IL-10 Restrains IL-17 to Limit Lung Pathology Characteristics following Pulmonary Infection with Francisella tularensis Live Vaccine Strain
2013; Elsevier BV; Volume: 183; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2013.07.008
ISSN1525-2191
AutoresSamantha Slight, Leticia Monin, Radha Gopal, Lyndsay Avery, Marci Davis, Hillary Cleveland, Tim D. Oury, Javier Rangel‐Moreno, Shabaana A. Khader,
Tópico(s)Poxvirus research and outbreaks
ResumoIL-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 is a facultative intracellular bacterium that activates both Th1 and Th17 protective immune responses. Herein, we report that IL-10–deficient mice (Il10−/−), despite having increased Th1 and Th17 responses, exhibit increased mortality after pulmonary infection with F. tularensis live vaccine strain. We demonstrate that the increased mortality observed in Il10−/−-infected mice is due to exacerbated IL-17 production that causes increased neutrophil recruitment and associated lung pathology. Thus, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis live vaccine strain, its production is tightly regulated by IL-10 to generate efficient induction of protective immunity without mediating pathology. These data suggest a critical role for IL-10 in maintaining the delicate balance between host immunity and pathology during pulmonary infection with F. tularensis live vaccine strain. 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 is a facultative intracellular bacterium that activates both Th1 and Th17 protective immune responses. Herein, we report that IL-10–deficient mice (Il10−/−), despite having increased Th1 and Th17 responses, exhibit increased mortality after pulmonary infection with F. tularensis live vaccine strain. We demonstrate that the increased mortality observed in Il10−/−-infected mice is due to exacerbated IL-17 production that causes increased neutrophil recruitment and associated lung pathology. Thus, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis live vaccine strain, its production is tightly regulated by IL-10 to generate efficient induction of protective immunity without mediating pathology. These data suggest a critical role for IL-10 in maintaining the delicate balance between host immunity and pathology during pulmonary infection with F. tularensis live vaccine strain. Francisella tularensis, a facultative intracellular bacterium, because of its infectious nature and the severe disease caused by low doses of airborne bacteria, has been classified as a 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 as a biological weapon: medical and public health management.JAMA. 2001; 285: 2763-2773Crossref PubMed Scopus (1198) Google Scholar Infection in humans is caused by two main subspecies, F. tularensis (type A) and Francisella holarctica (type B).2Conlan J.W. Vaccines against Francisella tularensis–past, present and future.Expert Rev Vaccines. 2004; 3: 307-314Crossref PubMed Scopus (36) Google Scholar An F. tularensis live vaccine strain (LVS) has been developed from the F. tularensis B strain as an experimental vaccine, but is not licensed for use in humans.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 as a biological weapon: medical and public health management.JAMA. 2001; 285: 2763-2773Crossref PubMed Scopus (1198) Google Scholar F. tularensis LVS has been used as a representative attenuated model to address the immune requirements for protection against Francisella. By using this model, the importance of IL-12 in driving interferon γ (IFN-γ) and T-helper cell 1 (Th1) responses in immunity to F. tularensis LVS infection is well described.3Anthony L.S. Ghadirian E. Nestel F.P. Kongshavn P.A. The requirement for gamma interferon in resistance of mice to experimental tularemia.Microb Pathog. 1989; 7: 421-428Crossref PubMed Scopus (84) Google Scholar, 4Elkins K.L. Cooper A. Colombini S.M. Cowley S.C. Kieffer T.L. In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70.Infect Immun. 2002; 70: 1936-1948Crossref PubMed Scopus (117) Google Scholar, 5Duckett N.S. Olmos S. Durrant D.M. Metzger D.W. Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain.Infect Immun. 2005; 73: 2306-2311Crossref PubMed Scopus (82) Google Scholar In contrast, IL-17 is generally thought to play a role in protection against extracellular, but not intracellular, pathogens.6Kolls J.K. Khader S.A. The role of Th17 cytokines in primary mucosal immunity.Cytokine Growth Factor Rev. 2010; 21: 443-448Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar However, we and others recently identified a protective role for IL-17 in the induction of cellular immunity to F. tularensis LVS pulmonary infection,7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 8Cowley S.C. Meierovics A.I. Frelinger J.A. Iwakura Y. Elkins K.L. Lung CD4- CD8- double-negative T cells are prominent producers of IL-17A and IFN-gamma during primary respiratory murine infection with Francisella tularensis live vaccine strain.J Immunol. 2010; 184: 5791-5801Crossref PubMed Scopus (89) Google Scholar, 9Markel G. Bar-Haim E. Zahavy E. Cohen H. Cohen O. Shafferman A. Velan B. The involvement of IL-17A in the murine response to sub-lethal inhalational infection with Francisella tularensis.PLoS One. 2010; 5: e11176Crossref PubMed Scopus (37) Google Scholar by driving the production of IFN-γ through IL-12 induction.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar IL-17 is a proinflammatory cytokine also known to induce chemokines, such as keratinocyte chemoattractant, macrophage inflammatory protein 2 (MIP-2), and granulocyte colony-stimulating factor (G-CSF), to mediate granulopoiesis, neutrophil recruitment, and inflammation.6Kolls J.K. Khader S.A. The role of Th17 cytokines in primary mucosal immunity.Cytokine Growth Factor Rev. 2010; 21: 443-448Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Accordingly, the absence of IL-17 during F. tularensis LVS pulmonary infection also results in decreased induction of G-CSF and MIP-2, as well as decreased accumulation of neutrophils and lung inflammation.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar Neutrophil depletion alone does not affect bacterial control after pulmonary infection with F. tularensis LVS,10Conlan J.W. KuoLee R. Shen H. Webb A. Different host defences are required to protect mice from primary systemic vs pulmonary infection with the facultative intracellular bacterial pathogen, Francisella tularensis.LVS Microb Pathog. 2002; 32: 127-134Crossref PubMed Scopus (80) Google Scholar suggesting that the role for IL-17 in driving Th1 responses, and not neutrophil recruitment, was the primary immune mechanism mediating protection in this model.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar These data together suggest that both IL-17 and IFN-γ are required for generating protective immunity to pulmonary F. tularensis LVS infection. IL-10 is an anti-inflammatory cytokine best studied for its inhibitory effects on IL-12 production and down-regulation of Th1 responses.11Saraiva M. O'Garra A. The regulation of IL-10 production by immune cells.Nat Rev Immunol. 2010; 10: 170-181Crossref PubMed Scopus (2176) Google Scholar Accordingly, IL-10–deficient mice show enhanced protection in models of intracellular bacterial infections, such as Mycobacterium tuberculosis12Redford P.S. Boonstra A. Read S. Pitt J. Graham C. Stavropoulos E. Bancroft G.J. O'Garra A. Enhanced protection to Mycobacterium tuberculosis infection in IL-10-deficient mice is accompanied by early and enhanced Th1 responses in the lung.Eur J Immunol. 2010; 40: 2200-2210Crossref PubMed Scopus (193) Google Scholar and Listeria monocytogenes.13Dai W.J. Kohler G. Brombacher F. Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10-deficient mice.J Immunol. 1997; 158: 2259-2267Crossref PubMed Google Scholar In addition, in a cutaneous model of F. tularensis LVS infection, IL-10–deficient mice exhibit increased protection, and this was reversed when IL-17 was depleted.14Metzger D.W. Salmon S.L. Kirimanjeswara G. Differing effects of interleukin-10 on cutaneous and pulmonary Francisella tularensis live vaccine strain infection.Infect Immun. 2013; 81: 2022-2027Crossref PubMed Scopus (17) Google Scholar In contrast to these published studies, in the current study, we report that after pulmonary infection with F. tularensis LVS, mice deficient in IL-10 (Il10−/−) exhibit increased mortality. We clearly demonstrate that the increased mortality in the Il10−/−-infected mice is not associated with loss of protective immunity, because bacterial burden between wild-type and Il10−/− mice is similar, but is caused by exacerbated inflammation and increased lung pathology. We demonstrate that the exacerbated inflammation observed in Il10−/−-infected mice is the result of unrestrained IL-17 production and IL-17–dependent recruitment of neutrophils and resulting lung pathology. These data together suggest that, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis LVS,7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 9Markel G. Bar-Haim E. Zahavy E. Cohen H. Cohen O. Shafferman A. Velan B. The involvement of IL-17A in the murine response to sub-lethal inhalational infection with Francisella tularensis.PLoS One. 2010; 5: e11176Crossref PubMed Scopus (37) Google Scholar IL-17 production is tightly regulated by anti-inflammatory cytokines, such as IL-10. Our studies highlight how inflammatory cytokines, such as IL-17, can be beneficial for host protection, but when produced unrestrained, can mediate host pathology. C57BL/6 (B6) and Il10−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Il17−/− mice15Nakae S. Komiyama Y. Nambu A. Sudo K. Iwase M. Homma I. Sekikawa K. Asano M. Iwakura Y. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses.Immunity. 2002; 17: 375-387Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar were crossed to the Il10−/− mice, and Il17−/−/Il-10−/− double-deficient mice were generated in house on the B6 background and were used in accordance with University of Pittsburgh Institutional Animal Care and Use Committee guidelines. F. tularensis LVS (BEI Resources, Manassas, VA) were grown in Mueller-Hinton (MH) broth or agar.5Duckett N.S. Olmos S. Durrant D.M. Metzger D.W. Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain.Infect Immun. 2005; 73: 2306-2311Crossref PubMed Scopus (82) Google Scholar For pulmonary infections, mice were infected with 1000 colony-forming units (CFUs) of F. tularensis LVS. On day 6 after infection, serial dilutions of the homogenized infected lungs were plated on MH agar plates and lung CFUs were determined. In some experiments, survival was monitored in infected B6- and gene-deficient mice. For depletion of neutrophils, mice were treated with 300 μg of Gr1-depleting antibody (clone IA8; BioXCell, West Lebanon, NH) or isotype control antibody (BioXCell) every 48 hours, as previously described.16Kang D.D. Lin Y. Moreno J.R. Randall T.D. Khader S.A. Profiling early lung immune responses in the mouse model of tuberculosis.PLoS One. 2011; 6: e16161Crossref PubMed Scopus (98) Google Scholar In some experiments, single-cell lung suspensions were prepared, as previously described, and used for flow cytometric analyses.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar Lungs from mice were inflated with 10% neutral-buffered formalin and embedded in paraffin. Lung sections were stained with H&E stain (Colorado Histo-Prep, Fort Collins, CO) and processed routinely for light microscopy. Slides were scored by one of the authors (T.D.O.), who was blinded to the sample groups. Briefly, every field in the entire lung was observed with a light microscope and scored for inflammation, as previously described.17Manni M.L. Epperly M.W. Han W. Blackwell T.S. Duncan S.R. Piganelli J.D. Oury T.D. Leukocyte-derived extracellular superoxide dismutase does not contribute to airspace EC-SOD after interstitial pulmonary injury.Am J Physiol Lung Cell Mol Physiol. 2012; 302: L160-L166Crossref PubMed Scopus (5) Google Scholar Scoring was based on the percentage of alveolar tissue with inflammation, according to the following scale: 0, no inflammation; 1, 1% to <25%; 2, 25% to <50%; 3, 50% to <75%; and 4, 75% to 100% inflammation. For immunofluorescence, paraffin was removed from the formalin-fixed lung sections, as previously described,7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar and samples were probed with biotinylated rat, anti-mouse GR1 (Rat AL-21; BD Pharmingen). Slow-fade gold antifade with DAPI (Molecular Probes, Grand Island, NY) was used to counterstain tissues and detect nuclei. Images were obtained with a Zeiss Axioplan 2 microscope (Carl Zeiss Microscopy, Jena, Germany) and were recorded with a Zeiss AxioCam digital camera (Carl Zeiss Microscopy). IL-10, IL-12, and IL-23 protein levels were measured using a Mouse Duoset enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). Other cytokine and chemokine protein levels were determined with a mouse Luminex assay (Linco/Millipore, Billerica, MA). Myeloperoxidase (MPO) chlorination and peroxidase activity were determined in lung homogenates using the MPO activity assay kit (Invitrogen, Grand Island, NY). Single-cell suspensions were stained with fluorochrome-labeled antibodies specific for CD11c (HL3), Gr1 (RB6-8C5), CD11b (M1/70), CD3 (145-2C11), CD4 (RM4-5), NK1.1 (PK136), γδ T cells (GL3), IFN-γ (XMG1.2), IL-10 (JES5-16E3), and IL-17 (TC11-18H10) or relevant isotype control antibodies. For analysis of intracellular cytokines, cells were stimulated with 50 ng/mL phorbol myristate acetate; 750 ng/mL ionomycin (Sigma Aldrich, St. Louis, MO) in the presence of Golgistop (BD Pharmingen, San Jose, CA) was surface stained, permeabilized with Cytofix-Cytoperm solution (BD Pharmingen), and stained for relevant cytokines. Cells were collected in a Becton Dickinson FACS Aria flow cytometer with FACS Diva software version 6.1.2. Cells were gated based on their forward-by-side scatter characteristics, and the frequency of specific cell types was calculated using FlowJo version 7.6.5 (Tree Star Inc., San Carlos, CA). CD11c+ cells with low autofluorescence were designated as lung dendritic cells (DCs), and CD11c+ cells with high autofluorescence were designated as lung macrophages, as previously described.16Kang D.D. Lin Y. Moreno J.R. Randall T.D. Khader S.A. Profiling early lung immune responses in the mouse model of tuberculosis.PLoS One. 2011; 6: e16161Crossref PubMed Scopus (98) Google Scholar, 18Khader S.A. Partida-Sanchez S. Bell G. Jelley-Gibbs D.M. Swain S. Pearl J.E. Ghilardi N. Desauvage F.J. Lund F.E. Cooper A.M. Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection.J Exp Med. 2006; 203: 1805-1815Crossref PubMed Scopus (252) Google Scholar, 19Slight S.R. Rangel-Moreno J. Gopal R. Lin Y. Fallert Junecko B.A. Mehra S. Selman M. Becerril-Villanueva E. Baquera-Heredia J. Pavon L. Kaushal D. Reinhart T.A. Randall T.D. Khader S.A. CXCR5+ T helper cells mediate protective immunity against tuberculosis.J Clin Invest. 2013; 123: 712-726PubMed Google Scholar Bone marrow dendritic cells (BMDCs) were generated from bone marrow.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar On day 7, nonadherent cells were infected with F. tularensis LVS (multiplicity of infection, 1:100) alone or in combination with 10 μg/mL αIL-10 antibody (clone: JES5-2A5; BioXCell) or isotype control antibody (Rat IgG1; BioXCell) in antibiotic-free Dulbecco's modified Eagle's medium for 48 hours. Naïve CD4+ T cells were isolated from OT-II T-cell receptor αβ Tg mice using magnetic CD4+ beads (GK1.5) (Miltenyi Biotec, Auburn, CA) and were cultured with 1 × 106 cells F. tularensis LVS-stimulated or unstimulated BMDCs/mL, with 5 μmol/L ovalbumin323-339 peptide for 6 days, as previously described.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar Culture supernatants were then analyzed by ELISA. Differences between the means of two experimental groups were analyzed using the two-tailed Student's t-test; a one-way analysis of variance test was used when more than two groups were analyzed. The log-rank test was used for statistical analyses for the survival studies. Differences were considered significant when P ≤ 0.05. During intracellular bacterial infections, IL-10 expression is detrimental,12Redford P.S. Boonstra A. Read S. Pitt J. Graham C. Stavropoulos E. Bancroft G.J. O'Garra A. Enhanced protection to Mycobacterium tuberculosis infection in IL-10-deficient mice is accompanied by early and enhanced Th1 responses in the lung.Eur J Immunol. 2010; 40: 2200-2210Crossref PubMed Scopus (193) Google Scholar, 13Dai W.J. Kohler G. Brombacher F. Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10-deficient mice.J Immunol. 1997; 158: 2259-2267Crossref PubMed Google Scholar because IL-10–deficient mice (Il10−/−) show enhanced protection and decreased bacterial burdens. However, the functional role for IL-10 in the context of F. tularensis LVS infection has not been completely explored. We found that BMDCs from B6 mice produced IL-10 and other polarizing cytokines, such as IL-12 and IL-23, on infection with F. tularensis LVS (Figure 1A). In addition, IL-10 protein was induced early in the lung, whereas IL-12 levels were induced at a later time point after pulmonary F. tularensis LVS infection (Figure 1B). Lung IL-23 levels were lower than detectable levels (data not shown). Lung CD11c+ DCs, but not lung macrophages, were one of the primary sources of IL-10 and accumulated at early time points after pulmonary infection (Figure 1C). Both Th1 and Th17 responses are required for protective immunity against F. tularensis LVS pulmonary infection.5Duckett N.S. Olmos S. Durrant D.M. Metzger D.W. Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain.Infect Immun. 2005; 73: 2306-2311Crossref PubMed Scopus (82) Google Scholar, 7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar To address the role of F. tularensis LVS-induced IL-10 on Th1 and Th17 responses, we treated F. tularensis LVS-infected B6 BMDCs with IL-10–neutralizing antibody in a DC:T-cell co-culture system and found that IL-10 neutralization resulted in enhanced IFN-γ and IL-17 production by CD4+ T cells (Figure 1, D and E, respectively). These data suggest that IL-10, induced after F. tularensis LVS infection, may limit both Th1 and Th17 responses. In vitro data suggest that IL-10 may limit both Th1 and Th17 responses, and its presence may be detrimental for protective immunity against F. tularensis LVS infection. Unexpectedly, when Il10−/− mice were infected intratracheally with F. tularensis LVS, they demonstrated decreased survival and increased mortality, when compared with control B6-infected mice (Figure 2A). Interestingly, Il10−/−-infected mice had similar levels of bacterial burden in the lungs at both early and later time points, suggesting that the decreased survival was not associated with increased bacterial burden (Figure 2B). We also found that Il10−/− mice displayed heightened pathology and severe pulmonary inflammation (Figure 2, C and D), suggesting that Il10−/−-infected mice die as a consequence of increased inflammation rather than loss of protective immunity. In vitro data using DC:T-cell co-cultures suggest that IL-10 production restrains both IL-17 and IFN-γ responses in CD4+ T cells (Figure 1, D and E). Therefore, we next addressed whether IL-10 also restrains IL-17 and IFN-γ responses in vivo, and whether dysregulated expression of these proinflammatory cytokines mediates pathology and inflammation during pulmonary tularemia. We found that, when compared with B6 F. tularensis LVS-infected lungs, Il10−/−-infected lungs expressed significantly higher levels of IL-17 (Figure 2E) and IFN-γ protein (Figure 2F). Both γδ T cells and CD4+ T cells are major producers of IL-17 in response to pulmonary infection with F. tularensis LVS.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 9Markel G. Bar-Haim E. Zahavy E. Cohen H. Cohen O. Shafferman A. Velan B. The involvement of IL-17A in the murine response to sub-lethal inhalational infection with Francisella tularensis.PLoS One. 2010; 5: e11176Crossref PubMed Scopus (37) Google Scholar Accordingly, although numbers of CD4+ and γδ T cells producing IL-17 were increased in B6-infected lungs compared with uninfected lungs, their numbers were further enhanced in Il10−/−-infected lungs (Table 1). These data suggest that, in addition to its known role in restraining Th1 responses,11Saraiva M. O'Garra A. The regulation of IL-10 production by immune cells.Nat Rev Immunol. 2010; 10: 170-181Crossref PubMed Scopus (2176) Google Scholar IL-10 also has a role in restraining Th17 responses after pulmonary infection with F. tularensis LVS.Table 1IL-17 Production on F. tularensis LVS InfectionType of cellUninfected lungsB6-infected lungsIl10−/−-infected lungsCD4+ IL-17+ T cells1.8 × 104 ± 6 × 1032.4 × 105 ± 9.2 × 104∗P ≤ 0.005 between B6-infected and uninfected mice.4.2 × 105 ± 1.5 × 104†P ≤ 0.05 between B6-infected and Il10−/−-infected mice.γδ+ IL-17+ T cells1.2 × 104 ± 6.2 × 1031.7 × 105 ± 7.4 × 104∗P ≤ 0.005 between B6-infected and uninfected mice.2.9 × 105 ± 6.1 × 104†P ≤ 0.05 between B6-infected and Il10−/−-infected mice.B6 and Il10−/− mice were left uninfected or were infected with 1000 CFUs F. tularensis LVS intratracheally. On day 6, single-cell lung suspensions were assayed for IL-17 production by intracellular staining and flow cytometry.∗ P ≤ 0.005 between B6-infected and uninfected mice.† P ≤ 0.05 between B6-infected and Il10−/−-infected mice. Open table in a new tab B6 and Il10−/− mice were left uninfected or were infected with 1000 CFUs F. tularensis LVS intratracheally. On day 6, single-cell lung suspensions were assayed for IL-17 production by intracellular staining and flow cytometry. The absence of IL-17 during pulmonary tularemia results in decreased inflammation in the infected lung.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar Therefore, we hypothesized that excess IL-17, rather than excess IFN-γ, caused the inflammation, and increased the mortality observed in the Il10−/−-infected mice. To address this, we generated IL-10/IL-17 double-deficient mice (Il10−/−/Il17−/−) and infected them with F. tularensis LVS via the pulmonary route. We found that absence of IL-17 significantly reversed the increased susceptibility seen in Il10−/− mice (Figure 2A). More importantly, we found that the heightened pathological characteristics observed in the lungs of infected Il10−/− mice were also reversed in lungs from Il10−/−/Il17−/−-infected mice (Figure 2, C and D). We have recently shown that IL-17 is protective and is required to drive IL-12–driven Th1-protective cell immunity against F. tularensis LVS.7Lin Y. Ritchea S. Logar A. Slight S. Messmer M. Rangel-Moreno J. Guglani L. Alcorn J.F. Strawbridge H. Park S.M. Onishi R. Nyugen N. Walter M.J. Pociask D. Randall T.D. Gaffen S.L. Iwakura Y. Kolls J.K. Khader S.A. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis.Immunity. 2009; 31: 799-810Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar This role for IL-17 is likely to overcome the inhibitory effects of IL-10 on IL-12 production, as demonstrated by us in another intracellular bacterial model (namely, Mycobacterium bovis bacillus Calmette-Guerin exposure).20Gopal R. Lin Y. Obermajer N. Slight S. Nuthalapati N. Ahmed M. Kalinski P. Khader S.A. IL-23-dependent IL-17 drives Th1-cell responses following Mycobacterium bovis BCG vaccination.Eur J Immunol. 2012; 42: 364-373Crossref PubMed Scopus (130) Google Scholar In support of a role for IL-17 in driving IFN-γ responses specifically to overcome IL-10–mediated inhibition, significantly higher levels of IFN-γ were found in lungs of both Il10−/− and Il10−/−/Il17−/− mice when compared with B6-infected lungs (Figure 2F). In addition, Il10−/−/Il17−/−-infected mice showed a similar bacterial burden to B6- and Il10−/−-infected mice at both early and later time points (Figure 2B). These data together suggest that when IL-10 is absent, IL-17 is not required for generation of Th1 responses or overall protective immunity against pulmonary F. tularensis LVS infe
Referência(s)