INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1β PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS
by
MAUSITA KARMAKAR
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Thesis Advisor: Eric Pearlman, Ph.D.
Department of Pathology
CASE WESTERN RESERVE UNIVERSITY
May, 2014
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Mausita Karmakar
candidate for the Ph. D. degree*
(Signed) George R. Dubyak (chair of the committee)
Amy G. Hise
Brian A. Cobb
Arne Rietsch
Clive R. Hamlin
Eric Pearlman
(Date) 1/31/2014
* We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Figures 3
List of Tables 4 List of Abbreviations 5
Acknowledgements 7
Abstract 9
Chapter 1: Introduction
1.1: Bacterial keratitis: A perspective on epidemiology 13
1.2: Other microbial keratitis 14 1.3: Clinical characteristics of bacterial keratitis and its outcome 16 1.4: Anti-bacterial defense at the ocular surface 18 1.5: Immune recognition by bacteria during corneal infection 22
1.6: Virulence factors of S. pneumoniae 27
1.7: Virulence factors of P. aeruginosa 30 1.8: Role of IL-1 family cytokines in inflammation 36 1.9: IL-1β in immune mediated diseases 37 39 1.10: Role of inflammasome in IL-1β processing 1.11: Activation of NLRP3 and NLRC4 inflammasome 43
1.12: Inflammasome independent IL-1β processing 48
1.13: The role of neutrophils in IL-1β processing 49
Chapter 2: IL-1β processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and Caspase-1
Abstract 53
Introduction 54 Materials and Methods 55 Results 62 Discussions 75
1 Chapter 3: Bacterial pneumolysin mediates NLRP3 activation and IL-1β processing by neutrophil during Streptococcus pneumoniae corneal infection
Abstract 81
Introduction 82
Materials and Methods 83 Results 92 Discussions 113
Chapter 4: Discussions and Future Directions
Research Summary 119
Regulation and resolution of inflammation during bacterial keratitis 121
Future Directions:
4.1: Role of ROS in activating NLRP3/ASC inflammasome in neutrophils 126 4.2: Role of ATP in amplifying NLRP3 activation in neutrophils 127 4.3: Role of AIM2/ASC inflammasome in IL-1β processing by neutrophils during 130 S. pneumoniae infection. 4.4: Role of S. aureus pore forming toxins in activating inflammasomes
by neutrophils 132 4.5: Pyroptotic cell death in neutrophils due to inflammasome activation 133 4.6: Inflammasome activation and autophagy 135
4.7: Mechanism of non-canonical IL-1β secretion by neutrophils 136 4.8: Microbial proteases in alternate IL-1β processing 137
4.9: Therapeutic implications 140
Concluding Remarks 141
Selected Publications 142 Bibliography 143
2 List of Figures:
Chapter 1: 1.1: Clinical characteristics of bacterial keratitis 17
1.2: Anatomy of eye and histology of corneal section 20
1.3: PAMP recognition by various TLRs 23 1.4: TLR structure and signaling cascade 24 1.5: Proposed sequence of events during P. aeruginosa corneal infection 26 1.6: NOD2 signaling by peptidoglycan (PGN) from Gram positive bacteria 28 1.7: Structure of pneumolysin mediated pore formation 31
1.8: Virulence factors of P. aeruginosa 33
1.9: Type III secretion system (T3SS) of P. aeruginosa 35 1.10: Signal 1 and signal 2 together mediate IL-1β processing 40
1.11: Domain organization of representative NOD like receptors 42 1.12: Mechanisms of NLRP3 activation 45
1.13: NLRC4 activation by bacteria T3SS and Flagellin 47
1.14: Cellular composition and protein expression in corneal ulcers of keratitis patients 51
Chapter 2:
2.1: IL-1β from bone marrow derived cells mediate cellular infiltration, corneal disease
and bacterial clearance 63 S2.1: Quantification of corneal opacification 64 2.2: Neutrophils are the major source of IL-1β in P. aeruginosa corneal infection 67 S2.2: Contribution of macrophages in IL-1β production during P. aeruginosa keratitis 69 2.3: Neutrophil mediated IL-1β processing in vivo is caspase-1 and NLRC4
independent 71 2.4: IL-1β processing by murine and human neutrophils is mediated by serine proteases 74 2.5: Proposed Model 78
3 Chapter 3: 3.1: IL-1β, NLRP3 and ASC regulate S. pneumoniae corneal infection 93
S3.1: Quantification of corneal opacification 94 S3.2: Histology of corneal sections 96 3.2: NLRP3 and ASC mediated IL-1β processing in the cornea is dependent on active caspase-1 produced by neutrophils 98 3.3: Active caspase-1 induction in murine neutrophils is dependent on NLRP3 and ASC, and requires active pneumolysin 100 S3.3: Quantification of FLICA-YVAD staining 102 3.4: Inducible NLRP3 expression in murine and human neutrophils 104 S3.4: Mapping of transcription factor binding sites in murine Nlrp3 promoter 105 3.5: Pneumolysin mediates IL-1β processing and release from human neutrophils 107 + 3.6: Pneumolysin-induced IL-1β secretion by neutrophils is mediated by K efflux and caspase-1 109 -/- 112 S3.5: IL-1β secretion by neutrophil elastase (NE) mice
Chapter 4: 4.1: ATP release by human neutrophils in response to S. pneumoniae infection 128 4.2: Expression of P2X4 receptors in human neutrophils 129 4.3: Working Model for ATP mediated amplification of NLRP3 activation 131 4.4: Proposed mechanism of non-classical IL-1β processing by neutrophil serine proteases 138
List of Tables
Chapter 1: 1.1: Streptococcus pneumoniae virulence factors and their role in colonization 29 Chapter 3: 3.1: Plasmids and primers used for generation of S. pneumoniae Δply 85
4 List of Abbreviations
AIM-2- Absent In Melanoma-2
AP-1- Activator Protein-1
CARD- Caspase Recruitment Domain
CXCL-1/8- Chemokine (C-X-C motif) ligand-1/8
HIN- hematopoietic interferon-inducible nuclear proteins
IKK- IκB Kinase
IL-1R1- Interleukin-1 Receptor 1
IL-1β- Interleukin-1β
IRAK-4- Interleukin-1 Receptor Associated Kinase4
IRF3- Interferon Regulatory Factor 3
JNK- c-Jun N-terminal kinase
LDH- Lactate Dehydrogenase
LPS- Lipo polysaccharide
LRR- Leucine Rich Repeat
MAPK- Mitogen Associated Protein Kinase
MD-2- Myeloid Differentiation-2
MDP- Muramyl Dipeptide
MyD88- Myeloid Differentiation 88
NAIP- Neuronal apoptosis inhibitor protein
NFκB- nuclear factor kappa enhancer Binding protein
NK cells- Natural Killer cells
NLR- NOD-like receptor
NLRC4- NLR family CARD domain-containing protein 4
5 NLRP3- NACHT, LRR and PYD domains-containing protein 3
PAMP- Pathogen Associated Molecular Pattern
PI3K- phosphoinositide 3-kinase
Ply- Pneumolysin
PMN – Polymorpho nuclear cells
PRR- Pathogen Recognition Receptor
PYD- Pyrin Containing Domain
TIR- Toll/Interleukin-1 Receptor
TIRAP- toll-interleukin 1 receptor domain containing adaptor protein
TLR- Toll like Receptor
TRIF- TIR-domain-containing adapter-inducing interferon-β
TXNIP- Thioredoxin Interacting Protein
UBC- Ubiquitin Conjugating Enzyme
6 ACKNOWLEDGEMENTS
Completing my PhD degree is probably by far the most challenging and exciting activity in my academic career. The constant support and encouragement of numerous people including my family, well-wishers, my friends, and colleagues have assisted me on my path towards this doctoral degree. At this moment of accomplishment, I would like to express my gratitude to all those who contributed in different ways to the success of this study and made it an unforgettable experience for me.
This thesis would have remained a dream had it not been for my mentor Dr. Eric
Pearlman who with his selfless guidance, understanding and patience provided me just the right platform for completing my graduate studies. His unflagging encouragement and advice helped me to proceed through the doctoral program and complete my dissertation.
I am not sure how many graduate students are given the opportunity to develop their own individuality and self-sufficiency by being allowed to work with such independence and freedom as I could in the Pearlman Lab. I am greatly indebted to him for this. The incredible members of the Pearlman Lab deserve special thanks as well. Since I joined the lab in 2009, my coworkers – Sixto, Steven and Sanhita and others has always been great friends and supported me all these years. Apart from being highly productive,
Pearlman Lab is also a ‘Fun lab’ and I believe individual members of this family play an important role in creating a happy and conducive work environment in the lab. I also extend my thanks to all the committee members – Drs. Dubyak, Hise, Rietsch, Cobb and
Hamlin whose guidance has served me well and I owe them my heartfelt appreciation. I am especially grateful to Dr. Dubyak who helped me understand the field of
7 ‘inflammasome biology’ and have guided me with helpful suggestions all the time.
Members of the Dubyak Lab – Michael, Christina and Caroline have assisted me with experiments and reagents whenever I needed and I couldn’t have asked for a better collaborating lab. All the members of VSRC core facility have been awesome. Scott
Howell at the microscopy core has provided immense help throughout and I thank him for that.
Lastly, and most importantly, no words are enough to thank my parents for all their encouragements and support through this entire journey. My mom and dad have ingrained in me and my little brother the importance of education ever since our childhood. It is for them only, that I could walk the long path to earn my PhD and my brother who is also currently pursuing his PhD in one of the premier institutes in India.
My best friend and husband– Arnab has been my lifeline here in USA and his unfailing support everyday made this journey seem easy and truly a memorable one.
8 INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1β PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS
Abstract
by
MAUSITA KARMAKAR
Bacterial infections of the cornea caused by Pseudomonas aeruginosa, Streptococcus
pneumoniae and Staphylococcus aureus are major causes of preventable blindness
worldwide as indicated by the World Health Organization (WHO). The major risk factors
for P. aeruginosa keratitis are contact lens wear and ocular injury, whereas S.
pneumoniae infection primarily occurs following corneal trauma. Microbial infections are
associated with a robust inflammatory response that includes infiltration of neutrophils
and macrophages to the cornea, and results in corneal opacity and loss of vision. The
series of studies described herein reveals an important role for neutrophils in producing
IL-1β during corneal infection by P. aeruginosa and S. pneumoniae and shows distinct
pathways for IL-1β processing by these cells.
In order to investigate the mechanism of IL-1β processing during P. aeruginosa
keratitis, we established a murine model of corneal infection and demonstrated that
neutrophils are the key cell type that produces IL-1β early during infection (at 24hr). At this time point, there are significantly fewer infiltrating macrophages (> 3%) compared to
9 neutrophils (< 20%) in the corneal stroma indicating a minimal contribution of
macrophages in IL-1β processing. Additionally, we showed that IL-1β processing in the
cornea is independent of NLRC4 inflammasome and caspase-1. Using highly purified
human and murine neutrophils, we demonstrated that blockade of neutrophil serine
proteases like elastase and cathepsin G blocks IL-1β processing and secretion. This was further supported by the observation that neutrophil elastase (NE)-/- mice have impaired
bacterial killing and reduced IL-1β in the cornea compared with C57BL/6 mice. Taken
together, these findings indicate that during acute P. aeruginosa corneal infection,
neutrophils are the predominant producers of IL-1β and that the processing relies on
neutrophil elastase instead of NLRC4/caspase-1 inflammasome.
In a similar model of murine S. pneumoniae corneal infection, we showed that
unlike P. aeruginosa keratitis, IL-1β processing by neutrophils is dependent on
NLRP3/ASC inflammasome and caspase-1. This was confirmed using purified murine
neutrophils, where IL-1β secretion was found to be dependent on NLRP3, ASC and caspase-1. Confocal microscopy identified caspase-1 and NLRP3 ‘specks’ in neutrophils
upon inflammasome induction. Additionally, S. pneumoniae cytolytic toxin –
pneumolysin (Ply) was found to activate NLRP3 in neutrophils, as a Δply mutant strain
(pneumolysin deficient bacteria) did not induce caspase-1 activation or IL-1β secretion by neutrophils. We also demonstrated that Ply - induced pore formation leads to loss of intracellular K+, which is a sufficient and necessary to signal inflammasome activation in
neutrophils. Blocking of pyroptotic cell death in neutrophils inhibited IL-1β secretion but did not block loss of intracellular K+ in response to S. pneumoniae, indicating that loss of
10 intracellular ion is a direct consequence of pore formation by Ply and not a secondary
response of pyroptosis.
In summary, these findings significantly advance our knowledge of neutrophil -
mediated IL-1β processing during infection with two major human bacterial pathogens.
Since neutrophils are the first responders that infiltrate the tissue in majority of microbial infections, these mechanisms of IL-1β processing by neutrophils might also play an important role in other inflammatory conditions.
11
CHAPTER 1 INTRODUCTION
12 1.1: Bacterial keratitis: A perspective on epidemiology
Bacterial keratitis is a painful infection of the cornea that can lead to visual impairment and blindness. Corneal ulcers caused by microbial infections have been described as a major cause of preventable blindness by the World Health Organization
(Al-Mujaini et al., 2009). Until recently, a majority of the cases of bacterial infection were associated with ocular trauma; however, widespread use of contact lenses has significantly increased the incidence of contact lens related bacterial keratitis over the past few years (Choy et al., 2008; Green et al., 2008; Robertson and Cavanagh, 2008).
The prevalence of contact lens related corneal ulcers in the general population has increased from almost 0% in the 1960s to 52% of the total number of bacterial keratitis cases in the 1990s. Extended wear soft contact lens users have a higher annual incidence of ulcerative keratitis than daily wear soft contact lenses. This was confirmed in a case control study of ulcerative keratitis in soft contact lens wearers, where overnight use of contact lenses were the most important risk factor for ulcerative keratitis (Poggio et al.,
1989). As there are over 140 million contact lens wearers world-wide, this form of keratitis has a significant clinical and economic impact. The most common bacterial pathogens associated with bacterial keratitis include Pseudomonas aeruginosa,
Streptococcus pneumoniae and Staphylococcus aureus. P. aeruginosa is mostly associated with contact lens related keratitis, P. aeruginosa and S. pneumoniae are the major cause of corneal ulcers in India and other developing countries (Bharathi et al.,
2007; Bharathi et al., 2009; Parmar et al., 2003; Upadhyay et al., 1991) accounting for more than 30% of all cases of bacterial keratitis. (Parmar et al., 2003). Methicillin resistant S. aureus (MRSA) have also become more prevalent in recent years, are more
13 difficult to treat, and are more virulent than methicillin sensitive strains (Chuang et al.,
2012; Elsahn et al., 2010).
1.2: Other microbial keratitis
Fungal pathogens include Fusarium, Aspergillus, and Candida species, and
parasitic causes include Acanthamoeba and the Onchocerca volvulus worm that causes river blindness. In the latter case, endosymbiotic Wolbachia bacteria play a critical role in corneal inflammation (Saint Andre et al., 2002; Tamarozzi et al., 2011; Turner et al.,
2009). In developed nations, fungal keratitis mostly occurs in immunosuppressed individuals with increased susceptibility to infection by the commensal yeast Candida
albicans. In contrast, in developing nations and hot and humid regions of industrialized nations, fungal keratitis occurs primarily in immunocompetent individuals with no preexisting conditions and is usually initiated by preexisting corneal trauma which gets
infected with vegetative matter bearing conidia from filamentous fungi. As moulds such
as Aspergillus and Fusarium species are plant saprophytes, and grow abundantly on
crops, the highest incidence and prevalence of fungal keratitis is in agricultural regions,
especially during harvest season where there is an abundance of airborne spores. Once in
a nutrient - rich environment such as the mammalian cornea, conidia germinate and
hyphae can penetrate throughout the corneal stroma. The hyphal tips contain several
proteases, including collagenases that facilitate migration of the hyphae. The general
direction of migration is towards the nutrient rich anterior chamber, and hyphae can
penetrate Descemet’s membrane. Once in the anterior chamber, there is a massive
14 neutrophil infiltrate (seen clinically as a hypopyon), which limits further penetration to
the posterior segment of the eye. On a global scale, fungal keratitis accounts for ~65% of
all corneal ulcers and are more common than bacterial infections. These infections are not
readily treatable, and tissue damage from the corneal ulcers results in >10% of infections requiring corneal transplantation (Thomas, 2003a, b).
Herpes simplex virus-1 (HSV-1) is also a major cause of infectious keratitis in the
USA. HSV-1 not only cause oral and genital lesions, but also herpes keratitis and infects
the eyelids, conjunctiva, cornea uveal tract and retina. Global incidence of HSV keratitis
is roughly 1.5 million, including 40,000 new cases of severe monocular visual
impairment or blindness each year (Farooq and Shukla, 2012). Like oral and genital
infection, herpes keratitis can occur repeatedly and can cause corneal scar formation.
Following primary infection, the virus enters the corneal neurons and migrates to the
trigeminal ganglia where it is maintained in the latent stage; however, infection is
triggered by reactivation of latent HSV-1 infection of neurons in the trigeminal ganglion,
with subsequent axonal transport of viral proteins or infectious virus into the corneal
stroma which causes herpes keratitis associated with pronounced neovascularization
(Gimenez et al., 2013; Suryawanshi et al., 2011). Apart from ultraviolet rays, immune
suppression also leads to reactivation of the virus, indicating an essential role of host
immune response to maintain latency of the virus.
Despite being a relatively rare disease in comparison with other forms of
infectious keratitis, Acanthameoba keratitis is an extremely painful and potentially
blinding infection. Infections are often associated with contact lens wear, and occur when
the cornea is exposed to high concentration of amoeba that is present in the water
15 supplies. Acanthamoeba keratitis outbreak in Chicago in 2007 was associated with
changes in the water treatment. Acanthamoeba have been recovered from soil, air,
chlorinated swimming pools, hot tubs, tap water and contact lens solutions. Wearing contact lenses while showering or swimming in fresh water ponds increases the risk of
infection too. Invasion of the cornea is due to expression of lectins and proteases and
inflammation may be exacerbated by the presence of intracellular bacteria including
Legionella pneumophilia and P. aeruginosa (Clarke and Niederkorn, 2006; Iovieno et al.,
2010). Chlamydia trachomatis bacteria that cause trachoma is another major cause of
blindness worldwide, with ~1.3 million people blind and an estimated 1.8 million people
with impaired vision. They are primarily located in epithelial cells of the palpebral
conjunctiva where they are present as reticulate bodies that do not have cell wall. They
also form spore like elementary bodies that is transmitted either person to person or by
flies that are attracted to the eyes. Infection of the conjunctival epithelium results in
inflammation and development of papillary conjunctivitis. Chronic conjunctival
inflammation can cause scarring and subsequent inward turned eyelashes which rub on
the cornea causing secondary corneal diseases.
1.3: Clinical Characteristics of Bacterial Keratitis and its outcome
The main etiologic agents for bacterial keratitis are P. aeruginosa and S.
pneumoniae as shown in Figure 1.1. P. aeruginosa forms a biofilm on silicone hydrogel
contact lenses, which enables them to penetrate the intact corneal epithelium (Tam et al.,
2010). Previous studies have shown that P. aeruginosa can invade the corneal stroma
within one hour of adhering to an injured corneal epithelium. Within 6-8 hours, it
16 Figure 1.1: Clinical characteristics of bacterial keratitis
A. Psseudomonas aeruginosa keratitis
C.
Aravind Eye Hospital, Madurai, India
B. Streptococcus pneumoniae keratitis
D.
Aravind Eye Hospital, Madurai, India
A. Eye image of patient with contact lens related P. a eruginosa keratitis and B. S. pneumoniae keratitis. C. Gram staining of corneal ulcer material showing Gram negative bacilli in P. aeruginosa keratitis paatient, and D. Gram positive diplococci and chains in S. pneumoniae keratitis patients. (Karthikeyan et al, 2013, PLoS One).
17 produces grayish superficial epithelial and stromal infiltration with edema. During the
next 18-24 hours, there is a severe anterior chamber inflammation with hypopyon. This is
associated with characteristic diffuse grayish, epithelial inflammation and cellular
infiltration. During the next 48-96 hours, if untreated, a ring infiltration develops with
scleral and corneal melting associated with greenish yellow mucopurulent discharge
leading to eventual perforation of cornea. (Hyndiuk, 1981; Ostler et al., 1978). Corneal ulceration and abscess formation due to S. pneumoniae is classically associated with a
deep, oval central stromal ulceration accompanied by neutrophil infiltration to the
anterior chamber (hypopyon) and corneal thinning. The treatment strategy involves moxifloxicin and related antibiotics, which kill the bacteria, but does not address the tissue damage and scarring of corneal stroma caused by bacterial toxins and infiltrating neutrophils. Corticosteroids such as dexamethasone are the standard anti-inflammatory treatment; however, steroids have non-specific effects during infection, and have off target effects that include increased ocular pressure. Despite all the advances in the diagnosis and treatment, bacterial keratitis remains very aggressive and destructive sight threatening form of corneal infection.
1.4: Anti-bacterial defense at the ocular surface
Figure 1.2A illustrates the anterior or posterior segments of a normal human eye.
Light rays are reflected off an object and enter the eyes through the transparent outer
layer of the eye – cornea. The cornea bends or refracts the rays allowing it to pass
through a round hole called the pupil. The light rays then pass through the lens, which
actually changes shape so it can further bend the rays and focus them on the retina at the
18 back of the eye. The retina is a thin layer of tissue at the back of the eye that contains
millions of photoreceptors that convert the light into electrical impulses. The optic nerve
sends these impulses to the brain where an image is produced and we can see. Thus,
transparency of the cornea is essential in accurate transmission of light and vision.
Cornea is transparent because 1. It is highly avascular. 2. Epithelial cells form a tight
external barrier and 3. passive diffusion of water and ions via the Na+/K+ ATPase from
the anterior chamber into the stroma helps maintain 80% hydration of the cornea, which
also is believed to play an important role in corneal transparency (Freegard, 1997).
Corneal inflammation due to microbial infection results in influx of inflammatory cells which causes edema resulting in corneal opacity and changes the refractive index of the cornea leading to impaired vision.
The cornea and ocular surface are protected from trauma and microbial infection
by physical and molecular defense mechanisms including 1. eyelid closure and blinking,
which protects the cornea from physical trauma, and removes microbes from the ocular
surface 2. ocular surface mucins and the tear film restrict pathogen interaction with the
corneal epithelium 3. antibacterial components present in human tears include β-
defensins, LL-37, calprotectin and lysozyme, histatins, human natriuretic peptides,
dermcidin, hepcidin/liver-expressed antimicrobial peptide (LEAP)-1, LEAP-2, HE2α and
HE2β1(McDermott, 2004). Tears also contain high levels of the iron-chelating protein
lactoferrin (Flanagan and Willcox, 2009) and siderophore-binding protein lipocalin
(Fluckinger et al., 2004). Though their role in tears is not fully understood, it is likely that
cation (Fe2+, Zn2+) sequestration in the ocular surface inhibits microbial growth, which
requires these essential metals (Fluckinger et al., 2004; Leal and Pearlman, 2012).
19 Figure 1.2: Anatomy of eye and histology of corneal sectioon
A. B. Epithelium
Stroma
Streilein et al., 2003, Nat Rev Immunologyy Endothelium
C. D. Resident cells in stroma
Resident cells in epithelium
Pearlman et al., 2013, Intl Rev Immunology
A. Anatomy of human eye (Streilein et al, 2003, Nature Reviews Immunology) and B. Histology of normal murine cornea after H&E staining. C. Confocal microscopy images of mouse corneal whole + mounts from C57BL/6 mice expressing MHC II (1) CD11c eYFP (2) or CX3CR1GFP mice (3) after incubating with antibodies to MHC class II or CD11b, counterstained with DAPI. Original magnification is x400. D. H&E stain of C57BL/6 cornea showing corneal infiltrates 24h after infection with P. aeruginosa (unpublished data) Original magnification is x20. Reprint permission obtained from the publisher.
20 Figure 1.2B shows an H&E stained section of a normal human cornea comprised of the outer epithelium (~50μm), the stroma (~500μm) and inner single layer of endothelium (~5 μm). The non-keratinized stratified corneal epithelial cells with tight junctions are a physical barrier limiting access of microbes into the corneal stroma.
Additionally, mucins and surfactants secreted by epithelial cells prevent microbe adhesion to the corneal epithelium (Ueta, 2008; Ueta and Kinoshita, 2010). Integrity of the corneal epithelium is essential because it restricts access of millions of live organisms to the corneal stroma even under stressful conditions such as long term contact lens wear
(Alarcon et al., 2011; Sun et al., 2010a). However, corneal trauma or regular use of contact lens might breach the corneal epithelium which then facilitates microbes like bacteria or fungus to gain access into the underlying corneal stroma. Once in the stroma, bacteria are recognized by pattern recognition receptors (PRRs) of resident dendritic cells and macrophages in the cornea (Brissette-Storkus et al., 2002; Chinnery et al., 2008;
Hamrah et al., 2003a). Figure 1.2C shows confocal microscopy images of resident dendritic cells in the epithelium and resident CX3CR1+ CD11b+ macrophages and
CD11c+ MHC II+ dendritic cells in the stroma of naïve C57BL/6 cornea (Chinnery et al.,
2007; Hamrah et al., 2003a). These cells are necessary for initiating inflammatory host defense responses in the cornea against bacteria since ablation of the resident macrophages and DCs leads to uncontrolled growth of bacteria in stroma resulting in corneal perforation (Sun et al., 2010b). Following recognition of bacteria by resident macrophages and DCs in the stroma, proinflammatory cytokines and chemokines are produced which recruit neutrophils from the peripheral, limbal blood vessels where they induce bacterial killing (Pearlman et al., 2013; Sun et al., 2010b). Figure 1.2D shows an
21 H&E stained section of C57BL/6 cornea with influx of inflammatory cells in the corneal stroma 24h post infection with P. aeruginosa.
1.5: Immune recognition by bacteria during corneal infection
Initiation of innate immune responses relies on the recognition of pathogen- associated molecular patterns (PAMPs) by the PRRs present on the immune cells.
Bacterial PAMPs are recognized by toll like receptors (TLRs) which are widely expressed by macrophages, dendritic cells, epithelial cells, fibroblasts and neutrophils.
Figure 1.3 shows different TLRs and their known ligands (Netea et al., 2012). Huang and
Hazlett et al reported a role for TLR4 and macrophages in regulating P. aeruginosa corneal infection, although they suggested that TLR4 activation is important for expression of antimicrobial peptides rather than neutrophil infiltration (Huang et al.,
2006; McClellan et al., 2003). Previous studies published by our group have shown that
TLR4 and TLR5 on macrophages recognize LPS and flagellin of P. aeruginosa and initiating signaling by recruitment of adaptor molecules MyD88 to the TIR domain of
TLR4 andTLR5, and recruitment of TIRAP and TRIF to TLR4, all of which lead to NF-
κB translocation to the nucleus, and expression of pro-inflammatory and chemotactic cytokines. These include CXCL1/KC, which recruits neutrophils from limbal capillaries to the corneal stroma, and IL-1α and IL-1β, which then activate the IL-1R1/MyD88 pathway in macrophages and resident corneal epithelial cells and keratocytes (Sun et al.,
2010b). The positive feedback signaling by IL-1R is a major contributing pathway for exacerbated inflammation and has been implicated in many inflammatory diseases including rheumatoid arthritis (Sims and Smith, 2010). Shown in Figure 1.4A is the
22 Figure 1.3: PAMP recognition by various TLRs
A. Membrane bound TLRs and B. endosomal TLRs with their known ligands. ssRNA- single stranded RNA, dsRNA-double stranded RNA. (O’Neill et al, 2012, Nature Reviews Immunology). Reprint permission obtained from the publisher.
23 Figure 1.4: TLR structure and signaling cascade
A. B.
A. TLRs and IL-1Rs have a conserved cytoplasmic (TIR) domain characterized by the presence of three homologous regions (known as boxes 1, 2 and 3). Despite the similarity of the cytoplasmic domains, their extracellular regions differ markedly: TLRs have tandem repeats of LRR, whereas IL-1Rs have three Ig-like domains. B. Activated TLRs associate with its adaptor MyD88 which in turn recruits IRAK4 and IRAK1. IRAK4 phosphorylates IRAK1 and facilitate binding of TRAF6. This complex then dissociates from the receptor and binds with TAK1, TAB1 and TAB2 at the plasma membrane (not shown), which induces the phosphorylation of TAB2 and TAK1. IRAK1 is degraded at the plasma membrane, and the remaining complex (consisting of TRAF6, TAK1, TAB1 and TAB2) translocate to the cytosol, where it associates with the ubiquitin ligases UBC13 and UEV1A. This leads to the ubiquitylation of TRAF6, which induces activation of TAK1 which in turn phosphorylates both MAPK and the IKK complex . The IKK complex then phosphorylates IκB, which leads to its ubiquitylation and subsequent degradation. This allows NF-κB to translocate to the nucleus and induce the expression of its target pro inflammatory genes (Akira et al, 2004, Nature Reviews Immunology). Reprint permission obtained from the publisher.
24 comparison between structures of TLR and IL-1R and Figure 1.4B illustrates the
signaling cascade downstream of TLR activation. Figure 1.5 demonstrates the sequence
of events that takes place during P. aeruginosa corneal infection (Pearlman et al., 2013;
Sun et al., 2010b). Although the mechanism of neutrophil mediated killing in bacterial keratitis has yet to be determined, NADPH oxidase mediated generation of reactive oxygen species (ROS) has a critical role in regulating hyphal growth during fungal keratitis (Leal et al., 2012). Although, macrophages are the major responders during P. aeruginosa corneal infection, recent studies by Roy et al. have shown that TLR4 on epithelial cells also contributes to cytokine responses and host defense during infection.
Using flow cytometry, it was shown that TLR4 is constitutively expressed on the surface of human corneal epithelial cells, however these cells are unresponsive to LPS due to lack of expression of TLR4 co-receptor MD-2 both at RNA or protein level. However,
IFN-γ produced by NK cells during P. aeruginosa corneal infection activates the
Jak2/STAT1 pathway and p-STAT1 binding to IFN-Gamma Activating Sites (GAS) on
the MD-2 promoter, resulting in MD-2 gene and surface expression and LPS
responsiveness (Roy et al., 2011). Additionally, CD14 mediates TLR4 internalization in
human corneal epithelial cells and macrophages, activation of syk and IRF3
phosphorylation and production of CCL5/RANTES and IFN-β, but not IL-8 (Roy et al.,
2013).
Neutrophil and macrophages induced killing of S. pneumoniae is mediated mostly
by opsonization by serotype specific antibody followed by phagocytosis. Mice that lack
Toll-like receptor 2 (TLR2), an initiator of the inflammatory responses upon recognition
of lipoteichoic acid and/or lipoproteins, exhibit delayed pneumococcal clearance (van
25 Figure 1.5: Proposed sequence of events during P. aeruginosa corneal infection
PRRs like TLR4/5 on innate immune cells can recognize LPS and flagella on the bacteria, which initiates downstream signaling cascade to activate the transcription factor NFκB and IRF3 leading to production of inflammatory cytokines and chemokines which recruits neutrophils in the corneal stroma. Of the cytokines produced, IL-1α and IL-1β can be secreted out of the immune cells and on a n autocrine and paracrine fashion binds to IL-1R and mediates positive feedback reaction. (Sun,Y et al, 2010, Journal of Immunology). Reprint permission obtained from the publisher.
26 Rossum et al., 2005). In a murine model of S. pneumoniae corneal infection, TLR2 play a critical role mainly by facilitating neutrophil infiltration and controlling the bacterial load in mice cornea (Tullos et al., 2013). Epithelial cells in culture respond to the
Streptococcus lytic toxin-pneumolysin, by activating the p38 MAPK pathway which results in increased cytokine and chemokine production and recruitment of neutrophils
(Ratner et al., 2006). Some reports also suggest pneumolysin mediated activation of
TLR4 aids host defense during S. pneumoniae infection (Kadioglu et al., 2008; Malley et al., 2003) whereas, some studies show no role of TLR4 (McNeela et al., 2010).
Intracellular receptor NOD2 has also been implicated in mediating the inflammatory responses in astrocytes and microglial cells in response to S. pneumoniae infection (Liu et al., 2010). Nod2 is an intracellular receptor that responds to muramyl dipeptide (MDP) - a break down product of peptidoglycan from gram-positive bacteria. The signaling mechanism downstream of Nod2 is illustrated and described in Figure 1.6 (Strober et al.,
2006).
1.6: Virulence factors of S. pneumoniae
The major virulence factors expressed by S. pneumoniae and their likely role in colonization in hosts are described in Table 1.1 (Kadioglu et al., 2008). Of all the virulence factors expressed by S. pneumoniae, pneumolysin (Ply) is particularly important for activating the inflammasome, the mechanism of which will be described in detail in Chapter 3. Ply is a member of the cholesterol-dependent cytolysin (CDC) family that attacks membranes containing cholesterol and form a ring shaped pores that mediate cell death (Kadioglu et al., 2008; Tilley et al., 2005). Unlike other cholesterol dependent
27 Figure 1.6. NOD2 signaling by peptidoglycan (PGN) from Gram positive bacteria
Peptidoglycan (PGN) potentially activates both cell-surface TLR2 and cytosolic NOD2 through the generation of muramyl dipeptide (MDP). Stimulation of TLR2 triggers association with MyD88 and subsequent activation of NFκB as described before in Figure 1.5B. TLR2 also triggers the activation of receptor-interacting serine/threonine kinase (RICK), which ubiquitylates the IKK subunit of the IKK complex. Concomitant activation of NOD2 by MDP leads to the activation of RICK and NFκB mediated proinflammatory responses (Strober W et al. 2006, Nature Reviews Immunology).
28 Table 1.1: Streptococcus pneumoniae virulence factors and their role in colonization
BgaA, β-galactosidase; CbpA, choline-binding protein A; ChoP, phosphorylcholine; Eno, enolase; Hyl, hyaluronate lyase; IgA, IgA1 protease; IgA1, immunoglobulin A1; LytA, autolysin A; Nan, neuraminidase; PavA, pneumococcal adhesion and virulence A; PiaA, pneumococcal iron acquisition A; PiuA, pneumococcal iron uptake A; Ply, pneumolysin; PsaA,pneumococcal surface antigen A; PspA, pneumococcal surface protein A; StrH, β- N-acetylglucosaminidase (Kadioglu et al, 2008, Nature Reviews Microbiology). Reprint permission obtained from the publisher.
29 cytolysins, Ply lacks a signal peptide for export and is thus released only upon autolysis of the bacteria. Ply is localized primarily in the cell wall compartment of multiple serotypes of S. pneumoniae and has active hemolytic property (Price and Camilli, 2009).
Using cryo-electron microscopy, Tilley et al. showed the structures of the pre-pore
(membrane surface bound) and inserted pore oligomer form. With an oligomer size of up to 44 subunits in the pore, Ply creates a transmembrane channel of 400Å in diameter which consists of 176 β strands (Tilley et al., 2005). The structure of Ply mediated pore formation is illustrated in Figure 1.7 (Tilley et al., 2005). Ply is expressed by virtually all clinical isolates of S. pneumoniae. Interestingly, the amino acid sequence of Ply is well conserved between all natural isolates with few variations only (Kirkham et al., 2006;
Lock et al., 1996). Clinical isolates obtained from 27 patients with S. pneumoniae corneal ulcers all expressed pneumolysin (Karthikeyan et al., 2013). However, strains that express non hemolytic versions of Ply have also been isolated from invasive cases of
Streptococcal infection (Kirkham et al., 2006). Apart from its cytolytic activity, non- hemolytic Ply can promote IFN-γ production by spleen cells (Baba et al., 2002).
1.7: Virulence factors of P. aeruginosa
The gram-negative flagellated bacterium Pseudomonas aeruginosa is a major human pathogen responsible for various diseases like pneumonia, urinary tract infections, cystic fibrosis and others. It is also a major cause of nosocomial infections for burnt- wound patients (Hauser, 2009; Lyczak et al., 2002). P. aeruginosa expresses multiple virulence factors that help them to establish an infection in the host system. Additionally, the ability of the bacteria to form biofilms also facilitates their capability to infect the
30 Figure 1.7: Structure of pneumolysin mediated pore formation
A. B. C.
A. Surface-contoured view of the pre pore on the extracted disk of liposome membrane. The prepore oligomer is 290 Å in diameter and the protein ring is 60 Å thick. B. Equivalent surface view of the 38-mer pore with a surrounding ring of membrane. The pore is 400 Å in diameter and the protein ring is 85 Å thick. C. Raw cryo-EM image of liposomes 5 mins after incubation with a 1:2000 molar ratio of PLY to lipid at 37°C. Examples of pre pore oligomers (inset, solid bars) and a membrane- inserted pore (open rectangle) are labeled. Two vesicles are joined by a pair of back-to-back pre pores (double line). Scale bars 350 Å. (Tilley et al, 2005, Cell). Reprint permission obtained from the publisher.
31 host system. Flagella and type IV pili expressed by P. aeruginosa are the main adhesins that mediate binding to host tissue. Along with lipopolysaccharide, flagella and pili are also highly inflammatory as they can activate pathogen recognition receptors. Once bacteria contact the host epithelia, the Type III secretion system (T3SS) – the major virulence factor of P. aeruginosa can inject effector toxin directly into the host cell.
Several virulence factors are secreted by P. aeruginosa and have varying effects on the host. Several proteases produced by the bacteria, can degrade host complement factors, mucins, and disrupt tight junctions between epithelial leading to dissemination of the bacteria. Lipases and phospholipases can target lipids in the surfactant as well as host cell membranes. Pyocyanin, a blue-green pigment, can interfere with host cell electron transport pathways and redox cycling. Pyoverdine captures Fe3+ to allow for a competitive edge in an environment in which free iron is scarce (Gellatly and Hancock,
2013; Kipnis et al., 2006). Figure 1.8 illustrates multiple virulence factors that are expressed by P. aeruginosa and how they alter functions of host epithelial cells.
Additionally, various PAMPs expressed by the bacteria are recognized by host immune cells to mount a proper inflammatory response against the P. aeruginosa. Recently, we and others have shown that LPS – a cell wall component of this Gram negative bacterium can be recognized by macrophages which then produce chemokines and cytokines to recruit other inflammatory cells to combat infection. Flagella of the bacteria are also recognized by TLR5 to mount similar inflammatory responses (Huang et al., 2006; Sun et al., 2010b).
The major virulence factor expressed by P. aeruginosa is the type III secretion system, which is a needle complex that is utilized by the bacteria to inject exotoxins
32 Figure 1.8: Virulence factors of P. aeruginosa
Virulence strategies utilized by P.aeruginosa to compromise the target host cell: Flagella and type IV pili are the main adhesins, capable of binding to host epithelial gangliosides – asialo GM1 and GM2. Along with lipopolysaccharide, these surface appendages are also highly inflammatory. Once contact with host epithelia has occurred, the T3SS is able to inject exotoxins directly into the host cell. Several proteases are produced, which can degrade host complement factors, mucins, and disrupt tight junctions between epithelium leading to cause dissemination of the bacteria. Lipases and phospholipases can target lipids in the surfactant as well as host cell membranes. Pyocyanin, a blue-green pigment, can interfere with host cell electron transport pathways and redox cycling. Pyoverdine captures 3+ Fe to allow for a competitive edge in an environment in which free iron is scarce. (Hancock et al, 2013, Pathogens and Disease). Reprint permission obtained from the publisher.
33 directly into the host cell. Figure 1.9A demonstrates the structure of T3SS of P.
aeruginosa. The needle-like appendage of the T3SS, which is evolutionarily related to
flagella, permits the translocation of effector proteins from the bacterium into the host
cell through a pore formed in the host cell membrane. Only four effectors have been
identified – ExoY, ExoS, ExoT, and ExoU (Hauser, 2009). The entire T3SS is
transcriptionally controlled by ExsA, a member of the AraC family of transcriptional activators (Yahr and Wolfgang, 2006). Nearly all clinical isolates express one of the two major exotoxins exoU or exoS but very rarely both (Hauser, 2009), while most strains express exoY and exoT. ExoS and ExoT are bifunctional, having N-terminal GTPase- activating protein activity and C-terminal ADP ribosyltransferase (ADPRT) activity as illustrated in Figure 1.9B. ExoU is a phospholipase that can cause rapid death of host eukaryotic cells due to loss of plasma membrane integrity. ExoY is an adenylate cyclase, which elevates the intracellular cAMP levels in cultured mammalian cells and causes actin cytoskeleton reorganization. The RhoGAP domains of ExoS and ExoT stimulate the reorganization of actin cytoskeleton through inactivation of Rho GTPases (RhoA, Rac1,
CDC 42), while the ADP-ribosyltransferase domains ADP-ribosylate distinct host proteins. ExoS can ADP ribosylate variety of host proteins including ezrin, radixin,
moesin, vimentin, cyclophilinA, RAS, Rac1, CDC42, RABs. However ExoT has limited
substrate specificity and ribosylates only phosphoglycerate kinase and CRK I & II (Sun
and Barbieri, 2003). Crks are SH2-SH3 domain containing proteins that function in
integrin mediated phagocytosis and focal adhesion. Thus bacteria that co expresses ExoS
and ExoT can modulate host cell physiology, because the spectrum of host proteins that
ExoS and ExoT target for ADP-ribosylation is unique. Our group has demonstrated that
34 Figure 1.9: Type III secretion system (T3SS) of P. aeruginosa
A.
B.
A. T3SS is functionally divided into 5 components: the needle complex, the translocation apparatus, the regulatory protein, the effector proteins and the chaperons. All these act in concert to create a pore which 2-6 nm in diameter and inject the effector toxins into host cell. B. The modular domains of ExoS, ExoT, ExoU and ExoY. ExoS is a bifunctional toxin that has both GTPase activating protein (GAP) and ADP ribosyl transferase (ADPRT) activity. Arg146 is required for the GAP activity and both Glu379 and Glu381 are required for efficient catalytic activity. ExoT is closely related to ExoS. ExoU is a acid protein that contains a patatin-like domain that is necessary for phospholipase A2 activity. ExoY is a adenylyl cyclase. Residues Lys81, Lys88, Asp212 and Asp214 are required for its activity and are thought to be necessary for interactions between ExoY and ATP. CBD: chaperone binding domain; CF: cofactor binding site; MLD: membrane localization domain; S: secretion signal (Hauser et al, 2009, Nature Rev Microbiology). Reprint permission obtained from the publisher.
35 functional T3SS is required for efficient disease manifestation during P. aeruginosa
corneal infection (Sun et al., 2012b). ADPRT activity of ExoS and ExoT mediates P.
aeruginosa keratitis by causing neutrophil apoptosis and promoting bacterial survival in
C57BL/6 mice. Interestingly, in a recent study, 84% of the clinical isolates from patients
with P. aeruginosa corneal infections expressed ExoS and ExoT rather than ExoU
indicating the importance of these effector proteins in causing corneal ulcers
(Karthikeyan et al., 2013).
1.8: Role of IL-1 family cytokines in inflammation
The interleukin family of cytokines is comprised of 11 proteins encoded by 11
distinct genes in humans and mice (Dunn et al., 2001; Sims et al., 2001). The major
function of the IL-1 family of cytokines is to regulate proinflammatory reactions in
response to tissue injury by PAMPs or DAMPs (Danger associated molecular pattern)
such as uric acid crystals, silica (Dinarello, 2009; Gaestel et al., 2009). The founding
members of this group of cytokines are IL-1α and IL-1β. These two cytokines have only
24% sequence homology but have largely identical biological functions (Dinarello, 1996;
Pizarro and Cominelli, 2007). Both IL-1α and IL-1β also induce expression of their own genes which serves as a positive feedback loop that amplifies IL-1 response in autocrine and paracrine fashion (Dinarello, 1996; Gaestel et al., 2009; Granowitz et al., 1992b;
Hoffmann et al., 2005). Thus IL-1signaling plays an important role in sustaining an inflammatory response. This concept if corroborated by studies where blocking IL-1R1 by using antagonists, like anakinra, demonstrated their major function in human auto inflammatory diseases (Dinarello, 2009). Murine models have shown that mice lacking
36 IL-1R antagonist develop spontaneous rheumatoid arthritis and lethal arthritis (Horai et
al., 2000; Nicklin et al., 2000). Although macrophages and monocytes are a major source
of IL-1 during active inflammation (Dinarello et al., 1987; Granowitz et al., 1992a; Netea
et al., 2009), other cell types like epithelial cells (Hoffmann et al., 2005), fibroblast
(Holzberg et al., 2003) and endothelial cells (Bandman et al., 2002) also produce IL-1α and IL-1β. Whereas IL-1β is secreted and circulates systemically, IL-1α is associated with plasma membrane of the secreting cells and thus acts locally (Dinarello, 1996, 2009;
Kurt-Jones et al., 1985). Moreover, IL-1β is produced mostly by monocytes and macrophages whereas IL-1α expression is more wide-spread and highly expressed by keratinocytes and endothelial cells (Dinarello, 1996, 2009). IL-1α has also been
implicated in T cell priming during contact hypersensitivity and for induction of serum
IgE. IL-1β which can circulate to the brain is more important for induction of fever
(Horai et al., 1998; Nakae et al., 2003a; Nakae et al., 2001). IL-1β also induces gene
expression and synthesis of cyclooxygenase2 (COX2), inducible nitric acid oxide
synthase (iNOS), platelet activating factor and NO. Another important proinflammatory
property of IL-1β is its ability to increase the expression of adhesion molecules such as
intercellular adhesion molecule-1 on mesenchymal cells and vascular cell adhesion
molecule−1 on endothelial cells promoting infiltration of inflammatory and immune
competent cells from the circulation into the extravascular space (Dinarello, 2009).
1.9: IL-1β and immune mediated diseases
Many human diseases are caused by dysregulation of immune system, resulting in
localized tissue damage. IL-1 family members, specifically IL-1β are critical in driving T
37 cell, B cell and innate immune cell responses. Not surprisingly these cytokines are
important contributors to human diseases. IL-1 is crucial for the development of
collagen-induced arthritis (CIA) in mice, mainly because it can drive TH17 cell
differentiation, enhance IL-17 production and mediate joint destruction. Inhibition of IL-
1 not only stops further disease progression but also reverses established disease (Joosten et al., 1999; Nakae et al., 2003b). Gout is an example of a crystal-mediated arthropathy, and there has been recent interest in the role of IL-1 in gout with the discovery that crystals of uric acid activate the inflammasome and lead to IL-1β release (Martinon et al.,
2006). Indeed, pilot clinical studies using anakinra showed considerable benefit for the
treatment of gout (So et al., 2007). In type 2 diabetes, either anakinra or monoclonal Abs
to IL-1β improves glycemic control. Asthma is a syndrome of reduced airway flow and
bronchial remodelling that is driven in part by unregulated and excessive pulmonary
inflammation. In mouse models of airway inflammation both IL-1α and IL-1β are
required for full development of disease. IL-1R antagonist administered at the allergen
challenge phase reduces inflammation and airway hyper responsiveness in murine models
of asthma (Wang et al., 2006). The expression of IL-1β by macrophages in the alveoli
and airway submucosa is increased in patients with asthma. IL-1β expression is also
enhanced in the inflamed intestinal mucosa of patients with inflammatory bowel disease
and inhibition of IL-1 is beneficial in several models of inflammatory bowel disease
(Casini-Raggi et al., 1995). In addition to sterile inflammation, IL-1β regulates
inflammatory responses in multiple microbial infections. For example, mice deficient in
IL-1β were found to be susceptible to infections with E. coli, Shigella, Salmonella, C.
38 albicans, S. aureus, Influenza virus and P. aeruginosa (Karmakar et al., 2012; Netea et al., 2010) indicating that IL-1β is required for host defense during infections.
1.10: Role of inflammasomes in IL-1β processing
IL-1β requires a two-step process before it gets secreted from cells. Initially
Signal1 induces transcription and translation of the precursor IL-1β in response to activation of TLR/IL-1R. NFκB activity downstream of the receptor leads to production of the 31kDa precursor IL-1β (pro form). Additionally, NFκB also induces the expression of NLRP3 and ‘primes’ the cell. This is referred to as Signal 1. However, the pro IL-1β formed in response to Signal 1 cannot be secreted until it is cleaved to a 17KDa mature form. This cleavage is mediated by Signal 2 which requires formation of a multi protein complex called the ‘inflammasome’ which serves as a platform for proteolytic cleavage of IL-1β to its mature secretory form as shown in Figure 1.10 (Anand et al., 2011).
Inflammasomes family is composed of four different kinds of receptor, three of which (NLRP1, NLRP3, NLRC4) belong to NOD like receptor (NLR) family and the
AIM2 inflammasomes is a receptor of the HIN family of proteins (Chen et al., 2009).
Structurally, NLRs are multidomain proteins that contain a C terminal region with multiple LRRs, a central nucleotide domain termed as NACHT domain and an N- terminal effector domain as shown in Figure 1.11. The leucine rich repeat (LRR) domain has a structural motif of 20-30 amino acids with a characteristic pattern rich in the hydrophobic amino acid leucine. The LRR domain has been implicated mainly in ligand sensing and auto regulation of NLRs, although the exact mechanism of how this is achieved is still unclear (Martinon et al., 2009). The NACHT domain is central to all
39 Figure 1.10: Signal 1 and signal 2 together mediate IL-1β processing
Signal 1
Signal 2
IL-1β is a two-step process where IL-1β and NLRP3 expression is induced by NFκB after TLR activation (Signal 1). Formation of the inflammasome complex is initiated after the cell receives various physical or chemical insults. (Signal 2). These include ATP, uric acid crystals, exogenous particles like alum, or pathogen derived pore forming toxins. Activation of inflammasome causes proteolytic cleavage of caspase-1 to its active form which then mediates IL-1β processing and secretion of the cytokine. (Kanneganti et al, 2011, Frontiers in Microbiology). Reprint permission obtained from the publisher.
40 NLRs and is crucial for dATP/ATP- dependent oligomerisation (Leipe et al., 2004).
Thus, the NACHT domain plays a crucial role in forming the high molecular weight oligomeric complex that is characteristic of inflammasome formation (Mariathasan and
Monack, 2007; Martinon et al., 2009). The N terminal effector domain which is essential for downstream signaling is a tripartite structure comprised of a caspase recruitment domain (CARD), a pyrin domain (PYD) or an acidic transactivating domain or baculovirus inhibitor repeat (BIR). The CARD domain is associated with proteins involved in apoptosis, such as caspase-1. The structure of the PYD domain is homologous to the CARD domain and provides homophilic interactions with other PYD containing proteins which are necessary for downstream signaling. Structures of various members of NLRs and HIN family of receptors are illustrated in Figure 1.11.
Formation of the inflammasome complex is initiated after the cell is stimulated with ATP, uric acid crystals, exogenous particles like alum, pathogen derived pore forming toxins or even parts of the pathogen itself. Upon sensing these danger signals, the PYD domain interacts with and recruits the adaptor ASC (apoptosis associated spec like protein containing a CARD domain) via PYD-PYD interaction. The PYD domain of
ASC then serves as a binding platform for CARD containing caspase-1 as shown in
Figure 1.11. Once all these molecules assemble, they form a functional ‘inflammasome’ complex. Caspase-1- which is bound to the complex as an inactive cysteine protease undergoes an autocatalytic processing to active caspase-1which is capable of cleaving pro
IL-1β to bioactive secretory form (Franchi et al., 2009; Gross et al., 2011; Schroder and
Tschopp, 2010).
41 Figure 1.11: Domain organization of representative NOD like receptors
NLRs are characterized by three distinct domains: the ligand-sensing carboxy terminal leucine-rich repeats (LRRs); Oligomerization is mediated by nucleotide-binding domain (NBD); and the effector domain, which consists of pyrin domain (PYD), CARD, or BIR domain. PYD-PYD and CARD-CARD homotypic interactions recruit either the adaptor ASC or the inflammatory caspases. IPAF/NLRC4 recruits caspase-1 directly via CARD-CARD interactions. NLRP1 contains an unique function-to-find domain (FIIND) which mediated inflammasome activation by auto proteolysis. Neuronal apoptosis inhibitory proteins (NAIPs) contain a baculovirus inhibitor of apoptosis repeat (BIR) domain. In absent in melanoma 2 (AIM2), the HIN-2– DNA binding domain is the sensory domain. (Power et al, 2014, Nature Reviews Neuroscience). Reprint permission obtained from the publisher.
42 1.11: Activation of NLRP3 and NLRC4 inflammasomes
The best studied of all are the NLRP3 inflammasomes and the mechanisms of its
activation are intensely debated. Currently, three models of NLRP3 activation that are not
mutually exclusive are proposed in the literature. However, all the proposed models agree
that the cytoplasmic K+ concentration regulates NLRP3 inflammasome activation. The
three models of NLRP3 activation that have been proposed are (Tschopp and Schroder,
2010b) : the channel model, the ROS model and the lysosome disruption model.
The channel model: Extracellular ATP released at the site of cellular injury or necrosis,
can activate the P2X7 ATP-gated ion channel, which then triggers rapid K+ efflux from
the cell (Ferrari et al., 2006). This also causes recruitment and pore formation of the
pannexin1 hemichannel (Kanneganti et al., 2007; Pelegrin and Surprenant, 2006). The
channel model of inflammasome activation proposes P2X7-dependent formation of the
hemichannel that allows extracellular inflammasome activators like bacterial products to
gain access to the cytoplasm and thus interact with and activate NLRP3 directly
(Kanneganti et al., 2007). Pore formation, as described in this model also supports the
NLRP3 activating property of bacterial pore forming toxins. Any pore in the plasma
membrane of macrophages, monocytes or DCs will lead to K+ efflux which has been
shown to be sufficient for activation of NLRP3 inflammasome in vitro (Petrilli et al.,
2007).
The ROS model: NLRP3 activators including ATP and particulate activators such as asbestos and silica trigger the generation of short-lived ROS, and treatment with ROS scavengers blocks NLRP3 activation (Dostert et al., 2008; Petrilli et al., 2007). ROS generation is often accompanied by K+ efflux (Kowaltowski et al., 2009) and ROS
43 production results in NLRP3 inflammasome activation through release of the ROS- sensitive NLRP3 ligand thioredoxin-interacting protein (TXNIP) from its inhibitor thioredoxin (TRX). Furthermore, TXNIP knockout or knockdown impairs caspase1 activation and IL-1β secretion in macrophages following stimulation by NLRP3 agonists
(Dostert et al., 2008; Zhou et al., 2010). Additionally, studies by Tschopp et al. demonstrated direct bindng of TXNIP to NLRP3 inflammasome further strengthening the validity of this model (Zhou et al., 2010).
The lysosome rupture model: This model takes into account the size of inflammasome activators. Large particulate activators (such as alum and silica) cause inefficient clearance of the activating particle following phagocytosis which leads to phagosomal destabilization and lysosome rupture. The ensuing release of the lysosomal protein cathepsin B into the cytoplasm triggers inflammasome activation either directly or indirectly through an uncharacterized pathway (Halle et al., 2008; Hornung et al., 2008).
Recent study showed direct interaction of cathepsin B to NLRP3, although how that leads to NLRP3 activation is still not clear (Bruchard et al., 2013). This model is also supported by the observations that cathepsin B inactivation in human cells by a cathepsin B inhibitor impairs NLRP3 inflammasome activation in response to particulate activators and that lysosome disruption can stimulate NLRP3 activity (Hornung et al., 2008).
Figure 1.12 illustrates these mechanisms of NLRP3 activation (Schroder and Tschopp,
2010; Tschopp and Schroder, 2010b).
The NLRC4/IPAF inflammasome is primarily activated by Gram negative bacteria possessing type III or type IV secretion systems, including Salmonella typhimurium, P. aeruginosa and Shigella flexneri (Schroder and Tschopp, 2010). The
44 Figure 1.12: Mechanisms of NLRP3 activation
The Channel Model: Extracellular ATP causes the opening on P2X7 channels. Additionally, bacterial toxins causes pore formation in plasma membranes. Both of + these lead to loss of intracellular K as well as entry of NLRP3 activators into the cytoplasm.
The Lysosome Disruption Model: Particulate NLRP3 activators can get phagocytosed and once in the cytoplasm they can cause lysosome disruption and release of cathepsin B into the cytoplasm. Bacterial toxins like pneumolysin have also been shown to cause release of cathepsin B. In the cytoplasm, cathepsin B interacts with NLRP3 and activates the inflammasome.
The ROS Model: ROS production results in NLRP3 inflammasome activation through release of the ROS-sensitive NLRP3 ligand thioredoxin-interacting protein (TXNIP) from its inhibitor thioredoxin (TRX). Although the exact mechanism is still not known.
(Schroder et al, 2011, Nature Reviews Immunology). Reprint permission obtained from the publisher.
45 CARD domain in NLRC4 allows it to directly recruit caspase-1 by CARD-CARD homotypic interaction. Cytosolic flagellin and bacterial type III secretion systems are inducers of the NLRC4 inflammasome (Franchi et al., 2007b; Miao et al., 2008; Miao et al., 2010b). The exact mechanism by which NLRC4 senses bacterial flagellin was not clear until recent studies implicated NAIP5 and 6 serve as receptors for recognizing flagellin in NLRC4 activation (Kofoed and Vance, 2011; Zhao et al., 2011). Also, flagellin binding promotes a physical association between NAIP5 and NLRC4, resulting in formation of an oligomeric NLRC4 inflammasome complex and leading to IL-1β production as shown in Figure 1.13. Thus NAIP5 serves as a receptor for bacterial flagellin and NLRC4 acts as an adaptor molecule to transmit the signal downstream to mediate caspase-1 activation and IL-1β processing. Additionally, since the bacterial flagellar apparatus and T3SS are evolutionary and functionally similar, it had been hypothesized that flagellin monomers are accidentally translocated into host cytosol through the T3SS. The NLRC4 inflammasome can also respond to the rod subunit of the type III secretion system, as has been observed with PrgJ of Salmonella SPI-1 in mouse macrophage (Miao et al., 2010b). Cytoplasmic delivery of purified recombinant type III secretion rod or needle can stimulate NLRC4-dependent caspase-1 activation and macrophage inflammation, indicating an inflammasome-stimulating activity of these secretion system components (Zhao et al., 2011). Similarly, T3SS rod proteins are recognized by NAIP2 in mice which directly binds to PrgJ of S. typhimurium or BsaK of
B. thailandensis (Kofoed and Vance, 2011; Zhao et al., 2011) and facilitates complex formation between NAIP2 and NLRC4 as illustrated in Figure 1.13.
46 Figure 1.13: NLRC4 activation by bacteria T3SS and flagellin
IL-1β processing
NAIP 2 and NAIP 5 as a receptor by binding directly to bacterial T3SS proteins and flagellin. NAIPs can then interact with NLRC4 which in turn recruits caspase-1 in this complex. Activated caspase-1 facilitates cleavage of pro IL-1β to mature secretory form. (Shao et al, 2012, Protein Cell). Reprint permission obtained from the publisher.
47 1.12: Inflammasome independent IL-1β processing
Although inflammasomes play an essential role in IL-1β processing in macrophages in culture, inflammasomes as well as caspase-1 are not always required for
IL-1β production in vivo models of microbial or sterile inflammation. In a sterile model of inflammation induced by turpentine or MSU crystals caspase-1 was found to have no role in IL-1β processing (Fantuzzi et al., 1997; Guma et al., 2009; Labow et al., 1997).
Furthermore, caspase-1 seems to be not required for host defense against certain microorganisms like Chlamydia trachomatis, P. aeruginosa and M. tuberculosis. (Cheng et al., 2008; Karmakar et al., 2012; Lu et al., 2000; Mayer-Barber et al., 2010). These observations demonstrate the existence of inflammasome independent activation of IL-
1β. Indeed, subsequent studies identified neutrophil and macrophage derived serine proteases like elastase, PR-3 and cathepsin-G as enzymes that can process pro-IL-1β to the mature bioactive forms (Hazuda et al., 1990). The inflammasome independent activation of proIL-1β is predominant in situations where neutrophils comprise of most of the inflammatory infiltrates rather than macrophages. In P. aeruginosa corneal infection, where neutrophils are the main cells that infiltrate the tissue during acute inflammation and neutrophil derived elastase is primarily responsible for cleaving IL-1β rather than the
NLRC4/caspase-1 inflammasome (Karmakar et al., 2012). In contrast during the chronic phases of inflammation where macrophages are the main components of the infiltrate, inflammasome mediated caspase-1 dependent cleavage of pro IL-1 predominate (Joosten et al., 2009).
In addition to neutrophil derived serine proteases, proteases from microorganisms also cleave pro IL-1β. Recent studies by Deslauriers et al. demonstrated that aspartyl
48 proteases from Candida albicans can cleave pro IL-1 to the mature bioactive form
(Beausejour et al., 1998). Considering the vast array of proteases secreted by microorganisms, it is possible that this mechanism of IL-1β processing might also play an important part in other microbial infections.
1.13: The role of neutrophils in IL-1β processing
Our current understanding of the role of inflammasomes in IL-1β production is
derived almost entirely from studies in macrophages and dendritic cells. Although
neutrophils are the predominant cell types in the tissue during multiple microbial
infections and sterile inflammation, very little information is available on the role of these cells in IL-1β production in vivo. Although neutrophils were long thought to be cells with limited life span and have little biosynthetic capacity, it is now appreciated that
neutrophils produce pro- and anti-inflammatory cytokines, chemokines and lipid
mediators, which can be induced by PRR ligands. This direct the recruitment, activation, and maturation of neutrophils and other immune cells at the site of infection (Cassatella,
1999; Levy et al., 2001; Scapini et al., 2000). Human and murine neutrophils secrete a
variety of proinflammatory cytokines, including TNF, IL-8, and IL-12. Although the
importance of neutrophil-derived cytokines in humans is difficult to ascertain, recent
studies have demonstrated critical roles of murine neutrophil derived proinflammatory
cytokines in vivo. For example, neutrophil-derived IL-1β and TNF drive S. aureus
abscess formation and shock (Cheung et al., 2011; Cho et al., 2012). Neutrophils were
also found to produce IFN-γ in the context of Toxoplasma gondii infection (Sturge et al.,
2013) and IL-17 in the context of fungal infections and autoimmunity. We recently
49 demonstrated that neutrophils not only produce IL-17, but that they express the RORγT transcription factor that was thought to be present exclusively in lymphoid cells (Taylor et al., 2013).
Our collaborators at the Aravind Eye Hospital, India demonstrated that during P. aeruginosa and S. pneumoniae corneal infection, 90% of the cellular infiltrates in the corneal ulcers of patients are comprised of neutrophils. These patients also have elevated expression of IL-1β and other inflammasome components like NLRP3, ASC and the
NLRC4 inflammasome in the cornea compared to normal healthy individuals as shown in
Figure 1.14. These observations indicate that neutrophils are primarily responsible for
IL-1β production in vivo during corneal infections. All these studies prompted us to
investigate the role of neutrophils in vivo in murine models of P. aeruginosa and S.
pneumoniae infections. Data represented in Chapter 2 and Chapter 3 of this dissertation
will describe in depth the mechanism of neutrophil mediated IL-1β processing in vivo.
Given that neutrophils are ubiquitous in the tissue during multiple microbial infections;
these mechanisms might also be true for other disease.
50 Figure 1.14: Cellular composition and protein expression in corneal ulcers of keratitis patients A.
B.
P. aeruginosa S. pneumoniae C.
A. Percent neutrophils and mononuclear cells were determined by counting cells from ten P. aeruginosa and ten S. pneumoniae patients. B. Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from P. aeruginosa or S. pneumoniae infected tissue. Original magnification is x400. C. RNA was extracted from corneal ulcers, reverse transcribed and processed for q-PCR. Data points represent individual patients infected with P. aeruginosa (closed circles) or S. pneumoniae (open circles), and the values presented are the log of relative gene expression [log(RQ)] in relation to uninfected donor corneas. (Karthikeyan et al, 2013, PLoS One).
51
CHAPTER 2
IL-1β processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and Caspase-1
52 ABSTRACT:
Inflammasome and caspase-1 dependent cleavage of IL-1β to the mature, active form is the predominant, but not sole mechanism of IL-1β processing. To investigate the role of IL-1β, IPAF/NLRC4 inflammasome and caspase-1 in bacterial keratitis, which is an important worldwide cause of blindness, mouse corneas were infected with
Pseudomonas aeruginosa strain PAO1 (expressing ExoS, not ExoU), and corneal inflammation and bacterial survival were examined. We found that IL-1β-/- mice had significantly fewer infiltrating neutrophils, and significantly higher CFU than C57BL/6 mice. In contrast, there was no difference in either corneal disease or bacterial survival between caspase-1-/-, IPAF-/- and C57BL/6 mice. In all mouse strains, we found that neutrophils are the major producers of IL-1β during corneal infection, and that although
IL-1β processing by macrophages was completely dependent on caspase-1, neutrophil mediated IL-1β processing in vivo was not impaired in caspase-1-/- or in IPAF1-/- mice. In vitro studies showed that inhibition of neutrophil elastase significantly inhibited IL-1β processing by human and mouse neutrophils. Taken together, these results demonstrate a novel caspase-1 independent mechanism of IL-1β processing by neutrophils during bacterial infection.
53 INTRODUCTION:
IL-1β is a critical player in the host response to microbial infections, including bacterial and fungal infections of the lung, gut, skin and cornea, as disruption of the IL-1β
/IL-1R1 pathway results in uncontrolled microbial growth. Production of functional IL-
1β is a two-step process requiring an initial signal for transcription of the 31kDa pro- form, and a second signal that mediates post-translation cleavage to the mature 17kDa active form that is secreted (reviewed in (Dinarello, 2009). Initial production is mediated by Toll like receptors induced by bacteria, and in macrophages, IL-1β cleavage depends on assembly of a multi-protein inflammasome complex which results in autocatalytic cleavage of Caspase-1, which can then cleave pro-IL-1β in addition to pro-IL-18 and pro-
IL-33 (Dinarello, 2009; Mariathasan et al., 2004; Martinon et al., 2002).
Studies from several groups demonstrated that IL-1β processing in isolated macrophages incubated with Pseudomonas aeruginosa flagellin is dependent on the IPAF
(NLRC4) inflammasome (Franchi et al., 2007b; Miao et al., 2008; Sutterwala et al.,
2007); however, only one study examined the role of IPAF in P. aeruginosa infection in vivo. Using a mouse model of P. aeruginosa lung infection, IL-1β was shown to be produced in alveolar macrophages and IPAF-/- mice had lower IL-1β production and impaired bacterial clearance at early time points (Franchi et al., 2007b). Similarly, earlier studies showed that caspase-1 has an important role in clearance of P. aeruginosa from the cornea during infection (Thakur et al., 2004a; Thakur et al., 2004b).
In the current study we examined the role of IPAF and caspase-1 in a related model of P. aeruginosa corneal infection, which has a similar TLR4/TLR5 dependence as shown in P. aeruginosa lung infections (Sun et al., 2010b). In contrast to the requirement
54 for IPAF and caspase-1 for IL-1β cleavage in isolated macrophages, we demonstrate that
neutrophils are the primary source of the pro- and mature forms of IL-1β in vivo, and that
IL-1β processing in vivo and in vitro was independent of IPAF and caspase-1. Instead,
we demonstrate that IL-1β processing is dependent on neutrophil serine proteases
including elastase and proteinase3. Given the ubiquitous presence of neutrophils in
bacterial infections and their ability to produce IL-1β, it is likely the role of these cells in
IL-1β dependent microbial infections has been underestimated.
MATERIALS AND METHODS:
Source of mice
C57BL/6 mice (6-12 weeks old) were purchased from The Jackson Laboratory
(Bar Harbor, ME). IL1β-/- mice were obtained from Dr. Iwakura (University of Tokyo,
Japan). Caspase1-/- mice were generated by R. Flavell (Yale University). IPAF-/- mice were generated by Millenium Pharmaceuticals. All animals were housed under specific pathogen-free condition in microisolator cages and treated according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research.
Bacterial strain and culture condition
P. aeruginosa strain PAO1 was maintained as a stock in Dr. Arne Rietsch’s
Laboratory (Case Western Reserve University). Bacteria were grown in Brain Heart
Infusion (BHI) media (BD Diagnostics, Sparks, MD) in 37°C shaker incubator to mid log
55 8 phase OD650 – 0.2 (1x10 bacteria/ml). Bacteria were washed and diluted in sterile PBS
(Hyclone, Logan, UT) and concentrated to 1 x 105 bacteria/ 2.5μL.
In vivo murine model of P. aeruginosa keratitis
Mice were anesthetized by intraperitoneal injection of 0.25mL 2,2,2- tribromoethanol in 0.9% saline (1.2%). Central corneas were then scarified with three parallel 1 mm in length abrasions using a 26 gauge needle. A 2.5 µl aliquot containing approximately 105 live P. aeruginosa PAO1 was applied to the scarified right cornea, and
mice remained in this position for 5 min. Sterile PBS was applied to the abraded left
cornea as a control.
Colony forming units (CFU) quantification from infected cornea
At 24 or 48h post infection, mice were euthanized by CO2 asphyxiation and whole
eye was homogenized under sterile condition in 1mL PBS using the Mixer Mill MM300
(Retsch, Inc., Newtown, PA) at 33 Hz for 4 min. Serial log dilutions of the eye
homogenate were prepared in sterile PBS and plated onto BHI agar and the plates were
incubated at 37°C for 18h. The colony forming units were then determined by direct
counting.
Imaging corneal opacity
At the set time points, mice were sacrificed by CO2 asphyxiation and positioned
in a three-point stereotactic mouse restrainer. Corneal opacification (bright field) and
cellular infiltration (eGFP: 488nm) was visualized in the intact eye using a high-
56 resolution stereo fluorescence MZFLIII microscope (Leica Microsystems) and Spot RT
Slider KE camera (Diagnostics Instruments). All images were captured using SpotCam software (RT Slider KE; Diagnostics Instruments).
Generation of chimera mouse
Bone marrow cells were isolated from C57BL/6-GFP and IL-1β-/- mice as
described before (Chinnery et al., 2009). Recipient C57BL/6 mice received 2x600 Gy
doses of full body irradiation 3h apart. Following the second irradiation mice were
reconstituted with 5x106 bone marrow cells by i.v injection in the tail. The mice were
allowed to reconstitute for 4 weeks in germ free conditions after which they were used
for corneal infection.
Immunohistochemistry
Infected eyes were enucleated and fixed in 10% phosphate buffered formalin
(Fisher) for 24h. The eyes were then embedded in paraffin and 5μm sections were cut
from the centre of the cornea. The sections were then deparaffinized by 3 changes in
xylene and 5 changes in ethanol. To stain for neutrophils, the sections were blocked in
1.5% normal rabbit serum in 1X PBS for 20 mins at RT followed by O/N incubation with
primary rat anti-mouse neutrophil antibody (NIMP-R14; Abcam, Cambridge, MA) at
4°C. The sections were washed and incubated 45 min with FITC conjugated rabbit anti-
rat antibody (Vector Laboratories, Burlingame, CA). Neutrophils in the corneal stroma
were visualized by fluorescence microscopy.
57 Western blot analysis
Infected or PBS control corneas were dissected and homogenized in 50μl ice cold
cell lysis buffer (Cell Signaling Technology, Beverly, MA). For peritoneal neutrophils,
treated cells were washed in ice cold PBS and lysed using 1X Cell Lysis Buffer (Cell
Signaling Technologiy). Amount of protein was quantified by standard BCA assay
(Pierce, Rockfort, IL). 30µg of protein was then separated in 12% SDS-PAGE,
transferred to nitrocellulose membrane and incubated with goat primary antibodies to IL-
1β (R&D systems, Minneapolis, MN) and β-actin (Cell Signaling Technology). Proteins
were then detected using HRP-conjugated secondary antibodies and developed with
Supersignal West Femto Maximum Sensitivity Substrate (Pierce, Rockfort, IL).
Flow Cytometry analysis of corneas
Corneas were excised from infected mice and incubated in typeI collagenase
(Sigma) approximately 82U/cornea for 2h at 37°C. Fc blocking was done for 20 mins at
RT with anti-mouse CD16/32 antibody (eBiosciences, San Diego, CA) followed by incubation with Alexa 488-NIMP-R14 (in-house) and PeCy5-F4/80 (eBiosciences) antibodies or isotype control antibodies to detect neutrophils and macrophages respectively. After staining, the cells were washed in 2mL of FACS Buffer (5% FBS in
1X PBS) and fixed in 0.5% PFA for analysis by flow cytometry using Accuri C6 Flow cytometer (BD).
For intracellular staining, collagenase digested corneal cells were treated with
Protein Transport Inhibitor Cocktail (ebioscience, San Diego, CA) O/N at 4ºC. The cells
were then fixed for 20mins in PFA followed by incubation in permeabilization buffer
58 (ebioscience) for 20 min. Intracellular staining was performed using APC-conjugated
anti-mouse IL-1β antibody (ebioscience). Stained cells were washed in FACS buffer and
fixed in 0.5% PFA for analysis by flow cytometry.
Confocal Microscopy
Images were collected using an UltraVIEW VoX spinning disk confocal system
(PerkinElmer, Waltham, MA) mounted on a Leica DMI6000B microscope equipped with a HCX PL APO 100x/1.4 oil immersion objective using a 0.2-micron step size. Images were then imported into Metamorph Image Analysis Software (Molecular Devices Corp.
Downington, PA) where maximum projections were generated from the original stacks and visualized following 2D deconvolution.
Peritoneal macrophage and neutrophil isolation and stimulation
For macrophages, mice were injected with i.p with 3% thioglycolate and 3days
later peritoneal exudates were collected by lavage. The cells were washed with 1X PBS
and red blood cells were lysed using 1X RBC lysis buffer (eBiosciences). Finally the
cells were resuspended in DMEM (Hyclone) and used for stimulation. For neutrophils,
mice were injected i.p with 3% thioglycolate 18h and 3h before collection of peritoneal
exudates. The cells were washed and re suspended in DMEM. The cell suspension was
then overlayed on 90% Percoll (Amersham) and centrifuged at 100,000g for 20min at
4°C. The overlying monocytic cell layer was aspirated and the underlying neutrophil
layer was carefully isolated, washed x3 in PBS and re suspended in DMEM (Hyclone).
Cell purity was assessed by Cytospin/Diff-quick staining followed by light microscopy.
59 This procedure routinely generates > 96% pure neutrophil population and cell purity was
checked for each experiment. Cells were then infected with P. aeruginosa PAO1 at a
50:1 ratio, or were incubated with 500ng/ml ultrapure E. coli LPS (Invivogen, San Diego,
CA).
Isolation of Human neutrophils from peripheral blood
Human neutrophils were isolated from normal donor blood using Ficoll density
centrifugation. This procedure was approved by the Institutional Review Board (IRB) at
CWRU. After informed consent was obtained in accordance with the Declaration of
Helsinki, 20ml of peripheral blood was collected from normal healthy individual.
Heparinized blood was mixed with 20ml of 3% dextran in PBS (Sigma) and incubated for
20 mins at RT to sediment the erythrocytes. The top clear layer containing predominantly
all the blood leukocytes were then transferred to fresh tube and cells were underlaid with
10mL of Ficoll-Paque Plus (GE Healthcare) and centrifuged at 500xg for 20mins at 4ºC.
The top layer containing PBMC was aspirated and neutrophil/RBC pellet was suspended in RBC lysis buffer (ebioscience) for 10 mins on ice. The cells were then washed in PBS
(Hyclone) and resuspended in DMEM (Hyclone). The purity of each cell preparation was confirmed to be more than 97% via Cytospin/Diff Quick staining followed by light microscopy.
Neutrophil Protease activity assay and inhibition
Treated neutrophils were lysed in lysis buffer (50mM Tris, 1% Triton-X100,
0.25% deoxycholate, 150mM NaCl, 1mM EGTA) and hydrolysis of MeOSuc-AAPV-
60 pNA (Calbiochem) was quantified in assay buffer (0.1M HEPES, 0.5M NaCl, 10%
DMSO) at 405nm on Synergy HT multimode microplate reader. For inhibition of neutrophil protease rhSLPI (R&D systems, Minneapolis, MN), Neutrophil elastase inhibitor III and IV (Calbiochem) and nonspecific 3, 4 DCIC (Sigma Aldrich) was used.
Detection of cytokines in the cornea
Infected and PBS treated corneas were excised using a 2 mm trephine and homogenized in 150μL 1X PBS using a Mixer Mill MM300 (Retsch) for 4 min at 33Hz.
For peritoneal neutrophils, cell supernatant was assayed directly. Cytokine levels were determined by sandwich ELISA according to manufacturer’s protocol (R&D systems,
Minneapolis, MN). Absorption was measured at 450 nm on a Synergy HT multimode microplate reader (Biotek Instruments, Inc.).
Statistical Analysis
Statistical analysis was performed using an either an unpaired t test or ANOVA with Tukey post-test analysis (Prism; GraphPad Software). P values less than 0.05 were considered significant.
61 RESULTS:
IL-1β from bone marrow derived cells mediates cellular infiltration to the cornea, corneal disease and bacterial clearance
We and others showed that IL-1β is produced following P. aeruginosa corneal infection with an ExoU expressing strain (Rudner et al., 2000; Sun et al., 2010b). To determine the role of IL-1β on CXC chemokine production and neutrophil recruitment to the cornea, IL-1β-/- and C57BL/6 mice were infected with the ExoS expressing PAO1
strain, and corneas were dissected and chemokines and neutrophils infiltration were
measured by ELISA and flow cytometry respectively. Figure 2.1A shows that IL-1β,
CXCL1 and CXCL2 are elevated compared with trauma (PBS) controls in C57BL/6
corneas within 6h of infection, and that CXCL2, but not CXCL1 levels are lower in IL-
1β-/- corneas. This is consistent with the observation that IL-1β-/- mice had impaired
neutrophil recruitment in the cornea compared with C57BL/6 as shown by NIMP-R14 staining of corneal section (peripheral cornea) after 6h of infection in Figure 2.1B.
Furthermore 24h after infection, the number of neutrophils is significantly elevated in
C57BL/6 corneas, whereas IL-1β-/- corneas showed impaired neutrophil recruitment as
demonstrated in Figure 2.1C. At this time point, the number of macrophages in the
cornea was lower than neutrophils, and there was no significant difference in
macrophages between IL-1β-/- and C57BL/6 mice.
As epithelial cells and fibroblasts can produce IL-1β in addition to myeloid cells
(Hoffmann et al., 2005; Holzberg et al., 2003), we generated chimeric mice to determine
the relative contribution of resident and myeloid cells in IL-1β production following P.
aeruginosa corneal infection. IL-1β-/-, C57BL/6 mice and chimeric mice were infected,
62 Figure 2.1: IL-1β from bone marrow derived cells mediates cellular infiltration to the cornea, corneal disease and bacterial clearance
CXCL2/MIP2 B. -/- A. IL-1β CXCL1/KC C57BL/6 IL-1β 250 8000
200 6000
150 Limbal 4000
(pg/ml) vessel 100 2000 50
0 0 -/- -/- / -/- / PBS C57BL/6 IL-1β PBSC57BL/6 IL-1β PBS C57BL/6IL-1β -/- GFP-B6 IL1β
C. D. -/- C57BL/6 IL-1β C57BL/6 C57BL/6
24h
48h E.
CFU/eye
-/- The role of IL-1β in P. aeruginosa corneal infection. C57BL/6 and IL-1β corneas 5 were infected with 1x10 PAO1. A. After 6 hr, corneas were homogenized, and cytokines were measured by ELISA and B. 5μm corneal sections were stained with NIMP-R14 Ab and visualized by fluorescence microscope to identify migrating + neutrophils in the peripheral cornea (original magnification x20). C. Total F4/80 and + NIMP-R14 cells were quantified by flow cytometry 24 h post infection. Data in (A) and (C) are mean ±SD for 5 mice per group. D. Bright fifield images of infected eyes -/- -/- and E. CFU of C57BL/6, IL-1β , C57BL/6/IL-1β (recipient/donor), and C57BL/6/GFP-B6 (recipient/donor) at 24 and 48 h post infection. Data points represent individual eyes. Results are represenntative of two independent experiments with five mice per group. Reprint permission obtained from the publisher.
63 Figure S2.1: Quantification of corneal opacification -/- GFP-B6 IL-1β A. -/- Naïve C57BL/6 IL-1β C57BL/6 C57BL/6
Corneal opacity
Corneal opacity: 50% 97.21% 57.4% 94.6% 7 7 7 8 Integrated Intensity: 2.5x10 9.6 x10 3.2x10 1.0x10
B.
C.
A. Representative corneas of naive and P. aeruginosa infected mice shown by bright field and false coloro . The % Area of Opacity and Integrated Intensity of Corneal Opacity was derived as described by (Sun et al, 2012). Color visualization of corneal opacity used a scale (in left) ranging from purple (no opacity) to red (maximum opacity) that corresponds to increasing pixel intensity. Areas of glare (red arrows) were demarcated and set to zero to eliminate glare during analysis. To set threshold for no opacity, we used images of naïve corneas that have regions with no disease (indiccated by purple and blue pseudocolors). Mean value obtained from naïve corneas was considered the threshold value. All values above this were included in analysis of % Corneal Opacity (= area over the threshold value / area of circle – glare x100). Total Corneal Opaccity (= integrated pixel intensity above the threshold) was calculated using Metamorph software. B. Percent corneal opacification and C. integrated intensity of opacification was determined from infected corneas. Reprint permission obtained from the publisher.
64 and corneal opacification and bacterial survival were assessed 24 and 48h post infection.
Figure 2.1D shows clinical disease in representative corneas after 24 and 48h post infection. C57BL/6 and C57BL/6-GFP/C57BL/6 (donor/recipient) show central corneal opacification at both time points. Interestingly the corneal opacity in the C57BL/6-
GFP/C57BL/6 chimeric mice exactly coincides with the GFP-bone marrow cells, indicating that bone marrow derived cells are the primary cause of corneal opacification at these time points. In contrast, IL1β-/- and IL1β-/-/C57BL/6 (donor/recipient) corneas have significantly less corneal opacity than C57BL/6 corneas at 24h, coincident with lower neutrophil infiltration shown in previous figure. Quantification of corneal opacity by image analysis software is shown in Supplemental Figure S2.1. To quantify the bacterial burden from these corneas, infected eyes were homogenized in PBS 24 and 48h after infection, and colony forming units (CFU) were counted manually. Figure 2.1E show significantly higher CFU in IL1β-/- cornea compared with C57BL/6 at 48h.
Similarly, CFU from C57BL/6 mice given IL1β-/- bone marrow cells was significantly higher than mice given GFP-C57BL/6 cells.
Taken together, these results indicate that IL-1β production by bone marrow derived cells regulates CXC chemokine production, cellular infiltration to the cornea and bacterial replication.
Neutrophils are the major source of IL-1β in P. aeruginosa corneal infection
Given that neutrophils are the predominant cell type recruited to the cornea during infection, we explored the possibility that neutrophils are a source of IL-1β. C57BL/6 mice were infected as described above, and infiltrating cells were recovered from the
65 cornea following collagenase incubation. Cells were incubated with NIMP-R14 and intracellular IL-1β antibody and examined by flow cytometry. Figure 2.2A shows a distinct population of IL-1β/NIMP-R14 double positive cells (17% total cells), and only
1% total IL-1β positive cells in the cornea were NIMP-R14 negative, indicating that almost all IL-1β positive cells were neutrophils. Gating on the entire NIMP-R14 cells showed that >80% neutrophils in the cornea were IL-1β positive as shown in Figure
2.2B. NIMP-R14 positive cells from infected corneas were also FACS sorted then permeabilized and stained for intracellular IL-1β and examined by confocal microscopy.
Figure 2.2C shows cells with characteristic polymorphonuclear appearance (Hoechst nuclear stain) that are NIMP-R14 positive and express intracellular IL-1β.
To determine the relative contribution of neutrophils in IL-1β production during infection, C57BL/6 mice were injected systemically with anti-NIMP-R14 antibody to deplete neutrophils. We demonstrated previously that this protocol selectively depletes neutrophils systemically and from the cornea following LPS induced inflammation (Lin et al., 2008). To further confirm that depletion of neutrophils did not alter bone marrow macrophage number, we isolated total bone marrow cells from mice treated with anti-
NIMP-R14 and IgG, and stained them with neutrophil and macrophage specific antibody.
Figure 2.2D demonstrates that depletion of bone marrow neutrophils using anti-NIMP-
R14 antibody did not have any effect on the number of macrophages in these mice.
Neutrophil depleted mice were then infected in the cornea with 105 CFU PAO1 and 24h later corneas were dissected and IL-1β was examined by ELISA and by western blot analysis. As shown in Figures 2.2E and 2.2F, mature IL-1β production in the cornea was completely ablated in neutrophil depleted mice.
66 Figure 2.2: Neutrophils are the majoor source of IL-1β in P. aeruginosa corneal infection
A. B. + C. Isotype Control C57BL/6 cornea NIMP- R14 cells Hoescht NIMP-R14 IL-1β 18.9% 0.5% 0.2% 4.2% 17.1% 81.1%
99.1% 0.2% 77.1% 1.0% NIMP- R14 NIMP-R14 IL-1β IL-1β IL-1β NIMP-R14 D. E. G. IgG control treated 78.3% 21.7% 72.3% 27.7% Bright field
(pg/ml) β RF mcherry Control IgG IL-1 H.
CFU/eye 96.7% 3.3% 70.5% 29.5%
F. IgG NIMP-R14 PBS conttrol treated Neutrophil Neutrophil depleted pro IL-1β mat IL-1β β-actin NIMP-R14 F4/80 I. J.
Role of neutrophils in IL-1β production in vivo. C57BL/6 mice were infected with P. aeruginosa and 24 h later corneal cells were stained for intracellular IL-1β and NIMP-R14. A,B. Flow cytometry showing NIMP-R14+ and IL-1β+ cells. C. Stained cells were sorted by flow cytometry and visualized by confocal microscopy (original magnification x100). D-J. Neutrophil depletion by i.p. injection of NIMP-R14Ab 18h prior to corneal infection. D. Total bone marrow cells from IgG and NIMP-R14 treated mice were stained with F4/80 or NIMP-R14 Ab and analyzed by flow cytometry. E. Secreted and F. processed IL-1β from infected corneas after 24h. G. Bright field and red fluorescence image of infected corneas and H. CFU after 24h. I, J. Quantification of corneal opacity. ELISA data are mean ±SD of five mice per group. Western blot shows tthree representative corneas from each group. Data points represent individual eyes. This experiment was repeated twice with similar results. Reprint permission obtained from the publisher.
67 The effect of neutrophil depletion on corneal disease and bacterial survival is
shown in Figures 2.2G-J. In these experiments, corneas were infected with PAO1
expressing mcherry so that the presence of bacteria in the cornea can be visualized by
fluorescence microscopy. Figure 2.2G shows clinical disease in representative corneas at
24h. The top panel shows bright field images of corneal opacification and lower panel
reveals the growth of red fluorescent bacteria in the corresponding corneas (RF mcherry).
Mice receiving IgG show corneal opacification suggesting efficient recruitment of
inflammatory cells in the cornea after infection whereas neutrophil depleted mice have
significantly reduced corneal opacity compared to the untreated group as shown in
Figure 2.2I and 2.2J. Corneas of infected, neutrophil depleted mice show elevated
mcherry expression in the cornea compared with control mice receiving IgG which was
further confirmed by CFU quantification where neutrophil depleted mice showed
significantly higher (3 logs fold) bacterial load than control mice as shown in Figure
2.2H. After 48h, bacterial replication in neutrophil depleted mice resulted in perforation
of the corneas (data not shown). Additionally, in order to understand the relative
contribution of macrophages during P.aeruginosa corneal infection, C57BL/6 mice were infected with PAO1 and 24, 48 and 72hrs after infection; corneas were isolated and stained with F4/80 Ab and intracellular IL-1β. As demonstrated in Supplemental Figure
S2.2, there are very few macrophages in the cornea that are IL-1β producers (6.2%) at
24hr. This is consistent with observation that neutrophils are much more abundant in the tissue during early inflammation and are the major contributor of IL-1β at that time point.
However, at later time points of 48 and 72 hrs, there is more macrophage infiltration in
68 Figure S2.2: Contribution of macrophages in IL-1β production during
P. aeruginosa keratitis
5 C57BL/6 mice were infected with 10 PAOI. Coorneas were isolated 24, 48 and 72h after infection and stained for IL-1β producing macrophages for quantification by flow cytometry. Reprint permission obtained from the publisher.
69 the cornea and they contribute more than 40% of total IL-1β producing cells in the
cornea.
Taken together, the neutrophil depletion studies demonstrate that neutrophils are not only the primary responder cells that limit bacterial growth early during inflammation and protect the cornea from perforating, but are also the major IL-1β producing cells in the cornea.
Neutrophil-mediated IL-1β processing in vivo is caspase-1 and NLRC4
independent
IL-1β processing by macrophages incubated with Gram negative bacteria is
dependent on the activity of the NLRC4/IPAF inflammasome and caspase-1 (Cerretti et
al., 1992; Li et al., 1995; Thornberry et al., 1992). To ascertain the role of caspase-1 and
NLRC4 in isolated neutrophils, peritoneal neutrophils and macrophages were isolated
from C57BL/6, caspase-1-/-, and NLRC4-/- mice and incubated at a 50:1 ratio with P. aeruginosa and IL-1β was quantified by ELISA. IL-1β production by C57BL/6 macrophages was increased in response to P. aeruginosa as shown in Figure 2.3A, and
as reported (Kofoed and Vance, 2011; Miao et al., 2010; Sutterwala et al., 2007), P.
aeruginosa induced IL-1β was ablated in caspase-1-/- macrophages and significantly
reduced in NLRC4-/- macrophages compared with C57BL/6 macrophages. IL-1β
secretion by caspase-1-/- and NLRC4-/- neutrophils was also significantly lower than
C57BL/6 neutrophils, although inhibition was partial as shown in Figure 2.3B and 2.3C.
These findings indicate that IL-1β secretion by neutrophils in vitro is partly dependent on caspase-1 and NLRC4 in contrast to IL-1β processing by macrophages, which is entirely
70 Figure 2.3: Neutrophil mediated IL-1β processing in vivo is caspase-1 and NLRC4 independent
The role of caspase-1 and NLRC4 in IL-1β processing. in vitro: secreted IL-1β from A. -/- -/- peritoneal macrophages and B. neutrophils from C57BL/6, caspase-1 , and NLRC4 mice after 3h of incubation with PAO1. C. Western blot of pro and mature (mat) IL-1β in
-/- C57BL/6 and caspase-1 neutrophils which are stimulated with PBS and PAO1. D-F. IL-
-/- -/- 1β production in C57BL/6, caspase-1 , and NLRC4 corneas 24 h post infection with PAO1 as measured by D. ELISA and E&F. Western blot. G-H. Bacterial CFU was quantified from -/- infected corneas after 24 and 48hrs. I. Caspase-1 mice were infected with PAO1 and corneal cells were stained for NIMP-R14 and intracellular IL-1β and quantified by flow
cytometry. ELISA data are mean ±SD of five mice per group; Western blot shows two representative corneas from each group. Experiments were repeated twice with similar results. Reprint permission obtained from the publisher.
71 caspase-1/NLRC4 dependent. To determine the role of caspase-1 and NLRC4 on IL-1β
production in vivo, C57BL/6 and caspase-1-/- mice were infected with P. aeruginosa
PAO1 as before. After 24h, corneas were dissected and homogenized, and IL-1β was
examined by ELISA and by western blot analysis. Figure 2.3D shows no difference in
total IL-1β production in infected C57BL/6, caspase-1-/- and NLRC4-/- mice. Similarly,
caspase-1-/- and NLRC4-/- corneas had similar levels of the pro-form and mature form of
IL-1β as evident by western blot shown in Figure 2.3E and 2.3F. Also, NIMP-R14
expressing neutrophils were the predominant IL-1β producing cells in infected caspase-1-
/- corneas as shown in Figure 2.3I, which is not significantly different from C57BL/6
neutrophils shown in Figure 2.2.
To determine if the phenotype of infected caspase-1-/- and NLRC4-/- mice is similar to C57BL/6 mice, corneas of these mice were infected with 1x105 PAO1 as
described above, and corneal opacification and bacterial CFU was examined 24 and 48h
post infection. Figure 2.3G and 2.3H shows that there were no differences in CFU
between C57BL/6 and either caspase-1-/- or NLRC4-/- mice.
Taken together, these data indicate that during P. aeruginosa infection, caspase-1 and NLRC4 have either no role or a redundant role in IL-1β processing in the cornea, thereby implying an alternate mechanism for IL-1β processing occurs in vivo.
IL-1β processing by murine and human neutrophils is mediated by serine proteases
As serine proteases have been shown to cleave cytokines and chemokines (Black
et al., 1988; Hazuda et al., 1990; Stehlik, 2009), we next examined if there is a role for
72 neutrophil serine proteases in IL-1β secretion. Elastase activity in human neutrophils
were inhibited by incubating cells for 30 min with the elastase inhibitor NEI III, a broad
serine protease inhibitor 3,4 DCIC, or the cathepsin G inhibitor rhSLPI. Live PAO1 or
flagellin was added as stimulants, and elastase activity and secreted IL-1β were measured
after 3 h of incubation. As shown in Figure 2.4A, elastase activity was not increased following incubation with PAO1 or Flagellin alone, indicating constitutive activity of the serine protease. However, elastase activity was significantly lower in the presence of NEI
III and 3,4 DCIC, whereas there was no difference after incubation with rhSLPI suggesting NEI III and 3,4 DCIC efficiently inhibited neutrophil elastase activity.
Consistent with this, IL-1β secretion was elevated after stimulation with PAO1 or
Flagellin which was severely impaired in presence of NEI III or 3,4 DCIC as shown in
Figure 2.4B. IL-1β production was significantly lower after incubation with the rhSLPI - cathepsin G inhibitor, although the effect was partial. Similarly, IL-1β secretion by P. aeruginosa stimulated mouse neutrophils was significantly lower in the presence of murine elastase inhibitor NEI IV or 3,4 DCIC as shown in Figure 2.4C. We also measure the cytotoxicity of the inhibitors on human neutrophils and found no cytotoxic effects even at the highest concentration of the inhibitors Figure 2.4D. To determine the role of neutrophil elastase in IL-1β processing during P. aeruginosa keratitis, C57BL/6 and
(neutrophil elastase) NE-/- mice corneas were infected with 105 PAO1, and mature IL-1β
in the cornea was examined 24h post infection. As shown in Figure 2.4E, mature IL-1β was lower in NE-/- corneas compared with C57BL/6 corneas, indicating a requirement for
neutrophil elastase in IL-1β processing. NE-/- mice also had significantly higher CFU,
indicating impaired bacterial clearance as demonstrated in Figure 2.4F. Corneal
73 Figure 2.4: IL-1β processing by murine and human neutrophils is mediated
by serine proteases
The role of serine proteases in IL-1β processing. A. Intracellular Neutrophil elastase (NE) activity of human neutrophils after incubation of 30mins with secretory leukocyte protease inhibitor (1μg/ml), NEI III (500μM), or 3,4 DCIC (100μM) and a 3h incubation with live PAO1 or purified flagellin. B. IL-1β production by human neutrophils under the same conditions. C. PAO1 or flagellin induced IL-1β secretion by C57BL/6 peritoneal neutrophils in the presence of serine protease inhibitors. D. LDH release assay from human neutrophils in presence of the inhibitors to quantitate their cytotoxic effects. E. IL-1β production in infected C57BL/6 and
-/- NE mice after 24hr of infection with PAO1. F. Brightfield image of infected eyes and corneal
-/- CFU 24hrs after infection of C57BL/6 and neutrophil elastase (NE) mouse. G. Quantification of corneal opacity G. Data points represents individual corneas and histograms are mean ±SD of five mice/samples per group. Reprint permission obtained from the publisher.
74 opacification was also higher in NE-/- corneas as shown by bright field images of the infected eyes and quantification of corneal opacity as shown in Figure 2.4G. Taken together, these findings demonstrate that neutrophil serine protease - elastase mediates
IL-1β processing and susceptibility to bacterial infection, consistent with impaired bacterial clearance during P. aeruginosa keratitis.
DISCUSSION:
Neutrophils have three main granule types - the primary or azurophilic, secondary or secretory, tertiary or gelatinase granules harboring MMP9. Serine proteases are the major component of primary granules as reviewed in (Pham, 2006). Serine proteases are produced as a pro form and require proteolytic cleavage by cathepsin to generate the active form. Following activation and degranulation, serine proteases remain on the plasma membrane where they continue to have activity.
Neutrophil serine proteases have direct anti-bacterial activity both within the primary granules, and following secretion. Neutrophil elastase was the first serine protease shown to specifically target bacterial virulence factors (Weinrauch et al., 2002), and was subsequently shown to digest the outer membrane porins of E. coli (OmpA)
(Pham, 2006), and P. aeruginosa (OmpF) (Hirche et al., 2008). Neutrophil elastase can also modulate the inflammatory response by cleaving IL-8 and other cytokines and chemokines to their active forms, and can degrade TNF-α and IL-6 (reviewed in (Pham,
2006). Addition of purified elastase or PR1 to purified IL-1β results in cleavage to the active form (Greten et al., 2007). In that study, neutrophil mediated IL-1β cleavage
(induced by LPS) was also shown to be caspase-1 independent. An additional study
75 showed in a mouse model of arthritis that IL-1β processing by neutrophils was independent of caspase-1, but dependent on elastase (Guma et al., 2009).
In contrast, reports on the role of IL-1β on bacterial infections have focused on inflammasomes and caspase-1, even though neutrophils comprise the earliest cellular infiltrate in most bacterial infections. Elegant in vitro studies clearly demonstrate that IL-
1β processing by macrophages in response to Gram-negative bacteria requires inflammasome activation and caspase-1 cleavage. Miao and colleagues also showed that
NLRC4 detects the Type III secretion basal body rod proteins of several bacteria, including P. aeruginosa (PscI) (Miao et al., 2010), and Vance and co-workers showed that Legionella pneumophila flagellin binds directly to the NAIP5 inflammasome, which then activates IPAF (Kofoed and Vance, 2011).
Although there are fewer reports describing IL-1β processing during infection,
Nunez and colleagues showed that NLRC4-/- mice with P. aeruginosa lung infection have reduced IL-1β levels and bacterial clearance (Franchi et al., 2007b), and Modlin et al. showed a role for the common ASC adaptor molecule in Staphylococcus aureus skin infection (Miller et al., 2007), and Hise and colleagues demonstrated an important role for NLRP3 and NLRC4 in oral candidiasis (Hise et al., 2009; Tomalka et al., 2011).
Further, in earlier studies of P. aeruginosa corneal infection, Hazlett and co-workers showed that caspase-1 inhibition or caspase-1-/- mice had less corneal disease and a lower neutrophil infiltrate (Thakur et al., 2004a; Thakur et al., 2004b).
In the current model of acute bacterial infection, we show that neutrophils rather than macrophages are the predominant source of IL-1β, and that production of mature IL-
1β in vivo is independent of caspase-1 and NLRC4, which also have either no role or a
76 redundant role in P. aeruginosa disease as we found no difference in IL-1β production, corneal opacity or CFU in caspase-1-/- and NLRC4-/- corneas compared with C57BL/6.
Although NLRC4 can be activated in a caspase-1 independent responses (Pereira et al.,
2011), we also found no role for NLRC4 in vivo.
In vitro, we found that neutrophils secrete ~3-fold more IL-1β than macrophages, and that IL-1β secretion by neutrophils in vitro is partially dependent on caspase-1, as caspase-1-/- neutrophils produce less mature IL-1β than C57BL/6 neutrophils. However, we also show that IL-1β cleavage by murine and human neutrophils is dependent on the activity of serine proteases, including elastase and proteinase 3 as summarized in Figure
2.5. A recent study by Mankan et al. showed that neutrophils are the predominant source of IL-1β in mouse bone marrow and human PBMCs; however, they reported that neutrophil mediated production of IL-1β in response to LPS and nigericin (as the second signal) was completely dependent on NLRP3 and caspase-1, and independent of serine proteases (Mankan et al., 2011). Although the difference between their in vitro findings and ours has yet to be determined, it is clear that in vivo, neutrophil production of IL-1β is independent of caspase-1. Sher and colleagues showed that IL-1β processing during chronic Mycobacteria tuberculosis infection is also independent of caspase-1, and that
IL-1β was produced by dendritic cells and a unique population of inflammatory monocyte-macrophages (Mayer-Barber et al., 2011; Mayer-Barber et al., 2010).
However, in acute and in early stages of bacterial infections, neutrophils are generally the first cells to arrive in large numbers. Given that neutrophils produce multiple cytokines and chemokines, including IL-1β [reviewed in (Mantovani et al., 2011)], it is likely that in addition to their phagocytic and anti-bacterial effects, neutrophils also regulate the
77 Figure 2.5: Proposed Model
Neutrophil mediated IL-1β production and processing in P. aeruginosa keratitis.
1&2. P. a eruginosa enters the corneal stroma following a breach in the corneal epithelium. LPS and flagellin of P. aeruginosa activates TLR4 and TLR5 respectively on the corneal epithelial cells as well as on resident macrophages. NLRC4/caspase-1 inflammasome mediates processing and seccretion of IIL-1β which then recruits neutrophils in the cornea by enhancing expression of adheesion molecules on vascular
endothelial cells. 3. Once neutrophils enter the cornea, activation of TLR4 and TLR5 mediates production of proIL-1β by neutrophils which is further cleaved to mature form by serine proteases, including elastase that is in primary granules of the neutrophils. 4.
Neutrophil mediated IL-1β serves as an important contributor in amplifying self- perpetuating inflammatory response.
78 inflammatory response at this stage of infection. Results from our previous work support this concept, as we showed that P. aeruginosa LPS and flagellin activate TLR4 and
TLR5, respectively, elevating IL-1α, IL-1β and CXC chemokines levels in the cornea, which mediate neutrophil recruitment and bacterial clearance (Sun et al., 2010b). We also found that P. aeruginosa survival in neutrophils is dependent on the ADP ribosyltransferase activity of ExoS and ExoT of the Type III secretion system (Sun et al.,
2012a); however, in contrast to studies on the role of type III secretion on NLRC4 activation in macrophages, type III secretion mutants are rapidly killed and therefore do not induce IL-1β production in the cornea. Results from the current study add to our understanding of P. aeruginosa infections by demonstrating an essential role for myeloid cell derived IL-1β in regulating chemokine production and neutrophil recruitment, and thereby limiting corneal opacification and loss of visual function.
79
CHAPTER 3
Neutrophil mediated IL-1β processing in
Streptococcus pneumonia corneal infection is dependent on the NLRP3/ASC inflammasome and
Caspase-1 activation
80 ABSTRACT:
Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the current study, we investigated the role of neutrophils in IL-1β processing using a murine model of Streptococcus pneumoniae
corneal infection. We demonstrated that neutrophils are the predominant IL-1β producing
cells in the cornea, and that production requires expression of NLRP3, ASC and caspase-
1. We also visualized caspase-1 specks in neutrophils in infected corneas. Furthermore,
using highly purified murine and human peripheral blood neutrophils, we show that
pneumolysin is required for NLRP3/caspase-1 activation and IL-1β processing. Ply also
mediates K+ efflux, which is not a consequence of neutrophil pyroptosis, but instead
serves as an early second signal which acts upstream of NLRP3/caspase-1 activation. In
contrast, there was no role for lysosomal destabilization or neutrophil elastase in
pneumolysin mediated IL-1β processing in neutrophils. Together, these findings clearly
indicate that neutrophils are a major source of IL-1β during S. pneumoniae infection and
identify the underlying mechanisms of IL-1β processing. This function of neutrophils is
likely occurring in other multiple causes of acute inflammation.
81 INTRODUCTION:
Neutrophils are broadly recognized as the major effector cells during inflammatory responses, and as the primary cells involved in clearing pathogenic bacteria and fungi. However, neutrophils are becoming widely recognized as a source of pro- inflammatory and regulator cytokines (Mantovani et al., 2011). A recent report showed that neutrophils are an important source of IFN-γ in Toxoplasma gondii infection (Sturge et al., 2013). Further, we recently identified a sub-population of human and murine neutrophils that both produce and respond to IL-17A, which results in increased production of reactive oxygen species and enhanced killing of Aspergillus fumigatus
(Taylor et al., 2014).
Production of bioactive IL-1β requires caspase-1 dependent cleavage of the 34kD pro-form to a 17kD processed form that can then be secreted (Latz et al., 2013). Further, enzymatically active caspase-1 is generated from a pro-form by autocatalysis following assembly of a large, multi-protein inflammasome complex (Latz et al., 2013). However, most of our understanding of this process comes from studies on monocytes, macrophages and dendritic cells, and the role of neutrophils in IL-1β production and the mechanisms of IL-1β processing are not well understood.
In the current study, we demonstrate that neutrophils are the predominant source of IL-1β in a murine model of acute S. pneumoniae corneal infection, and that IL-1β processing is dependent on expression of the Nod-like receptor protein 3 (NLRP3) inflammasome, the adaptor molecule apoptosis-associated speck like protein containing a caspase recruitment domain (ASC), and active caspase-1, which are detected as large, intracellular specks in neutrophils during infection. Further, using highly purified human
82 neutrophils, we show that S. pneumoniae – induced secretion of mature IL-1β is dependent on expression of bioactive pneumolysin, and that the mechanism requires a rapid efflux of K+ ions that precedes NLRP3 inflammasome activation and pyroptosis.
Finally, we demonstrate that pneumolysin induced NLRP3 activation in neutrophils do not require lysosomal disruption/cathepsin B release and also neutrophil elastase has no role in IL-1β processing during S. pneumoniae infection.
MATERIALS AND METHODS:
Source of mice
C57BL/6 mice (6-10 weeks old) were from The Jackson Laboratory (Bar Harbor,
ME), IL-1β-/- mice were obtained from Dr. Iwakura (University of Tokyo, Japan).
Caspase-1-/- mice were generated by Richard Flavell (Yale University, CT). NLRP3-/- and ASC-/- mice were generated by Millennium Pharmaceuticals (Cambridge, MA). All knockout mice were on C57BL/6 background. All animals were housed in pathogen free conditions in microisolator cages and were treated according to institutional guidelines after approval by the Case Western Reserve University IACUC.
Bacterial Strains and growth conditions
Wild type encapsulated Streptococcus pneumoniae TIGR4 (serotype IV) was used in this study. To generate Δply deletion mutant, the Ply coding region of TIGR4 was replaced with an antibiotic resistance cassette encoding chloramphenicol acetyltransferase (cat) via transformation of competent cells with a linear splicing by
83 overlap extension (SOE) PCR product. Approximately 1 kb of flanking sequence on either side of ply was amplified from TIGR4 genomic DNA using primer sets SP_1923
F1/SP_1923 R1 and SP_1923 F2/SP_1923 R2 (Table 3.1) for upstream and downstream sequences, respectively. The cat cassette was PCR amplified from pEVP3 using primer set Fcat/Rcat and all three products were spliced together by SOE using primers SP_1923
F1 and SP_1923 R2. Competent cells of TIGR4 were transformed with the resulting amplicon and chloramphenicol-resistant colonies were selected on Fluka Blood Agar
Base No. 2 supplemented with 5% defibrinated whole sheep blood (Sigma Aldrich) containing chloramphenicol (4μg/ml). PCR and DNA sequencing were carried out to confirm replacement of ply with the cat cassette. Both WT and Δply strains were routinely grown in Todd-Hewitt Broth (Neogen) supplemented with 0.5% yeast extract in
37ºC and 5% CO2 incubator. Bacteria were grown to mid exponential phase such that there is 108CFU/ml and diluted in sterile PBS to desired concentrations for corneal infection or in vitro stimulation. To generate heat killed bacteria (hkSP), TIGR4 was diluted in sterile PBS and heated at 95ºC for 5mins. Viability was confirmed by plating bacteria in trypticase soy agar plate containing 5% defibrinated sheep blood (BD).
Recombinant pneumolysin (Ply) was expressed in E. coli and purified as described (Gilbert et al., 1998; McNeela et al., 2010). Unless otherwise stated the specific haemolytic activity of Ply was 100,000 HU/mg. The toxin was passed three times through an EndoTrap endotoxin removal column (Profos AG, Germany) after which LPS was undetectable using the PyroGene Recombinant Factor C assay (Lonza; detection limit 0.01 EU/ml).
84
Table 3.1: Primer sequences for generation of Δply strain
85 Murine model of Streptococcus pneumoniae corneal infection
S. pneumoniae TIGR4 was grown in Todd-Hewitt Broth until mid-exponential phase (108CFU/ml). Bacteria were washed with sterile PBS and further diluted such that there are 105 CFU/ml in a 2μl volume. Subsequently, mice were anesthetized with 1.2%
2,2,2 tribromoethanol. The corneal epithelium of the mice was abraded using a 26-gauge needle through which a 2μl injection containing 105 TIGR4 was released into the corneal stroma using a 33-gauge Hamilton syringe and mice were allowed to recover from anesthesia. Infected mice were anesthetized after 24hrs and positioned in a three point stereotactic mouse restrainer to monitor corneal opacification using a stereomicroscope and Spot RT Slider KE camera (Diagnostics Instruments). Images were uploaded into
Metamorph Image analysis software (Molecular Devices Corp) and percent and total opacity was quantified as described in Figure S3.1.
Quantification of Streptococcal colony forming units (CFU)
For assessment of bacterial viability, infected mice were sacrificed by CO2 asphyxiation and whole eyes were isolated and homogenized in 1ml sterile PBS in Mixer
Mill MM300 (Retsch) at 33Hz for 3mins. Serial log dilutions of the bacteria were plated on blood agar plates, incubated in CO2 incubator at 37ºC for 18h and colony numbers were counted manually.
Immunohistochemistry of cornea sections
Infected eyes were enucleated and fixed overnight in 10% formalin buffer (Fisher
Scientific). 5μm section was cut from the center of the paraffin embedded cornea and
86 stained with Gill’s hematoxylin following deparaffinization.To stain for infiltrating neutrophils, fixed sections were stained with rat anti-mouse NIMP R14 IgG (in-house) and counter stained with Alexa-488 goat anti-rat (Invitrogen). Slides were mounted with
DAPI containing mounting media (Vector Laboratories) and imaged by fluorescence microscopy.
Flow Cytometry of corneas and Image Stream analysis
Corneas from infected mice were excised using a surgical microscissor and incubated in type I collagenase (Sigma) at 82U/cornea for 2h at 37°C. The cell suspension was then passed through 30μm filter to remove any undigested tissue. Fc receptors were blocked for 20 min at RT with anti-mouse CD16/32 antibody
(eBiosciences) followed by incubation with Alexa488-NIMP-R14 (in-house) and PE-
F4/80 (eBiosciences) antibodies to detect neutrophils and macrophages, respectively.
After staining, cells were washed in 2mL of FACS Buffer (1% FBS in PBS) and fixed in
0.5% PFA for analysis by flow cytometry using Accuri C6 Flow cytometer (Becton
Dickinson).
For intracellular cytokine staining, corneal cells were incubated at 4ºC overnight with 1X Protein Transport Inhibitor Cocktail (eBiosciences), fixed in 4% paraformaldehyde and permeabilized in 1X Perm Buffer (eBiosciences) for 10mins. Cells were then stained with APC-IL-1β antibody (eBiosciences), washed in FACS buffer and fixed in 0.5% PFA for analysis by flow cytometry or Multispectral Imaging Flow
Cytometry (Image stream-100, Amnis).
87 Inhibitors, recombinant protein and other reagents
NFκB inhibitor – JSH23 (Sigma Aldrich) and pJNK/AP-1 inhibitor – SP600125
(Tocris Bioscience) were dissolved in DMSO and used at assay dependent concentrations. Caspase-1 inhibitor YVAD (Bachem) and pan caspase inhibitor ZVAD
(ApexBio) were dissolved in DMSO and used at indicated concentration. Bafilomycin A
(LC laboratories), CA-074-Me (EMD Chemicals) and ZFA (Bachem) was dissolved according to manufacturer’s protocol. Nigericin (Calbiochem) was used at 10μM concentration. High potassium medium for neutrophils had the following composition:
130mM KCl, 1.5mM CaCl2, 1mM MgCl2, 25mM HEPES, 5mM glucose and 0.1% BSA at pH 7.4. For low potassium medium, we add 5mM KCl keeping other components the same.
Western blot analysis
To perform western blot from infected corneas, corneas were excised from the eyes using a microscissor. Any associated iris was removed and clean corneas were homogenized in 1X cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail. For in vitro experiment, 4x106 cells were lysed in cell lysis buffer after appropriate stimulation. 20-30μg of protein were fractionated in 12% SDS-
PAGE, transferred into nitrocellulose membrane and incubated with primary antibodies – mouse IL-1β (R&D Systems), mouse NLRP3 and ASC (Adipogen), human NLRP3
(Sigma Aldrich) and ASC (Adipogen) , mouse Caspase-1p10 (Santa Cruz), mouse pJNK
(T183/Y185) and total JNK (Cell Signaling Technology) and β actin (Sigma Aldrich).
88 Reactivity was determined using HRP-conjugated secondary antibodies (Santa Cruz) and developed with Supersignal West Femto Maximum Sensitivity Substrate (Pierce).
Isolation of human and mouse bone marrow neutrophils
Human neutrophils were isolated from the peripheral blood of healthy volunteers following informed consent as approved by the Institutional Review Board of University
Hospitals of Cleveland. Heparinized blood was incubated with 3% dextran in PBS
(Sigma Aldrich) for 20mins at RT. The top clear layer containing leukocytes was transferred to a fresh tube and the cells were underlaid with 10ml of Ficoll Paque Plus
(GE Healthcare). The cells were centrifuged at 500xg for 20mins. The overlying plasma and PBMC layer was aspirated and the neutrophil/RBC pellets were suspended in 1X
RBC lysis buffer (ebiosciences). Following this, cells were washed in sterile PBS and resuspended in RPMI+L-glutamine media (Hyclone) supplemented with 2% FBS
(Mediatech). Cell purity was determined for each experiment by Wright-Giemsa (Sigma
Aldrich) and routinely yielded >97% pure neutrophils.
For mouse bone marrow neutrophils, mice were sacrificed by CO2 asphyxiation and total bone marrow cells were collected from tibias and femurs. Neutrophils were isolated from the total bone marrow cells by negative selection using EasySep™ Mouse
Neutrophil Enrichment Kit (Stem Cell). After magnetic separation, cells were washed with PBS and resuspended in RPMI+L-glutamine with 1% FBS. This procedure routinely yielded >94% pure neutrophils as quantified by Wright-Giemsa stain.
89 Caspase-1 activation assay
Active caspase-1 was quantified by using FLICA-660 (660-YVAD-FMK) far red detection kit (Immunochemistry Technologies) according to manufacturer’s guidelines.
Stained cells were either quantified by flow cytometry (using unstained cells to set the gate) or are visualized by confocal microscopy for active caspase-1 oligomerization.
Immunofluorescence staining
Stimulated human and mouse neutrophils were fixed with 4% PFA (Fisher) at RT for 15mins and permeabilized with 0.1% Triton-X100 for 10mins. The cells were then blocked in 10% goat or rabbit serum (Vector Laboratories) in PBS for 1h at 22ºC followed by staining with goat anti-mouse NLRP3 (Abcam) or rabbit anti-mouse ASC
(Adipogen) for 1h at 37ºC. Cells were then washed 3X in PBS and counter stained with
Texas red rabbit anti-goat IgG (Vector Laboratories) and Alexa488 goat-anti mouse IgG
(Invitrogen) for 40 mins at RT. The cells were washed 3X in PBS and stained with DAPI before visualization by confocal microscopy.
Confocal Microscopy
Images were collected using UltraVIEW VoX spinning disk confocal system
(Perkin Elmer) mounted on a Leica DMI6000B microscope equipped with a HCX PL
APO 100 x/1.4 oil immersion objectives using a 0.2μm step size. Images were then imported into Metamorph Image Analysis Software (Molecular Devices Corp) where maximum projections were generated from the original stacks and visualized following
2D deconvolution.
90 Atomic Absorbance Spectroscopy
After stimulation of mouse or human neutrophils, the extracellular medium was aspirated and the cells were rapidly washed in potassium-free isotonic buffer (135mM sodium gluconate, 1.5mM CaCl2, 1mM MgCl2, and 25mM HEPES). The washed cell
+ pellets were then extracted into 1ml of 10% HNO3. K content in the nitric acid extracts was quantified by atomic absorbance spectrometry (Agilent 55B AA). Triplicate samples were run for all test conditions in each experiment.
Detection of cytokines by ELISA
For analysis cytokine analysis, cell culture supernatants were assayed directly.
Half well cytokine assays were performed using Duoset ELISA assay kit for IL-1β and
CXCL8/IL-8 according to manufacturer’s protocol (R and D Systems).
LDH release assay
After stimulation of neutrophils, supernatant was collected and LDH release was quantified using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s instructions. Percentage cytotoxity was calculated based on LDH release in total cell lysate.
Statistical Analysis
Student t test or ANOVA with Tukey post hoc analysis (Prism, Graphpad
Software) were used as indicated in the figure legends. A P value equal or less than 0.05 was considered significant.
91 RESULTS:
IL-1β, NLRP3 and ASC regulate S. pneumoniae corneal infection
The normal avascular mammalian cornea has resident macrophages and dendritic cells, whereas neutrophils are only detected following an infectious or inflammatory event when they are recruited from peripheral, limbal vessels together with infiltrating macrophages and dendritic cells (Hamrah et al., 2003b; Knickelbein et al., 2009). To examine the role of IL-1β and the NLRP3/ASC inflammasome in S. pneumoniae keratitis, corneas of C57BL/6, IL-1β-/-, NLRP3-/- and ASC-/- mice were infected by intrastromal injection with 1x105 CFU of the S. pneumoniae strain TIGR4, which expresses pneumolysin. After 24h, corneal opacity and bacterial growth were examined, and the number of infiltrating neutrophils and macrophages was quantified by flow cytometry using NIMP-R14 that recognizes Ly6G on neutrophils, and the F4/80 antibody that primarily binds to macrophages.
Representative bright field images of S. pneumoniae infected corneas showed lower central corneal opacity and increased number of colony forming units (CFU) in IL-1β-/-
(Figure 3.1A) and in NLRP3-/- and ASC-/- mice (Figure 3.1B) compared with C57BL/6 mice at 24h post-infection (quantification of corneal opacity in Supplementary Figure
S3.1). Conversely, there was significantly higher CFU in IL-1β-/-, NLRP3-/- and ASC-/- mice corneas compared with C57BL/6 corneas (Figure 3.1A,B). After 48h, infected corneas of IL-1β-/-, NLRP3-/- and ASC-/- mice were found to perforate as a result of bacterial growth, whereas C57BL/6 corneas remained intact (data not shown).
Quantification of cells in the cornea by flow cytometry showed significantly lower
92 Figure 3.1: IL-1β, NLRP3 and ASC regulate S. pneumoniae corneal infection
-/- A. B. -/- -/- C57BL/6 IL-1β C57BL/6 NLRP3 ASC C.
C.
D. E.
IL-1β, NLRP3 and ASC are required for bacterial clearance during S. pneumoniae infection. Corneas of C57BL/6, IL-1β-/-, NLRP3-/- and ASC-/- mice were infected with live S. pneumoniae TIGR4. A, B. Representative corneas 24h post-infection (original magnification x20) and CFU after 2 h (starting inoculum) and 24 h post infection; data points represent individual corneas. C, D. Total NIMP-R14+ neutrophils and F4/80+ macrophages in infected corneas quantified by flow cytometry. E. Intracellular IL-1β in NIMP-R14+ and F4/80+ cells from infected corneas after 24h. Gates were determined based on an isotype control. Results are representative of three independent experiments with at least five mice per group.
93 Figure S3.1: Quantification of corneal opacification
A. Representative corneas of naive
-/- and S. pneumoniae infected Naïve C57BL/6 IL-1β A. C57BL/6 and gene knockout mice
shown by bright field and false color
Corneal using Metamorph Imaging software opacity (Molecular Devices, Downington Corneal opacity: 59.260% 23% 7 7 Integrated Intensity: 6.2x10 2.5 x10 PA). The Percent Area of Opacity
B. and the Integrated Intensity of Corneal Opacity was derived as described by (Sun et al, 2012). Color
visualization of corneal opacity used a scale (shown on left) ranging from
purple (no opacity) to red (maximum C. opacity) that corresponds to increasing pixel intensity level. Areas of glare (red arrows) were also demarcated and set to zero, thereby
eliminating any glare during analysis. To set the threshold for no
opacity (transparency), we used
images of naïve corneas imaged at the same time, that have regions with no apparent disease (indicated by purple and blue pseudocolors). The mean value obtained from at least four naïve corneas was considered the threshold value. All values above this threshold were included in analysis of Percent Corneal Opacity (= area over the threshold value / area of circle – glare x100). Total Corneal Opacity (= integrated pixel intensity above the threshold) was calculated using Metamorph software. B, C. Percent corneal opacification and. integrated intensity of opacification of C57BL/6 and knockout mice was determined from infected corneas at 24h post infection.
94 neutrophil numbers in infected IL-1β-/- NLRP3-/- and ASC-/- compared with C57BL/6 corneas, whereas there was no significant difference in the number of macrophages in the corneal stroma (Figure 3.1C, D). The increased number of neutrophils in the corneal stroma of C57BL/6 compared with IL-1β-/- NLRP3-/- and ASC-/- mice was also detected by immunohistochemistry (Supplementary Figure S3.2). Cumulatively, these data are consistent with our earlier findings that cellular infiltration is the major cause of corneal opacity during bacterial keratitis (Sun et al., 2010b), and also demonstrate that IL-1β,
NLRP3 and ASC are required for clearance of S. pneumoniae.
To identify the cellular source of IL-1β in S. pneumoniae infection, total cells from infected C57BL/6 corneas were stained with NIMP-R14, F4/80 and intracellular IL-
1β, and examined by flow cytometry. As shown in Figure 3.1E, a distinct population of
IL-1β producing neutrophils was present, comprising more that 20% of the total corneal cells. Further, 85% of total IL-1β producing cells in the cornea were NIMP-R14+ neutrophils as shown in the histogram (top panel) compared with F4/80+ macrophages, which accounted for less than 15% of the total IL-1β producing cells (lower panel).
These data show that neutrophils are the primary responders during S. pneumoniae corneal infection and are also the major source of IL-1β.
NLRP3 and ASC mediated IL-1β processing in the cornea is dependent on active caspase-1 produced by neutrophils
To assess whether the NLRP3/ASC inflammasome is required for IL-1β processing in vivo, corneas of C57BL/6, NLRP3-/- and ASC-/- mice were infected with S. pneumoniae as described above, and after 24h, corneas were homogenized and processed
95 Figure S3.2: Histology of corneal sections
A. H&E NIMP-R14
Epi
C57BL/6 Stroma
- / - β 1 - IL
B. H&E NIMP-R14
C57BL/6
- / - 3 NLRP
- / - ASC
-/- -/- -/- C57BL/6, IL-1β , NLRP3 , ASC mice were infected with S. pneumoniae and 24h later eyes were isolated, fixed in paraformaldehyde and embedded in paraffin sections. 5μm corneal sections were stained with H&E (Hematoxylin and eosin) and NIMP-R14 antibody to detect infiltrating neutrophils
96 for western blot analysis. Figure 3.2A shows the p10 caspase-1 subunit and the processed p17 IL-1β bands in infected C57BL/6 corneas, whereas these bands were barely detected in NLRP3-/- and ASC-/- corneas. Further, NLRP3 was detected only after
S. pneumoniae infection, whereas ASC was present in the trauma controls, indicating that
NLRP3 expression is induced following infection, whereas ASC is expressed constitutively in resident cells in the cornea.
To determine if neutrophils are producing active caspase-1, S. pneumoniae infected C57BL/6 corneas were digested with collagenase, and total corneal cells were stained with NIMP-R14 and FLICA660-YVAD, a fluorescent peptide that binds to active caspase-1. We found that > 30% of total cells in the cornea were FLICA-660 YVAD positive neutrophils (Figure 3.2B, upper panel), and that of the total active caspase-1 producing cells in the cornea, over 75% were NIMP-R14+ neutrophils (Figure 3.2B, lower panel). Active caspase-1 in neutrophils was also detected as speck-like aggregates as shown by multispectral imaging flow cytometry and confocal microscopy (Figure 3.2
C, D).
Together, these data indicate that during acute S. pneumoniae corneal infection, neutrophils are the major cell type in the cornea that contributes to NLRP3/ASC- dependent caspase-1 activation and IL-1β processing.
Active caspase-1 induction in murine neutrophils is dependent on NLRP3 and
ASC, and requires active pneumolysin
To determine whether live S. pneumoniae can directly activate caspase-1 in neutrophils, bone marrow neutrophils from C57BL/6 mice were incubated for 3h with
97 Figure 3.2: NLRP3 and ASC mediated IL-1β processing in the cornea is dependent
on active caspase-1 produced by neutrophils
A. -/- -/- B. C57BL/6 NLRP3 ASC NIMP-R14/FLICA-660 PBS Pro 6.8% 30.6% 24.5% 75.5% Casp-1
p10
R14 - IL-1β p17 54% 8.6%
pro IL-1β NIMP FLICA NLRP3 NIMP-R14 660 ASC βactin
DAPI/NIMP-R14 DAPI/FLICA Merged C. D. Bright Field NIMP-R14 DAPI FLICA-660
Role of NLRP3 and ASC in producing active caspase-1 by neutrophils in vivo. Western blot of corneas from C57BL/6, NLRP3-/- and ASC-/- mice 24h after S. pneumoniae infection. Mature forms of Caspase 1 (p10) and IL-1β (p17) are indicated by arrow heads. B. Total cells from infected C57BL/6 corneas were incubated with NIMP- R14 and FLICA-660-YVAD to detect active caspase-1. C, D. NIMP-R14 (green) and FLICA-660-YVAD (red) cells were detected by multi spectral imaging flow cytometry (MIFC) (C), and by confocal microscopy (D). Original magnification for C is x60 and for D is x100. These experiments were repeated twice with similar results.
98 heat-killed S. pneumoniae (hkSP, Signal 1), followed by 2h stimulation with live TIGR4
S. pneumoniae or with an isogenic mutant deleted for the gene encoding Ply (∆ply)
(Signal 2), and active caspase-1 was quantified using FLICA660-YVAD.
Confocal microscopy showed fluorescent speck-like structures in neutrophils following incubation with S. pneumoniae TIGR4 strain expressing pneumolysin, but not with the Δply pneumolysin mutant or with heat killed S. pneumoniae TIGR4 (hkSP)
(Figure 3.3A). Quantification by flow cytometry revealed significantly higher percent of
FLICA 660 YVAD+ neutrophils incubated with live S. pneumoniae compared with neutrophils stimulated with the Δply mutants or hkSP (Figure 3.3B).
To determine if pneumolysin is sufficient for caspase-1 activation, bone marrow neutrophils from C57BL/6 mice were primed with heat killed S. pneumoniae as before, and incubated with either a highly purified pneumolysin (Ply) that has pore-forming activity, or with a mutant pneumolysin (PdB) that has a single amino acid substitution resulting in diminished hemolytic activity (McNeela et al., 2010). The percent FLICA
660 YVAD+ neutrophils was assessed by flow cytometry. Figure 3.3C shows a distinct population of FLICA 660 YVAD+ neutrophils following stimulation with hemolytic pneumolysin (Ply, left panel), but not with PdB (right panel). Quantification of FLICA
660 YVAD+ cells showed >80% caspase-1 expressing neutrophils in presence of Ply compared to <10% in PdB treated cells (Figure 3.3D).
To assess the role of the NLRP3 inflammasome in pneumolysin - induced caspase-1 and IL-1β processing, NLRP3-/- and ASC-/- neutrophils were primed with hkSP and stimulated with pneumolysin as before, and the cleaved p10 form of caspase-1 in cell supernatants was TCA-precipitated and detected by western blot analysis. Caspase-1 p10
99 Figure 3.3: Active caspase-1 induction in murine neutrophils is dependent on
NLRP3 and ASC, and requires active pneumolysin
A. unstimulated live TIGR4 live Δply hkTIGR B. 4
YVAD -
FLICA
YVAD - + DAPI FLICA D.
- / -
C. 1 -
- / -
unstim unstim - /
E. -
Ply-stim aspase ASC NLRP3 unstim C57BL/6 C PdB-stim C57BL/6 events Casp-1 p10 pneumolysin Nig
FLICA-YVAD
F. G. H.
Active caspase-1 induction in murine neutrophils is dependent on NLRP3 and ASC, and requires active pneumolysin. C57BL/6 neutrophils were incubated 2h with heat killed S. pneumoniae (hkSP) (Signal 1), 1.5 h with either live S. pneumoniae TIGR4 (WT), or the ∆ply mutant (Signal 2), and active caspase-1 was detected using FLICA660- YVAD. A. Representative neutrophils showing FLICA660-YVAD positive cells (red specs) (original magnification is x100). B. Percent FLICA660-YVAD positive
100 neutrophils. C, D. Representative flow cytometry profiles (C) and percent (D) FLICA660-YVAD positive C57BL/6 neutrophils primed with hkSP and further incubated with Ply or the non-hemolytic Ply (PdB). E. Mature p10 form of caspase-1 from supernatant of C57BL/6, NLRP3-/-, ASC-/- and caspase-1-/- neutrophils after priming with Ply or with nigericin as a positive control. F, G. Percent FLICA660-YVAD positive neutrophils from C57BL/6 and caspase-1-/- mice (F) and from NLRP3-/- and ASC-/- mice (G) following hkSP priming and stimulation with live WT or Δply mutants. H. IL-1β secretion after hkSP priming and stimulation with WT TIGR4, purified Ply (500 ng/ml) or nigericin. Data are representative of three repeat experiments.
was clearly detected in supernatants from stimulated compared with unstimulated
C57BL/6 neutrophils (Figure 3.3E); however, caspase-1 p10 expression was markedly less in the supernatant from NLRP3-/- and ASC-/- neutrophils compared with C57BL/6
neutrophils (Figure 3.3E). Similarly, there were significantly fewer FLICA 660 YVAD+ cells in caspase-1-/-, NLRP3-/- and ASC-/- compared with C57BL/6 neutrophils (Figure
3.3F, G), and less secreted IL-1β following stimulation with live S. pneumoniae or purified Ply (Figure 3.3H). Dose response and representative flow cytometry profiles are shown in Supplementary Figure S3.3.
Taken together, these data indicate that pneumolysin induces NLRP3/ASC- dependent caspase-1 activation and IL-1β secretion by neutrophils.
Inducible NLRP3 expression in murine and human neutrophils
As we detected active FLICA 660 YVAD+ caspase-1 in speck-like intracellular
structures in pneumolysin activated neutrophils, we next examined the expression pattern
101 Figure S3.3: Quantification of FLICA-YVAD staining
A. Unstained gate 99.8% 0.2%
Unstimulated Live TIGR4 MOI 5 Live TIGR4 MOI 15 Live TIGR4 MOI 30 Live TIGR4 MOI 50 93.4% 6.6% 93.2% 6.8% 84% 16% 67.3% 32.7% 52.7% 47.3%
Active caspase-1 (FLICA-660) B. C.
S3.3A,B. Murine C57BL/6 neutrophils were left untreated or primed for 2hr followed by treatment with live TIGR4 at different M.O.I for 1.5h and stained with FLICA660- YVAD for 45mins and quantified by flow cytometry. Unstained cells were used to set up the gate. S3.3C. Primed human neutrophils were treated live TIGR4, Δply, and heat killed TIGR4 and active caspase-1 producing cells were quantified by flow
cytometry.
102 of NLRP3 in these cells. Bone marrow neutrophils from C57BL/6 mice were incubated
for 3h with hkSP (Signal 1), and with WT TIGR4, Ply or PdB as Signal 2, and
intracellular NLRP3 was detected by confocal microscopy. NLRP3 expression was very
low in unstimulated neutrophils; however, following incubation with hkSP, there was
enhanced NLRP3 expression in the cytosol that did not overlap with the nuclear DAPI
stain (Figure 3.4A). After further incubation with live TIGR4 or active pneumolysin
(Ply), NLRP3 was detected as speck-like aggregates in neutrophils, which is consistent
with inflammasome activation. Specks were not detected in neutrophils incubated with
non-hemolytic PdB.
Consistent with these findings, western blot analysis showed low NLRP3
expression in unstimulated neutrophils, but increased NLRP3 expression over time after
hkSP incubation, whereas ASC was constitutively expressed in unstimulated C57BL/6
and NLRP3-/- neutrophils (Figure 3.4B). Similarly, in human peripheral blood
neutrophils, NLRP3 expression was increased after incubation with hkSP, whereas ASC
expression was constitutive (Figure 3.4C).
To identify the pathogen recognition receptor and signaling pathways mediating
NLRP3 expression, bone marrow neutrophils were isolated from C57BL/6 and TLR2-/- mice and stimulated for 3h with hkSP, and NLRP3 and ASC expression were detected by western blot analysis. Also, the NFκB and MAPK pathways in C57BL/6 neutrophils were blocked using specific inhibitors JSH-23 (for NFκB) and SP600125 for JNK.
Figure 3.4D shows decreased NLRP3 expression in TLR2-/- compared with C57BL/6
neutrophils, whereas expression of ASC remained unchanged. Further, NLRP3
expression was completely inhibited in the presence of JSH-23 and partially inhibited
103 Figure 3.4: Inducible NLRP3 expression in murine and human neutrophils
C57BL/6 -/- B. NLRP3 C. hkSP: 0 2 4 6 0 2 4 6 (hr) NLRP3 NLRP3
ASC ASC
βactin
D. -/- E. 0 5 25 50 JSH-23 F. C57BL/6 TLR2 0 5 25 50 SP600125 hkSP: - + + + + (μM) hkSP: - + + + + (μM) C hkSP C hkSP NLRP3 NLRP3 NLRP3 ASC ASC ASC βactin βactin p-JNK total JNK βactin
Inducible NLRP3 expression in murine and human neutrophils. A. NLRP3 expression in C57BL/6 bone marrow neutrophils after priming with hkSP followed by stimulation with WT TIGR4, Ply or a non-hemolytic Ply (PdB). Red is NLRP3 and blue is DAPI; scale bar is 10μm. B, C. NLRP3 protein expression by western blot of C57BL/6 bone marrow neutrophils (B) and human peripheral blood neutrophils (C) after incubation with hkSP. D. NLRP3 protein expression in hkSP- stimulated bone marrow neutrophils from C57BL/6 and TLR2-/- mice. E, F. NLRP3 expression in hkSP – stimulated C57BL/6 neutrophils incubated with either the NFκB inhibitor JSH-23 (E) or the JNK/AP-1 inhibitor SP600125 (F) at the indicated concentrations (μM) prior to western blot analysis for NLRP3, ASC, p-JNK, total JNK and β-actin. Data are representative of two repeat experiments
104 Figure S3.4: Mapping of transcription factor binding sites in murine Nlrp3 promoter
Core Sequence of a matrix is defined as the (usually 4) highest conserved, consecutive positions of the matrix.
Ci-value (consensus index vector) for the matrix represents the degree of conservation of each position within the matrix. The maximum Ci-value of 100 is reached by a position with total conservation of one nucleotide, whereas the minimum value of 0 only occurs at a position with equal distribution of all four nucleotides and gaps.
105 after incubation with SP600125, with no effect on ASC expression (Figure 3.4E, F) The
fact that we did not see complete inhibition with SP600125 is because it is a specific inhibitor of pJNK and other redundant pathways like p38 and ERK might still be contributing to NLRP3 expression in absence of JNK pathway. To confirm the regulation of NLRP3 by NFκB and AP-1 transcription factor, we performed promoter analysis of murine Nlrp3 gene using genomatix (matinspector) software. As demonstrated in
Supplementary Figure S3.4, two putative binding sites each for NFκB and AP-1 transcription factor were identified in Nlrp3 promoter region further strengthening our
observation that blockade of these transcription factors impairs NLRP3 transcription and
translation.
Together, these data indicate that S. pneumoniae - induced NLRP3 expression is
induced by TLR2 activation through the NFκB and to some extent the MAPK pathway.
Pneumolysin mediates IL-1β processing and release from human neutrophils
To ascertain if S. pneumoniae activation of the NLRP3 inflammasome in human neutrophils is dependent on Ply, we utilized a Ply deletion mutant (Δply) or highly
purified preparations of active hemolytic pneumolysin (Ply) and the non-hemolytic (PdB)
toxoid. Human peripheral blood neutrophils were primed with hkSP for 3h to induce expression of pro-IL-1β and NLRP3, and were then stimulated with live WT TIGR4 or with mutant Δply.
IL-1β production by neutrophils was increased following incubation with increasing MOI (multiplicity of infection) of WT S. pneumoniae, but not with the TIGR4
Δply mutant (Figure 3.5A). Similarly, neutrophils incubated with purified pneumolysin
106 Figure 3.5: Pneumolysin mediates IL-1β processing and release from human neutrophils
A. B.
(pg/ml) (pg/ml)
β β 1 1 - - IL IL
C. D. % cytotoxicity (LDH release) % cytotoxicity (LDH release)
Pneumolysin dependent caspase-1 activation and IL-1β secretion by human neutrophils. Human peripheral blood neutrophils were primed with hkSP followed by 2h stimulation with WT or Δply (A, C) or with Ply or PdB (B, D) and IL-1β in cell supernatant was quantified by ELISA (A, B). Cell death was assessed by LDH release compared with lysed cells (C, D). Histograms are mean ±SD of samples per group and data shown are representative of three independent experiments with different donors. ** p< 0.001, *** p< 0.0001. Nig: Nigericin incubated 30 min with neutrophils.
107 (Ply) produced IL-1β in a dose dependent fashion, whereas the non-hemolytic pneumolysin (PdB) induced significantly less IL-1β (Figure 3.5B). Neutrophils
incubated with either live S. pneumoniae or purified pneumolysin did not release lactose
dehydrogenase (Figure 3.5C, D); indicating that IL-1β secretion under these conditions is not due to cell lysis.
Taken together, these data indicate that IL-1β release from human neutrophils is
dependent on pneumolysin that has pore forming activity.
Pneumolysin-induced IL-1β secretion by neutrophils is mediated by K+ efflux and caspase-1, but not by cathepsin B or serine proteases
Activation of the NLRP3 inflammasome in macrophages and dendritic cells can
occur following a loss of cytosolic K+, or after lysosome rupture and activation of the
cathepsin B pathway (Hoegen et al., 2011; Hornung et al., 2008; McNeela et al., 2010;
Munoz-Planillo et al., 2013; Tschopp and Schroder, 2010b). We therefore examined the effect of pneumolysin on K+ efflux and cathepsin B in human neutrophils.
Primed human peripheral blood neutrophils were incubated for 2h with hemolytic
(Ply) or non-hemolytic (PdB) pneumolysin, and total intracellular K+ in cell lysates was
measured by atomic absorbance spectrometry. Figure 3.6A shows a dose dependent
decrease in cell-associated K+ following incubation with pneumolysin, consistent with enhanced K+ efflux. In marked contrast, intracellular K+ did not decrease when cells were
incubated with PdB, thereby indicating an essential role for pore forming activity in
induction of the K+ efflux response. Conversely, when neutrophils were incubated in high
extracellular K+, which prevents formation of a plasma membrane K+ gradient, the
108 Figure 3.6: Pneumolysin-induced IL-1β secretion by neutrophils is mediated by K+ efflux and caspase-1, but not by cathepsin B or serine proteases
A. B. C.
D. E. F. + IL-1β (pg/ml) Intracellular K
G. % LDH release
K+ efflux mediates caspase-1 - dependent IL-1β secretion by human neutrophils: A. Human peripheral blood neutrophils were hkSP primed and stimulated 2h with Ply or PdB. Total cell contents were extracted using 10% HNO3, and the cell associated K+ concentration was quantified by atomic absorbance spectroscopy. B. FLICA660-YVAD positive murine neutrophils quantified by flow cytometry after treatment with live TIGR4 in presence of increasing concentration of extracellular KCl.; C. hkSP primed neutrophils were stimulated 2 h with either Ply (500ng/ml) or WT TIGR4 (50:1) in the presence of 5 mM or 130 mM KCl, and IL-1β secretion was measured after 2 h. D. Primed human neutrophils were pretreated with bafilomycin (200nM), CA-074-Me (100μM) or ZFA (50 μM) prior to stimulating with live TIGR4 (50:1) or Ply (500 ng/ml) for 2 h and IL-1β release were quantified from the supernatant. E-G. Neutrophils were primed and
109 stimulated with Ply, or were stimulated with Nigericin in the presence of the pan-caspase inhibitor ZVAD or the caspase-1 inhibitor YVAD at the indicated concentration (μM), and examined for IL-1β secretion (E), intracellular K+ (F) and LDH (G). Histograms are mean ±SD of three samples per treatment condition, and are representative of two similar experiments with neutrophils from different donors. ** p< 0.001, *** p< 0.0001.
pneumolysin – induced caspase-1 activation and IL-1β secretion were completely ablated
(Figure 3.6B, C), indicating that K+ efflux is critical for caspase-1 activation and IL-1β
secretion by pneumolysin – stimulated neutrophils.
To examine the role of cathepsin B on NLRP3 inflammasome activation and IL-
1β release, neutrophils were incubated with pneumolysin or S. pneumoniae TIGR4 in the
presence of cathepsin B inhibitors CA-074-Me, ZFA, or with bafilomycin A, which
blocks lysosomal acidification by inhibiting H+-ATPase. Figure 3.6D shows that IL-1β
secretion by pneumolysin or S. pneumoniae TIGR4-stimulated neutrophils in the
presence of cathepsin B inhibitors or with bafilomycin A was not significantly different
from cells in the absence of inhibitors, indicating that neither cathepsin B nor lysosomal
destabilization are required for pneumolysin-induced IL-1β processing in neutrophils.
LDH release was not increased following incubation with these inhibitors, indicating that
they are not cytotoxic at the concentrations used (Supplementary Figure S3.5).
In addition to cleaving pro-IL-1β, caspase-1 mediates pyroptotic cell death, which
is characterized by loss of plasma membrane integrity and release of cytoplasmic
contents, including LDH (Fernandes-Alnemri et al., 2007; Sagulenko et al., 2013). We
therefore examined if pneumolysin-induced K+ efflux occurs as a secondary consequence of pyroptotic cell death, i.e., after caspase-1 activation, or if K+ efflux is a signal for
110 NLRP3 inflammasome assembly and IL-1β secretion, i.e., before caspase-1 activation.
Pneumolysin or nigericin stimulated human neutrophils were incubated with the caspase-
1 inhibitor YVAD or the pan-caspase inhibitor ZVAD, and IL-1β secretion, intracellular
K+ and LDH release were measured.
As shown in Figure 3.6E, IL-1β secretion by neutrophils stimulated with
pneumolysin or nigericin was completely inhibited in presence of YVAD or ZVAD,
indicating a requirement for caspase-1 in pneumolysin induced IL-1β secretion. In
contrast, pneumolysin induced K+ efflux (loss of intracellular K+) was not inhibited by caspase-1 inhibitors, indicating that K+ efflux occurs upstream of caspase-1 activation
(Figure 3.6F). Similarly, pneumolysin – induced LDH release in the presence of caspase-
1 inhibitors was not significantly different from pneumolysin alone, and therefore
indicating that neutrophils are not undergoing caspase-1 dependent pyroptosis (Figure
3.6 G).
Finally, as serine proteases such as neutrophil elastase can also cleave pro-IL-1β
to the mature form (Black et al., 1988; Hazuda et al., 1990; Karmakar et al., 2012;
Stehlik, 2009); however, we found no difference in IL-1β secretion or LDH release by S.
pneumoniae-stimulated bone marrow neutrophils from neutrophil elastase (NE)-/- mice compared with C57BL/6 neutrophils (Supplemental Figure S3.5B,C), indicating that there is no role for elastase in IL-1β processing.
Taken together, the results of these studies support the concept that pneumolysin induces IL-1β secretion in neutrophils by causing loss of intracellular K+ and activation of
the NLRP3 inflammasome and is not a result of pyroptosis, lysosomal destabilization or
serine protease activity.
111 Figure S3.5: IL-1β secretion by neutrophil elastase (NE)-/- mice
A.
B. C.
S3.5A. Percent LDH release from human neutrophils following incubation with inhibitors of cathepsin B and phagosome acidification; S3.5B. IL-1β production and -/- LDH release (S3.5C) by neutrophils from neutrophil elastase KO (NE ) compared with C57BL/6 mice
112 DISCUSSION:
Streptococcus pneumoniae is the causative organism of pneumococcal septicemia,
meningitis, and pneumonia, which results in infant mortality on a global scale (Denny
and Loda, 1986; Kadioglu et al., 2008). S. pneumoniae is also a leading cause of corneal
ulcers worldwide resulting in visual impairment and blindness, especially in developing
countries where the organisms are resident in the conjunctiva and enter the corneal
stroma following an abrasion (Bharathi et al., 2007; Parmar et al., 2003; Upadhyay et al.,
1991). Neutrophils comprise > 90% of the total cells in S. pneumoniae corneal ulcers, and expression of IL-1β, NLRP3 and ASC is significantly elevated compared with normal corneas (Karthikeyan et al., 2013).
Although four distinct inflammasomes have been identified, the NLRP3 inflammasome has been extensively studied in mononuclear cells activated by a diverse array of structurally distinct agents, including silica, asbestos, and uric acid crystals, and by bacterial toxins (Franchi et al., 2012; Latz et al., 2013). A common response to all
NLRP3 activating stimuli is a change in plasma membrane permeability that leads to efflux of intracellular potassium. This K+ efflux can be mediated by specific host cell ion
channels, such as the ATP-gated P2X7 receptor channel in macrophages and dendritic
cells, but not in neutrophils (Franchi et al., 2007a; Martel-Gallegos et al., 2010; Qu et al.,
2011). However, K+ efflux can also be induced by bacterial exotoxins such as S.
pneumoniae pneumolysin (Franchi et al., 2007a; Kadioglu et al., 2008).
Pneumolysin is an important virulence factor in S. pneumoniae infections, and is expressed in clinical isolates from S. pneumoniae infections, including corneal ulcers
(Kadioglu et al., 2008; Karthikeyan et al., 2013). Pneumolysin is part of a family of
113 cholesterol-dependent cytolysin toxins, and forms oligomers in the host cell plasma
membrane, (~40 monomers), resulting in formation of large (~400 A) transmembrane
pores that cause rapid cell lysis (Tilley et al., 2005). Although shown to be a cytoplasmic
protein that is released after autolysis (Kadioglu et al., 2008), pneumolysin is also
localized in the bacterial cell wall, and can be released by extracellular proteases where it
exhibits pore-forming activity (Price and Camilli, 2009; Price et al., 2012). Pneumolysin has been shown to activate the NLRP3 inflammasome in monocytes, macrophages and dendritic cells in the context of pneumococcal pneumonia (McNeela et al., 2010;
Witzenrath et al., 2011), and pneumolysin expressing strains also cause keratitis in animal models (Moore et al., 2009; Reed et al., 2005). In the current study, we showed a requirement for pneumolysin with hemolytic activity in caspase-1 activation and IL-1β processing in human and murine neutrophils, as IL-1β processing did not occur in the absence of pneumolysin (S. pneumoniae Δply mutant) or following incubation with the non-hemolytic PdB toxoid.
We further demonstrated that neutrophils are not only the predominant source of
IL-1β in the early phase of S. pneumoniae corneal infection, but neutrophils in infected corneas expresses active caspase-1 which can be detected as cytosolic specks, and that
IL-1β processing in vivo is dependent on NLRP3 and ASC. This finding is consistent with early stage bacterial keratitis in patients, where neutrophils are the predominant cell type in S. pneumoniae corneal ulcers and there is elevated expression of ASC and NLRP3 in addition to IL-1β (Karthikeyan et al., 2013). Neutrophils were also found to be the predominant source of IL-1β in murine models of arthritis and osteomyelitis, although
IL-1β processing was caspase-1 independent (Cassel et al., 2014; Guma et al., 2009).
114 However, our current findings are in agreement with Staphylococcus aureus induced IL-
1β expression in bone marrow neutrophils, which was impaired in ASC gene knockout mice or in the presence of NLRP3 and caspase-1 inhibitors (Cho et al., 2012).
IL-1β regulates the severity of infections and inflammatory diseases by
stimulating production of CXC chemokines and inducing expression of vascular
endothelial cell adhesion molecules, resulting in increased neutrophil infiltration
(Dinarello, 2009). We reported that IL-1β is required for chemokine production and
neutrophil recruitment to the cornea in a model of Pseudomonas aeruginosa keratitis,
although IL-1β processing was mediated by serine proteases and not caspase-1
(Karmakar et al., 2012). In the current study, we demonstrate that IL-1β processing by
neutrophils is dependent on the NLRP3/ASC inflammasome and caspase-1 activation. In
addition, we also identified the mechanism by which the NLRP3 inflammasome is
activated in murine and human neutrophils incubated with either live S. pneumoniae or
pneumolysin. Three distinct pathways of NLRP3 inflammasome activation have been
proposed (Tschopp and Schroder, 2010b): a) the channel mode activated by extracellular
ATP through P2X7 receptor channels or by pore-forming microbial exotoxins that result
in loss of cytosolic K+ through the channels/ pores; b) the lysosome rupture / cathepsin B pathway that is activated following phagocytosis of crystals, such as silica or alum, leading to rupture of lysosomes and release of cathepsin B into the cytoplasm; and c) the reactive oxygen species pathway that releases the thioredoxin interacting protein that can interact with NLRP3.
Our findings are consistent with the first pathway in which active pneumolysin forms a membrane pore, as we showed that pneumolysin - mediated activation of the
115 NLRP3 inflammasome is due to loss of intracellular K+ as measured by atomic
absorbance spectroscopy. Because caspase-1 inhibitors blocked IL-1β secretion, but did
not inhibit K+ efflux, we conclude that pneumolysin -induced K+ efflux occurs upstream
and independently of caspase-1 mediated IL-1β processing and is not a secondary consequence of caspase-1-driven pyroptosis. Further, although lysosomal destabilization and cathepsin B activation contributes to S. pneumoniae induced NLRP3 activation in
monocytes and macrophages (Hoegen et al., 2011), we found no role for cathepsin B in
neutrophil mediated IL-1β processing. However, our findings on the role of K+ efflux are consistent with a recent study showing K+ efflux is critical for activation of the NLRP3
inflammasome in bone marrow macrophages and dendritic cells (McNeela et al., 2010;
Munoz-Planillo et al., 2013).
IL-1β producing neutrophils have been reported in murine models of microbial
infection and autoimmunity, including Staphylococcus aureus skin disease and uric acid
– induced arthritis (Cho et al., 2012; Guma et al., 2009), indicating that IL-1β production
by neutrophils occurs in multiple disease situations. Although there was no role for
caspase-1 in the arthritis model and in a model of Pseudomonas aeruginosa keratitis
(Guma et al., 2009; Karmakar et al., 2012), NLRP3, ASC and caspase-1 were required
for IL-1β secretion by bone marrow neutrophils stimulated in vitro with S. aureus or with
LPS / nigericin (Cho et al., 2012; Mankan et al., 2012). Similarly, LPS+ATP induced IL-
1β secretion by human neutrophils was inhibited in presence of either protease inhibitors
or caspase-1 inhibitors (Gabelloni et al., 2013b), indicating a role for both pathways.
In the current study, we provide further insight to the role of caspase-1 in IL-1β
processing by neutrophils by demonstrating that: 1) Signal 1 (heat killed S. pneumoniae)
116 induce NLRP3 protein expression in bone marrow neutrophils that is dependent on
TLR2, NFκB and JNK; 2) The S. pneumoniae pore forming toxin pneumolysin functions as Signal 2, which induces assembly of the NLRP3/caspase-1 inflammasome and is
detected as distinct specks in neutrophils both in vivo and in vitro; 3) IL-1β processing by
human neutrophils is solely dependent on K+ efflux and not on lysosome disruption and
cathepsin B release. We also show no role for proteases as IL-1β production was not
impaired in pneumolysin-stimulated elastase-/- neutrophils.
In conclusion, results of the current study add substantially to our understanding
of the role of IL-1β in bacterial infections by demonstrating that neutrophils are not only
an important source of this cytokine in vivo, but also form NLRP3 inflammasome
aggregates with active caspase-1 and mediate IL-1β cleavage to the bioactive form which
is required for bacterial killing and to limit corneal disease. Given the diverse infectious
and inflammatory conditions where neutrophils infiltrate the tissues in large numbers, it seems highly likely that their role in IL-1β mediated inflammation will be found in multiple diseases.
117
CHAPTER 4
DISCUSSIONS
118 RESEARCH SUMMARY:
We established a murine model of corneal infection with P. aeruginosa to better understand the host-pathogen interaction during bacterial keratitis. Previous studies from our lab showed that TLR4 and TLR5 on resident macrophages recognize P. aeruginosa
LPS and flagellin, and initiate downstream signaling leading to translocation of the NFκB transcription factor to the nucleus (Sun, Y., Karmakar, M. et al, 2010), and expression of proinflammatory cytokines and chemokines. These include CXCL1/KC, which recruits neutrophils from the limbal capillaries to the corneal stroma. Once in the stroma, neutrophils engage in active killing of the bacteria using an arsenal of antimicrobial defense mechanisms, including ROS and proteases. These cytotoxic agents cause tissue destruction, which is the major cause of corneal opacification during keratitis. IL-1α and
IL-1β are also produced during P. aeruginosa corneal infection, which then feed back to the IL-1R in bone marrow derived cells in a paracrine and autocrine fashion. IL-
1R/MyD88 signaling then promotes a sustained inflammatory response during P. aeruginosa corneal infection. In the absence of IL-1β, IL-1R signaling is inhibited, and impedes recruitment of neutrophils to the stroma which leads to uncontrolled bacterial growth and corneal perforation.
In order to determine the source of IL-1β in the cornea during acute microbial keratitis, we used two different murine models of corneal infections using P. aeruginosa and S. pneumoniae. Flow cytometry analysis of corneas 24h post infection showed that
NIMP-R14 positive neutrophils rather than macrophages are the predominant source of
IL-1β in both Pseudomonas and Streptococcus keratitis. Our observation is consistent
119 with studies by our collaborators, where neutrophils constituted more than 90% of the cellular infiltrates and 10% are mononuclear cells in P. aeruginosa and S. pneumoniae infected corneal ulcers (Karthikeyan et al., 2013). They also demonstrated elevated RNA expression of inflammasome components like NLRP3, ASC and NLRC4 in the corneal ulcers compared to corneas from healthy donors. Since P.aeruginosa Type III secretion system and S. pneumoniae toxin pneumolysin can activate NLRC4 and NLRP3 inflammasomes, respectively (discussed in Chapter1), we investigated the role of these inflammasomes in our murine model of corneal infection. Our main observations are:
1) IL-1β processing during P. aeruginosa infection is independent of NLRC4 inflammasome and caspase-1. Rather, neutrophil derived serine proteases, like elastase are primarily responsible for processing pro IL-1β to mature p17 form in vivo. This was also confirmed in isolated human neutrophils which showed significantly low IL-1β in presence of elastase inhibitor compared to untreated neutrophils.
2) Unlike in P. aeruginosa infection, IL-1β processing during S. pneumoniae corneal infection requires NLRP3/ASC/Caspase-1 inflammasome. We also demonstrated active caspase-1 as distinct specks in neutrophils from infected C57BL/6 corneas. NLRP3 inflammasome aggregate were also identified in purified murine neutrophils.
3) NLRP3 activation in neutrophil is dependent on S.pneumoniae pore forming toxin- pneumolysin which causes K+ efflux upstream of inflammasome activation.
Although there is a marked difference in the mechanism of IL-1β processing by neutrophils in response to these two different bacteria, the exact reason is yet unknown.
One plausible explanation might be that neutrophils express very little to almost no
NLRC4 inflammasome which has been reported in multiple studies (Ceballos-Olvera et
120 al., 2011; Mankan et al., 2012; Miao et al., 2010a). In absence of inflammasome
component, pro IL-1β is cleaved by an alternate mechanism that utilizes neutrophil
derived enzymes like elastase present in neutrophil granules. Although, it is still not
known how cytosolic proIL-1β might be targeted to the primary vesicles of neutrophil to get cleaved by these enzymes. Thus future studies should focus on identifying the mechanisms of IL-1β processing by neutrophil derived proteases.
Unlike NLRC4, neutrophils abundantly express NLRP3, ASC and caspase-1 as shown in our study (Chapter 3) as well as by others (Bakele et al., 2014; Gabelloni et al.,
2013a; Mankan et al., 2012). Thus IL-1β processing by neutrophils during S. pneumoniae
infection is dependent more on these inflammasome components rather than on
neutrophil proteases and we thus we observe no role of elastase during this process.
In conclusion, these cumulative works have significantly advanced our knowledge
on differential IL-1β processing by neutrophil during acute bacterial infection. Since, neutrophils are the first cells to get recruited to the tissue during majority of the microbial infection, these mechanisms of IL-1β processing by neutrophils are likely important
during other inflammatory conditions as well. Thus targeting the neutrophils may
represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases,
such as cystic fibrosis, rheumatoid arthritis or sepsis.
Regulation and resolution of inflammation during bacterial keratitis
Bacterial and fungal infections of the cornea are a major worldwide cause of
blindness and visual impairment. However, the cornea is a unique tissue as it is considered immune privileged, primarily because it is avascular and has high levels of
121 soluble immunosuppressive cytokines and neuropeptides from the anterior chamber. The
anterior chamber, vitreous cavity and the sub retinal space are immune privileged as allogenic tumor can grow in these sites, whereas similar implants are rejected in non- privileged sites (Jiang et al., 1993; Wenkel et al., 1999). In order to achieve immune privilege, the eye and the immune system has developed various strategies to subvert innate and adaptive immune response in the eye which includes maintenance of intraocular immunosuppressive micro environment. Since blood-ocular barriers are not
absolute, innate and adaptive immune cells from circulation can gain access to the
internal compartment of the eye. Additionally, resident macrophage and dendritic cells in the corneal stroma raises the possibility of activation of these leukocytes and initiate
inflammation if the internal compartments are not immunosuppressed. In order to achieve
this, there are soluble and cell surface immune modulatory factors. Aqueous humor
(AqH)- intraocular fluid of the anterior chamber inhibits T-cell activation and
differentiation in vitro and also inhibits innate immune effector cells (Kaiser et al., 1989).
The neuropeptides vasoactive intestinal peptide (VIP) and somatostatin of normal AqH to
suppresses antigen and mitogen activated T-cell proliferation (Taylor et al., 1994; Taylor and Yee, 2003). α-MSH in AqH prevents activated T cells from secreting inflammatory cytokines including IFN-γ. Macrophage inhibitory factor (MIF) in AqH inhibits NK cells from lysing the target and inducing inflammation. Additionally, expression of CD95 ligand (CD95L) blocks CD95 induced apoptosis of CD95 positive T cells. Another immunomodulatory factor that contributes to immune suppression in the eye is the active
TGF-β2. Ocular inflammation induces rapid production of IL-6 which in turn activate the resident macrophages to convert latent TGF-β2 to its active form (Streilein, 2003a, b).
122 Despite all these immunosuppressive mechanisms, the eye is still continuously threatened
by environmental pathogens including bacteria, virus or fungus. Whereas the ocular
surface comes in direct contact with external pathogens, the internal compartments are
vulnerable to blood-borne pathogens. Although a strong blood-tissue barrier formed by
endothelial cell tight junction reduces the likelihood to minimize pathogen access to the
eye, these barriers are not always absolute. Additionally, preexisting corneal trauma or
breach in the corneal epithelium increases the likelihood of pathogen access to the
stroma, induce inflammation and cause sight-threatening ocular diseases.
However, our understanding of how acute inflammation, infection and recruited neutrophil resolve from an injured or stressed ocular tissue is still evolving. Resolution of inflammation during keratitis comprises of a series of coordinated and active processes which results in restoration of tissue integrity and function. 1. Protective lipid mediator circuit: Recent studies using gene knockout mice as loss of function models have
established an essential role of two prominent early response circuits in the resolution of
acute inflammation in the eye. These circuits include 15-lipoxygenase (15 LOX), which generates protective and pro-resolving lipid signals. Lipid autacoids are of particular interest as virtually every cell can generate specific and highly conserved bioactive lipid signals such as arachidonic acid. 15 LOX is an inducible gene in macrophages and its expression is enhanced by IL-13 and IL-4 that promotes macrophage differentiation to the M2 phenotype (Serhan et al., 2008). 15-LOX is also highly expressed in mucosal epithelial cells, corneal epithelium and retinal pigment epithelium and its expression is further enhanced during acute inflammation. 15-LOX is the key enzyme required for the formation of anti-inflammatory eicosanoid – lipoxin A4 (LXA4). Additionally, 15-LOX
123 can directly generate autocoids which restrain neutrophils and lymphocyte activation,
dendritic cell function, formation of proinflammatory chemokines. It also promotes
clearance of apoptotic neutrophils by macrophages and contributes to wound healing, and
thus is a critical mediator of resolution during ocular inflammation (Gronert, 2010).
2. Cleavage of keratan sulfate proteoglycan: Inflammatory cells that are recruited to
the cornea from the peripheral vessel migrate several millimeters through the dense
extracellular matrix which is mostly comprised of type I collagen fibrils separated by
keratan sulfate proteoglycans, primarily lumican and keratocan. Studies by Carlson et al.,
demonstrated that lumican and keratocan null mice have impaired neutrophil recruitment
to the cornea (Carlson et al., 2007). Additionally, keratocan is degraded by infiltrating
neutrophils and in the absence of these proteoglycans, the CXCL1/KC chemokine is not
retained in the cornea and accumulates in the anterior chamber. Disruption of the
proteoglycan/CXC chemokine gradient contributes to the resolution of inflammatory
responses. This observation was further supported by the recent study where lumican-/-
mice showed poor resolution of P. aeruginosa keratitis, increased bacterial survival,
reduced inflammatory cell infiltrates compared to wild type mice. Lumican-/- infected
corneas harbored lower levels of the CXCL1 compared to C57BL/6 corneas, which could
be due to poor retention, or increased degradation of the chemokine gradient (Shao et al.,
2013).
3. Neutrophil apoptosis: Resolution of inflammation requires a shift in the tissue
response such that neutrophil recruitment no longer occurs in order to reconstitute tissue homeostasis. Also, the lifespan of the neutrophils in the cornea can be modified by inducing apoptosis. Fas-L and TNF secreted by tissue macrophages induces neutrophil
124 apoptosis at higher concentration (van den Berg et al., 2001). During pneumococcal
keratitis, NLRP3 mediated inflammasome activation induces active caspase-1 mediated
pyroptotic cell death in neutrophils thus limiting their activity in the tissue. Additionally,
PI3K mediated reactive oxygen species production is a key event in promoting apoptosis
in neutrophils (Geering et al., 2011). In addition, apoptotic neutrophils secrete annexin-
1(Anx-1) that inhibits further neutrophil recruitment. Anx-1 translocates to the plasma
membrane of neutrophils and interacts with formyl peptide receptor-2 and alter leukocyte
adhesion and migration (Dalli et al., 2008). Apoptotic neutrophils promote their own
clearance by expressing receptors that are recognized by macrophages. These signals can
be either molecules exposed de novo at the cell membrane or existing ones that undergo
modification in course of cell death (Ortega-Gomez et al., 2013).
4. Macrophage switch from M1 to M2: Once macrophages engulf dead neutrophils,
they cease expression of proinflammatory cytokines and lipid mediators and instead
exhibit an anti-inflammatory phenotype (M2 macrophages). These M2 macrophages are
characterized by secretion of IL-10 and TGF-β. In P. aeruginosa keratitis, IL-33 promotes a Th2-type immune response and reduces inflammation by polarizing the macrophage to the M2 phenotype, and production of anti-inflammatory mediators including IL-10 leads to resolution of inflammation (Hazlett et al., 2010). M2 macrophages also secrete B and T cell chemokines that repopulate the inflammatory sites
+ + with B1, NK, γδT, CD4 CD25 T cells that promote resolution of inflammation. Tregs- a subset of CD4+CD25+ T-cells that expresses Foxp3 transcription factor regulate immune
response through secretion of immunosuppressive cytokines like IL-10. This was further
supported by studies from Rouse et al., who demonstrated that depletion of IL-10 or Tregs
125 during stromal keratitis leads to exacerbated inflammatory responses (Sarangi et al.,
2008).
FUTURE DIRECTIONS:
4.1: Role of Reactive oxygen species (ROS) in activating the NLRP3/ASC inflammasome in neutrophils
A future set of studies will examine the regulatory activity of ROS in
inflammasome activation by neutrophil. ROS functions as an alarm signal for multiple
signal transduction pathways including activation of the NLRP3 inflammasome. Zhou et
al. demonstrated that thioredoxin (TRX) - interacting protein (TXNIP) is a redox
sensitive component that associates with NLRP3. In unstimulated cells, TXNIP is
constitutively bound to and inhibited by the oxidoreductase thioredoxin (TRX). ROS
generation facilitates TRX-TXNIP dissociation, and facilitates NLRP3-TXNIP
association (Zhou et al., 2010). Given that neutrophils respond to pathogens by
generating ROS (Leal et al., 2012), it is possible that NLRP3-TXNIP interaction will
regulate inflammasome activity in these infected cells. Whether neutrophil derived ROS
serves as a positive regulator of NLRP3 can be tested by comparing the ability of wild
type and CGD (gp91phox-/- deficient and hence do not generate ROS through the
NADPH oxidase pathway) neutrophils to activate caspase-1 in response to NLRP3
activators. Furthermore, the role of TXNIP can be tested using neutrophils from TXNIP-/- mice and comparing caspase-1 activation and IL-1β secretion with C57BL/6 neutrophils in response to S. pneumoniae or other NLRP3 activators like nigericin. Additionally,
126 immunoprecipitation of NLRP3 should be performed from stimulated neutrophil to test
its physical interaction with TXNIP. These future studies will reveal any role of ROS in
activating NLRP3 inflammasomes in neutrophils.
4.2: Role of ATP in amplifying NLRP3 activation in neutrophils.
ATP is actively secreted from pannexin channels in stressed or infected cells.
Additionally, S. pneumoniae has been shown to induce ATP release from THP-1 cells
(Hoegen et al., 2011). Unpublished data from our lab demonstrated that human
neutrophils release ATP in response to S. pneumoniae as shown in Figure 4.1.
Extracellular ATP has an amplification role in activating the NLRP3 inflammasome.
Previous studies have shown that NLRP3 can be activated by P2X7 - an ATP gated ion
channel, which triggers rapid K+ efflux from macrophages (Martinon et al., 2009;
Schroder and Tschopp, 2010). Recent studies by Hung et al. showed that ATP induced
pore formation in gingival epithelial cells requires P2X7 along with the other purinergic
receptor P2X4 and that deletion of P2X4 completely blocks NLRP3 activation and IL-1β
secretion by these cells (Hung et al., 2013). Additionally, a similar role for P2X4 has
been shown on neurons and in diabetic neuropathy where P2X4 is the major ATP gated ion channel that drives NLRP3 activation (Chen et al., 2013). We (data not shown) and others have demonstrated that pannexin1 channels are constitutively expressed in human neutrophils (Bao et al., 2013). Unpublished data from our lab have demonstrated that human neutrophils express only P2X4 and not P2X7 shown in Figure 4.2. This is consistent with previous reports where the authors used electrophysiological recordings and western blot analysis to confirm the absence of P2X7 receptors in the human
127 Figure 4.1: ATP release by human neutrophils in response to S. pneumoniae
Relative Luciferin Units (RLU/sec) Relative
Time (mins)
Purified human neutrophils were preincubated for 30mins with luciferin and S. pneumoniae was added (MOI 50) to the cells. Extracellular release of ATP was measure in real time for 1hr. Digitonin was added at the end to release total intracellular ATP into the extracellular medium. (unpublished data) Red data points: Neutrophils only, Green data points: Neutrophils + S. pneumoniae, Blue data points: Bacteria only
128 Figure 4.2: Expression of P2X4 receptors in human neutrophils
A. B.
Human neutrophils were incubated for 2hrs with LPS, MDP, S. aureus or S. pneumoniae and western blot was ATP binding P2X and P2Y purinergic performed for P2X7R and P2X4R. receptors mediates their activation for BMDM: Bone marrow derived murine regulation of ionic fluxes. Only P2X +2 macrophages (unpublished data) channels allow Ca influx and K+ efflux required for NLRP3 assembly (Vitiello et al, 2012)
129 promyeloid cell line HL-60 (Martel-Gallegos et al., 2010). Future studies should thus focus on understanding the mechanism of how the purinergic receptor P2X4 drives
NLRP3 activation in neutrophils. Purified murine or human neutrophils can be treated with S. pneumoniae or with purified PLY in the presence of a) apyrase, which hydrolyzes secreted ATP to adenosine and is therefore a broad inhibitor; b) the broad acting P2 receptor antagonist - pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS); c)
Ivermectin, which is a P2X4 activator as illustrated in Figure 4.3. Additionally, since neutrophils are the major cell contributing to NLRP3 mediated IL-1β release during S. pneumoniae keratitis, the role of P2X4 in vivo can be examined by comparing NLRP3 activation and IL-1β secretion between C57BL/6 and P2X4-/- mice.
4.3: Role of AIM2/ASC inflammasome in IL-1β processing by neutrophils during S. pneumoniae infection
Data shown in Chapter 3 indicates that S. pneumoniae infected ASC-/- mice have an impaired ability to clear bacteria from the cornea compared to C57BL/6 mice. In addition to the NLRP3inflammasomes, the ASC adaptor molecule is an essential component of the AIM2 (Absent in Melanoma) inflammasome, and is activated by DNA from viruses and intracellular bacteria such as Francisella and Listeria (Fernandes-
Alnemri et al., 2010; Franchi et al., 2012; Rathinam et al., 2010; Sauer et al., 2010). The mechanism by which DNA from extracellular bacteria interacts with cytoplasmic AIM2 has not been determined, but is likely due to degradation and release from phagolysosomes, which is distinct from activation of TLR9 in endosomes. Furthermore,
AIM2-/- macrophages stimulated with S. pneumoniae are deficient in caspase-1 activation
130 Figure 4.3: Working Model for ATP mediated amplification of NLRP3 activation
Extracellular ATP released by Pannexin1 channels present on neutrophils during S. + pneumoniae infection can bind to P2X4 receptors and cause K efflux to further amplify NLRP3 activation. Apyrase and PPADS are inhibitors of ATP and P2X4 respectively and Ivermectin is activator of P2 receptors. (Diagram adapted from Cho et al, 2012, Nat Imm)
131 and IL-1β cleavage (Fang et al., 2011). Preliminary studies (data not shown) from our lab
confirmed the constitutive expression of AIM2 inflammasomes in human neutrophils
which is also consistent with a previous study (Mankan et al., 2012). In order to examine
role of AIM2 inflammasomes in neutrophils during S. pneumoniae infection, bone
marrow neutrophils from C57BL/6 and AIM2-/- mice can be incubated with LPS or
muramyl dipeptide (MDP) as Signal 1, and then with bacterial DNA as a positive control,
or with S. pneumoniae as Signal 2. Caspase-1 activation and IL-1β secretion from the
neutrophils will indicate the role of AIM2 inflammasomes during S. pneumoniae
infection. To further ascertain role of AIM2 during Streptococcal corneal infection,
corneas of C57BL/6 and AIM2-/- mice will be abraded and infected and 24h post
infection, neutrophil infiltration will be assessed by flow cytometry, caspase-1 activation
and IL-1β production can be quantified by western blot analysis.
4.4: Role of S. aureus pore forming toxins in activating inflammasomes by neutrophils
Staphylococcus aureus which is another major cause of bacterial keratitis
worldwide also produces hemolytic αβγ toxins. In S. aureus infections, the inflammatory
response in αβ hemolysin induced necrotizing pneumonia is attenuated in the absence of
the NLRP3 inflammasome (Craven et al., 2009; Kebaier et al., 2012). Also, αβγ
hemolysins activate NLRP3 and caspase-1 together with bacterial lipoproteins in murine
macrophages (Munoz-Planillo et al., 2009). Unpublished data from our lab demonstrated
NLRP3 dependent bacterial killing using the wild type strain S. aureus, indicating a requirement of NLRP3 in host defense. αβ hemolysin contributes to NLRP3 activation by
132 macrophages and in murine models of respiratory infection (McNeela et al., 2010; Miller
and Cho, 2011; Munoz-Planillo et al., 2009). Also, αβ hemolysin is a major virulence factor in keratitis caused by S. aureus (Callegan et al., 1994; Girgis et al., 2005); however the role of these toxins in NLRP3 activation and IL-1β processing by neutrophils in the cornea has not been examined. To examine this, C57BL/6 mice will be infected with
Staph 8325 αβ hemolysin mutants, and parent strains. After 24h, active caspase-1 and mature IL-1β in the cornea can be detected by western blot and CFU can be quantified after 24h, 48h and 72h post infection. Additionally, Staph αβ hemolysin mutants will be incubated with purified human neutrophils or with C57BL/6, NLRP3-/-, ASC-/- and caspase-1-/- neutrophils and mature IL-1β secreted by neutrophils can be measured by
ELISA or western blot analysis. Although αβ hemolysin has been shown to induce
NLRP3 activation in monocytes and macrophages, the mechanism of activation is not
well studied. Prior studies demonstrated that high extracellular potassium blocks IL-1β
release from the monocytes in response to hemolytic toxins of S. aureus indicating that
low intracellular potassium mediates activation of NLRP3 (Craven et al., 2009). Future
studies should thus focus on understanding the mechanism of how Streptococcal αβ
hemolysin mediates inflammasome activation in neutrophils.
4.5: Pyroptotic cell death in neutrophils due to inflammasome activation
Pyroptosis is a novel form of cell death induced mostly by microbial infection.
This pathway of cell death is uniquely dependent on caspase-1. Caspase-1 is not involved
in apoptotic cell death and caspase-1 deficient cells respond normally to most apoptotic
signals (Li et al., 1995). Caspase-1 activation in macrophages infected with Salmonella or
133 Shigella results in processing of these cytokines and death of the host cell (Hersh et al.,
1999). The mechanism and outcome of this form of cell death are distinctly different
from apoptosis- which actively inhibits inflammation (Fernandes-Alnemri et al., 2007).
Morphologically, pyroptosis is most notably characterized by loss of plasma membrane integrity and release of cytoplasmic content into the extracellular milieu. The pyroptotic plasma membrane appears to rupture, then rapidly reseal and swell, forming a “balloon- shaped” vesicle around the nucleus. Indeed, during pyroptosis, cells undergo a measurable size increase (Fink and Cookson, 2006; Sun et al., 2005). As the membrane swells, the nucleus also undergoes rounding and condensation (Fernandes-Alnemri et al.,
2007; Molofsky et al., 2006) but, unlike apoptosis, nuclear integrity is maintained.
Pyroptotic cells undergo DNA fragmentation and, like apoptotic cells, show positive
TUNEL staining. Although pyroptosis in macrophages during inflammasome activation has been well studied, very little is known about pyroptotic cell death in neutrophils.
In the current study, we observe IL-1β secretion by neutrophils prior to LDH release suggesting IL-1 release occurs prior to caspase-1 mediated pyroptosis. Future studies should thus focus on characterizing the mechanism and kinetics of caspase-1 dependent pyroptosis in neutrophils during Streptococcal as well as other microbial infections. Human or murine neutrophils can be incubated with live S. pneumoniae WT or Δply at different multiplicity of infection and uptake of propidium iodide (PI) can be measured in real time using a fluorescent detector plate reader along with quantification of IL-1β release. This will help us to understand the sequence of events during inflammasome activation in neutrophils as well as the role of lytic toxin-pneumolysin in mediating pyroptotic cell death in neutrophils.
134 4.6: Inflammasome activation and autophagy
Autophagy is a cytoprotective process by which the cell sequesters damaged proteins, organelles, or pathogens in a double membrane compartment, the autophagosome, which targets this cellular material for degradation in the lysosome, and recycles the constituent molecules. Although autophagy is a normal physiological process, it can be up-regulated
during cellular stress, starvation or other inflammatory conditions. Recent studies have
implicated a complex interplay between autophagy and inflammasome activation in
macrophages. Blockade of autophagy by inhibiting Atg16L or Atg7 promotes LPS
induced inflammasome activation suggesting autophagy negatively regulates
inflammasomes. The mechanism underlying autophagy-dependent inflammasome
inhibition is currently not clear but it has been suggested that autophagosomes may target
inflammasomes for degradation (Harris et al., 2009). However, since NLRP3
inflammasome activity is suppressed by ROS blockade (discussed in Chapter1) and
autophagy negatively regulates ROS generation (Bensaad et al., 2009; Dupont et al.,
2009; Saitoh et al., 2008), it is possible that autophagic suppression of ROS indirectly
inhibits inflammasome activity. Studies by Suzuki et al add an additional layer of
complexity by indicating that the inflammasome may also negatively regulates
autophagy. This study found that caspase-1 deficiency promotes autophagy in
macrophages infected with NLRC4 agonist S. flexneri (Suzuki and Nunez, 2008).
Although autophagy and inflammasome activation have been studied to some degree in
macrophage/monocytes, no information is currently available about regulation of
inflammasome activation in neutrophils by autophagy. Studies by Mitroulis et al.,
demonstrated that human neutrophils can induce autophagy in a phagocytosis-
135 independent (rapamycin, TLR agonists, PMA) or phagocytosis (Escherichia coli) - dependent manner. Additionally, ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis in neutrophils (Mitroulis et al., 2010). Future studies should thus emphasize on understanding the complex interplay between autophagy and NLRP3 activation in neutrophils during different microbial infections.
4.7: Mechanisms for non-canonical IL-1β secretion by neutrophils
IL-1β is a secretory protein that lacks a signal peptide and does not follow the classical endoplasmic reticulum-golgi pathway of secretion (Rubartelli et al., 1990).
Although most of the IL-1β precursor (pro IL-1β) localizes in the cytosol of monocytes and macrophages, a fraction of it is contained within vesicles that co-fractionate with late endosomes and early lysosomes on percoll density gradient (Andrei et al., 1999). This raises the possibility that these vesicles are a part of the IL-1β secretion system in which pro IL-1β translocates across their membrane, undergoes maturation and is finally released outside after the vesicle membrane fuses with the plasma membrane. Similar to
IL-1β, a number of leaderless secretory proteins in prokaryotes and eukaryotes are secreted by a non-canonical mechanism. Whereas the mechanism in eukaryotes is still under investigation, prokaryotes use an ATP binding cassette (ABC) transporter to mediate the secretion of most of the LLS proteins. The role of ABC transporters in LLS protein secretion in mammals is unknown, but sulfonylurea glibenclamide- a potent blocker of murine ABC transporters inhibits the secretion of IL-1β from human and murine monocytes (Hamon et al., 1997). However, the previous observation has been
136 challenged by the recent findings that glibenclamide also inhibits NLRP3 inflammasome
(Lamkanfi et al., 2009). Moreover, increase in vesicular pH by Bafilomycin A has been shown to prevent both secretion and accumulation of pro IL-1β suggesting that abolition of pH gradient between cytosol and vesicle lumen affects the entry of pro IL-1β into the vesicles and consequently IL-1β secretion (Andrei et al., 1999). Although the mechanism of non-classical IL-1β secretion has been studied in monocytes and macrophages, the mechanism of neutrophil serine protease (main constituent of primary granules) mediated
IL-1β processing in neutrophils has yet to be determined. Macrophage Inhibitory Factor
(MIF) release from neutrophils can be blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is active and functional in neutrophils (Daryadel et al., 2006). Moreover, the pH gradient between the cytoplasm and acidic pH of the primary granules in neutrophils
(Styrt and Klempner, 1982) may also furnish the necessary energy that in part might be responsible for translocation of IL-1β into the vesicle lumen where it is processed by serine proteases. Future studies should thus focus on understanding the mechanism of non-classical IL-1β processing as illustrated in the proposed model in Figure 4.4.
4.8: Microbial proteases in alternate IL-1β processing
Although the cysteine protease caspase-1 is primarily responsible for processing of pro IL-1β into the mature bioactive form, other serine proteinases such as neutrophil elastase and cathepsin G, mast cell chymase, and granzyme A can all generate active fragments from pro-IL-1β (Herzog et al., 2005; Irmler et al., 1995; Mizutani et al., 1991).
This property contributes to the outcome of diseases including rheumatoid arthritis as
137 Figure 4.4: Proposed mechanism of non-classical IL-1β processing by neutrophil serine proteases
Step1: Translocation Step 2: Exocytosis
PMN
BafA pH
Glibenclamide
pro IL-1β IL-1β ABC transporter Glibenclamide Neutrophil serine protease
Two step model for IL-1β secretion showing vesicle-mediated transport from the cytosol to the extracellular space, The translocation step can be blocked by glibenclamide or by increasing the pH of the vesicle lumen using specific drug – bafilomycin A. Once in the granule lumen, pro IL-1β can be processed by neutrophil serine proteases like elastase or cathepsin G and secreted out of the cell by fusion of vesicle membrane with plasma membrane (Adapted from Andrei et al, 1997, Mol Cell Biol)
138 discussed in Chapter 1. However, proteinases secreted by microbes can also mediate IL-1
cleavage, including Aspartyl protease (Sap) secreted by Candida albicans, which cleaves
pro IL-1β to the mature 17kda-19kda forms. However, unlike caspase-1, Sap generated high molecular (25-30kda) fragments also. Prolonged incubation with C. albicans showed that the mature 17kda form is resistant to further proteolytic cleavage and is the predominant end product (Beausejour et al., 1998). Other than C. albicans, Streptococcus
pyogenes secretes an extracellular cysteine protease - Exotoxin B (Spe B) which can also
cleave pro IL-1β to a biologically active form (Kapur et al., 1993). Two additional naturally occurring Spe B variants cleave pro IL-1β in a similar fashion. Mature IL-1β resulting from cleavage of pro IL-1β by Spe B induced nitric oxide synthase activity in vascular smooth muscle cells and killed human melanoma A375 cells, indicating that this product is biologically functional. P. aeruginosa secretory protease elastase B (LasB), is an elastolytic zinc metalloproteinase encoded by the lasB gene of P. aeruginosa
(Morihara, 1964). Also known as pseudolysin, LasB is an important virulence factor and is thought to damage host tissues through hydrolysis of extracellular matrix proteins and
disrupting endothelial and epithelial barriers by targeting intercellular tight junctions
(Azghani, 1996; de Bentzmann et al., 2000). Under in vitro experimental conditions, Las
B degrades TNF-α, IFN-γ, IL-2 and IL-8 (Horvat et al., 1989; Leidal et al., 2003;
Parmely et al., 1990; Theander et al., 1988). Whether P. aeruginosa elastase can cause proteolysis of pro IL-1β to its mature form is still unknown, but it is possible that this
enzyme can mediate cleavage of IL-1β. This can be addressed by using a lasB mutant
strain along with wild type strain of P. aeruginosa and comparing their ability to cleave
IL-1β in vitro. Future studies should also focus on understanding whether these enzymes
139 from microbes mediate breakdown of pro IL-1β in tissues and orchestrate the outcome of the disease during various microbial infections.
4.9: Therapeutic implications
The NLRP3 inflammasome has attracted considerable attention due to its association
with human inflammatory diseases. Mutant NLRP3 is the cause of a group of
inflammatory diseases known as cryopyrin-associated periodic syndrome (CAPS).
Several point mutations target the NACHT domain of NLRP3 resulting in gain of
function mutations of NLRP3 and causes familial cold auto inflammatory syndrome
(FCAS), Muckle-Wells Syndrome (MWS) and neonatal onset multi-systemic inflammatory disease (NOMID) (Agostini et al., 2004; Masters et al., 2009). Strikingly,
treating these patients with anakinra, a non-glycosylated recombinant form of the
naturally occurring IL-1 receptor antagonist (IL-1Ra) which blocks inflammasome-
dependent IL-1β signaling, resulted in a complete cessation of clinical symptoms and biochemical changes within hours of administration. Canakinumab is a fully humanized monoclonal antibody against IL-1β with a half-life of about 4 weeks that has proven safe and effective in treating CAPS patients. In addition to its central role in auto inflammatory diseases, NLRP3 activation has recently emerged as an unexpected sensor for metabolic diseases like type2 diabetes and obesity induced insulin resistance. (Donath and Shoelson, 2011; Schroder et al., 2010). Other human pathological conditions like cancer, Alzheimer’s disease and gout are showing increasing association with NLRP3
hyperactivity. In the skin, NLRP3 inflammasome activation has been linked to UVB
induced damage and contact hypersensitivity (Feldmeyer et al., 2007; Watanabe et al.,
140 2007). Recent studies have demonstrated that haemozoin – a crystal produced by the
Plasmodium species during malarial infection activates the NLRP3 inflammasomes
(Dostert et al., 2009; Shio et al., 2009). Also, NLRP3 inflammasome contribute to host defense during bacterial infections. However, chronic inflammation accompanied by dysregulated inflammasome activation during persistent infection may result in extensive tissue damage, detrimental effects and immunopathologies. In addition, many pathogenic bacteria subvert the NLRP3 inflammasome pathways, often through inhibition or interaction with, the inflammasome core machinery by virulence factors (Brodsky et al.,
2010). Future studies of inflammasome activation and regulation by pathogens or microbial products should also use animal models to understand how pathogens subvert host inflammasome signaling pathways, and will contribute to development of
interventions to control bacterial infection and provide protective immunity.
CONCLUDING REMARKS
In conclusion, the cumulative studies described here enhance our current understanding
of how neutrophils regulate IL-1β processing in vivo during microbial infections by two
major human pathogens. Given that neutrophils are the first and most abundant cells to
enter the tissue following infection with pathogenic bacteria and fungi, these mechanisms
of IL-1β processing are likely relevant to other human infections as well. Data from the
current study will therefore lead to novel therapeutic interventions for treating microbial
infections in the eye and other inflammatory diseases.
141 SELECTED PUBLICATIONS
1. Sun Y, Karmakar M, Roy S, Ramadan RT, Williams SR, et al. (2010) TLR4 and
TLR5 on corneal macrophages regulate Pseudomonas aeruginosa keratitis by
signaling through MyD88-dependent and -independent pathways. J Immunol 185:
4272-4283
2. Sun Y, Karmakar M, Taylor P, Rietsch A, Pearlman E (2012) ExoS and ExoT ADP-
ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting
neutrophil apoptosis and bacterial survival. J Immunol 188(4):1884-95
3. Karmakar M, Sun Y, Hise AG, Rietsch A, 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. J Immunol 189: 4231-
4235
4. Pearlman E, Sun Y, Roy S, Karmakar M, Hise AG, et al. (2013) Host defense at the
ocular surface. Int Rev Immunol 32: 4-18
5. Roy S, Karmakar M, Pearlman, E (2014) CD14 mediates TLR4 endocytosis and
Syk and IRF3 activation in epithelial cells, and impairs neutrophil infiltration and
Pseudomonas aeruginosa killing in vivo. J Biol Chem 289: 1174-1182
6. Mausita Karmakar, Michael Katsnelson, Neil G Greene, Hesham A. Malak, Scott
Howell, Amy G. Hise, Andrew Camilli, Aras Kadioglu, George R. Dubyak, Eric
Pearlman. Neutrophil mediated IL-1β processing in Streptococcus pneumonia corneal
infection is dependent on the NLRP3/ASC inflammasome and Caspase-1 activation.
Manuscript under revision.
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