Bacterial Superinfection of Influenza Protein-2 Expression Drives Illness in Dysregulated Macrophage-Inflammatory

Dysregulated Macrophage-Inflammatory Protein-2 Expression Drives Illness in Bacterial Superinfection of Influenza

This information is current as Caleb C. J. Zavitz, Carla M. T. Bauer, Gordon J. Gaschler, of October 3, 2021. Katie M. Fraser, Robert M. Strieter, Cory M. Hogaboam and Martin R. Stampfli J Immunol published online 11 January 2010 http://www.jimmunol.org/content/early/2010/01/11/jimmun ol.0903304 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 11, 2010, doi:10.4049/jimmunol.0903304 The Journal of Immunology

Dysregulated Macrophage-Inﬂammatory Protein-2 Expression Drives Illness in Bacterial Superinfection of Inﬂuenza

Caleb C. J. Zavitz,* Carla M. T. Bauer,* Gordon J. Gaschler,* Katie M. Fraser,† Robert M. Strieter,‡ Cory M. Hogaboam,x and Martin R. Stampﬂi†,{

Influenza virus infection is a leading cause of death and disability throughout the world. Influenza-infected hosts are vulnerable to secondary bacterial infection, however,and an ensuing bacterial pneumonia is actually the predominant cause of influenza-attributed deaths during pandemics. A number of mechanisms have been proposed by which influenza may predispose to superinfection with an unrelated or heterologous pathogen, but the subsequent interaction between the host, virus, and bacteria remains an understudied area. In this study, we develop and examine a novel model of heterologous pulmonary infection in which an otherwise subclinical Bordetella parapertussis infection synergizes with an influenza virus infection to yield a life-threatening secondary pneumonia. Despite a profound pulmonary inflammatory response and unaltered viral clearance, bacterial clearance was significantly impaired Downloaded from in heterologously infected mice. No deficits were observed in pulmonary or systemic adaptive immune responses or the viability or function of infiltrating inflammatory cells to explain this phenomenon, and we provide evidence that the onset of severe pulmonary inflammation actually precedes the increased bacterial burden, suggesting that exacerbated inflammation is independent of bacterial burden. To that end, neutralization of the ELR+ inflammatory chemokine MIP-2 (CXCL2/GRO-b) attenuated the inflammation, weight loss, and clinical presentation of heterologously infected mice without impacting bacterial burden. These data suggest that pulmonary inflammation, rather than pathogen burden, is the key threat during bacterial superinfection of influenza and that http://www.jimmunol.org/ selective chemokine antagonists may be a novel therapeutic intervention in cases of bacterial superinfection of influenza. The Journal of Immunology, 2010, 184: 000–000.

espite the widespread application of vaccines, antibiotics, It has long been acknowledged that a substantial fraction of and other therapies, infectious disease remains a leading influenza-related death and disability results not from primary viral D threat to human health. The World Health Organization disease but from secondary infection with bacteria unrelated estimates that over 4 million lives are lost each year to lower re- (heterologous) to the virus. In fact, recent analysis points to sec- spiratory tract infections—more than are attributable to the widely ondary bacterial pneumonia, not primary viral disease, as the recognized HIV, malaria, or tuberculosis pandemics (1). Among predominant cause of death in the three 20th century influenza by guest on October 3, 2021 lung infections, influenza virus remains a persistent threat; in non- pandemics (5) and suggests that the remarkable lethality of the pandemic years, it infects over 1 billion people worldwide, causing 1918 influenza strain may be due to its ability to drive a secondary 3–5 million cases of serious illness and an estimated 500,000 deaths bacterial pneumonia (6). Deaths to secondary bacterial infection (2), whereas during pandemics, it has eclipsed all other causes of commonly result from immunopathology, rather than bacterial death combined. The 1918 influenza pandemic depressed world- infection per se, and once initiated, immunopathology in the form wide population growth for 10 y and killed 40 million people in just of sepsis and septic shock may progress independently of the one year—the single greatest loss of life in human history (3, 4). bacterial burden itself. Specifically, even if hosts suffering from heterologous infections are treated with sterilizing antibiotics, *Medical Sciences Program, †Department of Pathology and Molecular Medicine, they still frequently succumb to pathologic inflammation (7, 8). Center for Gene Therapeutics, and {Department of Medicine, McMaster University, Several clinical trials using corticosteroids (9), G-CSF (10), he- Hamilton, Ontario, Canada; ‡Division of Pulmonary Critical Care, Department of x mofiltration (11), or neutralizing Abs against endotoxin or TNF-a Medicine, University of Virginia, Charlottesville, VA 22908; and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (12) to modulate the inflammatory response have largely failed to Received for publication October 13, 2009. Accepted for publication December 7, prevent patients’ progression to septic shock, failed to reduce 2009. mortality, and raised concerns about the risks of nonspecifically This work was supported in part by the Canadian Institutes of Health Research, the suppressing an already infected host’s immune defenses. AllerGen Network of Centers of Excellence, and AstraZeneca. C.C.J.Z. and G.J.G. A number of mechanisms have been proposed through which hold Ontario graduate scholarships. C.C.J.Z. and C.M.T.B. hold Ashley Tobacco studentships. C.M.T.B. holds a Canadian Respiratory Health Professional fellowship viral infection may predispose to bacterial infection. Viruses may and a Scotiabank Ontario Graduate Scholarship in Medical Sciences. M.R.S. holds act first by lysing epithelial cells and disrupting the respiratory a new investigator award from the Canadian Institutes of Health Research. epithelium, thus exposing the basement membrane and permitting Address correspondence and reprint requests to Dr. Martin R. Stampfli, MDCL 4010, bacterial attachment as shown during times of influenza epidemics McMaster University, Hamilton, Ontario L8N 3Z5, Canada. E-mail address: stampfli@ mcmaster.ca (13–15) and in experimental models of disease (16). In epithelial The online version of this article contains supplemental material. cells that escape direct lysis, influenza and other viruses may then Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; AM, alveolar mac- increase the expression of receptors to which bacteria adhere (16) rophage; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; and impair ciliary function, reducing early mucocilliary clearance FSC, forward light scatter; MEF, mouse embryonic fibroblast; MPO, myeloperoxidase; of bacteria (17, 18). Additionally, viral insult may impair macro- SSC, side scatter; VILI, ventilator-induced lung injury; VSV, vesicular stomatitis virus. phage responsiveness to bacterial TLR ligands by reducing NF-kB Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 activity (19) and reduce macrophage antibacterial function (20).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903304 2 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION

Viral induction of type I IFNs may then compromise innate an- animals were bled retro-orbitally with nonheparinized Pasteur pipets, and tibacterial immunity by inducing granulocyte apoptosis (21) and serum was obtained by incubating whole blood for 30 min at 37˚C, followed 2 reducing the production of inflammatory chemokines (22). Fi- by centrifugation. Samples were stored at 20˚C until assayed. Lungs were removed, tracheas were cannulated, and bronchoalveolar nally, activation of the adaptive antiviral system may compromise lavage (BAL) was obtained by lavaging twice with PBS (250 and 200 ml). host defense against subsequent bacterial challenge via an IFN-g– Total cell counts were determined by hemocytometer. BAL supernatants dependent impairment of alveolar macrophage (AM) function (23, were stored at 220˚C until analysis of inflammatory mediators. Cytospins 24). Whether the life-threatening pneumonia seen in heterolo- for differential cell counts were prepared by resuspending cell pellets in PBS, followed by cytocentrifugation at 10 3 g for 2 min. Cells were gously infected individuals simply represents an appropriate im- stained by Hema 3 (Biochemical Sciences), and differentials were de- mune response to a magnified bacterial burden or is the result of termined by counting at least 300 cells. Standard hemacytological criteria a fundamental alteration in immunity brought on by two divergent were used to classify mononuclear cells, neutrophils, and eosinophils. For immune insults remains to be elucidated. histology, lung lobes were inflated at 20 cm H2O pressure with 10% for- In this study, we report on a novel model of severe secondary malin, embedded in paraffin, and 3-mm-thick sections were stained with H&E. Images were collected with OpenLab software (version 3.0.3; Im- bacterial pneumonia in H1N1 influenza A-infected mice, with ex- provision, Guelph, Ontario, Canada) via a Leica camera and microscope acerbated weight loss, clinical presentation, and mortality following (Leica Microsystems, Richmond Hill, Ontario, Canada). For determination administration of an otherwise subclinical dose of Bordetella par- of lung bacterial burdens and viral titers, lung lobes were collected in 1 ml apertussis bacteria. We report that viral infection does not alter PBS and then homogenized with a Polytron PT 1200C (Kinematica, Lit- tau-Lucerne, Switzerland). bacterial adherence or the early events of bacterial clearance but does lead to significantly impaired bacterial clearance both 1 and 2 Bacterial burdens and viral titers wk postinfection. Even before bacterial burden becomes exacer- Twenty-five microliters of serially diluted fresh homogenates in PBS was Downloaded from bated, heterologously infected mice are afflicted by a severe pul- plated onto Bordet Gengou blood agar plates and grown for 3 d at 37˚C to monary inflammation driven by the neutrophil chemokine MIP-2, enumerate viable bacteria. Homogenates were stored at 280˚C prior to which is not seen in animals given either pathogen alone. We determination of viral titers on Madin-Darby canine kidney cell mono- provide evidence that pulmonary neutrophilic inflammation, rather layers. Madin-Darby canine kidney cells grown to confluence in 6-well than pathogen burden, is the key determinant of illness during tissue culture plates were rinsed twice with warm PBS to remove serum, and 200 ml serially diluted samples were added. Plates were rocked and bacterial superinfection of influenza virus and that specific im- incubated at 37˚C for 30 min, after which 2 ml 0.65% agarose (BioShop munoneutralization of MIP-2 results in reduced weight loss and Canada, Burlington, Ontario, Canada) in MEM supplemented with 1% http://www.jimmunol.org/ symptom scores, without reducing bacterial burden. In contrast, L-glutamine, 1% penicillin and streptomycin, and 1 mg/ml trypsin was CXCR2 blockade resulted in severely impaired antibacterial im- added to each well. Once the agarose solidified, plates were incubated at 37˚C for 2 d. Cells were fixed using Carnoy’s fixative and plaques were munity and worsened clinical performance. These results suggest enumerated with Giemsa stain. a role for targeted antichemokine interventions in the treatment of heterologous pulmonary infection. Measurement of type I IFN by plaque reduction assay IFN bioactivity was measured in BAL samples by plaque reduction assay as Materials and Methods described previously (31). Briefly, BAL samples were assessed for their Animals ability to protect primary IFN regulatory factor-3–deficient mouse embryonic fibroblasts (MEFs) (provided by Dr. K. Mossman, McMaster by guest on October 3, 2021 Female C57BL/6 mice (6–8 wk old) were purchased from Charles River University, Hamilton, Ontario, Canada) from infection with a vesicular Laboratories (Montreal, Quebec, Canada) and kept in specific pathogen- stomatitis virus expressing GFP under an endogenous viral promoter free conditions on a 12-h light-dark cycle with food and water provided ad (VSV–GFP; provided by Dr. B. Lichty, McMaster University). The rep- libitum. Cages, food, and bedding were autoclaved. Animals were handled lication efficiency of VSV is decreased, because type I IFN present in BAL in class II biosafety cabinets by gloved, gowned, and masked staff. Ex- is able to produce an antiviral state in MEFs. IFN regulatory factor-3– periments were approved by the McMaster University Animal Research deficient MEFs are incapable of producing IFN; however, they are capable Ethics Board. After initial experiments in which animals succumbed to of responding to exogenous IFN to produce an antiviral state. infection, additional hydration and feeding measures were instituted to Cells were removed from BAL samples by centrifugation, and mono- prevent mortality in subsequent experiments. layers of MEFs were incubated with serial dilutions of supernatants for 24 h Infectious agents in 24-well dishes. Subsequently, supernatants were removed, and MEFs were infected with 4 3 103 PFU VSV–GFP in serum-free a-MEM. Viral B. parapertussis strain 12822 (American Type Culture Collection, Manassas, inocula were replaced with DMEM containing 1% methylcellulose fol- VA)has previously been sequenced (25), studied microbiologically (26), and lowing 40 min of incubation at 37˚C. GFP fluorescence intensity was studied in the murine respiratory tract (27). Bacteria were maintained on measured 24 h later on a Typhoon Trio (GE Healthcare, Piscataway, NJ) Bordet-Gengou agar (Difco, Oakville, Ontario, Canada) containing 15% imager and quantified using ImageQuantTL (GE Healthcare) software. defibrinated sheep blood (Lampire Biological Laboratories, Pipersville, PA). Liquid culture bacteria were grown at 37˚C overnight on a shaking incubator Flow cytometry in Stainer-Scholte broth (28). The H1N1 influenza A (A/FM/1/47) virus used in this study is a fully sequenced, plaque-purified preparation that is bi- Lungs were cut into ∼2-mm pieces, shaken for 1 h at 37˚C in 150 U/ml ologically characterized with respect to mouse lung infections (29). collagenase III (Invitrogen Life Technologies, Burlington, Ontario, Cana- da) in HBSS, pressed through nylon mesh, washed twice in HBSS, and Mouse infection and monitoring counted using a hemocytometer. Cells (2 3 106) were plated in 96-well, round-bottom plates, blocked for 15 min on ice with 50 ml anti-CD16/ Isoflurane-anesthetized mice were infected intranasally with 2.5 3 105 PFU 3 CD32 (purified, clone 93; eBioscience, San Diego, CA), then stained for 1 influenza virus in 35 ml PBS vehicle. Five days later, mice were given 5 h at room temperature with a PE-conjugated H2Db tetramer loaded with 105 CFU log-phase B. parapertussis in 35 ml PBS in the same manner. influenza A/FM/1/47 NP366–374 (Baylor College of Medicine Protein Core Mice were individually weighed before infection and throughout the Laboratory, Houston, TX). Cells were washed and resuspended in staining course of experimentation, and health status was scored by a blinded in- mixture for 30 min on ice. Abs used in this study include anti-CD11c vestigator as reported previously (30). (FITC, HL3; BD Biosciences, San Jose, CA), anti-CD11b (PE, M1/70; Tissue and cell collection and isolation eBioscience), anti-CD4 (PE-610, RM4-5; Invitrogen, Carlsbad, CA), anti- CD69 (PerCP-Cy5.5, [1H].2F3; BD Biosciences), anti-NK1.1 (PE-Cy7, At sacrifice, blood was collected by retro-orbital bleeding using heparin- PK136; eBioscience), anti-MHC class II (APC, M5/114.15.2; eBio- coated capillary tubes (Fisher Scientific, Pittsburgh, PA). Total leukocytes science), anti–Gr-1 (Alexa Fluor 700, RB6-8C5; eBioscience), anti- were counted after lysing RBCs, and blood smears were prepared and stained CD45R (APC-Alexa Fluor 750, RA3-6B2; eBioscience), anti-CD3 (Pacific with Hema 3 (Biochemical Sciences, Swedesboro, NJ) for differential cell Blue, eBio500A2; eBioscience), and anti-CD8 (Pacific Orange, 5H10; counts by counting a minimum of 300 cells per slide. To isolate serum, Invitrogen). For flow cytometric determination of apoptosis, cells were The Journal of Immunology 3 further stained with biotinylated Annexin V (BD Biosciences) and strep- Hercules, CA), both according to the manufacturer’s instructions. To neu- tavidin-Qdot800 (Invitrogen) and 7-aminoactinomycin D (7-AAD) (BD tralize MIP-2 in vivo, mice were given an i.p. administration of 25 mg pu- Biosciences), or with the in situ cell death detection kit according to the rified Ig once daily, commencing 3 d post-B. parapertussis infection, and manufacturer’s protocol (Roche, Laval, Quebec, Canada). Cells were continuing throughout the period of observation. washed, and 3 3 104–3 3 105 forward light scatter (FSC)/side scatter Goat anti-murine CXCR2 serum was generated as described in Ref. 34. (SSC)-gated events were collected per sample on an LSRII flow cytometer Briefly, goats were immunized at multiple intradermal sites with a peptide for flow cytometric analysis, or a FACSVantage digital cell sorter for cy- encompassing the ligand-binding domain of the murine CXCR2 (Met- tochemistry. Data were analyzed using FlowJo (Tree Star, Ashland, OR). Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-Asn-Ile-Glu-Asp-Phe-Phe-Ser-Gly), described in Ref. 35 in CFA. To block CXCR2 ligand binding, 0.5 ml Myeloperoxidase assay anti-CXCR2 immune serum was administered i.p. on the same schedule as for anti–MIP-2. Control mice were given a 0.5 ml daily i.p. administration Myeloperoxidase (MPO) was measured as previously describedinRef.30using of normal goat serum. a modification of the technique described in Ref. 32. Briefly, 0.5 ml lung ho- mogenate was mixed with 0.5 ml 5 mg/ml hexadecyltrimethylammonium bro- Ag-specific Ig ELISA mide (Sigma-Aldrich, Oakville, Ontario, Canada) in potassium phosphate buffer (pH 6.0) and centrifuged at 12,000 rpm for 2 min. Twenty microliters of su- Influenza- and B. parapertussis-specific Abs were detected in the BAL or pernatant was then added to 200 ml the reaction mixture (16.7 mg o-dianisidine serum using a minor modification of the procedure described previously [Sigma-Aldrich], 90 ml dH O, 10 ml potassium-phosphate buffer, and 50 ml1% (31). Briefly, MaxiSorp plates (Nalge Nunc International, Rochester, 2 NY) were coated overnight at 4˚C with a lysate from influenza-infected H2O2) in a 96-well plate. ODs were obtained by spectrophotometer at 1-min intervals at 450 nm. MPO activity was expressed as units per gram lung tissue, HELA cells or with a lysate from log-phase B. parapertussis bacteria. where 1 U MPO was defined as the amount of enzyme capable of degrading Coated wells were blocked with 1% casein in PBS for 2 h at room 1 mmol H O /min at room temperature. temperature. After washing, serum and BAL samples serially diluted in 2 2 PBS were incubated overnight at 4˚C, washed, and developed with bi- otin-labeled anti-mouse IgG or IgA (Southern Biotechnology Associates,

Cytokine measurements Downloaded from Birmingham, AL), respectively. Plates were washed and incubated with Bead array-based analysis of cytokines was performed by Rules Based alkaline-phosphatase streptavidin for 1 h at room temperature. The color Medicine with the Rodent multi-Analyte Profile (Austin, TX). The least reaction was developed with p-Nitrophenyl phosphate tablets (Sigma- detectable dose was determined as the mean + 3 SDs of 20 blank readings. Aldrich) in Diethanolamine buffer. Results below the least detectable dose were assigned a value of 0. Means of four independent mice in each treatment group and time were visualized in Data analysis a heat map using Excel (Microsoft, Seattle, WA). Individual scales were Data are expressed as the means 6 SEMs. All statistical analysis was applied to account for the large numerical differences between the mediators. performed using SigmaStat version 2.03 (Systat Software, Chicago, IL), http://www.jimmunol.org/ Levels of key cytokines were confirmed by DuoSet ELISAs (R&D Systems, with tests and results as indicated in figure legends. Differences were Minneapolis, MN) according to the manufacturer’s specifications. considered statistically significant at p , 0.05. MIP-2 neutralization and CXCR2 blockade Results Anti–MIP-2 Abs were generated as previously described (33) and have previously been characterized in models of infectious disease (34). Briefly, Influenza infection impairs host defense against B. parapertussis rabbits were immunized with multiple intradermal injections of 20 pg rMIP- To model heterologous infection, mice were first infected in- 2 emulsified with CFA, then boosted 10 d later. The rabbit was bled 10 d later tranasally with influenza virus and were then given an intranasal with antiserum isolated and heat inactivated. Antiserum was passed over Protein A columns (Pierce Biotechnology, Fischer Canada, Nepean, On- administration of B. parapertussis 5 d later. To ensure clarity, time by guest on October 3, 2021 tario, Canada), and purified Igs were quantified by Bradford assay (Bio-Rad, points throughout this article are expressed as the number of days of

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10 0 D5+7 D5+14 FIGURE 1. Influenza virus impairs host defense against Bordetella parapertussis. A and B, Mice were given sterile PBS (gray symbols) or influenza virus (black symbols) intranasally and then 5 d later sterile PBS (n)orB. parapertussis (triangular icons). Heterologously infected mice lost more body weight at the peak of illness and showed reduced survival following bacterial challenge when compared with single-infection control mice; n = 5–10/group. C, Mice were given PBS (N) or influenza virus (n), then challenged with B. parapertussis 5 d later. Seven (left) and 14 (right) d postchallenge, bacterial burden was significantly greater in homogenized lungs from heterologously infected animals than from B. parapertussis-only ones; n = 4–5/group. Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA for weights and bacterial burdens and by Mantel-Cox log-rank test for survival curves. p,†, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. Data are representative of at least two independent experiments. 4 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION

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B.paraper 5.0×10 102 BAL neutrophi BAL Lung * 100 0 PBS Influenza PBS Influenza FIGURE 2. Heterologous infection alters aspects of the adaptive immune response without impairing protective immune memory. A, Mice were given + sterile PBS or influenza virus intranasally and then 5 d later sterile PBS (N)orB. parapertussis (n). Influenza NP366–374-specific CD8 T cells were enumerated following a 7-wk convalescence by flow cytometry in enzymatically digested lungs and found to be reduced in heterologously infected mice. B, Seven weeks postchallenge, influenza (top panels)- and B. parapertussis-specific (bottom panels) Abs were determined by ELISA in the serum (left panels) and BAL (right panels) in mice given sterile PBS (gray symbols) or influenza virus (black symbols) intranasally and then 5 d later sterile PBS (squares) or B. parapertussis (triangles). Convalescent mice were rechallenged with influenza (top panels)orB. parapertussis, and pathogen burden (C) and BAL neutrophilia (D) were determined 5 d later for influenza and 7 d later for B. parapertussis, showing comparable protection against rechallenge in heterologous and single-infection mice; n = 4–7/group. Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA. p, †, ‡, and x denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, influenza + PBS, and influenza + B. parapertussis-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. Data are representative of two independent experiments. The Journal of Immunology 5

Table I. Flow cytometric analysis of lymphocyte subsets in the lungs of mice given PBS or inﬂuenza virus on day 0, PBS or B. parapertussis on day 5, and then sacriﬁced 7 d later

PBS + Influenza + PBS + PBS B. parapertussis PBS Influenza + B. parapertussis B cells 3.8 6 0.2 2.7 6 0.5 4.1 6 0.6 3.9 6 0.4 (3106 cells/lung) CD69+ 7.7 6 0.4 5.3 6 1.2 8.2 6 1.4 6.3 6 0.6 (3104) NK cells 2.1 6 0.6 2.3 6 0.2 1.6 6 0.2 1.9 6 0.3 (3106) CD69+ 5.2 6 2.1 7.4 6 0.3 2.5 6 0.9 2.1 6 1.3 (3104)† T cells 4.7 6 0.6 4.0 6 1.1 7.3 6 3.9 12.9 6 2.1 (3106)† CD4+ 2.3 6 0.2 1.7 6 0.2 2.3 6 0.9 3.5 6 0.5 (3106) CD25+ 1.9 6 0.2 1.6 6 0.3 2.0 6 0.5 3.0 6 0.3 (3105) CD69+ 5.5 6 0.8 4.6 6 1.1 4.6 6 2.0 10.0 6 1.6 (3104) CD8+ 1.9 6 0.4 1.3 6 0.6 4.4 6 2.3 7.3 6 1.4 (3106)†††,‡ CD25+ 7.0 6 3.8 5.3 6 3.8 22.3 6 10.4* 36.3 6 6.9 (3104)†††,‡‡ CD69+ 5.5 6 1.2 3.0 6 1.3 10.0 6 5.5 13.8 6 2.2 (3104)† Tetramer+ 0.0 6 1.0 0.1 6 2.3 6.8 6 6.0* 14.9 6 3.6 (3105)†††,‡ Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA. B cells were defined by FSC/SSC/CD45R profile, NK cells FSC/SSC/Nk1.1, and T cells by FSC/SSC/CD3; n = 4 /group. p, †, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01; three denote p , 0.001. Data are representative of two independent experiments. Downloaded from influenza infection (d 5), followed by the number of days of sec- control mice, whereas mice infected with influenza virus lost 22.1 ondary bacterial infection (i.e., “day 5+3” denotes 5 d of influenza 6 2.3% (n = 8) of their original body weight at the peak of illness infection, followed by an additional 3 d of secondary bacterial in- and began recovering 8 d postinfluenza infection. Mice infected fection). Mice infected with B. parapertussis alone did not become with both influenza virus and B. parapertussis lost significantly overtly ill and did not lose body weight when compared with more body weight (30.4 6 1.6%; n = 8) and had a more prolonged http://www.jimmunol.org/

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Influenz 01×5.2 6 †† ‡ 0 SBP azneulfnI PaSB zneulfnI PaSB zneulfnI FIGURE 3. Heterologous infection leads to severe pulmonary inflammation. Mice were given sterile PBS or influenza virus intranasally as indicated and then 5 d later sterile PBS (N)orB. parapertussis (n). A, Representative flow cytometry plots showing an increased proportion of Gr-1bright cells in the lung digests of heterologously infected animals on d 5+7, as quantified in B; n =4/group.C, Representative cytospin preparations of Gr-1bright cells isolated by FACS sorting from lung digests of mice on d 5+7 (original magnification 3400; .95% pure neutrophils by standard cytological criteria). D,Representative H&E-stained histological cross-sections from mouse lungs on d 5+7, showing severe neutrophilic inflammation and an inflammatory exudate in the lumen in heterologously infected mice (original magnification 3200 [images] and 3400 [insets]. E, BAL inflammation on day showing increased total cells and neutrophils (PMNs) in heterologously infected mice on d 5+7 (top panel), and total cells and mononuclear cells (MNCs) on d 5+14 (bottom panel)compared with mice given B. parapertussis or influenza alone. Inset in the center bottom panel shows the same data on a smaller scale. Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA; n = 3–4/group. p, †, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. Data are representative of at least two independent experiments. 6 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION

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B. parapertussis 10 25 ‡‡‡ % GFP Intensity Lung by guest on October 3, 2021 10 0 0 D5+1 D5+3

6 C 10 100 ‡‡‡ ‡‡‡ 75 10 4 P= 0.053 50 D5+3 (pfu/mL) 10 2

Lung InfluenzaLung 25 ‡‡‡ % GFP Intensity % GFP

10 0 0 D5+1 D5+3 1:10 1:100 1:500 1:3000 Dilution FIGURE 4. Increased inflammation is independent of pathogen burden in heterologous infection. Mice were given sterile PBS or influenza virus intranasally and then 5 d later sterile PBS (N)orB. parapertussis (n). A, BAL inflammation in mice given an intranasal administration of PBS or influenza as indicated, challenged intranasally with PBS (N)orB. parapertussis (n) 5 d later, and then sacrificed 1 (top panels)or3(bottom panels) d after challenge; n = 4 mice/group, showingnochangeininflammation1dpostchallengebutsignificantlyincreasedinflammationinheterologouslyinfectedmiceanadditional2dlater.Inset in the center top panel represents the same data on a smaller scale. Bacterial (B)andviral(C) burdens in the lungs of the same mice, showing unchanged pathogen loads on both d 5+1 and d 5+3. D, Type I IFN production was determined in the BAL of heterologously infected mice (n) and mice given influenza alone (N)ond5+1and d 5+3 by VSV–GFP plaque reduction assay, revealing reduced type I IFN expression in heterologously infected mice. GFP intensity was calculated as a percentage of the mean signal observed in nonprotected (“mock”), VSV–GFP-infected cells (arbitrarily set at 100% intensity). Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA; n = 4/group. p, †, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. Data are representative of two independent experiments. duration of symptoms (Fig. 1A and our unpublished data) than did infection mice suffered no mortality (Fig. 1B). In all subsequent mice given either pathogen alone. Moreover, 25% of heterologously experiments, heterologous mice were provided with additional infected mice succumbed to infection, whereas control and single- feeding and hydration measures to prevent mortality. Although The Journal of Immunology 7 heterologous infection did not impact mice’s ability to completely and pulmonary inflammation (Fig. 2D). On rechallenge, the dif- clear influenza virus from the lungs by d 5+7 (12 d postinfluenza ferences in bacterial burden between influenza- or heterologously infection; our unpublished data), it did significantly impair infected mice compared with PBS- or B. parapertussis-treated B. parapertussis clearance when measured on d 5+7 (3.7 6 2.6 3 mice was not statistically significant after allowing for the effects 104 B. parapertussis CFU/ml in bacteria-only mice versus 2.6 6 of prior B. parapertussis infection (p = 0.101). In similar fashion, 0.42 3 106 CFU/ml in heterologously infected mice) and 5+14 the effect of prior Bordetella infection (either alone or as part of (2.2 6 1.2 3 102 versus 5.9 6 1.2 3 103 CFU/ml) (Fig. 1C). a heterologous infection) on bacterial burden following rechallenge These findings confirm that influenza infection has a long-lasting was not significant after accounting for influenza’s effects (p = deleterious impact on antibacterial host defense, as has previously 0.058). Collectively these data suggested that although heterolo- been reported with respect to other bacteria (19). Of note, greater gous infection impacted aspects of the adaptive immune response, doses of B. parapertussis than these do not cause mortality, pro- it did not compromise overall immune protection. longed illness, or weight loss when administered to naive mice (our unpublished data), suggesting that increased bacterial burden Heterologous infection significantly worsens inflammatory was not the independent cause of the observed pathology. responses in the murine lung To further examine the early host defense response in heterologous Heterologous infection impacts aspects of adaptive immune infection, mice were given an intranasal administration of PBS or responses without impairing immune protection influenza virus and then challenged 5 d later with PBS or B. par- We questioned whether the deleterious effects of heterologous apertussis. Visual examination of heterologously infected lungs 7 d infection might extend beyond the acute phase of infection and postchallenge (d 5+7) revealed discoloration and overt hemorrhage Downloaded from impact the formation of immune memory. When mice were allowed (our unpublished data). Examination by flow cytometry revealed to convalesce for 7 wk following infection, numbers of cells a modest but significant increase in CD8 T cells in heterologously specific for influenza nucleoprotein residues 366–374 (NP366–374, infected mice (Table I). We further observed a dramatic increase in influenza’s immunodominant CD8 T cell epitope) in the lungs the frequency of Gr-1bright (Ly-6Gbright) cells in the lungs of het- were decreased in heterologously infected mice, as were in- erologously infected mice (52.0 6 6.3%), which was not observed in control mice, or mice infected with B. parapertussis or influenza fluenza-specific IgG titres in the serum (which was principally due http://www.jimmunol.org/ to a decrease in influenza-specific IgG3 [our unpublished data]) alone (9.8 6 0.5, 9.6 6 1.2, and 11.8 6 0.6%, respectively) (Fig. and IgA titers in the BAL (Fig. 2A,2B). In contrast, B. para- 3A,3B). Gr-1bright cells have been classified as neutrophils but may pertussis-specific Ab titers were unchanged between bacteria-only also represent populations of monocytes and/or dendritic cells. and heterologously infected mice in the serum and were un- Therefore, we isolated these cells by FACS and found them to detectable in the BAL (Fig. 2B). Any alterations observed in Ag- be .95% pure neutrophils by standard cytologic criteria (Fig. 3C). specific immune responses did not appear to have functional On histological examination of the lungs, mild and moderate consequences, because when convalescent mice were rechallenged perivascular and peribronchiolar inflammation was noted in mice with influenza virus or B. parapertussis, mice that had previously infected with either B. parapertussis or influenza virus, re- been given a heterologous infection were equivalently protected spectively, whereas heterologously infected mice had a greater by guest on October 3, 2021 against bacterial or viral rechallenge compared with their single- extent of both types of inflammation, along with extensive in- infection controls, both in terms of pathogen clearance (Fig. 2C) flammatory cell infiltration into the alveoli and the conducting

0.01 0.01 A B neutrophil of (% *** 7AAD ††† 5.7 ‡‡ 5.7

† + ** *** neutroph 0.5 ††† † 0.5 neutrophils

+ ‡‡‡ EL g f neutrophil gate) neutrophil f ils ate) N 5.2 * 5.2 o TU (% 0.0 0.0 azneulfnI - - + + - - + + - - + + - - + + - - + + - - + + sissutreparap .B sissutreparap - + - + - + - + - + - + - + - + - + - + - + - + 1+5D 3+5D 7+5D 1+5D 3+5D 7+5D C 005 ††† ‡‡‡ 573

052 tivity (U/mL) tivity c

521 MPO a MPO

0 SBP azneulfnI FIGURE 5. Neutrophil apoptosis is not impacted by heterologous infection. A, Apoptosis was measured by TUNEL-staining using flow cytometry of Gr-1bright neutrophils from lung digests of mice given an intranasal administration of PBS or influenza virus as indicated, challenged 5 d later with PBS (N)orB. parapertussis (n), and then sacrificed 1, 3, or 7 d later, showing no reduction in neutrophil apoptosis in heterologously infected mice. B, Neutrophil death was determined by 7- AAD staining and flow cytometry; n = 4/group. C, MPO activity was determined in lung homogenates on d 5+7, showing elevated MPO levels in heterologously infected mice; n = 8–9/group. Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA. p, †, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. 8 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION

A D5+1 D5+3 D5+7 D5+14 issutre sissutre s issutre issutre issutre ps arap sissutre arap sissutre ra rap sissutre SBP + a + SBP SBP + a + SBP SBP + a + SBP SBP + a + SBP pa . . . .B r r r Bp a Bp Bp + a + + a + + a + + a + ap ap s ap ap s p ar pa B B B PS B. PS B. p a PS B. B P SB B. p a B B B zneulfnI zneulfnI zneulfnI zneulfnI zneulfnI zneulfnI zneulfnI zneulfnI + SB + + SBP + + SB + + SBP + P SBP SBP + + + SB SBP + + PP PP PP MIP-2 (Macrophage Inflammatory Protein-2) IL-6 (Interleukin-6) MMP-9 (Matrix Metalloproteinase-9) MPO (Myeloperoxidase) KC/GROalpha (Melanoma Growth Stimulatory Activity Protein) IL-1beta (Interleukin-1beta) IP-10 (Inducible Protein-10) TIMP-1 (Tissue Inhibitor of Metalloproteinase Type-1) IL-17 (Interleukin-17) IL-1alpha (Interleukin-1alpha) IL-11 (Interleukin-11) MCP-1 (Monocyte Chemoattractant Protein-1) IFN-gamma (Interferon-gamma) MIP-1alpha (Macrophage Inflammatory Protein-1alpha)

TNF-alpha (Tumor Necrosis Factor-alpha) Downloaded from GCP-2 (Granulocyte Chemotactic Protein-2) Myoglobin MCP-3 (Monocyte Chemoattractant Protein-3) OSM (Oncostatin M) IL-7 (Interleukin-7) MIP-1gamma (Macrophage Inflammatory Protein-1gamma) IL-12p70 (Interleukin-12p70) MIP-1beta (Macrophage Inflammatory Protein-1beta) MIP-3beta (Macrophage Inflammatory Protein-3beta) http://www.jimmunol.org/ GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor) Fibrinogen LIF (Leukemia Inhibitory Factor) IL-10 (Interleukin-10) CRP (C Reactive Protein) FGF-basic (Fibroblast Growth Factor-basic) IL-3 (Interleukin-3) TPO (Thrombopoietin) SAP (Serum Amyloid P) Tissue Factor IL-2 (Interleukin-2) SCF (Stem Cell Factor) by guest on October 3, 2021 IL-4 (Interleukin-4) M-CSF (Macrophage-Colony Stimulating Factor) MCP-5 (Monocyte Chemoattractant Protein-5) CD40 Ligand Endothelin-1 Lymphotactin FGF-9 (Fibroblast Growth Factor-9) VCAM-1 (Vascular Cell Adhesion Molecule-1) IL-18 (Interleukin-18) EGF (Epidermal Growth Factor) Eotaxin vWF (von Willebrand Factor) VEGF (Vascular Endothelial Cell Growth Factor) MDC (Macrophage-Derived Chemokine) CD40 RANTES (Regulation Upon Activation, Normal T-Cell Expressed and Secreted) SGOT (Serum Glutamic-Oxaloacetic Transaminase) Factor VII IL-5 (Interleukin-5) GST-alpha (Glutathione S-Transferase alpha)

D5+1 D5+3 D5+7 ††† ††† 500 B 2500 ‡‡‡ C 500 ‡‡‡

)Lm/gp( )Lm/gp( ††† )Lm/ )Lm/gp( CK )Lm/gp( 2000 375 375 ‡‡‡ 1500 g 250 250 p( CK p( 1000 2-PIM †††*** *** * 125 *** 125 500

0 0 0 PBS Influenza PBS Influenza PBS Influenza PBS Influenza PBS Influenza ††† 2000 ‡‡‡ )Lm/g 1500 †† p ( 2-PIM ( 1000 ‡‡‡ 500 *

0 PBS Influenza PBS Influenza PBS Influenza FIGURE 6. Increased chemokine expression in the lungs of heterologously infected mice. Mice were given an intranasal administration of PBS or influenza as indicated, challenged intranasally with PBS or B. parapertussis 5 d later, and sacrificed 1, 3, 7, and 14 d later. A, Heat map of bead array-based analysis of cytokines in lung homogenates of animals in treatments and time points as indicated. Blue cells denote low values for a given cytokine and red denote high ones, with gradations through black indicating intermediate values. Cytokines are sorted from greatest increase in expression between The Journal of Immunology 9 airways. These inflammatory exudates were replete with neu- Because reduced apoptosis did not appear to drive the accu- trophils on d 5+7 and mononuclear cells on d 5+14 (Fig. 3D and mulation of neutrophils in the lungs of heterologously infected our unpublished data). Consistent with this, BAL inflammation mice, we questioned whether increased recruitment might instead. was significantly greater in heterologously infected mice than in We conducted a bead array-based analysis of cytokine and che- mice that received either infection alone. On d 5+7, heterolo- mokine expression in lung homogenates taken from all groups of gously infected mice had significantly elevated numbers of total animals on d 5+1, d 5+3, d 5+7, and d 5+14 (Fig. 6A and Sup- cells as compared with B. parapertussis-infected, influenza-in- plemental Tables 1–4). Over this time course, a number of me- fected, or vehicle-only mice, with neutrophils accounting for the diators were induced by influenza or B. parapertussis alone, and vast majority of this increase (Fig. 3E, top panels). An additional 7 many were further elevated following heterologous infection. d later, BAL inflammation remained elevated in heterologously Most striking was the intense increase in the expression of the infected mice as compared with B. parapertussis-infected, in- CXCR2 ligands MIP-2 (elevated by a factor of 33 with respect to fluenza-infected, or vehicle-treated mice, and this increase was influenza-only mice and a factor of 23 compared with B. para- composed principally of mononuclear cells (Fig. 3E). pertussis-only mice on d 5+7) and KC (10- and 3-fold increased, compared with influenza or B. parapertussis alone), both of which Increased inflammation precedes increased bacterial burden in are potent neutrophil chemoattractants. We confirmed the ex- heterologous infection pression of these mediators by ELISA (Fig. 6B) and noted that, in agreement with our bead array data, MIP-2 but not KC was in- To investigate whether the increased inflammation observed in creased at the d 5+3 time point, when inflammation in heterolo- heterologously infected hosts on d 5+7 was a response to an in- gously infected mice was first beginning to rise. We extended our creased bacterial burden early in infection, mice were sacrificed 1 Downloaded from analysis and found that both MIP-2 and KC were significantly and 3 d postbacterial challenge. BAL inflammation was equivalent between heterologous and influenza-only mice on d 5+1, but on elevated in the BAL of heterologously infected mice on d 5+7 D d 5+3, heterologously infected mice had a greater pulmonary (Fig. 6 ). We did not detect systemic production of MIP-2 in the serum, and whereas B. parapertussis infection resulted in in- inflammatory response with significantly increased numbers of neutrophils but not mononuclear cells (Fig. 4A). Importantly, creased serum KC levels compared with naive animals, expression bacterial burdens were unchanged at both of these time points, was not significantly different between heterologous mice and B. parapertussis http://www.jimmunol.org/ indicating that exacerbated inflammation is not the result of an their -only controls (our unpublished data). This increased bacterial burden (Fig. 4B). Equivalent bacterial burdens pointed to locally produced MIP-2 as a potential cause for the early postinfection also suggested that the increased bacterial profound neutrophilia in heterologously infected mice, and we burdens in heterologously infected animals on d 5+7 and d 5+14 therefore considered it as a therapeutic target. are not the result of increased adherence or decreased muco- Critical role of MIP-2 in heterologous pulmonary inflammatory cilliary clearance early in infection. Increased inflammation did pathology not appear to be a response to increased viral burdens either, because viral burdens were equivalent between influenza-only To examine neutralization of MIP-2 as a therapeutic avenue in the and heterologously infected mice at both time points (Fig. 4C), treatment of the immunopathology that ensues following heter- by guest on October 3, 2021 despite significant reductions in type I IFN production in the BAL ologous infection, mice were infected with influenza and challenged at both time points (Fig. 4D). with B. parapertussis 5 d later. Commencing on d 5+3, a time point when heterologous mice can be differentiated from influenza-only Increased recruitment, not decreased apoptosis, drives pulmonary mice on the basis of weight loss and/or pulmonary inflammation, we inflammation in heterologously infected lungs administered a daily i.p. injection of neutralizing anti–MIP-2 Abs or Because type I IFNs play an important role in regulating in- control IgG. Anti–MIP-2 treatment resulted in a significant im- flammation through the induction of granulocyte apoptosis, we provement in animals’ clinical scores on d 5+7, as well as decreased questioned whether reduced type I IFNs might result in decreased weight loss over the course of treatment (Fig. 7A,7B). Neutraliza- neutrophil apoptosis and consequent accumulation of neutrophils tion of MIP-2 had no impact on bacterial burden in these mice but in the lungs of heterologously infected mice. No reductions were did significantly reduce pulmonary neutrophilia (Fig. 7C,7D), noted in the number of cells staining positive in the TUNEL re- pointing to inflammation as a cause of heterologous infection-reaction—a marker of late apoptosis at any time point examined lated illness, rather than simply a response to increased pathogen (Fig. 5A). To exclude the possibility that necrotic neutrophils were burdens. This beneficial impact did not appear to be mediated by accumulating in the lungs, neutrophil viability was assessed by 7- reductions in TNF-a production, because anti–MIP-2–treated, AAD exclusion; although a statistically significant increase was heterologously infected mice had similar levels of TNF-a in their detected in neutrophil death on d 5+7, the increase was small lungs on d 5+7 when compared with heterologously infected mice (,1.5% compared with influenza-only mice and ,1% compared treated with control rabbit IgG (38 6 10 versus 42 6 6 pg/ml; n =5 with B. parapertussis-only infected mice [Fig. 5B]). Concordantly, and 9, respectively). MIP-2 neutralization did not significantly at- MPO levels in lung homogenates on d 5+7 (Fig. 5C) reflected the tenuate pulmonary neutrophilia in mice infected with B. para- numbers of neutrophils observed in those lungs (Fig. 3B), sug- pertussis or influenza alone (data not shown), which is unsurprising, gesting that the neutrophils in the lungs of heterologously infected given the modesty of pulmonary neutrophilia that exists in these mice were indeed viable and producing effector molecules. animals on d 5+7. Because granulocyte chemotactic protein-2 and

heterologous- and influenza-only infected mice on d 5+7 to greatest decrease in expression. Each cell represents the mean of homogenates taken from four independent animals. B, Expression of KC (top panels) and MIP-2 (bottom panels) in lung homogenates as confirmed by ELISA on d 5+1, d 5+3, and d 5+7 (left to right) in mice given PBS or influenza as indicated, then challenged 5 d later with PBS (N)orB. parapertussis (n). C, Levels of KC (left) and MIP-2 (right) in the BAL 7 d postchallenge; n = 3–4/group. Data are expressed as the means 6 SEMs and are analyzed by two-way ANOVA. p, †, and ‡ denote p , 0.05 compared with PBS + PBS, PBS + B. parapertussis, and influenza + PBS-exposed mice, respectively. Two symbols denote p , 0.01 and three denote p , 0.001. ELISA data are representative of at least two independent experiments. 10 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION

A 8 B 021 §§ A 021 B 01 8 §§ n §§ 6 01 6 001 001

4 01 4 ore at Sacrifice at ore §§§ weight iginal c 08 08 (cfu/ml) 2 01 2 % of original weight % of or Lung bacterial burde 0 Clinical S Clinical 0 06 06 01 GgI tbr GgI 2-PIM-itna mureS 2RCXC-itna mureS a 2RCXC-itn GgI tbr GgI 2PIM-itna FIGURE 8. Immunoblockade of CXCR2 impairs clinical performance 81.0=P and antibacterial immunity during heterologous infection. Mice were given 8 6 C 01 D 5.1×0 001×05 an intranasal administration of inﬂuenza virus and then challenged intranasally 5 d later with B. parapertussis and treated i.p. with normal goat 01 6 01×8.3 6 serum (black symbols) or anti-CXCR2 (gray symbols). Increased weight loss (A) and bacterial burden (B) at the time of sacriﬁce (d 5+7) in mice 01 4 01×5.2 6 § treated with a daily administration of anti-CXCR2 commencing on d 5+3;

(cfu/ml) n = 10 mice/group. Data are expressed as the means 6 SEMs and are (cells/ml) 01 2 01×3.1 6 analyzed by two-way ANOVA. xx denotes p , 0.01 compared with in- BAL neutrophils

ung bacterial burden fluenza + B. parapertussis-exposed mice. L 01 0 0 Downloaded from GgI tbr GgI 2-PIM-itna tbr GgI 2-PIM-itna phlococcus aureus, and Haemophilus influenzae (5) are com- FIGURE 7. Therapeutic neutralization of MIP-2 reduces pulmonary mensal in humans but not in specific pathogen-free mice. They are neutrophilia and improves clinical presentation and weight loss in heterologously infected mice. A, Symptom scores on d 5+7 for mice given an cleared quite quickly from the murine respiratory tract and induce intranasal administration of influenza virus and then challenged in- substantial disease on their own, even without a concomitant viral tranasally 5 d later with B. parapertussis and treated beginning on d 5+3 infection, which makes them less than ideal models of this par- with a daily i.p. injection of control IgG (black symbols) or anti–MIP-2 ticular human pathology. To overcome this, we developed a model http://www.jimmunol.org/ (gray symbols). Mice were scored by a blinded investigator on a scale from of heterologous pulmonary infection in which mice were infected 0 (no distress) to 8 (severe distress). Each symbol represents an individual with influenza virus, then challenged 5 d later with the Gram- animal. B, Body weight change at the time of sacrifice on d 5+7 for het- negative bacterium B. parapertussis. B. parapertussis was chosen erologously infected mice given i.p. rabbit IgG (black) or anti–MIP-2 because at the dose given in this study, infection with B. para- (gray) commencing on d 5+3; n = 15 and 17 mice, respectively. C, pertussis on its own is a subclinical event that causes no apparent Equivalent lung bacterial burdens on d 5+7 in heterologously infected mice illness or weight loss and only minimal inflammation. An In- treated with IgG (black) or anti–MIP-2 (gray); n = 6 and 7 mice, respectively. D, Reduced neutrophilia on d 5+7 in the lungs of mice treated fluenza-Bordetella coinfection model also has clinical relevance because this combination is observed in the clinic (36, 37). with anti–MIP-2 compared with treatment with control IgG; n = 14 and 17 by guest on October 3, 2021 mice, respectively. Data are representative of at least two independent Moreover, the Bordetellae are challenging to culture from bi- experiments. Data are expressed as the means 6 SEMs and are analyzed ological specimens and difficult to discriminate from other highly by two-way ANOVA. x denotes p , 0.05 compared with influenza + B. similar cocci by traditional microbiological techniques. Thus, parapertussis-exposed mice. Two symbols denote p , 0.01 and three Bordetella infections may indeed account for some of the sec- denote p , 0.001. ondary bacterial pneumonia seen in pandemics. Lungs from mice heterologously infected with influenza virus KC (both of which are ELR+ neutrophil chemokines that share the and B. parapertussis were hemorrhagic on gross examination, CXCR2 receptor with MIP-2) were also elevated in heterologously whereas microscopic and flow cytometric examination revealed infected mice, we questioned whether blockading CXCR2, rather a BAL and tissue inflammation that was substantially greater than than just one ligand, might provide a more pronounced therapeutic would be anticipated based on the sum of the Bordetella- and effect. On the contrary, a blocking Ab against CXCR2 resulted in influenza-induced responses on their own. This inflammation was dramatically increased bacterial burdens and reduced weight re- composed principally of neutrophils 7-d post-Bordetella infection covery when administered on the same treatment regimen as the and mononuclear cells an additional week later. Viral burdens anti-MIP2 treatment (Fig. 8). In parallel experiments, administra- were unaffected by heterologous infection, and although bacterial tion of anti-CXCR2 to mice infected only with B. parapertussis burdens were increased late in the course of infection, they were resulted in similar significant impairments to bacterial clearance in within the range of inoculums we have delivered to naive animals the lungs (our unpublished data). without inducing any significant pathology (our unpublished data). Furthermore, we have observed that a primary infection with live Discussion B. parapertussis protects against subsequent influenza virus During natural human influenza infection, bacterial pneumonia challenge (our unpublished data), and others have similarly found rather than viral disease is the predominant cause of death. Notably, that exposure to bacterial ligands such as FimH (38) or CpG DNA the bacteria most commonly associated with these bacterial (39, 40) or viral ligands such as poly(I:C) (41) protects against superinfections are not pathogens on their own but are instead subsequent viral challenge. Whereas extracellular bacteria, bac- common, commensal microbes, which suddenly become prob- terial ligands, and viral ligands are all potent activators of innate lematic when superimposed over viral infections (5). Although immune responses and may provide local protection against a later several mechanisms have been proposed by which a viral infection viral challenge, infection with live intracellular pathogens have can predispose to bacterial infection, the mechanism by which been proposed to establish a disadvantageous environment for a commensal bacterium becomes a threat remains an understudied antibacterial host defense. Evidence suggests that viral infection area. The bacteria most commonly associated with natural cases of results in lysis of epithelial cells, exposing the relatively “sticky” bacterial superinfection such as Streptococcus pneumonia, Sta- basement membrane to which bacteria may adhere, and in The Journal of Immunology 11 upregulation of bacterial adhesion factors on surviving cells (16), phil depletion did not damage the antipneumococcal response any so we questioned whether early changes in bacterial burdens further than influenza infection already had (48). might drive this pulmonary neutrophilia. In contrast, we found no The question “what drives MIP-2 overexpression only in heter- evidence of an increased bacterial burden in the first 3 d post- ologously infected mice?” remains an important one. The pulmonary infection and instead noticed that inflammation arose prior to defenses must concern themselves not only with the prevention of differences in bacterial burdens. infection, but also with the avoidance of inflammatory disease which We go on to show that severe pulmonary inflammation correlated would compromise gas exchange. Consequently, a number of with increased production of a wide range of mediators but spe- mechanisms have evolved that maintain a high degree of ignorance cifically attribute it to an overproduction of the inflammatory and tolerance to foreign invaders. To shift the “immune rheostat” chemokine MIP-2/CXCL2/GRO-b. We demonstrate that thera- (reviewed in Ref. 49) toward a pathological state of inflammation like peutic immunoneutralization of MIP-2, even beginning 3 d post- that observed in our hands, these mechanisms must be overcome. challenge, is an effective therapy in heterologously infected mice, Recent work by Shahangian et al. (22) may provide a clue to explain which improves mice’s clinical presentation, weight recovery, and this phenomenon. They showed that mice with a genetic deficiency in neutrophilia, without altering bacterial burdens. By showing that the IFN-a receptor produce more KC and MIP-2 and mount more inflammatory immunopathology is the real threat to life in heter- robust inflammatory responses during viral-bacterial superinfection ologously infected hosts, we recapitulate a notion that has been than do wild-type controls (22). In that regard, one of our most im- proposed in prior influenza pandemics and the present H1N1 swine portant findings is that heterologously infected mice have reduced influenza outbreak (42). Importantly, anti–MIP-2 was also given to pulmonary IFN titers compared with influenza-only mice. Because influenza- or B. parapertussis-only mice without apparent delete- early reductions in type I IFNs were noted in heterologously infected rious effect. In contrast, blockade of the entire CXCR signaling axis animals, this may have permitted the later increases in MIP-2 ex- Downloaded from with an antagonistic anti-CXCR2 resulted in dramatically impaired pression. One notable difference between Shahangian’s study and antibacterial immunity in bacteria-only and heterologous mice, as ours was that in their model, acute influenza infection attenuated well as reduced weight recovery in heterologously infected ones. inflammatory responses to bacterial challenge, rather than exacer- Others have made analogous findings. In a model of pulmonary bating it, so the administration of MIP-2 and KC, rather than their Pseudomonas aeruginosa infection, CXCR2 blockade attenuated removal, was used as a therapy. Although Didierlaurent et al. (19) pulmonary neutrophilia but impaired bacterial clearance and re- have shown a similar phenomenon, which begins weeks and con- http://www.jimmunol.org/ duced survival, whereas neutralization of MIP-2 significantly re- tinues for months after the resolution of influenza infection, to our duced neutrophilia, without compromising host defense (43). knowledge, reduced lung inflammation is not a feature of acute In other models of severe lung pathology, more complete in- bacterial superinfections of influenza in humans. In such cases, terference with the CXCR2 signaling axis has shown a positive a surfeit of inflammation, rather than a deficit, is commonly noted effect; genetic deletion or immunoneutralization of CXCR2 or its and is implicated in disease pathogenesis. ligands attenuates the pathology of ventilator-induced lung injury That such an intense and prolonged inflammatory response is (VILI) in mice (44). VILI is an aseptic cause of acute respiratory incapable of clearing the pulmonary bacterial load is an intriguing distress syndrome (ARDS), a disease that kills 26–44% of those it observation. Although neutrophils are required for B. parapertussis by guest on October 3, 2021 afflicts (45, 46). VILI-minimizing strategies reduce mortality in clearance (27), it is important to note that influenza infection may ARDS by minimizing CXCR2 signaling, reducing neutrophil re- impair neutrophil function to the extent that even partial depletion cruitment and neutrophillic injury (44, 47). In contrast, in cases of of neutrophils from influenza-infected mice does not further ARDS resulting from infectious causes such as heterologous damage antibacterial host defense (48). In addition, AMs are pneumonia, the merits of preventing immunopathology must be second only to respiratory epithelial cells as a target for influenza, weighed against the potentially deleterious effects of dampening and direct viral infection may further compromise their antibac- the antiviral and antibacterial host defense responses. We find that terial function, as has been reported in other models (20, 24). although overexpression of MIP-2 may drive inflammatory im- Moreover, the presence of neutrophils, whether functional or not, munopathology, clearly some signaling through CXCR2 is re- in the influenza-infected airway at the time of bacterial challenge quired for adequate antibacterial immunity. is a key difference between bacterial-only and heterologously Taken together, the disparity between our findings with MIP-2 infected mice. These cells must be cleared by AMs, which also neutralization and CXCR2 blockade suggest important pathological play a crucial role in the early pulmonary antibacterial response. and protective roles for CXCR2 ligands in pulmonary host defense. Efferocytosis has been shown to compromise their antibacterial Clinically, BAL levels of the CXCR ligands IL-8 and KC correlate function by inducing the release and autocrine activity of PGE2 with the severity of pulmonary neutrophilia in ARDS and pneu- (50), so having to clear the influenza-associated neutrophilia may monia (47). In this regard, we show that KC is highly expressed in place these macrophages at an immediate disadvantage when next heterologously infected mice, as is another CXCR2 ligand, gran- tasked with clearing a bacterial infection. A self-reinforcing vi- ulocyte chemotactic protein-2. When MIP-2 is neutralized, these cious circle may then arise, in which ever-increasing recruitment and other CXCR2 ligands, as well as CCR ligands (which have actually impairs resident antibacterial host defense cells, until evolved in mice to compensate for a species-wide deficiency in ultimately the adaptive immune system arrives to clear the path- CXCR1), may partially overcome the loss of MIP-2 and allow ogen. That the formation of immune memory was not sub- coordination of a response, which is effective for host defense, but stantially impacted by heterologous infection is consistent with does not cause immunopathology. In our model of heterologous the finding that survivors of the 1918 influenza pandemic, many of infection, the bacterial pathogen used does not cause major disease whom presumably suffered from bacterial superinfections of their on its own. Whether neutralization of MIP-2 will provide benefit in influenza, demonstrate robust B cell responses against influenza models of heterologous infection that use more virulent pathogens Ags even some 80 y later (51). The key issue seems to be that the such as S. pneumonia remains to be seen; there is admittedly a risk host must survive the initial pneumonia for long enough to arm that attenuating pulmonary neutrophilia might unacceptably impair and use the adaptive arm of immunity. host defense. Although we cannot wholly discount this possibility, It has been commonly stated that the “Spanish” influenza virus, in a mouse model of heterologous infection, Ab-mediated neutro- which swept the globe in 1918–1919, was remarkably lethal. Elegant 12 ROLE OF MIP-2 IN INFLUENZA SUPERINFECTION work by McAuley et al. (6) has recently called that notion into 8. McCullers, J. A. 2004. Effect of antiviral treatment on the outcome of secondary bacterial pneumonia after influenza. J. Infect. 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