ng IL-110 100 10ng/mL IL-1 10ng/mL 10ng/mL IL-1 10ng/mL 100ng/mL LPS 100ng/mL 100ng/mL IL-1 100ng/mL 100ng/mL IL-1 100ng/mL 10ng/mL IL-1 10ng/mL 100ng/mL IL-1 100ng/mL
Figure 4-13. Bone marrow-derived macrophages are unresponsive to IL-1 treatment. (A) BMDMs from WT or IL-1R-/- mice were left untreated with or infected with the Lp02 ΔflaA strain of L. pneumophila at an MOI = 5, infected for 4 hours, loaded with CCF4-AM, and stained for TNF. (B) BMDMs from WT or IL-1R-/- mice were infected with the Lp02 ΔdotA or ΔflaA strain of L. pneumophila at an MOI = 5 and IL-6 production was measured by ELISA 24 hours PI. (C) BMDMs were treated with 100-100,000 pg/mL of recombinant IL- 1α, IL-1β, or IL-1α and IL-1β combined, or 100ng/mL LPS for 24 hours. TNF and IL-6 were then measured by ELISA. (D) NIH/3T3 cells were treated with 10-100ng/mL of recombinant IL-1α, IL-1β, or IL-1α and IL-1β combined, or 100ng/mL LPS for 24 hours and IL-6 was measured by ELISA. Graphs show mean ± S.E.M. Representative of two independent experiments.
89 A! Untx! LPS! PAM3CSK4! IL-1α! IL-1β! IL-1α+β! ! CD11c 19.119.1 ! 40.040 ! 32.232.2 ! 26.326.3 ! 27.427.4 ! 27.627.6 !
CD86! B! Untx! LPS! PAM3CSK4! IL-1α! IL-1β! IL-1α+β! !
CD11c 0.94 26.4 7.8 3.77 11.1 11.8 0.9! 26.4! 7.8! 3.8! 11.1! 11.8!
TNF! C! BM DCs, 6 hours PT BM DCs, 6 hours PT D! BM DCs, 6 hours PT BM DCs, 6 hours PT 5050! 5050! ns! ns! 5,0005000! 16,00016000! ***! ***!******!******!******! ***! *! *! 4000 ! * ! ! 4040! 4040! 3,0003000! 12,00012000! ! 2000 /mL) /mL) 3030! 3030! 1,0001000! 8,0008000! pg 100 pg 200200! 20 20 20! % TNF + 20! %TNF+ 80
% CD86 hi 80! IL-6 (pg/mL) IL-6 %CD86 hi TNF (pg/mL) TNF
60 IL-6( TNF ( 100 1010! 1010! 4040! 100! 20 ! ! ! ! ! ! ! !
0 ! 0 0 ! 0 ! ! β! β! β! β! α! β! α! β! α! β! α β /β α β /β α β /β α α! β β! /β + + + α α + α α Untx LPS PAM Untx LPS PAM Untx LPS PAM Untx LPS PAM α α α α LPS LPS LPS LPS Untx Untx Untx Untx IL-1 IL-1 IL-1 IL-1 IL-1 IL-1 IL-1 IL-1 100ng IL-1100ng IL-1 100ng IL-1100ng IL-1 100ng IL-1100ng IL-1 100ng IL-1100ng IL-1 IL-1 IL-1 IL-1 100ng IL-1 100ng IL-1 IL-1 100ng IL-1 100ng IL-1 PAM3CSK4 PAM3CSK4 PAM3CSK4 PAM3CSK4 Figure 4-14. Bone marrow-derived dendritic cells produce cytokines and express CD86 in response to IL-1 treatment. (A & B) BMDCs were treated with LPS, PAM3CSK4, or 100ng/mL IL-1α, IL-1β, or both for 6 hours. 3 hours prior to harvest, cells were treated with BFA. Cells were then fixed and stained for CD86 (A) or TNF (B). Representative flow plots are shown. (C) Quantification of plots shown in (A & B). (D) BMDCs were treated as in (A & B) for 24 hours. Supernatants were then harvested and TNF and IL-6 production were measured by ELISA. Graphs show individual wells and mean. ANOVA was performed and statistics show significance between untreated cells and the appropriate condition as determined by Tukey post test. *p<0.05, **p<0.005, ***p<0.0005, ns=not significant.
90 A! Untx! LPS! IL-1α! IL-1β! IL-1α+β! ! SSC-A
1.81.77 ! 55.755.7 ! 4.84.82 ! 4.84.79 ! 6.05.99 ! TNF! B! Untx! LPS! IL-1α! IL-1β! IL-1α+β! ! SSC-A
2.82.76 ! 57.757.7 ! 0.70.687 ! 0.60.552 ! 0.80.828! Data 1 TNF! Data 1 C! 70 7070! WTWT! 60 WT 6060! IL-1RIL-1R-/--/-! 50 IL-1R-/- 10 5050! ! 1010! 8 8! 6 % TNF +
%TNF+ 6! 4% TNF + 4! 2 2! 0 !
0 ! ab β! Untx LPS betaα! β! alpha ab +
LPS beta α
Untx LPS Untx alpha IL-1 IL-1 IL-1 Figure 4-15. Alveolar macrophages produce TNF in response to IL-1 treatment. (A & B) Alveolar macrophages from WT (A) or IL-1R-/- (B) mice were isolated and seeded in culture at 10,000 cells per well in 150µl DMEM + 10% FBS. Cells were then treated with 100ng/mL LPS, IL-1α, IL-1β, or both for 6 hours. 3 hours prior to harvest, BFA was added to the wells. Cells were then fixed and then stained for TNF. (C) Quantification of (A & B). Graphs show mean of two independent mice.
91 A! Untx! LPS! IL-1α! IL-1β! IL-1α+β! 0.2970.3! 0.10.0156! 0.4290.4! 0.10.107! 0.4160.4! 0.00! 0.3310.3! 0.10.0331! 0.8550.9! 0.10.0238! ! CD86
99.3 ! 0.30.437! 38.438.5! 61.061! 98.9 ! 0.70.705! 98.8 ! 0.80.812! 98.198.0! 1.01.02! TNF! B! Untx! LPS! IL-1α! IL-1β! IL-1α+β! 0.2110.2! 0.00! 0.6970.7! 0.10.0634! 0.330.3! 0.10.0695! 0.430.4! 0.10.0159! 0.3190.3! 0.10.0159! ! CD86
99.1 ! 0.70.698! 42.5 ! 56.756.8! 98.5 ! 1.11.06! 99.3 ! 0.20.302! 99.1 ! 0.50.526! TNF! Data 1 iMCs CD86 C! iMCs TNF D! 7070! 70 5! CD45+Ter119- Cells! Ly6Chi Ly6G- Cells! WTWT! WT 5050! 60 WT ! 4! IL-1RIL-1R-/--/-! IL-1R-/- 3030! 50 IL-1R-/- 97.697.6 ! ! !
1010! 10 ! 3! 88! 8 Ly6G %CD86hi CD11b
%TNF+ 66! 6 2! % TNF + % TNF + % CD86hi 44! 4 1! 22! 2 95.395.3!
0 ! ! 0 ! 0 ! Ly6C! Ly6C! β! β! α! β! α! β! + ab + ab ab LPS LPS LPS beta α beta betaα Untx Untx Untx LPS LPS
Untx alpha Untx alpha alpha IL-1 IL-1 IL-1 IL-1 IL-1 IL-1 Figure 4-16. Inflammatory monocytes do not respond to IL-1 treatment. (A & B) inflammatory monocytes were isolated from the bone marrow of WT (A) or IL-1R-/- (B) mice. Cells were then treated with 100ng/mL LPS, IL-1α, IL-1β, or both for 6 hours. 3 hours prior to harvest, BFA was added to the wells. Cells were then fixed and then stained for TNF and CD86. (C) Quantification of (A & B). (D) Gating strategy for isolated inflammatory monocytes. Monocytes are Ly6Chi, CD11bint to CD11bhi and do not express Ly6G. Monocytes were 90-95% pure. Graphs show mean of two independent mice. Representative of two independent experiments.
92 CHAPTER 5
DISCUSSION
A. Alveolar macrophages and neutrophils are targeted by the type IV secretion system of L. pneumophila
Legionella pneumophila uses its T4SS to inject a large number of effector proteins into the cytosol of host phagocytes (Chen et al., 2010). The T4SS is necessary for intracellular replication and pathogenesis as L. pneumophila mutants lacking a functional
T4SS fail to establish a replicative niche and do not cause pathology in mice (Barry et al., 2013; LeibundGut-Landmann et al., 2011; Marra et al., 1992; Roy et al., 1998). In addition to being required for L. pneumophila pathogenesis, T4SS activity potently activates multiple cytosolic immunosurveillance pathways (Casson and Shin, 2013;
Losick and Isberg, 2006; Moltke et al., 2013; Shin et al., 2008). Thus, cells that interact with L. pneumophila and receive T4SS-translocated effectors serve as a potential replicative niche, but may also contribute to the immune response against L. pneumophila. However, the precise identity of such cells is unknown. I, therefore, set out to identify host cells that receive T4SS-translocated effectors during infection with L. pneumophila. BlaM reporter systems have been used during in vivo infection with
Yersinia pseudotuberculosis (Harmon et al., 2010; Maldonado-Arocho et al., 2013),
Yersinia pestis (Pan et al., 2009; Pechous et al., 2013), Yersinia entercolitica (Köberle et al., 2009), Salmonella typhimurium (Geddes et al., 2007; Yoon et al., 2011), and
Pseudomonas aeruginosa (Diaz and Hauser, 2010; Kung et al., 2012) to detect the translocation of effectors into host cells by the type III and type IV secretion systems. In chapter 3, I demonstrate that by using β-lactamase (BlaM) translationally fused to the
T4SS-translocated effector protein RalF, I can successfully track injection by the T4SS
93 into host cells during both in vitro and in vivo infection, and I describe the first use of this
BlaM reporter during in vivo pulmonary infection with L. pneumophila.
I observed robust T4SS-mediated injection into alveolar macrophages at four hours post infection, consistent with previous observations that these cells are the primary cell type infected by L. pneumophila during pulmonary infection in human patients (Nash et al.,
1984). At later time points post infection, I find that in addition to alveolar macrophages, a large number of the cells injected by L. pneumophila in vivo are neutrophils (Figure 5-
1). This is likely due to the influx of neutrophils into the lungs and airway space during infection (Ang et al., 2012; Berrington et al., 2010; Brieland et al., 1994; Frutuoso et al.,
2010; Winn and Myerowitz, 1981; Winn et al., 1978). Other researchers have shown that neutrophils contain intracellular L. pneumophila in a non-permissive mouse model of pulmonary infection, but it was not examined whether L. pneumophila could survive within neutrophils or whether neutrophils are capable of receiving T4SS-translocated effectors (LeibundGut-Landmann et al., 2011). In vitro studies have suggested that
Legionella species are resistant to the highly bactericidal activity of neutrophils but cannot replicate within these cells (Horwitz and Silverstein, 1981; Weinbaum et al.,
1983). Thus, I initially presumed that although neutrophils might be injected during in vivo infection, the majority of bacteria would eventually be cleared due to a failure to replicate in these cells, and I would not be able to detect any appreciable number of viable bacteria within these cells. I obtained viable L. pneumophila in numbers that roughly corresponded with the frequency of injection seen with our reporter system, suggesting that L. pneumophila can survive within neutrophils during in vivo infection.
Unexpectedly, given the large number of neutrophils that enter the lung, the total number of L. pneumophila CFUs harbored by neutrophils is greater than the total number of L. pneumophila CFUs found within the alveolar macrophage population 24 and 48 hours
94 PI. Given the large numbers of infected neutrophils that I observed, it would be of interest to determine whether L. pneumophila could establish an ER-derived vacuole and successfully replicate within neutrophils, as this could represent another intracellular niche for L. pneumophila. Most bacteria are not thought to survive or replicate within neutrophils, but there are a few exceptions, including Neisseria gonorrheae (Simons et al., 2005), Anaplasma phagocytophilum (Chen et al., 1994; Ohashi et al., 2002), and pathogenic Escherichia coli (Nazareth et al., 2007).
B. Non-phagocytic cells are not targeted by L. pneumophila for injection
As I was only able to detect robust T4SS-dependent injection into alveolar macrophages and neutrophils, I conclude that phagocytic cells in the airway space are the primary recipients of T4SS-translocated effectors during pulmonary L. pneumophila infection.
Whether or not T4SS-injected cells survive infection and traffic to other organs including lymph nodes is unknown. However, previous studies have reported that alveolar macrophages do traffic to lymph nodes when given allergic stimuli (Kirby et al., 2009).
Thus, it would be of interest to investigate whether T4SS-injected alveolar macrophages either induce adaptive immunity or participate in the dissemination of infection to other organs (Kirby et al., 2009). Surprisingly, I was unable to detect T4SS-injected conventional dendritic cells during in vivo infection. Dendritic cells undergo rapid apoptosis in response to L. pneumophila T4SS activity (Nogueira et al., 2009), which could account for why I do not detect as robust injection in dendritic cells as compared to macrophages in vitro and in vivo, although the number of DCs increased during infection. Further experiments to determine whether injection could be detected in DCs lacking apoptotic regulators such as BAX and BAK, thus, resistant to L. pneumophila- induced apoptosis, could be fruitful. However, C57BL/6-derived macrophages undergo rapid pyroptosis in response to WT L. pneumophila infection, yet I still detect injection in
95 this cell type, suggesting that L. pneumophila-induced cell death is an insufficient explanation to account for the lack of detectable injection in DCs (Case et al., 2009).
Given that phagocytosis is required for T4SS-mediated translocation, another possibility is that dendritic cells in the lung may not efficiently phagocytose L. pneumophila
(Charpentier et al., 2009). Alternatively, pulmonary dendritic cells and L. pneumophila may be spatially separated during in vivo infection. Infection may occur primarily in the airway space itself, and thus only neutrophils and alveolar macrophages are available targets for translocation. To what extent infection occurs in the lung tissue itself has not been fully investigated. Histological studies would help elucidate the temporal and spatial constraints present during L. pneumophila infection.
Although I could only detect T4SS-mediated translocation in pulmonary phagocytic cells, other infectious models using different pathogens have observed translocation into a larger variety of cells. For instance, studies using an intraperitoneal model of Yersinia pseudotuberculosis demonstrate translocation mediated by the type three secretion system into splenic phagocytes as well as splenic B and T cells (Köberle et al., 2009). In an infectious model using the parasite Toxoplasma gondii that does not encode a secretion system but does inject proteins into infected cells, injects phagocytic cells, non-immune cells, and even injects neurons (Koshy et al., 2012). Interestingly, in the T. gondii model, injection is detected in cells that do not harbor intracellular parasites. I did not assay whether L. pneumophila also injects cells it does not invade, but the data obtained via imaging flow cytometry suggest that nearly all injected cells contain L. pneumophila and it has not been demonstrated that L. pneumophila can egress from an infected host cells without subsequent death of the cell.
96 The experiments performed in this study looked at cells targeted by the T4SS of L. pneumophila in immunocompetent hosts. Infections of immunocompromised mice, such as mice lacking IFNγ and MyD88, result in higher bacterial burdens, bacterial dissemination to peripheral organs, and sometimes death of the host organism (Archer et al., 2009; Spörri et al., 2006). Higher bacterial burdens would require increased bacterial burdens in alveolar macrophages and neutrophils either in the frequency of infected cells, or an increase in the number of viable bacteria in a single cell. However,
MyD88-deficient mice fail to mount an appropriate immune response and have delayed and reduced recruitment of cells such as neutrophils to the site of infection (Archer and
Roy, 2006). Whether other cells types, such as epithelial or dendritic cells, harbor viable
L. pneumophila in immunocompromised hosts remains to be seen. For dissemination, other cell types, such as tissue resident macrophages in the spleen and liver, may harbor viable L. pneumophila; however, I did not look at a model of dissemination. It will be important to investigate targeting of the T4SS in these models as they mimic the increased bacterial burdens and dissemination seen in immunocompromised human patients, which would lead to a better understanding of clinical disease (Lowry and
Tompkins, 1993).
Using the A549 alveolar epithelial cell line during in vitro infection, I detected a low percentage of T4SS-injected cells under conditions using both nonmotile and motile bacteria. Although the L. pneumophila were centrifuged onto A549 cells in vitro, motility may alter the contact between host cells and L. pneumophila in a way that facilitates bacterial entry. Many researchers utilize A549 cells as a model for L. pneumophila infection and can detect productive bacterial replication within these cells (Maruta et al.,
1998). However, these studies either use higher MOIs than those used in this study or opsonize the bacteria prior to infection. These discrepancies in technique may explain
97 why I am unable to detect higher percentages of injected alveolar epithelial cells during in vitro infection. I was also unable to detect robust T4SS-dependent translocation into airway epithelial cells or other non-phagocytic cells during in vivo infection. Utilizing a non-permissive model of C57BL/6 mice infected with WT L. pneumophila, other researchers found that lung epithelial cells did not appear to contain L. pneumophila
(LeibundGut-Landmann et al., 2011). Our data argue against a direct role for airway epithelial cells in cytosolic sensing of L. pneumophila T4SS activity during pulmonary infection. Airway epithelial cells have been shown to indirectly respond to L. pneumophila infection by producing the chemokine CXCL1 in response to IL-1 produced by macrophages (LeibundGut-Landmann et al., 2011).
C. Alveolar macrophages and neutrophils produce cytokines in response to virulent L. pneumophila infection
I found that alveolar macrophages secreted TNF and IL-1α four hours PI in vivo. Both
TNF and IL-1α are important for controlling L. pneumophila infection. The inflammasome-regulated cytokines IL-1α and IL-1β are critical for neutrophil recruitment to the lung airway during L. pneumophila infection through a mechanism involving the IL-
1R-dependent induction of CXCL1 from alveolar epithelial cells (Barry et al., 2013;
Casson et al., 2013; LeibundGut-Landmann et al., 2011). It is unclear whether other cells in the lung produce cytokines so early during infection. However, as IL-1α production in vivo is T4SS-dependent and I could only detect T4SS injection into alveolar macrophages at 4 hours post-infection, our data would suggest that during the first few hours of infection, alveolar macrophages are the primary source of IL-1α, consistent with another study indicating that hematopoietic cells are an early source of
IL-1α (Barry et al., 2013).
98 At 24 hours post-infection, I found that neutrophils recruited to the lungs of mice infected with ΔflaA L. pneumophila secrete the proinflammatory cytokines TNF and IL-1α, but not mice infected with ΔdotA L. pneumophila. These data indicate that, like alveolar macrophages, neutrophils secrete cytokines in response to cytosolic sensing of T4SS- translocated bacterial products. It has previously been reported that neutrophils can secrete cytokines, but the signaling pathways that control cytokine production and secretion in neutrophils are poorly understood. Neutrophils are known to release TNF- containing granules in response to a variety of stimuli, including various bacterial infections (Bennouna et al., 2003; Tsuda et al., 2004). Previous research has demonstrated that neutrophils can release IL-1α in a model of sterile inflammation or IL-
1β independently of caspase-1 and caspase-11 in a mouse model of arthritis and during bacterial infection (Guma et al., 2009; Karmakar et al., 2012; Rider et al., 2011). In an intravenous infection model of L. pneumophila infection, splenic neutrophils were shown to produce IL-18, an IL-1 family cytokine, which induces IFN-γ production from NK cells
(Spörri et al., 2008). Previous studies demonstrated that IL-1α secretion is regulated by both inflammasome-dependent and –independent pathways during in vivo WT L. pneumophila infection (Barry et al., 2013; Casson et al., 2013), but it is unknown which of these pathways are used by macrophages and neutrophils to secrete IL-1α in vivo. It would be of interest to determine the host and bacterial components required for release of IL-1 and other cytokines from macrophages and neutrophils in response to in vivo infection with L. pneumophila.
Overall, our study is the first to define the cell types that receive T4SS-translocated effectors during pulmonary L. pneumophila infection. I reveal that both alveolar macrophages and neutrophils receive translocated effector proteins, harbor viable bacteria, and respond to infection by producing inflammatory cytokines. Collectively, our
99 data indicate that alveolar macrophages and neutrophils not only provide an intracellular reservoir for L. pneumophila, but also provide an important source of proinflammatory cytokines that contribute to a successful host immune response during pulmonary L. pneumophila infection.
D. Bystander cytokine production by uninjected cells
Infection with L. pneumophila leads to robust pro-inflammatory cytokine production.
Much of this cytokine response is dependent on cytosolic sensing of T4SS activity
(Fontana et al., 2011; Shin et al., 2008), although paradoxically, several T4SS effector proteins introduced into infected cells potently block host protein synthesis (Barry et al.,
2013; Belyi et al., 2006; 2008; Fontana et al., 2012; Ivanov and Roy, 2013; McCusker et al., 1991; Shen et al., 2009). In chapter 4, I set out to examine how a robust cytokine response is generated despite this translational block. Our data indicate that cells receiving T4SS effectors can synthesize IL-1α and IL-1β, but are poor producers of the key immune proteins TNF, IL-6, IL-12, and CD86. Instead, bystander immune cells that have not received T4SS effectors produce the majority of these immune signals in vitro and in vivo. Furthermore, IL-1 signaling is required for robust production of TNF and other immune proteins by bystander cells. Thus, our data suggest that detection of
PAMPs is inadequate to trigger a robust immune response in bystander cells. Instead, a cytokine released by infected cells signals to uninfected bystander cells to bypass the pathogen-imposed translational block and enable production of a robust inflammatory cytokine response.
Why TLR signaling alone is insufficient to drive cytokine responses in vivo is a conundrum. In vitro and in vivo, the use of purified TLR ligands can induce robust cytokine production and cellular recruitment to the site of delivery. It must be noted,
100 however, that many LPS instillation protocols use an amount of LPS that can equate to a number of bacteria on the order of 1010 CFUs (if 0.1ng of LPS = 105 CFU of E. coli, then
10-100µg of LPS would be equivalent to nearly one million times more bacteria) (Arndt et al., 2005; Raetz, 1986; Szarka et al., 1997). Assuming equivalent calculations for L. pneumophila, this dose is equal to a 10,000-fold increase in the number of L. pneumophila we detect even during the peak of virulent pulmonary infection. Thus, TLR signaling can induce cytokine production alone, but perhaps only in response to large amounts of TLR ligands. L. pneumophila LPS is a poor stimulator of TLR4 (Lettinga et al., 2002) and infection itself does not appear to induce the required amount of TLR stimulation to drive cytokine responses in vivo.
Beyond the total amount of TLR ligands present, tolerance to TLR-mediated signaling, specifically tolerance to LPS or endotoxin is a well-described, multi-faceted phenomenon
(Biswas and Lopez-Collazo, 2009). Upon repeated, low exposure to LPS, cells exhibit milder and milder cytokine and recruitment responses. This tolerance is mediated by a variety of mechanisms, including increased expression of negative regulators of TLR signaling (Liew et al., 2005; Nimah et al., 2005), decreased expression of TLRs and downstream signaling pathways (Biswas and Tergaonkar, 2007; Medvedev et al., 2000), and changes in the chromatin containing proinflammatory genes (Chan et al., 2005;
Foster et al., 2007). Cells of the innate immune system patrol barrier sites and cells such as alveolar macrophages are exposed to the outside of the body, these cells may be in contact with a low, constant level of TLR ligands that may induce and maintain a state of tolerance in these cells. Indeed, a role for TLR tolerance in maintaining intestinal homeostasis has been demonstrated (Abreu et al., 2002; Singh et al., 2005). Likewise, research has demonstrated that innate immune cells of the lamina propria do not produce TNF or IL-6 in response to TLR stimulation, suggesting that cells at other barrier
101 sites may behave in a similar fashion (Franchi et al., 2012). As these cells may be tolerized, other signals such as IL-1 may be required either to act synergistically with
TLR and other PRR signaling to overcome this tolerance or may sensitize innate immune cells to the presence of bacterial PAMPs. It would be of interest to investigate whether TLR signaling is enhanced by the presence of IL-1. Treating cells with purified
TLR ligands or avirulent T4SS-deficient L. pneumophila in the presence or absence of
IL-1 will help determine whether synergistic effects occur with this combination of stimuli.
E. Injected cells produce IL-1α and IL-1β during protein translations inhibition
Cells targeted by the T4SS of L. pneumophila still transcribe many proinflammatory genes, such as Il6, Tnf, Il1a, and Il1b. These cells, however, are unable to translate IL-6 and TNF during virulent infection, but are able to translate IL-1α and IL-1β (Figure 5-2).
The inability of injected cells to produce TNF is partly due to effector-mediated protein translation inhibition, as cells injected by mutants of L. pneumophila lacking either 5 or 7 of the protein translation inhibiting effectors were able to produce TNF during infection.
At later times, however, injected cells infected with these mutants produced just as little
TNF and IL-6 as their WT infected counterparts. These data indicate that part of the inability of injected cells to produce certain proinflammatory cytokines is effector driven.
Why injected cells are unable to produce TNF and IL-6 later in infection remains to be elucidated. Given the highly redundant nature of the T4SS effectors, other effectors that inhibit host protein translation may exist. Alternatively, later protein translation may be a host-driven response depending on mTOR or some other stress-induced pathway.
Future experiments will focus on altering mTOR signaling during L. pneumophila infection and assaying for cytokine production from infected cells to determine whether a mTOR-mediated host response is responsible for the block in protein translation.
Alternatively, infections using T4SS-deficient L. pneumophila in combination with mTOR
102 inhibitors such as rapamycin may elucidate whether translation inhibition during L. pneumophila infection is host cell-driven.
It is intriguing that infected cells are able to effectively translate IL-1 but not other cytokines that I assayed. Unlike conventional cytokines, IL-1α and IL-1β are translated by cytosolic ribosomes in a pro-form and are secreted via an unknown mechanism requiring the activation of the inflammasome (Rathinam et al., 2012; Stevenson et al.,
1992). MyD88 and altered mTOR signaling participate in the selective translation of proinflammatory cytokines during L. pneumophila infection via unknown or poorly understood mechanisms (Asrat et al., 2014; Brieland et al., 1995; 1998; Byrd and
Horwitz, 1989; Casson et al., 2013; Copenhaver et al., 2014; Ivanov and Roy, 2013;
LeibundGut-Landmann et al., 2011; Spörri et al., 2006). Whether MyD88 and altered mTOR signaling occurs directly in infected cells or influences cytokine production in bystander cells remains to be determined. Previous research has suggested that mRNA abundance may determine which transcripts are selected for translation when translation is inhibited (Asrat et al., 2014; Ivanov and Roy, 2013). My data, however, do not demonstrate a large difference in the total abundance between transcripts that are translated (Il1a, Il1b) and transcripts that are not (Il6, Tnf). Thus, it is unlikely that mRNA fully explains the mechanism of selective translation.
During periods of protein synthesis blockade, not all proteins remain untranslated. Host cells target key mRNAs to ribosomes during protein synthesis inhibition induced during the unfolded protein response, which allows for at least minimal translation of key proteins. Targeting of key mRNAs to the ribosome during protein synthesis inhibition requires the presence of unique 5’ and 3’ untranslated regions (UTR) on the mRNA of interest (Vivinus et al., 2001). Host cells also segregate cytosolic non-essential mRNAs
103 into structures called stress granules (Thomas et al., 2011). These granules associate mRNAs with the mRNA degradation machinery, depleting the cell of non-essential mRNAs to translate. Sequestration and clearance of non-key mRNAs prevents their translation and decreases the competition key mRNAs have to overcome to be translated. Thus, even when global protein production is decreased, cells can preferentially target certain mRNAs for translation. It is known that the mRNAs encoding the proinflammatory cytokines IL-6 and TNF have AU-rich UTRs, but these UTRs have been implicated in the instability of these mRNAs (Villarino et al., 2011). In contrast, the mRNA encoding IL-1β has been previously shown to be more stable than the mRNA for
TNF (Chen et al., 2006). This raises the possibility that inflammasome-related mRNAs, due to their unique UTR sequences, are preferentially translated during L. pneumophila infection. Future work will investigate whether certain cytokine mRNAs, such as IL-1α and IL-1β contain regulatory elements in their UTRs that spare them from the effects of global suppression of protein synthesis and if mRNA regulation of IL-1 is important for its production during infection with L. pneumophila.
F. IL-1 induces TNF production and CD86 expression in dendritic cells and alveolar macrophages
My data indicate that uninfected bystander cells play a critical role in host defense during
L. pneumophila infection. These cells both directly or indirectly sense the IL-1 released by infected cells and respond by producing TNF and other immune proteins. IL-1 signaling utilizes MyD88 and leads to the activation of NF-κB as well as MAPK. In epithelial cells, IL-1 induces IL-6 production as well as chemokine production. TNF is not induced in response to IL-1 by epithelial cells; however. These data indicate that epithelial cells are incapable of producing TNF to a variety of stimuli that induce the production of other cytokines. IL-1-induced TNF production is not a well-characterized
104 phenomenon, but human PBMCs have been shown to produce TNF in response to treatment with IL-1α as well as IL-1β (Kim et al., 2013). As these experiments were performed with whole PBMCs, it is unclear which cell types specifically respond to IL-1, but it implies at least one cell type expresses the IL-1R and can produce TNF in response to IL-1 signaling.
Expression of the IL-1R in vivo is poorly characterized. Epithelial cells and T cells are known to express the IL-1R and the response to IL-1 stimulation is best characterized in these cell types (Dinarello, 1996). Bone marrow-derived macrophages fail to respond to
IL-1 either alone or in combination with other signals. Bone marrow-derived macrophages also do not express TLR5 and do not respond to extracellular flagellin, even though various subsets of macrophages in vivo do (Means et al., 2003). I detected a low, but evident production of TNF by alveolar macrophages in response to IL-1 stimulation ex vivo. It is possible that ex vivo culture diminishes the responsiveness of cells to IL-1 stimulation, although cells were able to produce robust amounts of TNF in response to LPS, ruling out broad inability of the cells to function. These data indicate that alveolar macrophages express the IL-1R. IL-1 stimulation is known to activate alveolar macrophages in vivo, but TNF production in response to IL-1 has never been assayed until this study (Hussell and Bell, 2014). Only a small frequency of alveolar macrophages produced TNF in response to IL-1 stimulation ex vivo. It is unclear whether all alveolar macrophages express the IL-1R ubiquitously or if only a subset of cells express the receptor. Although an antibody exists to stain for the IL-1R1 (CD121a) chain of the IL-1R, staining for this receptor in vivo has proved difficult and not definitive in mice (data not shown), but staining of human T cells demonstrates heterogeneous IL-1R expression on subsets of activated T cells (Dower et al., 1986; Lee et al., 2010).
Likewise, it is unclear whether TLR signaling or some other infectious cue can induce IL-
105 1R expression on alveolar macrophages. Activation of T cells can increase expression of the IL-1R on the surface, suggesting that similar activation of other cell types might also result in increased receptor expression and responsiveness to IL-1 (Lee et al., 2010).
Future work will focus on assaying IL-1R expression on various innate immune cell types during L. pneumophila infection to address whether receptor expression is altered during infection.
Bone marrow-derived dendritic cells, like alveolar macrophages, also responded to IL-1 stimulation. These cells produced TNF and increased surface expression of CD86.
Again, only a small portion of dendritic cells produced TNF in vitro. These data suggest that bone marrow-derived dendritic cells express the IL-1R at a resting state. It would be of interest to determine whether dendritic cells from the lung or other organs also express the IL-1R and respond in a similar fashion. Of note, dendritic cells transferred into the lungs of mice increase expression of CD86 upon repeated intranasal treatment with IL-1 which indicates that dendritic cells may express the IL-1R and are activated upon IL-1 stimulation (Pang et al., 2013).
Inflammatory monocytes isolated from the resting bone marrow pool did not respond to
IL-1 stimulation ex vivo. This is in stark contrast to the massive reduction in TNF- producing monocytes in IL-1R-/- mice. It is still unclear whether inflammatory monocytes express the IL-1R at a basal state or express it during infection or tissue egress.
Experiments in this study use bone marrow-derived monocytes, which are considered a resting pool of monocytes. Many recent advances in the description and analysis of inflammatory monocytes have described several subsets of monocytes that express different chemokine receptors, including CCR2 and CX3CR1 and vary in their functional capacities (Hohl et al., 2009; Narni-Mancinelli et al., 2011; Yang et al., 2014). Thus,
106 future work should determine the phenotype of the inflammatory monocytes recruited to the lung during L. pneumophila infection as well as their expression of various receptors, including IL-1R. If inflammatory monocytes do express the IL-1R, then their lack of a response to IL-1 stimulation alone could indicate that they require additional signals, such as TLR signaling or chemokines, to produce cytokines. It is also unclear whether inflammatory monocytes are responding directly to IL-1R signaling in a cell-intrinsic manner, or are responding to a signal produced in response to IL-1 made by another cell type. Chimeric mice, in which a mix of WT and IL-1R-deficient innate immune cells exist, will be useful to determine the direct and indirect role of IL-1 signaling on inflammatory monocytes as well as other innate immune cell types. There is some evidence for this cell-extrinsic response to IL-1 as neutrophils are recruited to sites of inflammation by chemokine production from epithelial cells induced by IL-1 signaling. Thus, we find that
IL-1R signaling is necessary, but not sufficient, to drive optimal cytokine production from monocytes during L. pneumophila infection.
I was unable to determine a role for IL-1 signaling directly on neutrophils during infection.
Neutrophils were originally thought to migrate towards IL-1 and do express some inducible level of IL-1R (Fasano et al., 1991). However, instead of migrating directly in response to IL-1, neutrophils respond to the aforementioned chemokine products of IL-1 signaling on epithelial cells (Tateda et al., 2001b). Neutrophils are a source of IL-1α during L. pneumophila infection, but unfortunately, we were unable to induce cytokine production from neutrophils isolated from the resting bone marrow pool ex vivo in response to any stimuli, including LPS (data not shown). It is possible that neutrophils do respond to IL-1, but as my culturing systems are unable to demonstrate neutrophil activation in response to any stimuli, I am currently unable to determine a direct role IL-1 signaling on neutrophils during infection.
107
Together, these data indicate that in certain cell types, IL-1 signaling is sufficient to drive a low-level cytokine response. However, not all cells are activated directly by IL-1 and IL-
1 does not induce robust cytokine production. Yet, a lack of IL-1R affects cytokine production from neutrophils, alveolar macrophages, dendritic cells, and inflammatory monocytes. These data argue for a complex model of IL-1 signaling in vivo during L. pneumophila infection (Figure 5-3). It is unclear what other signaling pathways might be important for bystander cytokine production in vivo.
Unlike other models, my data indicate IL-1 signaling is partially required for the bystander cytokine response to L. pneumophila in vivo. There are multiple possible benefits to regulating cytokine production in this way. First, it ensures the production of pro-inflammatory cytokines during infection with a pathogen that limits host protein translation. Second, it may be a strategy that the immune system uses to avoid inappropriate responses to avirulent bacteria and instead respond only to virulent pathogens when it encounters a second signal, such as IL-1, that is specifically released in response to virulent pathogens (Fontana and Vance, 2011; Franchi et al., 2012).
Third, cytokine production by bystander cells may provide a means of amplifying early immune signals, allowing for a more rapid and robust response. Bystander activation is likely to be a common strategy employed by the immune system for overcoming pathogen virulence mechanisms, and multiple mechanisms may exist for activating bystander cells by different cell types (Ablasser et al., 2013; Dolowschiak et al., 2010;
Dreux et al., 2012; Kasper et al., 2010; Patel et al., 2009). Notably, during influenza A virus infection, IL-1 signaling activates bystander dendritic cells and enables subsequent priming of naïve CD8+ T cells (Pang et al., 2013). This study, along with our finding that uninfected bystander cells upregulate the costimulatory molecule CD86 (Figures 3 &
108 S5), has implications for understanding how a T cell response is generated during L. pneumophila infection. This implies that bystander cells may be key antigen presenting cells, perhaps following uptake of dead or dying infected cells (Trunk and Oxenius, 2012;
Yrlid and Wick, 2000).
Our findings also elucidate another critical function for IL-1 in early innate immune defense. In addition to the well-established role of IL-1 in eliciting production of neutrophil-attracting chemokines and the subsequent recruitment of neutrophils to the site of infection (Barry et al., 2013; Casson et al., 2013; LeibundGut-Landmann et al.,
2011; Mascarenhas et al., 2015), my data indicate that IL-1 mediates production of cytokines by bystander innate immune cells (Figure 4-9 & 4-10). Immune signals other than IL-1 also likely contribute to T4SS-dependent proinflammatory cytokine production in vivo, as IL-1 signaling is required for the maximal production of TNF and IL-12, but is dispensable for IL-6 production during L. pneumophila infection (Figure 4). The nature of these other immune signals and whether they can compensate for the absence of IL-1 in certain infection settings remains to be determined. Future work will focus whether other
IL-1 family members, including IL-18 and IL-36 also contribute to bystander responses to
L. pneumophila infection (Spörri et al., 2008; Towne et al., 2011; Vigne et al., 2011).
Both IL-18 and IL-36 signal via MyD88 downstream of their receptors (Dinarello, 2013).
It is of interest to determine whether these other MyD88-dependent signals contribute to the immune response against L. pneumophila infection as it is currently unclear which signal or signals contribute to the defect in MyD88-deficient mice.
G. Bystander signaling occurs in other models of infection
Other pathways to activate immune responses have been described for other pathogens. The proinflammatory form of cell death known as necroptosis may activate
109 immune responses to Y. pseudotuberculosis in vivo from uninfected bystander cells
(Philip et al., 2014). Necroptosis has not been demonstrated during L. pneumophila infection as inhibiting pyroptosis prevents cell death (Casson et al., 2013). Inhibiting the production of reactive oxygen species (ROS) in intestinal epithelial cells infected with
Listeria monocytogenes prevents CXCL2 production by neighboring uninfected cells
(Dolowschiak et al., 2010). These data indicate that bystander epithelial cells are important for immune responses to L. monocytogenes and that ROS activates uninfected bystander cells. Similar experiments performed in macrophages infected with
L. pneumophila, however, did not produce similar results (data not shown). These data suggest that ROS production does not play a role in bystander activation during L. pneumophila infection. Gap junction signaling is important for the propagation of bystander responses to S. flexneri and vaccinia virus infection in epithelial cells
(Ablasser et al., 2013; Kasper et al., 2010). Inhibition of gap junction signaling during L. pneumophila infection in vitro, however, did not alter bystander cytokine production in macrophages (data not shown). Thus, a variety of signaling pathways initiated by infected cells are able to activate uninfected bystander cells to produce cytokines or chemokines important for controlling infection. These pathways are not important for all infections and others pathways may exist to induce bystander activation (Dolowschiak et al., 2010).
In vitro, TNF and IL-6 are still produced by bystander bone marrow-derived macrophages in the absence of IL-1 signaling, indicating that other signals are released by injected cells to activate bystanders. Since injected cells, in spite of translational inhibition, synthesize IL-1α and IL-1β, other cytokines may be translated in injected cells.
It remains to be seen whether IL-1α and IL-1β are the sole proteins produced robustly by injected cells. IL-18 is also released by the inflammasome, but the reagents for detecting
110 IL-18 are poor and often do not recognize all forms of IL-18 released from cells (Shida et al., 2001). Importantly, many inflammasome components are poorly expressed in resting cells and require NF-κB signaling for their expression (Lamkanfi and Dixit, 2009). As IL-
1α and IL-1β require the inflammasome for their release, these inflammasome components must be present in injected cells, indicating that these proteins may be translated in injected cells. Experiments to determine which proteins injected cells translate will be performed, including experiments to isolate polysomes to sequence which transcripts are associated with active ribosomes for translation.
H. Final Conclusions
I have identified a system used by the innate immune system to ensure the generation of proinflammatory cytokines during infection with an intracellular bacterial pathogen. My findings demonstrate the existence of heterogeneity in the production of critical protective cytokines by innate immune cells during the early response to bacterial infection and define a collaboration between infected and uninfected cells that enables the immune system to bypass protein synthesis inhibition and generate a robust immune response against L. pneumophila. As a variety of pathogens, including Yersinia spp., V. parahaemolyticus, S. flexneri, and others limit immune responses by inhibiting host cells signaling or protein translation, uninfected bystander cells may be required to carry out and amplify the early innate signals that eventually confer protective immunity (Fontana and Vance, 2011; Krachler et al., 2011). These bystander responses can be mediated by a number of pathways, including gap junctions, ROS production, cell death, and IL-1
(Dolowschiak et al., 2010; Kasper et al., 2010; Philip et al., 2014). IL-1 is produced in response to a variety of infectious pathogens, and beyond its known role in recruiting inflammatory cells to sites of infection, may orchestrate the production of bystander cytokines in response to these pathogens (Dinarello, 1996). Therefore, the ability of IL-1
111 to induce bystander cytokine production and cell activation may be a key pathway to bypass pathogen-induced manipulation of host cell processes during immune responses, ensuring the production of a successful immune response and eventual control of infection.
112 4 hours PI! 24 hours PI!
Airway epithelium Airway epithelium
Airway space! Airway space!
AMΦ! NΦ! ! ! AMΦ! AMΦ! NΦ! NΦ!
B! B! AMΦ! DC! DC! T! T! EΦ! EΦ! NK! NK! iMC!
Endothelium! Endothelium!
NΦ! NΦ! NΦ! NΦ! iMC! iMC! Blood! Blood!
Figure 5-1. Alveolar macrophages and neutrophils are targeted by the T4SS of L. pneumophila and contain viable bacteria during pulmonary infection. A variety of cells types exist within the mammalian lung including alveolar macrophages (AMΦ), dendritic cells (DC), natural killer (NK) cells, B cells, T cells, eosinophils (EΦ), and inflammatory monocytes (iMC) and neutrophils (NΦ). At 4 hours PI, alveolar macrophages in the airway space contain viable L. pneumophila and are targeted by the T4SS for injection. Activation of the immune response to virulent infection recruits inflammatory monocytes to the lung tissue and neutrophils to the tissue and airway space. Neutrophils, like alveolar macrophages, also contain vialbe bacteria at later times PI and are injected by the T4SS; whereas, inflammatory monocytes and DCs are not.
113 ! Infected alveolar macrophage or neutrophil! PRR !
Adaptor Lgt 1/2/3!
SidI/L, Pkn5! Caspase 1/11! ! NF-κB! Lpg1489! MAPK! ! IL-1α! Ribosome! IL-1β!
Il6, Tnf, Il1a, Il1b! !
IL-1α! IL-1β! And ?!
Figure 5-2. T4SS effectors block the production of cytokines in infected cells, yet IL-1α and IL-1β are still translated during infection. Cells infected with L. pneumophila activate several signaling pathways that induce the activation of NF-κB and MAPK. These pathways lead to the transcription of proinflammatory genes such as Il6, Tnf, Il1a, and Il1b. The T4SS effectors Lgt1, Lgt2, Lgt3, SidI, SidL, Pkn5, and Lpg1489 block early translation of TNF, but do not inhibit synthesis of TNF and IL-6 later post infection. Instead, TNF and IL-6 are inhibited by an unknown mechanism, either another bacterial effector or a host-driven response. IL-1α and IL-1β, however, are translated and activation of caspase-1 as well as capase-11 leads to the release of active IL-1 from infected cells. Whether infected cells release other inflammatory cues is unknown.
114 Infected Bystanders:" Bystanders:" cells" direct model" indirect model"
!CD86" iMC" TNF !CD86" DC" IL-12" AMΦ" IL-1R
NΦ" " IL-1α" IL-1β" " NΦ" " IL-1R IL-1R AMΦ" AEC" ?" Intermediate signal"
TNF KC" IL-12"
Figure 5-3. Direct and indirect IL-1 signaling induces bystander cytokine production during L. pneumophila infection from uninfected innate host cells. Alveolar macrophages (AMΦ) and neutrophils (NΦ) infected with virulent L. pneumophila produce IL-1α and IL- 1β, but do not produce TNF, IL-6, IL-12, or express CD86. Certain bystander cell types, such as uninfected alveolar macrophages and dendritic cells, can directly respond to IL- 1 signaling in a cell intrinsic manner and produce TNF and potentially IL-12 as well as express CD86. Other cell types, such as uninfected neutrophils and inflammatory monocytes (iMC), do not demonstrably respond directly to IL-1, yet the absence of IL-1R signaling during infection impairs their ability to produce cytokines. Other cells, such as airway epithelia cells (AEC) produce chemokines like KC in response to IL-1 signaling and may produce other signals that act to stimulate cytokine production from inflammatory monocytes and neutrophils. This indirect response to IL-1 may explain the requirement for IL-1R signaling from these cell types. Whether other signals are involved in the activation of bystander cells during L. pneumophila infection has not been determined.
115 BIBLIOGRAPHY
Aachoui, Y., Leaf, I.A., Hagar, J.A., Fontana, M.F., Campos, C.G., Zak, D.E., Tan, M.H., Cotter, P.A., Vance, R.E., Aderem, A., et al. (2013). Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978.
Ablasser, A., Schmid-Burgk, J.L., Hemmerling, I., Horvath, G.L., Schmidt, T., Latz, E., and Hornung, V. (2013). Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530–534.
Abreu, M.T., Arnold, E.T., Thomas, L.S., Gonsky, R., Zhou, Y., Hu, B., and Arditi, M. (2002). TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells. J. Biol. Chem. 277, 20431–20437.
Abu Kwaik, Y., Eisenstein, B.I., and Engleberg, N.C. (1993). Phenotypic modulation by Legionella pneumophila upon infection of macrophages. Infection and Immunity 61, 1320–1329.
Akhter, A., Caution, K., Abu Khweek, A., Tazi, M., Abdulrahman, B.A., Abdelaziz, D.H.A., Voss, O.H., Doseff, A.I., Hassan, H., Azad, A.K., et al. (2012). Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization. Immunity 37, 35–47.
Amer, A.O., and Swanson, M.S. (2005). Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 7, 765–778.
Amer, A., Franchi, L., Kanneganti, T.-D., Body-Malapel, M., Ozören, N., Brady, G., Meshinchi, S., Jagirdar, R., Gewirtz, A., Akira, S., et al. (2006). Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf. J. Biol. Chem. 281, 35217–35223.
Amor, J.C. (2004). The Structure of RalF, an ADP-ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site. Journal of Biological Chemistry 280, 1392–1400.
Ang, D.K.Y., Oates, C.V.L., Schuelein, R., Kelly, M., Sansom, F.M., Bourges, D., Boon, L., Hertzog, P.J., Hartland, E.L., and van Driel, I.R. (2010). Cutting edge: pulmonary Legionella pneumophila is controlled by plasmacytoid dendritic cells but not type I IFN. The Journal of Immunology 184, 5429–5433.
Ang, D.K.Y., Ong, S.Y., Brown, A.S., Hartland, E.L., and van Driel, I.R. (2012). A method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry. BMC Res Notes 5, 448.
Arbibe, L., Kim, D.W., Batsche, E., Pedron, T., Mateescu, B., Muchardt, C., Parsot, C., and Sansonetti, P.J. (2007). An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses. Nature Immunology 8, 47–56.
116 Archer, K.A., and Roy, C.R. (2006). MyD88-Dependent Responses Involving Toll-Like Receptor 2 Are Important for Protection and Clearance of Legionella pneumophila in a Mouse Model of Legionnaires' Disease. Infection and Immunity 74, 3325–3333.
Archer, K.A., Ader, F., Kobayashi, K.S., Flavell, R.A., and Roy, C.R. (2010). Cooperation between Multiple Microbial Pattern Recognition Systems Is Important for Host Protection against the Intracellular Pathogen Legionella pneumophila. Infection and Immunity 78, 2477–2487.
Archer, K.A., Alexopoulou, L., Flavell, R.A., and Roy, C.R. (2009). Multiple MyD88- dependent responses contribute to pulmonary clearance of Legionella pneumophila. Cell Microbiol 11, 21–36.
Aringer, M., Houssiau, F., Gordon, C., Graninger, W.B., Voll, R.E., Rath, E., Steiner, G., and Smolen, J.S. (2009). Adverse events and efficacy of TNF-alpha blockade with infliximab in patients with systemic lupus erythematosus: long-term follow-up of 13 patients. Rheumatology (Oxford) 48, 1451–1454.
Arndt, P.G., Young, S.K., and Worthen, G.S. (2005). Regulation of lipopolysaccharide- induced lung inflammation by plasminogen activator Inhibitor-1 through a JNK-mediated pathway. J. Immunol. 175, 4049–4059.
Asrat, S., Dugan, A.S., and Isberg, R.R. (2014). The frustrated host response to Legionella pneumophila is bypassed by MyD88-dependent translation of pro- inflammatory cytokines. PLoS Pathog 10, e1004229.
Auron, P.E., Webb, A.C., Rosenwasser, L.J., Mucci, S.F., Rich, A., Wolff, S.M., and Dinarello, C.A. (1984). Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc. Natl. Acad. Sci. U.S.a. 81, 7907–7911.
Bardill, J.P., Miller, J.L., and Vogel, J.P. (2005). IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Molecular Microbiology 56, 90–103.
Bardy, S.L., Ng, S.Y.M., and Jarrell, K.F. (2003). Prokaryotic motility structures. Microbiology (Reading, Engl.) 149, 295–304.
Barry, K.C., Fontana, M.F., Portman, J.L., Dugan, A.S., and Vance, R.E. (2013). IL-1α Signaling Initiates the Inflammatory Response to Virulent Legionella pneumophila In Vivo. The Journal of Immunology.
Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A., and Gordon, J.I. (2005). Host- bacterial mutualism in the human intestine. Science 307, 1915–1920.
Beck, J.M., Young, V.B., and Huffnagle, G.B. (2012). The microbiome of the lung. Transl Res 160, 258–266.
Beckers, M.C., Yoshida, S., Morgan, K., Skamene, E., and Gros, P. (1995). Natural resistance to infection with Legionella pneumophila: chromosomal localization of the Lgn1 susceptibility gene. Mamm. Genome 6, 540–545.
117 Beigel, F., Jürgens, M., Filik, L., Bader, L., Lück, C., Göke, B., Ochsenkühn, T., Brand, S., and Seiderer, J. (2009). Severe Legionella pneumophila pneumonia following infliximab therapy in a patient with Crohn's disease. Inflamm. Bowel Dis. 15, 1240–1244.
Belyi, Y., Jank, T., and Aktories, K. (2013). Cytotoxic glucosyltransferases of Legionella pneumophila. Curr. Top. Microbiol. Immunol. 376, 211–226.
Belyi, Y., Niggeweg, R., Opitz, B., Vogelsgesang, M., Hippenstiel, S., Wilm, M., and Aktories, K. (2006). Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A. Proc. Natl. Acad. Sci. U.S.a. 103, 16953–16958.
Belyi, Y., Tabakova, I., Stahl, M., and Aktories, K. (2008). Lgt: a family of cytotoxic glucosyltransferases produced by Legionella pneumophila. J. Bacteriol. 190, 3026– 3035.
Bennouna, S., Bliss, S.K., Curiel, T.J., and Denkers, E.Y. (2003). Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. J. Immunol. 171, 6052–6058.
Berger, K.H., and Isberg, R.R. (1993). Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Molecular Microbiology 7, 7–19.
Berrington, W.R., Iyer, R., Wells, R.D., Smith, K.D., Skerrett, S.J., and Hawn, T.R. (2010). NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila. Eur. J. Immunol. 40, 3519–3527.
Biswas, S.K., and Lopez-Collazo, E. (2009). Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol. 30, 475–487.
Biswas, S.K., and Tergaonkar, V. (2007). Myeloid differentiation factor 88-independent Toll-like receptor pathway: Sustaining inflammation or promoting tolerance? Int. J. Biochem. Cell Biol. 39, 1582–1592.
Black, D.S., and Bliska, J.B. (1997). Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions. Embo J. 16, 2730–2744.
Black, R.A., Kronheim, S.R., Cantrell, M., Deeley, M.C., March, C.J., Prickett, K.S., Wignall, J., Conlon, P.J., Cosman, D., and Hopp, T.P. (1988). Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor. J. Biol. Chem. 263, 9437–9442.
Boer, Den, J.W., Nijhof, J., and Friesema, I. (2006). Risk factors for sporadic community- acquired Legionnaires' disease. A 3-year national case-control study. Public Health 120, 566–571.
Bohn, E., and Autenrieth, I.B. (1996). IL-12 is essential for resistance against Yersinia enterocolitica by triggering IFN-gamma production in NK cells and CD4+ T cells. J. Immunol. 156, 1458–1468.
118 Borella, P., Bargellini, A., Marchesi, I., Rovesti, S., Stancanelli, G., Scaltriti, S., Moro, M., Montagna, M.T., Tatò, D., Napoli, C., et al. (2008). Prevalence of anti-legionella antibodies among Italian hospital workers. J. Hosp. Infect. 69, 148–155.
Borok, Z., Liebler, J.M., Lubman, R.L., Foster, M.J., Zhou, B., Li, X., Zabski, S.M., Kim, K.-J., and Crandall, E.D. (2002). Na transport proteins are expressed by rat alveolar epithelial type I cells. Am. J. Physiol. Lung Cell Mol. Physiol. 282, L599–L608.
Boshuizen, H.C., Nagelkerke, N.J.D., Boer, Den, J.W., De Melker, H., Schellekens, J.F.P., Peeters, M.F., Van Vliet, H., and Conyn-Van Spaendonck, M.A.E. (2006). Estimation of minimum infection rates with Legionella pneumophila in an exposed population. Epidemiol. Infect. 134, 579–584.
Brieland, J.K., Jackson, C., Hurst, S., Loebenberg, D., Muchamuel, T., Debets, R., Kastelein, R., Churakova, T., Abrams, J., Hare, R., et al. (2000). Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection. Infection and Immunity 68, 6567–6573.
Brieland, J.K., Remick, D.G., Freeman, P.T., Hurley, M.C., Fantone, J.C., and Engleberg, N.C. (1995). In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide. Infection and Immunity 63, 3253–3258.
Brieland, J.K., Remick, D.G., LeGendre, M.L., Engleberg, N.C., and Fantone, J.C. (1998). In vivo regulation of replicative Legionella pneumophila lung infection by endogenous interleukin-12. Infection and Immunity 66, 65–69.
Brieland, J., Freeman, P., Kunkel, R., Chrisp, C., Hurley, M., Fantone, J., and Engleberg, C. (1994). Replicative Legionella pneumophila lung infection in intratracheally inoculated A/J mice. A murine model of human Legionnaires' disease. Am. J. Pathol. 145, 1537– 1546.
Brodsky, I.E., and Medzhitov, R. (2008). Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence. PLoS Pathog 4, e1000067.
Brodsky, I.E., Palm, N.W., Sadanand, S., Ryndak, M.B., Sutterwala, F.S., Flavell, R.A., Bliska, J.B., and Medzhitov, R. (2010). A Yersinia effector protein promotes virulence by preventing inflammasome recognition of the type III secretion system. Cell Host and Microbe 7, 376–387.
Broz, P., Moltke, von, J., Jones, J.W., Vance, R.E., and Monack, D.M. (2010). Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing. Cell Host and Microbe 8, 471–483.
Burstein, D., Zusman, T., Degtyar, E., Viner, R., Segal, G., and Pupko, T. (2009). Genome-scale identification of Legionella pneumophila effectors using a machine learning approach. PLoS Pathog 5, e1000508.
Busse, W.W., and Sedgwick, J.B. (1992). Eosinophils in asthma. Ann Allergy 68, 286– 290.
119 Byrd, T.F., and Horwitz, M.A. (1989). Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin. Invest. 83, 1457–1465.
Byrne, B.G., Dubuisson, J.-F., Joshi, A.D., Persson, J.J., and Swanson, M.S. (2013). Inflammasome components coordinate autophagy and pyroptosis as macrophage responses to infection. MBio 4, e00620–12.
Casadevall, A., and Pirofski, L.-A. (2003). The damage-response framework of microbial pathogenesis. Nat. Rev. Microbiol. 1, 17–24.
Case, C.L., Shin, S., and Roy, C.R. (2009). Asc and Ipaf Inflammasomes Direct Distinct Pathways for Caspase-1 Activation in Response to Legionella pneumophila. Infection and Immunity 77, 1981–1991.
Case, C.L., Kohler, L.J., Lima, J.B., Strowig, T., de Zoete, M.R., Flavell, R.A., Zamboni, D.S., and Roy, C.R. (2013). Caspase-11 stimulates rapid flagellin-independent pyroptosis in response to Legionella pneumophila. Proc. Natl. Acad. Sci. U.S.a. 110, 1851–1856.
Casson, C.N., and Shin, S. (2013). Inflammasome-mediated cell death in response to bacterial pathogens that access the host cell cytosol: lessons from legionella pneumophila. Front Cell Infect Microbiol 3, 111.
Casson, C.N., Copenhaver, A.M., Zwack, E.E., Nguyen, H.T., Strowig, T., Javdan, B., Bradley, W.P., Fung, T.C., Flavell, R.A., Brodsky, I.E., et al. (2013). Caspase-11 Activation in Response to Bacterial Secretion Systems that Access the Host Cytosol. PLoS Pathog 9, e1003400.
Ceballos-Olvera, I., Sahoo, M., Miller, M.A., Del Barrio, L., and Re, F. (2011). Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1β is deleterious. PLoS Pathog 7, e1002452.
Centers for Disease Control and Prevention (CDC) (2011). Legionellosis --- United States, 2000-2009. MMWR Morb. Mortal. Wkly. Rep. 60, 1083–1086.
Chan, C., Li, L., McCall, C.E., and Yoza, B.K. (2005). Endotoxin tolerance disrupts chromatin remodeling and NF-kappaB transactivation at the IL-1beta promoter. J. Immunol. 175, 461–468.
Chandler, F.W., Hicklin, M.D., and Blackmon, J.A. (1977). Demonstration of the agent of Legionnaires' disease in tissue. N. Engl. J. Med. 297, 1218–1220.
Charpentier, X., Gabay, J.E., Reyes, M., Zhu, J.W., Weiss, A., and Shuman, H.A. (2009). Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila. PLoS Pathog 5, e1000501.
Chen, C., Banga, S., Mertens, K., Weber, M.M., Gorbaslieva, I., Tan, Y., Luo, Z.-Q., and Samuel, J.E. (2010). Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii. Proc. Natl. Acad. Sci. U.S.a. 107, 21755–21760.
120 Chen, C.-J., Kono, H., Golenbock, D., Reed, G., Akira, S., and Rock, K.L. (2007). Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 13, 851–856.
Chen, S.M., Dumler, J.S., Bakken, J.S., and Walker, D.H. (1994). Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595.
Chen, Y.-L., Huang, Y.-L., Lin, N.-Y., Chen, H.-C., Chiu, W.-C., and Chang, C.-J. (2006). Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells. Biochem. Biophys. Res. Commun. 346, 160–168.
Choy, A., Dancourt, J., Mugo, B., O'Connor, T.J., Isberg, R.R., Melia, T.J., and Roy, C.R. (2012). The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076.
Coers, J., Vance, R.E., Fontana, M.F., and Dietrich, W.F. (2007). Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways. Cell Microbiol 9, 2344–2357.
Copenhaver, A.M., Casson, C.N., Nguyen, H.T., Fung, T.C., Duda, M.M., Roy, C.R., and Shin, S. (2014). Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion. Infection and Immunity 82, 4325–4336.
Cornelis, G.R. (2006). The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811– 825.
Creasey, E.A., and Isberg, R.R. (2012). The protein SdhA maintains the integrity of the Legionella-containing vacuole. Proc. Natl. Acad. Sci. U.S.a. 109, 3481–3486.
Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S., and Albina, J.E. (2008). Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. Journal of Leukocyte Biology 83, 64–70.
De Haan, L., and Hirst, T.R. (2004). Cholera toxin: a paradigm for multi-functional engagement of cellular mechanisms (Review). Mol. Membr. Biol. 21, 77–92. de Jesus, A.A., Canna, S.W., Liu, Y., and Goldbach-Mansky, R. (2015). Molecular Mechanisms in Genetically Defined Autoinflammatory Diseases: Disorders of Amplified Danger Signaling. Annu. Rev. Immunol.
Delwart, E. (2013). A roadmap to the human virome. PLoS Pathog 9, e1003146.
Demuth, A., Goebel, W., Beuscher, H.U., and Kuhn, M. (1996). Differential regulation of cytokine and cytokine receptor mRNA expression upon infection of bone marrow-derived macrophages with Listeria monocytogenes. Infection and Immunity 64, 3475–3483.
Derré, I., and Isberg, R.R. (2004). Macrophages from mice with the restrictive Lgn1 allele exhibit multifactorial resistance to Legionella pneumophila. Infection and Immunity 72, 6221–6229.
121 Dewhirst, F.E., Chen, T., Izard, J., Paster, B.J., Tanner, A.C.R., Yu, W.-H., Lakshmanan, A., and Wade, W.G. (2010). The human oral microbiome. J. Bacteriol. 192, 5002–5017.
Diaz, M.H., and Hauser, A.R. (2010). Pseudomonas aeruginosa Cytotoxin ExoU Is Injected into Phagocytic Cells during Acute Pneumonia. Infection and Immunity 78, 1447–1456.
Dietrich, W.F., Damron, D.M., Isberg, R.R., Lander, E.S., and Swanson, M.S. (1995). Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13. Genomics 26, 443–450.
Diez, E., Lee, S.-H., Gauthier, S., Yaraghi, Z., Tremblay, M., Vidal, S., and Gros, P. (2003). Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila. Nat. Genet. 33, 55–60.
Dinarello, C.A. (1996). Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147.
Dinarello, C.A. (2013). Overview of the interleukin-1 family of ligands and receptors. Semin. Immunol. 25, 389–393.
Dinarello, C.A., and van der Meer, J.W.M. (2013). Treating inflammation by blocking interleukin-1 in humans. Semin. Immunol. 25, 469–484.
Dolowschiak, T., Chassin, C., Ben Mkaddem, S., Fuchs, T.M., Weiss, S., Vandewalle, A., and Hornef, M.W. (2010). Potentiation of epithelial innate host responses by intercellular communication. PLoS Pathog 6, e1001194.
Domínguez, A., Alvarez, J., Sabrià, M., Carmona, G., Torner, N., Oviedo, M., Cayla, J., Minguell, S., Barrabeig, I., Sala, M., et al. (2009). Factors influencing the case-fatality rate of Legionnaires' disease. Int. J. Tuberc. Lung Dis. 13, 407–412.
Dower, S.K., Call, S.M., Gillis, S., and Urdal, D.L. (1986). Similarity between the interleukin 1 receptors on a murine T-lymphoma cell line and on a murine fibroblast cell line. Proc. Natl. Acad. Sci. U.S.a. 83, 1060–1064.
Dreux, M., Garaigorta, U., Boyd, B., Décembre, E., Chung, J., Whitten-Bauer, C., Wieland, S., and Chisari, F.V. (2012). Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity. Cell Host and Microbe 12, 558–570.
Dunbar, T.L., Yan, Z., Balla, K.M., Smelkinson, M.G., and Troemel, E.R. (2012). C. elegans Detects Pathogen-Induced Translational Inhibition to Activate Immune Signaling. Cell Host and Microbe 11, 375–386.
Ensminger, A.W., and Isberg, R.R. (2009). Legionella pneumophila Dot/Icm translocated substrates: a sum of parts. Curr. Opin. Microbiol. 12, 67–73.
Ensminger, A.W., Yassin, Y., Miron, A., and Isberg, R.R. (2012). Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival. PLoS Pathog 8, e1002731.
122 Fasano, M.B., Cousart, S., Neal, S., and McCall, C.E. (1991). Increased expression of the interleukin 1 receptor on blood neutrophils of humans with the sepsis syndrome. J. Clin. Invest. 88, 1452–1459.
Feeley, J.C., Gibson, R.J., Gorman, G.W., Langford, N.C., Rasheed, J.K., Mackel, D.C., and Baine, W.B. (1979). Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10, 437–441.
Fernandes-Alnemri, T., Yu, J.-W., Datta, P., Wu, J., and Alnemri, E.S. (2009). AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513.
Fields, B.S. (1996). The molecular ecology of legionellae. Trends in Microbiology 4, 286–290.
Fleming, R.E., Moxley, M.A., Waheed, A., Crouch, E.C., Sly, W.S., and Longmore, W.J. (1994). Carbonic anhydrase II expression in rat type II pneumocytes. American Journal of Respiratory Cell and Molecular Biology 10, 499–505.
Fleming, T.J., Fleming, M.L., and Malek, T.R. (1993). Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte- differentiation antigen (Gr-1) detects members of the Ly-6 family. J. Immunol. 151, 2399–2408.
Fontana, M.F., and Vance, R.E. (2011). Two signal models in innate immunity. Immunol. Rev. 243, 26–39.
Fontana, M.F., Banga, S., Barry, K.C., Shen, X., Tan, Y., Luo, Z.-Q., and Vance, R.E. (2011). Secreted Bacterial Effectors That Inhibit Host Protein Synthesis Are Critical for Induction of the Innate Immune Response to Virulent Legionella pneumophila. PLoS Pathog 7, e1001289.
Fontana, M.F., Shin, S., and Vance, R.E. (2012). Activation of host mitogen-activated protein kinases by secreted Legionella pneumophila effectors that inhibit host protein translation. Infection and Immunity 80, 3570–3575.
Foster, S.L., Hargreaves, D.C., and Medzhitov, R. (2007). Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 447, 972–978.
Franchi, L., Kamada, N., Nakamura, Y., Burberry, A., Kuffa, P., Suzuki, S., Shaw, M.H., Kim, Y.-G., and Nuñez, G. (2012). NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense. Nature Immunology 13, 449–456.
Fraser, D.W., Tsai, T.R., Orenstein, W., Parkin, W.E., Beecham, H.J., Sharrar, R.G., Harris, J., Mallison, G.F., Martin, S.M., McDade, J.E., et al. (1977). Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297, 1189–1197.
Friedman, H., Yamamoto, Y., and Klein, T.W. (2002). Legionella pneumophila pathogenesis and immunity. Semin Pediatr Infect Dis 13, 273–279.
123 Frutuoso, M.S., Hori, J.I., Pereira, M.S.F., Junior, D.S.L., Sônego, F., Kobayashi, K.S., Flavell, R.A., Cunha, F.Q., and Zamboni, D.S. (2010). The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila. Microbes Infect. 12, 819–827.
Gal-Mor, O., Zusman, T., and Segal, G. (2002). Analysis of DNA regulatory elements required for expression of the Legionella pneumophila icm and dot virulence genes. J. Bacteriol. 184, 3823–3833.
Galyov, E.E., Brett, P.J., and DeShazer, D. (2010). Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis. Annu. Rev. Microbiol. 64, 495–517.
Garau, J., Fritsch, A., Arvis, P., and Read, R.C. (2010). Clinical efficacy of moxifloxacin versus comparator therapies for community-acquired pneumonia caused by Legionella spp. J Chemother 22, 264–266.
Ge, J., Xu, H., Li, T., Zhou, Y., Zhang, Z., Li, S., Liu, L., and Shao, F. (2009). A Legionella type IV effector activates the NF-kappaB pathway by phosphorylating the IkappaB family of inhibitors. Proc. Natl. Acad. Sci. U.S.a. 106, 13725–13730.
Geddes, K., Cruz, F., and Heffron, F. (2007). Analysis of cells targeted by Salmonella type III secretion in vivo. PLoS Pathog 3, e196.
Girardin, S.E., Boneca, I.G., Carneiro, L.A.M., Antignac, A., Jéhanno, M., Viala, J., Tedin, K., Taha, M.-K., Labigne, A., Zähringer, U., et al. (2003a). Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584–1587.
Girardin, S.E., Travassos, L.H., Hervé, M., Blanot, D., Boneca, I.G., Philpott, D.J., Sansonetti, P.J., and Mengin-Lecreulx, D. (2003b). Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278, 41702–41708.
Goody, R.S., and Itzen, A. (2013). Modulation of small GTPases by legionella. Curr. Top. Microbiol. Immunol. 376, 117–133.
Greenfeder, S.A., Nunes, P., Kwee, L., Labow, M., Chizzonite, R.A., and Ju, G. (1995). Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J. Biol. Chem. 270, 13757–13765.
Grice, E.A., and Segre, J.A. (2011). The skin microbiome. Nat. Rev. Microbiol. 9, 244– 253.
Guma, M., Ronacher, L., Liu-Bryan, R., Takai, S., Karin, M., and Corr, M. (2009). Caspase 1-independent activation of interleukin-1beta in neutrophil-predominant inflammation. Arthritis Rheum. 60, 3642–3650.
Guo, Y., Martinez-Williams, C., Gilbert, K.A., and Rannels, D.E. (1999). Inhibition of gap junction communication in alveolar epithelial cells by 18alpha-glycyrrhetinic acid. Am. J. Physiol. 276, L1018–L1026.
Hagar, J.A., Powell, D.A., Aachoui, Y., Ernst, R.K., and Miao, E.A. (2013). Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science
124 341, 1250–1253.
Hamon, M.A., Ribet, D., Stavru, F., and Cossart, P. (2012). Listeriolysin O: the Swiss army knife of Listeria. Trends in Microbiology 20, 360–368.
Harmon, D.E., Davis, A.J., Castillo, C., and Mecsas, J. (2010). Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis. Antimicrob. Agents Chemother. 54, 3241–3254.
Hasenberg, M., Stegemann-Koniszewski, S., and Gunzer, M. (2013). Cellular immune reactions in the lung. Immunol. Rev. 251, 189–214.
Hawn, T.R., Berrington, W.R., Smith, I.A., Uematsu, S., Akira, S., Aderem, A., Smith, K.D., and Skerrett, S.J. (2007). Altered inflammatory responses in TLR5-deficient mice infected with Legionella pneumophila. J. Immunol. 179, 6981–6987.
Hawn, T.R., Smith, K.D., Aderem, A., and Skerrett, S.J. (2006). Myeloid differentiation primary response gene (88)- and toll-like receptor 2-deficient mice are susceptible to infection with aerosolized Legionella pneumophila. J. Infect. Dis. 193, 1693–1702.
Hawn, T.R., Verbon, A., Lettinga, K.D., Zhao, L.P., Li, S.S., Laws, R.J., Skerrett, S.J., Beutler, B., Schroeder, L., Nachman, A., et al. (2003). A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease. J. Exp. Med. 198, 1563–1572.
Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng, J.K., Akira, S., Underhill, D.M., and Aderem, A. (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103.
Heath, L., Chrisp, C., Huffnagle, G., LeGendre, M., Osawa, Y., Hurley, M., Engleberg, C., Fantone, J., and Brieland, J. (1996). Effector mechanisms responsible for gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts. Infection and Immunity 64, 5151–5160.
Hirche, T.O., Benabid, R., Deslee, G., Gangloff, S., Achilefu, S., Guenounou, M., Lebargy, F., Hancock, R.E., and Belaaouaj, A. (2008). Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa. The Journal of Immunology 181, 4945–4954.
Hoffmann, C., Dollive, S., Grunberg, S., Chen, J., Li, H., Wu, G.D., Lewis, J.D., and Bushman, F.D. (2013). Archaea and fungi of the human gut microbiome: correlations with diet and bacterial residents. PLoS ONE 8, e66019.
Hohl, T.M., Rivera, A., Lipuma, L., Gallegos, A., Shi, C., Mack, M., and Pamer, E.G. (2009). Inflammatory monocytes facilitate adaptive CD4 T cell responses during respiratory fungal infection. Cell Host and Microbe 6, 470–481.
Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D.R., Latz, E., and Fitzgerald, K.A. (2009). AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518.
125 Horwitz, M.A. (1983). Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319– 1331.
Horwitz, M.A., and Silverstein, S.C. (1980). Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J. Clin. Invest. 66, 441–450.
Horwitz, M.A., and Silverstein, S.C. (1981). Interaction of the Legionnaires' disease bacterium (Legionella pneumophila) with human phagocytes. I. L. pneumophila resists killing by polymorphonuclear leukocytes, antibody, and complement. J. Exp. Med. 153, 386–397.
Howard, A.D., Kostura, M.J., Thornberry, N., Ding, G.J., Limjuco, G., Weidner, J., Salley, J.P., Hogquist, K.A., Chaplin, D.D., and Mumford, R.A. (1991). IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha. J. Immunol. 147, 2964–2969.
Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf, S.F., O'Garra, A., and Murphy, K.M. (1993). Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260, 547–549.
Hubber, A., and Roy, C.R. (2010). Modulation of Host Cell Function by Legionella pneumophilaType IV Effectors. Annu. Rev. Cell Dev. Biol. 26, 261–283.
Hussell, T., and Bell, T.J. (2014). Alveolar macrophages: plasticity in a tissue-specific context. Nat Rev Immunol 14, 81–93.
Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., and Steinman, R.M. (1992). Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176, 1693–1702.
Inohara, Chamaillard, McDonald, C., and Nuñez, G. (2005). NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev. Biochem. 74, 355– 383.
Ivanov, S.S., and Roy, C.R. (2013). Pathogen signatures activate a ubiquitination pathway that modulates the function of the metabolic checkpoint kinase mTOR. Nature Immunology 14, 1219–1228.
Janeway, C.A., and Medzhitov, R. (2002). Innate immune recognition. Annu. Rev. Immunol. 20, 197–216.
Jesus, A.A., and Goldbach-Mansky, R. (2014). IL-1 blockade in autoinflammatory syndromes. Annu. Rev. Med. 65, 223–244.
Jinno, S., Pulido, S., and Pien, B.C. (2009). First reported United States case of Legionella pneumophila serogroup 1 pneumonia in a patient receiving anti-tumor necrosis factor-alpha therapy. Hawaii Med J 68, 109–112.
Johnson, M.D., Widdicombe, J.H., Allen, L., Barbry, P., and Dobbs, L.G. (2002). Alveolar
126 epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis. Proc. Natl. Acad. Sci. U.S.a. 99, 1966–1971.
Jones, M.R., Quinton, L.J., Simms, B.T., Lupa, M.M., Kogan, M.S., and Mizgerd, J.P. (2006). Roles of interleukin-6 in activation of STAT proteins and recruitment of neutrophils during Escherichia coli pneumonia. J. Infect. Dis. 193, 360–369.
Juhas, M., Crook, D.W., and Hood, D.W. (2008). Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence. Cell Microbiol 10, 2377–2386.
Kahn, R.A., Fu, H., and Roy, C.R. (2002). Cellular hijacking: a common strategy for microbial infection. Trends Biochem. Sci. 27, 308–314.
Kamada, N., Chen, G.Y., Inohara, N., and Nuñez, G. (2013a). Control of pathogens and pathobionts by the gut microbiota. Nature Immunology 14, 685–690.
Kamada, N., Seo, S.-U., Chen, G.Y., and Nuñez, G. (2013b). Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol 13, 321–335.
Karmakar, M., Sun, Y., Hise, A.G., Rietsch, A., and Pearlman, E. (2012). Cutting edge: IL-1β processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and caspase-1. The Journal of Immunology 189, 4231–4235.
Kasper, C.A., Sorg, I., Schmutz, C., Tschon, T., Wischnewski, H., Kim, M.L., and Arrieumerlou, C. (2010). Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection. Immunity 33, 804– 816.
Kau, A.L., Ahern, P.P., Griffin, N.W., Goodman, A.L., and Gordon, J.I. (2011). Human nutrition, the gut microbiome and the immune system. Nature 474, 327–336.
Kayagaki, N., Wong, M.T., Stowe, I.B., Ramani, S.R., Gonzalez, L.C., Akashi-Takamura, S., Miyake, K., Zhang, J., Lee, W.P., Muszyński, A., et al. (2013). Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246– 1249.
Kim, B., Lee, Y., Kim, E., Kwak, A., Ryoo, S., Bae, S.H., Azam, T., Kim, S., and Dinarello, C.A. (2013). The Interleukin-1α Precursor is Biologically Active and is Likely a Key Alarmin in the IL-1 Family of Cytokines. Front Immunol 4, 391.
Kim, D.W., Lenzen, G., Page, A.-L., Legrain, P., Sansonetti, P.J., and Parsot, C. (2005). The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes. Proc. Natl. Acad. Sci. U.S.a. 102, 14046– 14051.
Kirby, A.C., Coles, M.C., and Kaye, P.M. (2009). Alveolar Macrophages Transport Pathogens to Lung Draining Lymph Nodes. The Journal of Immunology 183, 1983– 1989.
127 Kirnbauer, R., Köck, A., Schwarz, T., Urbanski, A., Krutmann, J., Borth, W., Damm, D., Shipley, G., Ansel, J.C., and Luger, T.A. (1989). IFN-beta 2, B cell differentiation factor 2, or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines. J. Immunol. 142, 1922–1928.
Knapp, T., Hare, E., Feng, L., Zlokarnik, G., and Negulescu, P. (2003). Detection of beta-lactamase reporter gene expression by flow cytometry. Cytometry A 51, 68–78.
Kofoed, E.M., and Vance, R.E. (2011). Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477, 592–595.
Kohase, M., May, L.T., Tamm, I., Vilcek, J., and Sehgal, P.B. (1987). A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1. Mol. Cell. Biol. 7, 273–280.
Kool, J.L., Fiore, A.E., Kioski, C.M., Brown, E.W., Benson, R.F., Pruckler, J.M., Glasby, C., Butler, J.C., Cage, G.D., Carpenter, J.C., et al. (1998). More than 10 years of unrecognized nosocomial transmission of legionnaires' disease among transplant patients. Infect Control Hosp Epidemiol 19, 898–904.
Koshy, A.A., Dietrich, H.K., Christian, D.A., Melehani, J.H., Shastri, A.J., Hunter, C.A., and Boothroyd, J.C. (2012). Toxoplasma co-opts host cells it does not invade. PLoS Pathog 8, e1002825.
Köberle, M., Klein-Günther, A., Schütz, M., Fritz, M., Berchtold, S., Tolosa, E., Autenrieth, I.B., and Bohn, E. (2009). Yersinia enterocolitica targets cells of the innate and adaptive immune system by injection of Yops in a mouse infection model. PLoS Pathog 5, e1000551.
Krachler, A.M., Woolery, A.R., and Orth, K. (2011). Manipulation of kinase signaling by bacterial pathogens. The Journal of Cell Biology 195, 1083–1092.
Kramer, R.W., Slagowski, N.L., Eze, N.A., Giddings, K.S., Morrison, M.F., Siggers, K.A., Starnbach, M.N., and Lesser, C.F. (2007). Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation. PLoS Pathog 3, e21.
Kubori, T., Shinzawa, N., Kanuka, H., and Nagai, H. (2010). Legionella metaeffector exploits host proteasome to temporally regulate cognate effector. PLoS Pathog 6, e1001216.
Kung, V.L., Khare, S., Stehlik, C., Bacon, E.M., Hughes, A.J., and Hauser, A.R. (2012). An rhs gene of Pseudomonas aeruginosa encodes a virulence protein that activates the inflammasome. Proc. Natl. Acad. Sci. U.S.a. 109, 1275–1280.
Lamkanfi, M., and Dixit, V.M. (2009). Inflammasomes: guardians of cytosolic sanctity. Immunol. Rev. 227, 95–105.
Lee, W.-W., Kang, S.W., Choi, J., Lee, S.-H., Shah, K., Eynon, E.E., Flavell, R.A., and Kang, I. (2010). Regulating human Th17 cells via differential expression of IL-1 receptor. Blood 115, 530–540.
128 Leemans, J.C., Cassel, S.L., and Sutterwala, F.S. (2011). Sensing damage by the NLRP3 inflammasome. Immunol. Rev. 243, 152–162.
LeibundGut-Landmann, S., Weidner, K., Hilbi, H., and Oxenius, A. (2011). Nonhematopoietic cells are key players in innate control of bacterial airway infection. The Journal of Immunology 186, 3130–3137.
León, B., Martínez del Hoyo, G., Parrillas, V., Vargas, H.H., Sánchez-Mateos, P., Longo, N., López-Bravo, M., and Ardavín, C. (2004). Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8- and CD8+ splenic dendritic cells. Blood 103, 2668–2676.
Lettinga, K.D., Florquin, S., Speelman, P., van Ketel, R., van der Poll, T., and Verbon, A. (2002). Toll-like receptor 4 is not involved in host defense against pulmonary Legionella pneumophila infection in a mouse model. J. Infect. Dis. 186, 570–573.
Li, H., Xu, H., Zhou, Y., Zhang, J., Long, C., Li, S., Chen, S., Zhou, J.-M., and Shao, F. (2007). The phosphothreonine lyase activity of a bacterial type III effector family. Science 315, 1000–1003.
Lichtman, A.H., Chin, J., Schmidt, J.A., and Abbas, A.K. (1988). Role of interleukin 1 in the activation of T lymphocytes. Proc. Natl. Acad. Sci. U.S.a. 85, 9699–9703.
Liew, F.Y., Xu, D., Brint, E.K., and O'Neill, L.A.J. (2005). Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 5, 446–458.
Lightfield, K.L., Persson, J., Brubaker, S.W., Witte, C.E., Moltke, von, J., Dunipace, E.A., Henry, T., Sun, Y.-H., Cado, D., Dietrich, W.F., et al. (2008). Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin. Nature Immunology 9, 1171–1178.
Lippmann, J., Müller, H.C., Naujoks, J., Tabeling, C., Shin, S., Witzenrath, M., Hellwig, K., Kirschning, C.J., Taylor, G.A., Barchet, W., et al. (2011). Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice. Cell Microbiol 13, 1668–1682.
Losick, V.P., and Isberg, R.R. (2006). NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila. J. Exp. Med. 203, 2177–2189.
Lowry, P.W., and Tompkins, L.S. (1993). Nosocomial legionellosis: a review of pulmonary and extrapulmonary syndromes. Am J Infect Control 21, 21–27.
Maldonado-Arocho, F.J., Green, C., Fisher, M.L., Paczosa, M.K., and Mecsas, J. (2013). Adhesins and Host Serum Factors Drive Yop Translocation by Yersinia into Professional Phagocytes during Animal Infection. PLoS Pathog 9, e1003415.
March, C.J., Mosley, B., Larsen, A., Cerretti, D.P., Braedt, G., Price, V., Gillis, S., Henney, C.S., Kronheim, S.R., and Grabstein, K. (1985). Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature 315, 641– 647.
129 Mariathasan, S., Weiss, D.S., Newton, K., McBride, J., O'Rourke, K., Roose-Girma, M., Lee, W.P., Weinrauch, Y., Monack, D.M., and Dixit, V.M. (2006). Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232.
Marra, A., and Shuman, H.A. (1989). Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J. Bacteriol. 171, 2238–2240.
Marra, A., Blander, S.J., Horwitz, M.A., and Shuman, H.A. (1992). Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc. Natl. Acad. Sci. U.S.a. 89, 9607–9611.
Martinon, F., Burns, K., and Tschopp, J. (2002). The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10, 417–426.
Maruta, K., Miyamoto, H., Hamada, T., Ogawa, M., Taniguchi, H., and Yoshida, S. (1998). Entry and intracellular growth of Legionella dumoffii in alveolar epithelial cells. Am. J. Respir. Crit. Care Med. 157, 1967–1974.
Mascarenhas, D.P.A., Pereira, M.S.F., Manin, G.Z., Hori, J.I., and Zamboni, D.S. (2015). Interleukin 1 Receptor-Driven Neutrophil Recruitment Accounts to MyD88-Dependent Pulmonary Clearance of Legionella pneumophila Infection In Vivo. J. Infect. Dis. 211, 322–330.
Mayer-Barber, K.D., Andrade, B.B., Barber, D.L., Hieny, S., Feng, C.G., Caspar, P., Oland, S., Gordon, S., and Sher, A. (2011). Innate and Adaptive Interferons Suppress IL-1α and IL-1β Production by Distinct Pulmonary Myeloid Subsets during Mycobacterium tuberculosis Infection. Immunity 35, 1023–1034.
McCusker, K.T., Braaten, B.A., Cho, M.W., and Low, D.A. (1991). Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells. Infection and Immunity 59, 240–246.
McDade, J.E., Shepard, C.C., Fraser, D.W., Tsai, T.R., Redus, M.A., and Dowdle, W.R. (1977). Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease. N. Engl. J. Med. 297, 1197–1203.
McEwan, D.L., Kirienko, N.V., and Ausubel, F.M. (2012). Host translational inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an immune response in Caenorhabditis elegans. Cell Host and Microbe 11, 364–374.
Means, T.K., Hayashi, F., Smith, K.D., Aderem, A., and Luster, A.D. (2003). The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol. 170, 5165–5175.
Medvedev, A.E., Kopydlowski, K.M., and Vogel, S.N. (2000). Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression. J. Immunol. 164, 5564–5574.
130 Mizgerd, J.P. (2002). Molecular mechanisms of neutrophil recruitment elicited by bacteria in the lungs. Semin. Immunol. 14, 123–132.
Mody, C.H., Paine, R., Shahrabadi, M.S., Simon, R.H., Pearlman, E., Eisenstein, B.I., and Toews, G.B. (1993). Legionella pneumophila replicates within rat alveolar epithelial cells. J. Infect. Dis. 167, 1138–1145.
Molmeret, M., Alli, O.A.T., Zink, S., Flieger, A., Cianciotto, N.P., and Kwaik, Y.A. (2002). icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infection and Immunity 70, 69–78.
Molofsky, A.B., Byrne, B.G., Whitfield, N.N., Madigan, C.A., Fuse, E.T., Tateda, K., and Swanson, M.S. (2006). Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203, 1093–1104.
Moltke, von, J., Ayres, J.S., Kofoed, E.M., Chavarría-Smith, J., and Vance, R.E. (2013). Recognition of bacteria by inflammasomes. Annu. Rev. Immunol. 31, 73–106.
Monroe, K.M., McWhirter, S.M., and Vance, R.E. (2009). Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila. PLoS Pathog 5, e1000665.
Mouchtouri, V.A., Goutziana, G., Kremastinou, J., and Hadjichristodoulou, C. (2010). Legionella species colonization in cooling towers: risk factors and assessment of control measures. Am J Infect Control 38, 50–55.
Mukherjee, S., Keitany, G., Li, Y., Wang, Y., Ball, H.L., Goldsmith, E.J., and Orth, K. (2006). Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science 312, 1211–1214.
Musher, D.M., and Thorner, A.R. (2014). Community-acquired pneumonia. N. Engl. J. Med. 371, 1619–1628.
Nagai, H. (2002). A Bacterial Guanine Nucleotide Exchange Factor Activates ARF on Legionella Phagosomes. Science 295, 679–682.
Nagai, H., and Kubori, T. (2011). Type IVB Secretion Systems of Legionella and Other Gram-Negative Bacteria. Front Microbiol 2, 136.
Nagai, H., Cambronne, E.D., Kagan, J.C., Amor, J.C., Kahn, R.A., and Roy, C.R. (2005). A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells. Proc. Natl. Acad. Sci. U.S.a. 102, 826–831.
Nagelkerke, N.J.D., Boshuizen, H.C., de Melker, H.E., Schellekens, J.F.P., Peeters, M.F., and Conyn-van Spaendonck, M. (2003). Estimating the incidence of subclinical infections with Legionella Pneumonia using data augmentation: analysis of an outbreak in The Netherlands. Stat Med 22, 3713–3724.
Nakamura, H., Yoshimura, K., Jaffe, H.A., and Crystal, R.G. (1991). Interleukin-8 gene expression in human bronchial epithelial cells. J. Biol. Chem. 266, 19611–19617.
131 Narni-Mancinelli, E., Soudja, S.M., Crozat, K., Dalod, M., Gounon, P., Geissmann, F., and Lauvau, G. (2011). Inflammatory monocytes and neutrophils are licensed to kill during memory responses in vivo. PLoS Pathog 7, e1002457.
Nash, T.W., Libby, D.M., and Horwitz, M.A. (1984). Interaction between the legionnaires' disease bacterium (Legionella pneumophila) and human alveolar macrophages. Influence of antibody, lymphokines, and hydrocortisone. J. Clin. Invest. 74, 771–782.
Nauseef, W.M. (2007). How human neutrophils kill and degrade microbes: an integrated view. Immunol. Rev. 219, 88–102.
Nazareth, H., Genagon, S.A., and Russo, T.A. (2007). Extraintestinal pathogenic Escherichia coli survives within neutrophils. Infection and Immunity 75, 2776–2785.
Neild, A.L., and Roy, C.R. (2003). Legionella Reveal Dendritic Cell Functions that Facilitate Selection of Antigens for MHC Class II Presentation. Immunity 18, 813–823.
Neild, A.L., and Roy, C.R. (2004). Immunity to vacuolar pathogens: What can we learn from Legionella? Cell Microbiol 6, 1011–1018.
Neild, A., Murata, T., and Roy, C.R. (2005). Processing and major histocompatibility complex class II presentation of Legionella pneumophila antigens by infected macrophages. Infection and Immunity 73, 2336–2343.
Neville, B.A., Sheridan, P.O., Harris, H.M.B., Coughlan, S., Flint, H.J., Duncan, S.H., Jeffery, I.B., Claesson, M.J., Ross, R.P., Scott, K.P., et al. (2013). Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans. PLoS ONE 8, e68919.
Newton, C.A., Perkins, I., Widen, R.H., Friedman, H., and Klein, T.W. (2007). Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice. Infection and Immunity 75, 146–151.
Nguyen, T.M.N., Ilef, D., Jarraud, S., Rouil, L., Campese, C., Che, D., Haeghebaert, S., Ganiayre, F., Marcel, F., Etienne, J., et al. (2006). A community-wide outbreak of legionnaires disease linked to industrial cooling towers--how far can contaminated aerosols spread? J. Infect. Dis. 193, 102–111.
Nimah, M., Zhao, B., Denenberg, A.G., Bueno, O., Molkentin, J., Wong, H.R., and Shanley, T.P. (2005). Contribution of MKP-1 regulation of p38 to endotoxin tolerance. Shock 23, 80–87.
Ninio, S., and Roy, C.R. (2007). Effector proteins translocated by Legionella pneumophila: strength in numbers. Trends in Microbiology 15, 372–380.
Nogueira, C.V., Lindsten, T., Jamieson, A.M., Case, C.L., Shin, S., Thompson, C.B., and Roy, C.R. (2009). Rapid Pathogen-Induced Apoptosis: A Mechanism Used by Dendritic Cells to Limit Intracellular Replication of Legionella pneumophila. PLoS Pathog 5, e1000478.
132 O'Connor, T.J., Adepoju, Y., Boyd, D., and Isberg, R.R. (2011). Minimization of the Legionella pneumophila genome reveals chromosomal regions involved in host range expansion. Proc. Natl. Acad. Sci. U.S.a. 108, 14733–14740.
Obert, S., O'Connor, R.J., Schmid, S., and Hearing, P. (1994). The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex. Mol. Cell. Biol. 14, 1333–1346.
Ohashi, N., Zhi, N., Lin, Q., and Rikihisa, Y. (2002). Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agents. Infection and Immunity 70, 2128–2138.
Palmer, L.E., Hobbie, S., Galán, J.E., and Bliska, J.B. (1998). YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF-alpha production and downregulation of the MAP kinases p38 and JNK. Molecular Microbiology 27, 953– 965.
Pan, N.J., Brady, M.J., Leong, J.M., and Goguen, J.D. (2009). Targeting type III secretion in Yersinia pestis. Antimicrob. Agents Chemother. 53, 385–392.
Pan, X., Luhrmann, A., Satoh, A., Laskowski-Arce, M.A., and Roy, C.R. (2008). Ankyrin Repeat Proteins Comprise a Diverse Family of Bacterial Type IV Effectors. Science 320, 1651–1654.
Pang, I.K., Ichinohe, T., and Iwasaki, A. (2013). IL-1R signaling in dendritic cells replaces pattern-recognition receptors in promoting CD8⁺ T cell responses to influenza A virus. Nature Immunology 14, 246–253.
Parr, A., Whitney, E.A., and Berkelman, R.L. (2014). Legionellosis on the Rise: A Review of Guidelines for Prevention in the United States. J Public Health Manag Pract.
Patel, S.J., King, K.R., Casali, M., and Yarmush, M.L. (2009). DNA-triggered innate immune responses are propagated by gap junction communication. Proc. Natl. Acad. Sci. U.S.a. 106, 12867–12872.
Pechous, R.D., Sivaraman, V., Price, P.A., Stasulli, N.M., and Goldman, W.E. (2013). Early Host Cell Targets of Yersinia pestis during Primary Pneumonic Plague. PLoS Pathog 9, e1003679.
Philip, N.H., Dillon, C.P., Snyder, A.G., Fitzgerald, P., Wynosky-Dolfi, M.A., Zwack, E.E., Hu, B., Fitzgerald, L., Mauldin, E.A., Copenhaver, A.M., et al. (2014). Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling. Proc. Natl. Acad. Sci. U.S.a.
Phin, N., Parry-Ford, F., Harrison, T., Stagg, H.R., Zhang, N., Kumar, K., Lortholary, O., Zumla, A., and Abubakar, I. (2014). Epidemiology and clinical management of Legionnaires' disease. Lancet Infect Dis.
Piao, Z., Sze, C.C., Barysheva, O., Iida, K.-I., and Yoshida, S.-I. (2006). Temperature- regulated formation of mycelial mat-like biofilms by Legionella pneumophila. Appl Environ Microbiol 72, 1613–1622.
133 Plumlee, C.R., Lee, C., Beg, A.A., Decker, T., Shuman, H.A., and Schindler, C. (2009). Interferons direct an effective innate response to Legionella pneumophila infection. Journal of Biological Chemistry 284, 30058–30066.
Pore, D., Mahata, N., and Chakrabarti, M.K. (2012). Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide. Journal of Biological Chemistry 287, 12589– 12601.
Prashar, A., Bhatia, S., Tabatabaeiyazdi, Z., Duncan, C., Garduño, R.A., Tang, P., Low, D.E., Guyard, C., and Terebiznik, M.R. (2012). Mechanism of Invasion of Lung Epithelial Cells by Filamentous Legionella pneumophila. Cell Microbiol.
Puljic, R., Benediktus, E., Plater-Zyberk, C., Baeuerle, P.A., Szelenyi, S., Brune, K., and Pahl, A. (2007). Lipopolysaccharide-induced lung inflammation is inhibited by neutralization of GM-CSF. Eur. J. Pharmacol. 557, 230–235.
Qiu, J., and Luo, Z.-Q. (2013). Effector translocation by the Legionella Dot/Icm type IV secretion system. Curr. Top. Microbiol. Immunol. 376, 103–115.
Raetz, C.R. (1986). Molecular genetics of membrane phospholipid synthesis. Annu. Rev. Genet. 20, 253–295.
Rathinam, V.A.K., Vanaja, S.K., and Fitzgerald, K.A. (2012). Regulation of inflammasome signaling. Nature Immunology 13, 333–342.
Ren, T., Zamboni, D.S., Roy, C.R., Dietrich, W.F., and Vance, R.E. (2006). Flagellin- Deficient Legionella Mutants Evade Caspase-1- and Naip5-Mediated Macrophage Immunity. PLoS Pathog 2, e18.
Rider, P., Carmi, Y., Guttman, O., Braiman, A., Cohen, I., Voronov, E., White, M.R., Dinarello, C.A., and Apte, R.N. (2011). IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation. The Journal of Immunology 187, 4835– 4843.
Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Zähringer, U., Seydel, U., and Di Padova, F. (1994). Bacterial endotoxin: molecular relationships of structure to activity and function. Faseb J. 8, 217–225.
Roberts, T.L., Idris, A., Dunn, J.A., Kelly, G.M., Burnton, C.M., Hodgson, S., Hardy, L.L., Garceau, V., Sweet, M.J., Ross, I.L., et al. (2009). HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science 323, 1057–1060.
Rolando, M., Sanulli, S., Rusniok, C., Gomez-Valero, L., Bertholet, C., Sahr, T., Margueron, R., and Buchrieser, C. (2013). Legionella pneumophila Effector RomA Uniquely Modifies Host Chromatin to Repress Gene Expression and Promote Intracellular Bacterial Replication. Cell Host and Microbe 13, 395–405.
Roy, C.R., Berger, K.H., and Isberg, R.R. (1998). Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Molecular Microbiology 28, 663–674.
134 Roy, C.R., and Mocarski, E.S. (2007). Pathogen subversion of cell-intrinsic innate immunity. Nature Immunology 8, 1179–1187.
Sandvig, K., and van Deurs, B. (1996). Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin. Physiol. Rev. 76, 949–966.
Sansonetti, P.J., Phalipon, A., Arondel, J., Thirumalai, K., Banerjee, S., Akira, S., Takeda, K., and Zychlinsky, A. (2000). Caspase-1 activation of IL-1beta and IL-18 are essential for Shigella flexneri-induced inflammation. Immunity 12, 581–590.
Schesser, K., Spiik, A.K., Dukuzumuremyi, J.M., Neurath, M.F., Pettersson, S., and Wolf-Watz, H. (1998). The yopJ locus is required for Yersinia-mediated inhibition of NF- kappaB activation and cytokine expression: YopJ contains a eukaryotic SH2-like domain that is essential for its repressive activity. Molecular Microbiology 28, 1067–1079.
Scholtze, D., Varga, Z., and Imhof, A. (2011). Post-infection immunocomplex glomerulonephritis and Legionnaires“ disease in a patient with adult Still”s disease during treatment with interleukin 1 receptor antagonist anakinra: a case report. J Med Case Rep 5, 299.
Schröder, J.M., Sticherling, M., Henneicke, H.H., Preissner, W.C., and Christophers, E. (1990). IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1/IL-8- related neutrophil chemotactic proteins in human dermal fibroblasts. J. Immunol. 144, 2223–2232.
Segal, G., Purcell, M., and Shuman, H.A. (1998). Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. U.S.a. 95, 1669–1674.
Selyunin, A.S., Sutton, S.E., Weigele, B.A., Reddick, L.E., Orchard, R.C., Bresson, S.M., Tomchick, D.R., and Alto, N.M. (2011). The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold. Nature 469, 107–111.
Serbina, N.V., Jia, T., Hohl, T.M., and Pamer, E.G. (2008). Monocyte-mediated defense against microbial pathogens. Annu. Rev. Immunol. 26, 421–452.
Shen, X., Banga, S., Liu, Y., Xu, L., Gao, P., Shamovsky, I., Nudler, E., and Luo, Z.-Q. (2009). Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response. Cell Microbiol 11, 911–926.
Shi, J., Zhao, Y., Wang, Y., Gao, W., Ding, J., Li, P., Hu, L., and Shao, F. (2014). Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192.
Shida, K., Shiratori, I., Matsumoto, M., Fukumori, Y., Matsuhisa, A., Kikkawa, S., Tsuji, S., Okamura, H., Toyoshima, K., and Seya, T. (2001). An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies. J. Immunol. 166, 6671–6679.
Shin, S. (2012). Innate Immunity to Intracellular Pathogens: Lessons Learned from Legionella pneumophila. Adv. Appl. Microbiol. 79, 43–71.
135 Shin, S., and Roy, C.R. (2008). Host cell processes that influence the intracellular survival of Legionella pneumophila. Cell Microbiol 10, 1209–1220.
Shin, S., Case, C.L., Archer, K.A., Nogueira, C.V., Kobayashi, K.S., Flavell, R.A., Roy, C.R., and Zamboni, D.S. (2008). Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila. PLoS Pathog 4, e1000220.
Simons, M.P., Nauseef, W.M., and Apicella, M.A. (2005). Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes. Infection and Immunity 73, 1971–1977.
Singh, J.C.I., Cruickshank, S.M., Newton, D.J., Wakenshaw, L., Graham, A., Lan, J., Lodge, J.P.A., Felsburg, P.J., and Carding, S.R. (2005). Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G514–G524.
Singh, N., Muder, R.R., Yu, V.L., and Gayowski, T. (1993). Legionella infection in liver transplant recipients: implications for management. Transplantation 56, 1549–1551.
Sironi, M., Breviario, F., Proserpio, P., Biondi, A., Vecchi, A., Van Damme, J., Dejana, E., and Mantovani, A. (1989). IL-1 stimulates IL-6 production in endothelial cells. J. Immunol. 142, 549–553.
Sminia, T., van der Brugge-Gamelkoorn, G.J., and Jeurissen, S.H. (1989). Structure and function of bronchus-associated lymphoid tissue (BALT). Crit. Rev. Immunol. 9, 119– 150.
Somerville, D.A. (1969). The normal flora of the skin in different age groups. Br. J. Dermatol. 81, 248–258.
Spörri, R., Joller, N., Albers, U., Hilbi, H., and Oxenius, A. (2006). MyD88-dependent IFN-gamma production by NK cells is key for control of Legionella pneumophila infection. J. Immunol. 176, 6162–6171.
Spörri, R., Joller, N., Hilbi, H., and Oxenius, A. (2008). A novel role for neutrophils as critical activators of NK cells. The Journal of Immunology 181, 7121–7130.
Stanley, E., Lieschke, G.J., Grail, D., Metcalf, D., Hodgson, G., Gall, J.A., Maher, D.W., Cebon, J., Sinickas, V., and Dunn, A.R. (1994). Granulocyte/macrophage colony- stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc. Natl. Acad. Sci. U.S.a. 91, 5592– 5596.
Stetson, D.B., and Medzhitov, R. (2006). Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24, 93–103.
Stevens, W.W., Kim, T.S., Pujanauski, L.M., Hao, X., and Braciale, T.J. (2007). Detection and quantitation of eosinophils in the murine respiratory tract by flow cytometry. Journal of Immunological Methods 327, 63–74.
136 Stevenson, F.T., Torrano, F., Locksley, R.M., and Lovett, D.H. (1992). Interleukin 1: the patterns of translation and intracellular distribution support alternative secretory mechanisms. J. Cell. Physiol. 152, 223–231.
Sunderkötter, C., Nikolic, T., Dillon, M.J., van Rooijen, N., Stehling, M., Drevets, D.A., and Leenen, P.J.M. (2004). Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J. Immunol. 172, 4410–4417.
Sung, S.-S.J., Fu, S.M., Rose, C.E., Gaskin, F., Ju, S.-T., and Beaty, S.R. (2006). A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J. Immunol. 176, 2161–2172.
Sutherland, M.C., Binder, K.A., Cualing, P.Y., and Vogel, J.P. (2013). Reassessing the role of DotF in the Legionella pneumophila type IV secretion system. PLoS ONE 8, e65529.
Sutherland, R.E., Olsen, J.S., McKinstry, A., Villalta, S.A., and Wolters, P.J. (2008). Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. The Journal of Immunology 181, 5598–5605.
Sutterwala, F.S., Mijares, L.A., Li, L., Ogura, Y., Kazmierczak, B.I., and Flavell, R.A. (2007). Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome. Journal of Experimental Medicine 204, 3235–3245.
Symons, J.A., Young, P.R., and Duff, G.W. (1995). Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of IL-1 beta precursor and loses affinity for IL-1 receptor antagonist. Proc. Natl. Acad. Sci. U.S.a. 92, 1714–1718.
Szarka, R.J., Wang, N., Gordon, L., Nation, P.N., and Smith, R.H. (1997). A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of Immunological Methods 202, 49–57.
Tateda, K., Moore, T.A., Deng, J.C., Newstead, M.W., Zeng, X., Matsukawa, A., Swanson, M.S., Yamaguchi, K., and Standiford, T.J. (2001a). Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia. J. Immunol. 166, 3355–3361.
Tateda, K., Moore, T.A., Newstead, M.W., Tsai, W.C., Zeng, X., Deng, J.C., Chen, G., Reddy, R., Yamaguchi, K., and Standiford, T.J. (2001b). Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory cells. Infection and Immunity 69, 2017–2024.
Thomas, M.G., Loschi, M., Desbats, M.A., and Boccaccio, G.L. (2011). RNA granules: The good, the bad and the ugly. Cellular Signalling 23, 324–334.
Tilney, L.G., Harb, O.S., Connelly, P.S., Robinson, C.G., and Roy, C.R. (2001). How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. Journal of Cell Science 114, 4637–4650.
Ting, J.P.-Y., Kastner, D.L., and Hoffman, H.M. (2006). CATERPILLERs, pyrin and
137 hereditary immunological disorders. Nat Rev Immunol 6, 183–195.
Towne, J.E., Renshaw, B.R., Douangpanya, J., Lipsky, B.P., Shen, M., Gabel, C.A., and Sims, J.E. (2011). Interleukin-36 (IL-36) ligands require processing for full agonist (IL- 36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity. Journal of Biological Chemistry 286, 42594–42602.
Trosky, J.E., Li, Y., Mukherjee, S., Keitany, G., Ball, H., and Orth, K. (2007). VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases. J. Biol. Chem. 282, 34299–34305.
Trunk, G., and Oxenius, A. (2012). Innate Instruction of CD4+ T Cell Immunity in Respiratory Bacterial Infection. The Journal of Immunology.
Tsuda, Y., Takahashi, H., Kobayashi, M., Hanafusa, T., Herndon, D.N., and Suzuki, F. (2004). Three different neutrophil subsets exhibited in mice with different susceptibilities to infection by methicillin-resistant Staphylococcus aureus. Immunity 21, 215–226.
Uliczka, F., Kornprobst, T., Eitel, J., Schneider, D., and Dersch, P. (2009). Cell invasion of Yersinia pseudotuberculosis by invasin and YadA requires protein kinase C, phospholipase C-gamma1 and Akt kinase. Cell Microbiol 11, 1782–1801. van der Poll, T., Keogh, C.V., Guirao, X., Buurman, W.A., Kopf, M., and Lowry, S.F. (1997). Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J. Infect. Dis. 176, 439–444.
Vance, R.E., Isberg, R.R., and Portnoy, D.A. (2009). Patterns of Pathogenesis: Discrimination of Pathogenic and Nonpathogenic Microbes by the Innate Immune System. Cell Host and Microbe 6, 10–21.
Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., and Stockinger, B. (2006). TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24, 179–189.
Viboud, G.I., and Bliska, J.B. (2005). Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu. Rev. Microbiol. 59, 69–89.
Vigne, S., Palmer, G., Lamacchia, C., Martin, P., Talabot-Ayer, D., Rodriguez, E., Ronchi, F., Sallusto, F., Dinh, H., Sims, J.E., et al. (2011). IL-36R ligands are potent regulators of dendritic and T cells. Blood 118, 5813–5823.
Villarino, A.V., Katzman, S.D., Gallo, E., Miller, O., Jiang, S., McManus, M.T., and Abbas, A.K. (2011). Posttranscriptional Silencing of Effector Cytokine mRNA Underlies the Anergic Phenotype of Self-Reactive T Cells. Immunity 34, 50–60.
Vincent, C.D., Friedman, J.R., Jeong, K.C., Buford, E.C., Miller, J.L., and Vogel, J.P. (2006). Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Molecular Microbiology 62, 1278–1291.
Vivinus, S., Baulande, S., van Zanten, M., Campbell, F., Topley, P., Ellis, J.H., Dessen, P., and Coste, H. (2001). An element within the 5' untranslated region of human Hsp70
138 mRNA which acts as a general enhancer of mRNA translation. Eur. J. Biochem. 268, 1908–1917.
Vogel, J.P., Andrews, H.L., Wong, S.K., and Isberg, R.R. (1998). Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873–876.
Vogel, J.P., Roy, C., and Isberg, R.R. (1996). Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann. N. Y. Acad. Sci. 797, 271–272.
Way, S.S., Borczuk, A.C., Dominitz, R., and Goldberg, M.B. (1998). An essential role for gamma interferon in innate resistance to Shigella flexneri infection. Infection and Immunity 66, 1342–1348.
Weber, A., Wasiliew, P., and Kracht, M. (2010). Interleukin-1 (IL-1) pathway. Science Signaling 3, cm1.
Weber, S.S., Joller, N., Küntzel, A.B., Spörri, R., Tchang, V.S., Scandella, E., Rösli, C., Ludewig, B., Hilbi, H., and Oxenius, A. (2012). Identification of protective B cell antigens of Legionella pneumophila. The Journal of Immunology 189, 841–849.
Weinbaum, D.L., Bailey, J., Benner, R.R., Pasculle, A.W., and Dowling, J.N. (1983). The contribution of human neutrophils and serum to host defense against Legionella micdadei. J. Infect. Dis. 148, 510–517.
Westphalen, K., Gusarova, G.A., Islam, M.N., Subramanian, M., Cohen, T.S., Prince, A.S., and Bhattacharya, J. (2014). Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity. Nature 506, 503–506.
Wickremasinghe, M.I., Thomas, L.H., and Friedland, J.S. (1999). Pulmonary epithelial cells are a source of IL-8 in the response to Mycobacterium tuberculosis: essential role of IL-1 from infected monocytes in a NF-kappa B-dependent network. J. Immunol. 163, 3936–3947.
Wilson, B.A., and Collier, R.J. (1992). Diphtheria toxin and Pseudomonas aeruginosa exotoxin A: active-site structure and enzymic mechanism. Curr. Top. Microbiol. Immunol. 175, 27–41.
Winn, W.C., and Myerowitz, R.L. (1981). The pathology of the Legionella pneumonias. A review of 74 cases and the literature. Hum. Pathol. 12, 401–422.
Winn, W.C., Glavin, F.L., Perl, D.P., Keller, J.L., Andres, T.L., Brown, T.M., Coffin, C.M., Sensecqua, J.E., Roman, L.N., and Craighead, J.E. (1978). The pathology of Legionnaires' disease. Fourteen fatal cases from the 1977 outbreak in Vermont. Arch. Pathol. Lab. Med. 102, 344–350.
Wright, E.K., Goodart, S.A., Growney, J.D., Hadinoto, V., Endrizzi, M.G., Long, E.M., Sadigh, K., Abney, A.L., Bernstein-Hanley, I., and Dietrich, W.F. (2003). Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila. Curr. Biol. 13, 27–36.
139 Yang, J., Zhang, L., Yu, C., Yang, X.-F., and Wang, H. (2014). Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark Res 2, 1.
Yirmiya, R., Avitsur, R., Donchin, O., and Cohen, E. (1995). Interleukin-1 inhibits sexual behavior in female but not in male rats. Brain Behav. Immun. 9, 220–233.
Yoon, H., Ansong, C., Adkins, J.N., and Heffron, F. (2011). Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages. Infection and Immunity 79, 2182–2192.
Yrlid, U., and Wick, M.J. (2000). Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells. J. Exp. Med. 191, 613–624.
Yu, V.L., Greenberg, R.N., Zadeikis, N., Stout, J.E., Khashab, M.M., Olson, W.H., and Tennenberg, A.M. (2004). Levofloxacin efficacy in the treatment of community-acquired legionellosis. Chest 125, 2135–2139.
Yuan, M., Deleuil, F., and Fällman, M. (2005). Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb. J. Mol. Microbiol. Biotechnol. 9, 214–223.
Zamboni, D.S., Kobayashi, K.S., Kohlsdorf, T., Ogura, Y., Long, E.M., Vance, R.E., Kuida, K., Mariathasan, S., Dixit, V.M., Flavell, R.A., et al. (2006). The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection. Nature Immunology 7, 318–325.
Zlokarnik, G., Negulescu, P.A., Knapp, T.E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K., and Tsien, R.Y. (1998). Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279, 84–88.
140