IL-10 Restrains IL-17 to Limit Lung Pathology Characteristics following Pulmonary Infection with Francisella tularensis Live Vaccine Strain

• DOI: 10.1016/j.ajpath.2013.07.008
• Publication Date: 2013-09-03T07:46:38Z
• Authors: Samantha R. Slight; Leticia Monin; Radha Gopal; Lyndsay Avery; Marci Davis; Hillary Cleveland; Tim D. Oury; Javier Rangel-Moreno; Shabaana A. Khader
• Keywords: Tularemia; Attenuated vaccine; Francisella; Strain (injury); Intracellular parasite

IL-10 production during intracellular bacterial infections is generally thought to be detrimental because of its role in suppressing protective T-helper cell 1 (Th1) responses. Francisella tularensis a facultative bacterium that activates both Th1 and Th17 immune Herein, we report IL-10–deficient mice (Il10−/−), despite having increased responses, exhibit mortality after pulmonary infection with F. live vaccine strain. We demonstrate the observed Il10−/−-infected due exacerbated IL-17 causes neutrophil recruitment associated lung pathology. Thus, although required for immunity against strain, tightly regulated by generate efficient induction without mediating These data suggest critical maintaining delicate balance between host pathology tularensis, bacterium, infectious nature severe disease caused low doses airborne bacteria, has been classified as category A select bioterrorism agent.1Dennis D.T. Inglesby T.V. Henderson D.A. Bartlett J.G. Ascher M.S. Eitzen E. Fine A.D. Friedlander A.M. Hauer J. Layton M. Lillibridge S.R. McDade J.E. Osterholm M.T. O'Toole T. Parker G. Perl T.M. Russell P.K. Tonat K. Tularemia biological weapon: medical public health management.JAMA. 2001; 285: 2763-2773Crossref PubMed Scopus (1198) Google Scholar Infection humans two main subspecies, (type A) holarctica B).2Conlan J.W. Vaccines tularensis–past, present future.Expert Rev Vaccines. 2004; 3: 307-314Crossref (36) An strain (LVS) developed from B an experimental vaccine, but not licensed use humans.1Dennis LVS used representative attenuated model address requirements protection Francisella. By using this model, importance IL-12 driving interferon γ (IFN-γ) responses well described.3Anthony L.S. Ghadirian Nestel F.P. Kongshavn P.A. The requirement gamma resistance tularemia.Microb Pathog. 1989; 7: 421-428Crossref (84) Scholar, 4Elkins K.L. Cooper A. Colombini S.M. Cowley S.C. Kieffer T.L. In vivo clearance LVS, dependent on p40 subunit interleukin-12 (IL-12) p70.Infect Immun. 2002; 70: 1936-1948Crossref (117) 5Duckett N.S. Olmos S. Durrant D.M. Metzger D.W. Intranasal treatment respiratory strain.Infect 2005; 73: 2306-2311Crossref (82) contrast, play extracellular, intracellular, pathogens.6Kolls J.K. Khader S.A. cytokines primary mucosal immunity.Cytokine Growth Factor Rev. 2010; 21: 443-448Abstract Full Text PDF (141) However, others recently identified cellular infection,7Lin Y. Ritchea Logar Slight Messmer Rangel-Moreno Guglani L. Alcorn J.F. Strawbridge H. Park Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Kolls Interleukin-17 T helper pathogen tularensis.Immunity. 2009; 31: 799-810Abstract (237) 8Cowley Meierovics A.I. Frelinger J.A. Elkins Lung CD4- CD8- double-negative cells are prominent producers IL-17A IFN-gamma murine strain.J Immunol. 184: 5791-5801Crossref (89) 9Markel Bar-Haim Zahavy Cohen O. Shafferman Velan B. involvement response sub-lethal inhalational tularensis.PLoS One. 5: e11176Crossref (37) IFN-γ through induction.7Lin proinflammatory cytokine also known induce chemokines, such keratinocyte chemoattractant, macrophage inflammatory protein 2 (MIP-2), granulocyte colony-stimulating factor (G-CSF), mediate granulopoiesis, recruitment, inflammation.6Kolls Accordingly, absence results decreased G-CSF MIP-2, accumulation neutrophils inflammation.7Lin Neutrophil depletion alone does affect control LVS,10Conlan KuoLee Shen Webb Different defences protect systemic vs pathogen, tularensis.LVS Microb 32: 127-134Crossref (80) suggesting was mechanism model.7Lin together generating infection. anti-inflammatory best studied inhibitory effects down-regulation responses.11Saraiva O'Garra regulation cells.Nat 10: 170-181Crossref (2176) show enhanced models infections, Mycobacterium tuberculosis12Redford P.S. Boonstra Read Pitt Graham C. Stavropoulos Bancroft G.J. Enhanced tuberculosis IL-10-deficient accompanied early lung.Eur J 40: 2200-2210Crossref (193) Listeria monocytogenes.13Dai W.J. Kohler Brombacher Both innate acquired monocytogenes mice.J 1997; 158: 2259-2267Crossref addition, cutaneous infection, protection, reversed when depleted.14Metzger Salmon Kirimanjeswara Differing interleukin-10 infection.Infect 2013; 81: 2022-2027Crossref (17) contrast these published studies, current study, deficient (Il10−/−) mortality. clearly loss immunity, burden wild-type Il10−/− similar, inflammation result unrestrained IL-17–dependent resulting that, LVS,7Lin cytokines, IL-10. Our studies highlight how IL-17, can beneficial produced unrestrained, C57BL/6 (B6) were purchased Jackson Laboratory (Bar Harbor, ME). Il17−/− mice15Nakae Komiyama Nambu Sudo Iwase Homma I. Sekikawa Asano Antigen-specific sensitization impaired IL-17-deficient mice, causing suppression allergic humoral responses.Immunity. 17: 375-387Abstract (927) crossed Il17−/−/Il-10−/− double-deficient generated house B6 background accordance University Pittsburgh Institutional Animal Care Use Committee guidelines. (BEI Resources, Manassas, VA) grown Mueller-Hinton (MH) broth or agar.5Duckett For infected 1000 colony-forming units (CFUs) LVS. On day 6 serial dilutions homogenized lungs plated MH agar plates CFUs determined. some experiments, survival monitored B6- gene-deficient mice. neutrophils, treated 300 μg Gr1-depleting antibody (clone IA8; BioXCell, West Lebanon, NH) isotype (BioXCell) every 48 hours, previously described.16Kang D.D. Lin Moreno J.R. Profiling mouse tuberculosis.PLoS 2011; 6: e16161Crossref (98) single-cell suspensions prepared, described, flow cytometric analyses.7Lin Lungs inflated 10% neutral-buffered formalin embedded paraffin. sections stained H&E stain (Colorado Histo-Prep, Fort Collins, CO) processed routinely light microscopy. Slides scored one authors (T.D.O.), who blinded sample groups. Briefly, field entire microscope inflammation, described.17Manni M.L. Epperly M.W. Han W. Blackwell T.S. Duncan Piganelli J.D. Oury Leukocyte-derived extracellular superoxide dismutase contribute airspace EC-SOD interstitial injury.Am Physiol Cell Mol Physiol. 2012; 302: L160-L166Crossref (5) Scoring based percentage alveolar tissue according following scale: 0, no inflammation; 1, 1% <25%; 2, 25% <50%; 3, 50% <75%; 4, 75% 100% inflammation. immunofluorescence, paraffin removed formalin-fixed sections, described,7Lin samples probed biotinylated rat, anti-mouse GR1 (Rat AL-21; BD Pharmingen). Slow-fade gold antifade DAPI (Molecular Probes, Grand Island, NY) counterstain tissues detect nuclei. Images obtained Zeiss Axioplan (Carl Microscopy, Jena, Germany) recorded AxioCam digital camera Microscopy). IL-10, IL-12, IL-23 levels measured Mouse Duoset enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). Other chemokine determined Luminex (Linco/Millipore, Billerica, MA). Myeloperoxidase (MPO) chlorination peroxidase activity homogenates MPO kit (Invitrogen, NY). Single-cell fluorochrome-labeled antibodies specific CD11c (HL3), Gr1 (RB6-8C5), CD11b (M1/70), CD3 (145-2C11), CD4 (RM4-5), NK1.1 (PK136), γδ (GL3), (XMG1.2), (JES5-16E3), (TC11-18H10) relevant antibodies. analysis stimulated 50 ng/mL phorbol myristate acetate; 750 ionomycin (Sigma Aldrich, St. Louis, MO) presence Golgistop (BD Pharmingen, San Jose, CA) surface stained, permeabilized Cytofix-Cytoperm solution Pharmingen), cytokines. Cells collected Becton Dickinson FACS Aria cytometer Diva software version 6.1.2. gated their forward-by-side scatter characteristics, frequency types calculated FlowJo 7.6.5 (Tree Star Inc., Carlos, CA). CD11c+ autofluorescence designated dendritic (DCs), high macrophages, 18Khader Partida-Sanchez Bell Jelley-Gibbs Swain Pearl Ghilardi Desauvage F.J. Lund F.E. Interleukin 12p40 migration priming infection.J Exp Med. 2006; 203: 1805-1815Crossref (252) 19Slight Gopal Fallert Junecko B.A. Mehra Selman Becerril-Villanueva Baquera-Heredia Pavon Kaushal Reinhart T.A. CXCR5+ tuberculosis.J Clin Invest. 123: 712-726PubMed Bone marrow (BMDCs) bone marrow.7Lin 7, nonadherent (multiplicity 1:100) combination 10 μg/mL αIL-10 (clone: JES5-2A5; BioXCell) IgG1; antibiotic-free Dulbecco's modified Eagle's medium hours. Naïve CD4+ isolated OT-II T-cell receptor αβ Tg magnetic beads (GK1.5) (Miltenyi Biotec, Auburn, cultured × 106 LVS-stimulated unstimulated BMDCs/mL, 5 μmol/L ovalbumin323-339 peptide days, described.7Lin Culture supernatants then analyzed ELISA. Differences means groups two-tailed Student's t-test; one-way variance test more than analyzed. log-rank statistical analyses studies. considered significant P ≤ 0.05. During expression detrimental,12Redford 13Dai burdens. functional context completely explored. found BMDCs other polarizing IL-23, (Figure 1A). induced lung, whereas at later time point 1B). lower detectable (data shown). DCs, sources accumulated points 1C). infection.5Duckett 7Lin To LVS-induced LVS-infected IL-10–neutralizing DC:T-cell co-culture system neutralization resulted D E, respectively). may limit vitro Unexpectedly, intratracheally they demonstrated mortality, compared B6-infected 2A). Interestingly, had similar points, 2B). displayed heightened C D), die consequence rather immunity. co-cultures restrains E). Therefore, next addressed whether vivo, dysregulated mediates tularemia. lungs, expressed significantly higher 2E) 2F). major LVS.7Lin numbers producing uninfected further (Table 1). addition restraining responses,11Saraiva LVS.Table 1IL-17 Production InfectionType cellUninfected lungsB6-infected lungsIl10−/−-infected lungsCD4+ IL-17+ cells1.8 104 ± 1032.4 105 9.2 104∗P 0.005 mice.4.2 1.5 104†P 0.05 mice.γδ+ cells1.2 6.2 1031.7 7.4 mice.2.9 6.1 mice.B6 left intratracheally. 6, assayed staining cytometry.∗ mice.† Open table new tab cytometry. tularemia lung.7Lin hypothesized excess IFN-γ, this, IL-10/IL-17 (Il10−/−/Il17−/−) them via route. susceptibility seen More importantly, pathological characteristics Il10−/−/Il17−/−-infected D). have shown drive IL-12–driven Th1-protective This likely overcome production, us another (namely, bovis bacillus Calmette-Guerin exposure).20Gopal Obermajer Nuthalapati Ahmed Kalinski P. IL-23-dependent drives Th1-cell BCG vaccination.Eur 42: 364-373Crossref (130) support specifically IL-10–mediated inhibition, Il10−/−/Il17−/− showed absent, generation overall infe