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• W2478651599 abstract "Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI−/−IL17RA−/− (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA−/− mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus. Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI−/−IL17RA−/− (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA−/− mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus. The complex interplay between the effector program of Th1 cells, controlled by interferon (IFN)-γ signaling, and the Th17 program predominated by IL-17 signaling, remains an active area of investigation. To investigate the relation between IFN-γ and IL-17A in our mouse model of autoimmune heart disease, we generated double-knockout mutants lacking both IFN-γ and IL-17A and reported that these mice develop a particularly severe, fatal eosinophilic form of autoimmune heart disease bearing hallmarks of Th2 deviation.1Barin J.G. Baldeviano G.C. Talor M.V. Wu L. Ong S. Fairweather D. Bedja D. Stickel N.R. Fontes J.A. Cardamone A.B. Zheng D. Gabrielson K.L. Rose N.R. Cihakova D. Fatal eosinophilic myocarditis develops in the absence of IFN-gamma and IL-17A.J Immunol. 2013; 191: 4038-4047Crossref PubMed Scopus (40) Google Scholar As part of these studies, we set out to concurrently delete the cognate receptors for these cytokines—IFNγRI and IL17RA, respectively. In the process of establishing IFNγRI−/−IL17RA−/− (GRAKO) double-knockout mice, we found that these mice began spontaneously developing oral abscesses which, on testing, were determined to be uncontrolled infections with Staphylococcus aureus. S. aureus is a Gram-positive member of the Firmicutes phylum, commonly found at cutaneous and mucosal surfaces in healthy mammalian hosts as a commensal organism. However, acquisition of virulence factors or immune compromise of the host can lead to opportunistic invasion and infection.2Lowy F.D. Staphylococcus aureus infections.N Engl J Med. 1998; 339: 520-532Crossref PubMed Scopus (4599) Google Scholar Patient infections with S. aureus in hospital and community settings are a growing problem, particularly given the ongoing emergence of antibiotic resistance.3Tong S.Y. Davis J.S. Eichenberger E. Holland T.L. Fowler Jr., V.G. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.Clin Microbiol Rev. 2015; 28: 603-661Crossref PubMed Scopus (2379) Google Scholar Clear roles for IL-17 signaling and associated pathways in staphylococcal infections have been well established in experimental infection models.4Miller L.S. Cho J.S. Immunity against Staphylococcus aureus cutaneous infections.Nat Rev Immunol. 2011; 11: 505-518Crossref PubMed Scopus (282) Google Scholar In these studies, expression of IL-17 by cutaneous γδ cells was essential in supporting rapid local recruitment of neutrophils, which in turn supported Th17 differentiation through IL-1β, forming a positive feed-forward circuit at the site of infection.5Cho J.S. Pietras E.M. Garcia N.C. Ramos R.I. Farzam D.M. Monroe H.R. Magorien J.E. Blauvelt A. Kolls J.K. Cheung A.L. Cheng G. Modlin R.L. Miller L.S. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice.J Clin Invest. 2010; 120: 1762-1773Crossref PubMed Scopus (478) Google Scholar, 6Cho J.S. Guo Y. Ramos R.I. Hebroni F. Plaisier S.B. Xuan C. Granick J.L. Matsushima H. Takashima A. Iwakura Y. Cheung A.L. Cheng G. Lee D.J. Simon S.I. Miller L.S. Neutrophil-derived IL-1beta is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.PLoS Pathog. 2012; 8: e1003047Crossref PubMed Scopus (165) Google Scholar However, unambiguous protective roles for IFN-γ from S. aureus have not been as clearly delineated, in part because many of these studies preceded the Th17 paradigm.7Nakane A. Okamoto M. Asano M. Kohanawa M. Minagawa T. Endogenous gamma interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice.Infect Immun. 1995; 63: 1165-1172Crossref PubMed Google Scholar, 8Sasaki S. Nishikawa S. Miura T. Mizuki M. Yamada K. Madarame H. Tagawa Y.I. Iwakura Y. Nakane A. Interleukin-4 and interleukin-10 are involved in host resistance to Staphylococcus aureus infection through regulation of gamma interferon.Infect Immun. 2000; 68: 2424-2430Crossref PubMed Scopus (76) Google Scholar, 9Sasaki S. Tagawa Y. Iwakura Y. Nakane A. The role of gamma interferon in acquired host resistance against Staphylococcus aureus infection in mice.FEMS Immunol Med Microbiol. 2006; 46: 367-374Crossref PubMed Scopus (17) Google Scholar, 10Zhao Y.X. Tarkowski A. Impact of interferon-gamma receptor deficiency on experimental Staphylococcus aureus septicemia and arthritis.J Immunol. 1995; 155: 5736-5742PubMed Google Scholar The spontaneous staphylococcal disease of our IFNγRI−/−IL17RA−/− model pointed us toward a novel role for IFN-γ in this host defense process with a requisite interaction with IL-17–dependent signaling. The purpose of this work was to first characterize the disease of IFNγRI−/−IL17RA−/− double-knockout mice and then to examine their immunologic phenotype and function to elucidate the mechanisms underlying the severe spontaneous oral infections of IFNγRI−/−IL17RA−/− mice. From these studies, we find evidence for novel collaborative interactions between neutrophils and macrophages, as effectors of immunity driven by IL-17 and IFN-γ signaling, respectively. Wild-type BALB/cJ mice and founder IFNγRI−/− BALB/c mice were commercially sourced (The Jackson Laboratory, Bar Harbor, ME). IL17RA−/− BALB/c founder mice were the generous provision of Amgen (Thousand Oaks, CA), through Dr. Jay Kolls (University of Pittsburgh, Pittsburgh, PA). To establish the IFNγRI−/−IL17RA−/− double-knockout strain, IFNγRI−/− and IL17RA−/− mice were intercrossed for one generation. [F1] hybrid progeny were further backcrossed to the IFNγRI−/− background to fix the Ifngr1 locus at homozygosity of the knockout allele, then intercrossed to generate IFNγRI−/−IL17RA−/− double-knockouts. Following emergence of the disease phenotype, the colony was maintained at hemizygosity at the Il17ra locus to limit disease among breeders. For some experiments, Thy1.1+ BALB/c congenic mice were substituted for wild-type controls, and IL17RA−/−CD45.1+ mice for IL17RA−/− mice. All mice were maintained in the Johns Hopkins University School of Medicine specific pathogen–free vivarium. Experiments were conducted in compliance with the Animal Welfare Act and the principles set forth in the Guide for the Care and Use of Laboratory Animals.11Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar All methods and protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University. For mononuclear phagocyte system (MPS) depletions, clodronate- and phosphate-buffered saline-loaded liposomes (ClondronateLiposomes.com, Amsterdam, the Netherlands) were administered intraperitoneally, 200 μL per mouse, twice per week, for up to 6 weeks. For neutrophil depletions, IFNγRI−/− mice were administered 500 μg of anti-Ly6G monoclonal antibody (mAb) 1A8 (or isotype control rat IgG2a mAb 2A3; Bio X-Cell, West Lebanon, NH) intraperitoneally on days 0 and 2, followed by 250 μg maintenance doses b.i.d. for 4 weeks. Pathology, cytometry, and microbiology were performed at termination of the experiment. Tissues were fixed in SafeFix (Thermo Fisher Scientific, Waltham, MA) and embedded, and 5-μm step sections were cut and stained with hematoxylin and eosin (H&E), or Brown & Brenn (Histoserv, Germantown, MD). Scoring was based on a weighted composite score of six criteria, each graded on a 0 to 4 scale: presence of abscess, bacteria, infiltration with polymorphonuclear or mononuclear cells, and inflammatory involvement of epidermis or dermis—the first two weighted doubly, then added, resulting in a 1 to 32 scale. Oral tissues were digested in 10 mg Liberase TL (Roche, Basel, Switzerland), 100 mg Dispase (Roche), 100 mg collagenase II (Worthington Biochemical Corporation, Lakewood, NJ), and 0.1 mg/mL DNase I (Worthington Biochemical Corporation) for 90 minutes at 37°C, followed by suspension in gentle MACS C Tubes (Miltenyi Biotec, Gladbach, Germany). Lymph node (LN) cells and splenocytes were also extracted into single-cell suspension, and red blood cells were lyzed by <5 minutes of incubation in ammonium-chloride-potassium lysis buffer (Quality Biological, Gaithersburg, MD). Before surface staining, viability was determined by LIVE/DEAD staining according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Cells were washed, and FcγRII/III was blocked with αCD16/32 mAb 93 (eBioscience, San Diego, CA). Surface markers were stained with fluorochrome-conjugated mAbs: CD11b mAb M1/70, CD45RB mAb C363-16A, Siglec F mAb E50-2440 (BD Biosciences, San Jose, CA); CD4 mAb RM4-5, CD8α mAb 53-6.7, CD54/ICAM1 mAb YN1/1.7.4, CD62L mAb MEL-14, CD64 mAb X54-5/7.1, CD183/CXCR3 mAb CXCR3-173, CD206/MφMR mAb C068C2, FcεRIα mAb MAR1, Ly6C mAb HK1.4, Ly6G mAb 1A8 (BioLegend, San Diego, CA); CD3ε mAb 145-2C11, CD44 mAb IM7, F4/80 mAb BM8, TCRβ mAb H57-597, TCRγδ mAb GL3 (eBioscience); CD45 mAb MCD4517, and CD45R/B220 mAb RA3-6B2. After washing, cells were fixed (BD Biosciences), or for intracellular, intranuclear staining with RORγt mAb Q31-378 (BD Biosciences), fixed and permeabilized with transcription factor-specialized buffers (eBioscience). Samples were acquired on a four laser custom LSR II cytometer running FACSDiva 6.0 (BD Immunocytometry, San Jose, CA). Data were analyzed with FlowJo version 10.8 (TreeStar Software, Ashland, OR). Superficial cervical LNs (scLNs) were resected, made into single-cell suspensions, and washed generously. LN cells (2 × 106) were plated in complete Dulbecco's modified Eagle's medium with 2.5 × 106 heat-killed S. aureus (InvivoGen, San Diego, CA). Supernatants were collected after 48 hours and stored at −80°C before interrogation by Linco multiplex cytokine assays (Millipore, Billerica, MA) or enzyme-linked immunosorbent assay (ELISA). Tissues were snap-frozen, stored at −80°C, homogenized in minimal essential medium (MEM) + 2% fetal bovine serum FBS, and stored at −80°C until used in ELISA or Linco assays. Homogenate cytokine levels were normalized to wet sample weights. Linco multiplex cytokine assays (Millipore) were used according to the manufacturer's instructions and acquired on a Luminex (Austin, TX) xMAP reader. Total serum IgG and IgE (BD Biosciences) and mouse myeloperoxidase (Abcam, Cambridge, MA) were determined by quantitative sandwich ELISA against standard curves, according to manufacturers' instructions. Oral swabs were obtained with Copan eSwab kits and were subjected to broth-enrichment culture for Staphylococcus spp. as previously described.12Davis M.F. Baron P. Price L.B. Williams D.L. Jeyaseelan S. Hambleton I.R. Diette G.B. Breysse P.N. McCormack M.C. Dry collection and culture methods for recovery of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains from indoor home environments.Appl Environ Microbiol. 2012; 78: 2474-2476Crossref PubMed Scopus (18) Google Scholar Isolates from these swabs were subjected to species identification and strain characterization by PCR.13Garcia-Alvarez L. Holden M.T. Lindsay H. Webb C.R. Brown D.F. Curran M.D. Walpole E. Brooks K. Pickard D.J. Teale C. Parkhill J. Bentley S.D. Edwards G.F. Girvan E.K. Kearns A.M. Pichon B. Hill R.L. Larsen A.R. Skov R.L. Peacock S.J. Maskell D.J. Holmes M.A. Methicillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study.Lancet Infect Dis. 2011; 11: 595-603Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 14Shopsin B. Gomez M. Montgomery S.O. Smith D.H. Waddington M. Dodge D.E. Bost D.A. Riehman M. Naidich S. Kreiswirth B.N. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains.J Clin Microbiol. 1999; 37: 3556-3563Crossref PubMed Google Scholar, 15Sasaki T. Tsubakishita S. Tanaka Y. Sakusabe A. Ohtsuka M. Hirotaki S. Kawakami T. Fukata T. Hiramatsu K. Multiplex-PCR method for species identification of coagulase-positive staphylococci.J Clin Microbiol. 2010; 48: 765-769Crossref PubMed Scopus (290) Google Scholar, 16Zhang K. McClure J.A. Elsayed S. Louie T. Conly J.M. Novel multiplex PCR assay for simultaneous identification of community-associated methicillin-resistant Staphylococcus aureus strains USA300 and USA400 and detection of mecA and Panton-Valentine leukocidin genes, with discrimination of Staphylococcus aureus from coagulase-negative staphylococci.J Clin Microbiol. 2008; 46: 1118-1122Crossref PubMed Scopus (95) Google Scholar, 17Poyart C. Quesne G. Boumaila C. Trieu-Cuot P. Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target.J Clin Microbiol. 2001; 39: 4296-4301Crossref PubMed Scopus (242) Google Scholar For in vitro killing assay, primary bone marrow neutrophils isolated by paramagnetic negative selection (Miltenyi Biotec) were co-cultured for up to 4 hours with live S. aureus at MOI = 1. Total colony counts of water-lyzed wells from each time point were plated for enumerating colony-forming units. Statistical analyses were performed on multiple group comparisons as described in the figure legends (GraphPad Prism version 6.0, San Diego, CA; StatPlus:mac Pro; AnalystSoft, Walnut, CA; Microsoft Excel 2016 version 15.24, Microsoft, Redmond, WA). We observed periocular blepharitis developing in our IL17RA−/− colony with varying penetrance, beginning at roughly 3 to 4 months of age (Figure 1, A and E). Similar phenotypes have been described in patients with defects in IL17RA or Act1.18Puel A. Cypowyj S. Bustamante J. Wright J.F. Liu L. Lim H.K. Migaud M. Israel L. Chrabieh M. Audry M. Gumbleton M. Toulon A. Bodemer C. El-Baghdadi J. Whitters M. Paradis T. Brooks J. Collins M. Wolfman N.M. Al-Muhsen S. Galicchio M. Abel L. Picard C. Casanova J.L. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity.Science. 2011; 332: 65-68Crossref PubMed Scopus (783) Google Scholar, 19Boisson B. Wang C. Pedergnana V. Wu L. Cypowyj S. Rybojad M. Belkadi A. Picard C. Abel L. Fieschi C. Puel A. Li X. Casanova J.L. An ACT1 mutation selectively abolishes interleukin-17 responses in humans with chronic mucocutaneous candidiasis.Immunity. 2013; 39: 676-686Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar Veterinary clinical calls from the Research Animal Resources division of Johns Hopkins University School of Medicine had alerted us to the presence of facial growths developing in our newly established IFNγRI−/−IL17RA−/− mouse colony (Figure 1, B–F). Over time, the abscesses of IFNγRI−/−IL17RA−/− mice led to cachexia (Figure 1G), presumably because the abscesses interfered with normal feeding. IFNγRI−/−IL17RA−/− mice further demonstrated splenomegaly at the time of sacrifice compared with controls (Figure 1H), probably an indicator of their uncontrolled spontaneous infection. The time course of onset for the oral abscesses is depicted in Figure 1I. IFNγRI−/−IL17RA−/− mice began manifesting gross lesions between 6 to 10 weeks of age, increasing in severity over time. Comparing the slope of this regression with the control genotypes demonstrated significant progression of oral lesions in the IFNγRI−/−IL17RA−/− strain (P = 0.0015), in a manner not shared by the progression of the orbital lesions (not shown). At ages >20 weeks, abscesses were often severe enough to warrant euthanasia of IFNγRI−/−IL17RA−/− mice on consultation with veterinarians. Both male and female mice were affected with comparable incidence and severity (Supplemental Figure S1, A and B) and time course (not shown). Methicillin-susceptible S. aureus was isolated and PCR-confirmed from the oral mucosa of nine of nine symptomatic mice tested (Figure 1J).12Davis M.F. Baron P. Price L.B. Williams D.L. Jeyaseelan S. Hambleton I.R. Diette G.B. Breysse P.N. McCormack M.C. Dry collection and culture methods for recovery of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains from indoor home environments.Appl Environ Microbiol. 2012; 78: 2474-2476Crossref PubMed Scopus (18) Google Scholar, 15Sasaki T. Tsubakishita S. Tanaka Y. Sakusabe A. Ohtsuka M. Hirotaki S. Kawakami T. Fukata T. Hiramatsu K. Multiplex-PCR method for species identification of coagulase-positive staphylococci.J Clin Microbiol. 2010; 48: 765-769Crossref PubMed Scopus (290) Google Scholar All isolates were negative for mecA/C, USA300, and luk-PV genes.16Zhang K. McClure J.A. Elsayed S. Louie T. Conly J.M. Novel multiplex PCR assay for simultaneous identification of community-associated methicillin-resistant Staphylococcus aureus strains USA300 and USA400 and detection of mecA and Panton-Valentine leukocidin genes, with discrimination of Staphylococcus aureus from coagulase-negative staphylococci.J Clin Microbiol. 2008; 46: 1118-1122Crossref PubMed Scopus (95) Google Scholar, 20Bae I.G. Tonthat G.T. Stryjewski M.E. Rude T.H. Reilly L.F. Barriere S.L. Genter F.C. Corey G.R. Fowler Jr., V.G. Presence of genes encoding the panton-valentine leukocidin exotoxin is not the primary determinant of outcome in patients with complicated skin and skin structure infections due to methicillin-resistant Staphylococcus aureus: results of a multinational trial.J Clin Microbiol. 2009; 47: 3952-3957Crossref PubMed Scopus (78) Google Scholar In a subset of three confirmed methicillin-susceptible S. aureus isolates selected for testing, all were scn positive and were spa-type t774. Although the identified strain did not match the dominant human community clone (PVL+ USA300) circulating in the United States, the presence of the scn gene in tested isolates indicates likely human origin and not a mouse-adapted strain.21Holtfreter S. Radcliff F.J. Grumann D. Read H. Johnson S. Monecke S. Ritchie S. Clow F. Goerke C. Broker B.M. Fraser J.D. Wiles S. Characterization of a mouse-adapted Staphylococcus aureus strain.PLoS One. 2013; 8: e71142Crossref PubMed Scopus (48) Google Scholar We further found expression of superoxide dismutase A (sodA) by quantitative real-time PCR from bulk genomic DNA in oral tissues (Figure 1K). Single-knockout IL17RA−/− and IFNγRI−/− controls, as well as wild-type mice, lacked similar pathology (Figure 1, E–K) (representative gross pathology for controls is not shown). Together, these data support that defects of both IFN-γ and IL-17 signaling pathways are essential contributors to the disease pathology of the IFNγRI−/−IL17RA−/− strain. Moreover, these pathways were collaborative in a manner in which their combined defect was sufficient for opportunistic infection. We examined net Ig levels in the sera of these mice, as general markers of immune status, but more specifically, due to previous evidence of Th2 deviation in our investigations of IFNγ−/−IL17A−/− mice.1Barin J.G. Baldeviano G.C. Talor M.V. Wu L. Ong S. Fairweather D. Bedja D. Stickel N.R. Fontes J.A. Cardamone A.B. Zheng D. Gabrielson K.L. Rose N.R. Cihakova D. Fatal eosinophilic myocarditis develops in the absence of IFN-gamma and IL-17A.J Immunol. 2013; 191: 4038-4047Crossref PubMed Scopus (40) Google Scholar By absolute quantitation through sandwich ELISA methods, we observed elevated serum IgG levels in IFNγRI−/−IL17RA−/− and IL17RA−/− mice (Figure 1L). However, quantitation of serum Ig demonstrated specific elevation of total IgE only in IFNγRI−/−IL17RA−/−mice (Figure 1M). From these data, we conclude that spontaneous staphylococcal infection of IFNγ−/−IL17A−/− mice recapitulates the susceptibility of hyper IgE patients to staphylococcal infection, possibly presenting a novel model of immunodeficiency processes in that disease. Histopathologic examination of oral tissues in control wild-type or IFNγRI−/− mice revealed no or limited oral inflammation (Figure 2, A, B, E and F). IL17RA−/− control mice, demonstrated signs of inflammation in the dermis that appeared to be consistent with folliculitis (Figure 2, C and G). The oral abscesses in IFNγRI−/−IL17RA−/− mice demonstrated extensive infiltration with mononuclear and polymorphonuclear cells, generally surrounding large heterogeneous eosin-staining material featuring large filamentous hematoxylin-staining inclusions (Figure 2, D and H). Generally, lesions appeared to be more associated with the oral mucosa of these tissues than the keratinaceous epidermal layers. Quantitative, subjective measurement of the histopathologic severity of these abscesses supported severely uncontrolled infection of oral tissues in IFNγRI−/−IL17RA−/− mice (Figure 2I). Brown & Brenn Gram-staining of affected oral tissues in IFNγRI−/−IL17RA−/− mice revealed clusters of Gram-positive cocci deep within the abscess (Figure 2, J–L). These clusters also stained positive with Grocott's metheneamine silver (not shown). Serial section staining with H&E indicated these Gram-positive bacterial clusters were immediately bound by extensive polymorphonuclear and mononuclear inflammatory infiltrates (Figure 2, N–P). In addition, diffuse, isolated Gram-staining cocci could be seen elsewhere in the inflamed region (Figure 2M), further indicative of insufficient control of bacterial invasion and outgrowth in the oral mucosa of IFNγRI−/−IL17RA−/− mice. We expanded our histopathologic analysis to other tissues, to determine whether staphylococcal infection had trafficked to other tissues or become systemic. We did not observe clear signs of disseminated infection in lung, liver, or spleen (Supplemental Figure S1, C–G). In mice with highly advanced severe oral lesions, we observed limited periportal hepatic inflammatory foci (Supplemental Figure S1, C and D) and pyogranulomatous infiltrates in lung (Supplemental Figure S1G). These signs of advancing systemic inflammation or disseminating infection generally appeared only in older mice with more severe oral pathology. We interpreted the involvement of these organs as spillover from severe oral infections. In addition to the perioral lesions, we also observed smaller purulent abscesses developing in the forelimbs of IFNγRI−/−IL17RA−/− mice (Supplemental Figure S1, H–M) and none of the control genotypes (not shown). Generally, these forelimb manifestations were not as prevalent, appearing largely in older mice with more severe perioral abscesses. Forelimb abscesses were often bilateral, but not necessarily symmetric. Histopathologic examination revealed Gram-positive abscesses developed in the subdermis, without invasion into the underlying musculoskeletal structures or synovia. It should also be noted that these lesions do not appear in our colony of IFNγ−/−IL17A−/− mice,1Barin J.G. Baldeviano G.C. Talor M.V. Wu L. Ong S. Fairweather D. Bedja D. Stickel N.R. Fontes J.A. Cardamone A.B. Zheng D. Gabrielson K.L. Rose N.R. Cihakova D. Fatal eosinophilic myocarditis develops in the absence of IFN-gamma and IL-17A.J Immunol. 2013; 191: 4038-4047Crossref PubMed Scopus (40) Google Scholar indicating that an IL-17 family member other than IL-17A is the necessary ligand in native defense to S. aureus invasion in the oral mucosa. We used cytometric methods to interrogate the infiltrate of affected perioral tissues, with particular attention toward effector populations. Clear infiltration of these tissues by CD45+ leukocytes was evident in IFNγRI−/−IL17RA−/− mice (Figure 3A). Consistent with histopathologic data, there was also evidence of mild inflammatory infiltration of perioral tissues in IL17RA−/− mice, although not to the extreme degree of IFNγRI−/−IL17RA−/− mice. By far, the most abundant infiltrating leukocyte was Ly6Ghi neutrophils (Figure 3B), consistent with the extensive polymorphonuclear infiltrates observable by histopathology. Other granuloid cell types, including Siglec F+ eosinophils (Figure 3C) and FcεRIα+ mast cells and basophils (Figure 3D), were also elevated in the affected oral tissues of IFNγRI−/−IL17RA−/− mice, although not to the same extreme degree as neutrophils and not significantly in comparison with IL17RA−/− controls. The extensive neutrophil infiltration of infected oral tissues of IFNγRI−/−IL17RA−/− mice was at odds with well-described roles for neutrophils in control of experimental infection with S. aureus.6Cho J.S. Guo Y. Ramos R.I. Hebroni F. Plaisier S.B. Xuan C. Granick J.L. Matsushima H. Takashima A. Iwakura Y. Cheung A.L. Cheng G. Lee D.J. Simon S.I. Miller L.S. Neutrophil-derived IL-1beta is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.PLoS Pathog. 2012; 8: e1003047Crossref PubMed Scopus (165) Google Scholar, 22Kim M.H. Granick J.L. Kwok C. Walker N.J. Borjesson D.L. Curry F.R. Miller L.S. Simon S.I. Neutrophil survival and c-kit(+)-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.Blood. 2011; 117: 3343-3352Crossref PubMed Scopus (82) Google Scholar The abundance of neutrophils despite ongoing infection suggested functional defects (Figure 3, E–H). To address the role of neutrophils in native oral defense to S. aureus, IFNγRI−/− mice were depleted of neutrophils by extended treatment over 4 weeks with the mAb 1A8. At the end of this neutrophil depletion course, mild subdermal inflammation was observed (Supplemental Figure S2), resembling the mild inflammation observed in IL17RA−/− controls. As an indicator of neutrophil function in the affected perioral tissues, we interrogated homogenates of cheek tissues by quantitative sandwich ELISA for the canonical neutrophil granule product myeloperoxidase (MPO). Like many of the other cytokine analytes (Supplemental Figure S3), MPO levels were highest in IFNγRI−/−IL17RA−/− mice (Figure 3I), a probable indicator of persistent infection. These data were normalized to sample weight, similar to the cytometric analyses presented in Figure 3B. Because of this common normalization factor, we could approximate a ratio of MPO levels to neutrophil numbers—as an indicator of how much MPO each neutrophil might be producing in situ. From this calculation, we observed diminished per-cell equivalents of MPO in the oral tissues of IFNγRI−/−IL17RA−/− and IL17RA−/− mice (Figure 3J). Experimental in vitro co-culture assays with live S. aureus (Figure 3K) did not demonstrate defective killing with IFNγRI−/−IL17RA−/− bone marrow neutrophils. Altogether, these data support that IL-17 signaling predominates control of neutrophilic recruitment and function in host defense to Staphylococcus. Neutrophil recruitment is a canonical function for IL-17 signaling, but it fails to explain the involvement of IFN-γ in defense against Staphylococcus, or why IFNγRI−/−IL17RA−/− mice progress to uncontrolled infection. To better understand the mechanism underlying the failure of IFNγRI−/−IL17RA−/− mice to control staphylococcal infection, we examined the expression of various cytokine and chemokine mediators by interrogating homogenates of perioral tissues by multiplex cytokine array. By and large, most cytokine analytes were highest in the IFNγRI−/−IL17RA−/− mice (Supplemental Figure" @default.
• W2478651599 created "2016-08-23" @default.
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• W2478651599 date "2016-09-01" @default.
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• W2478651599 title "Collaborative Interferon-γ and Interleukin-17 Signaling Protects the Oral Mucosa from Staphylococcus aureus" @default.
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