IL-17–Induced Pulmonary Pathogenesis during Respiratory Viral Infection and Exacerbation of Allergic Disease
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.003
ISSN1525-2191
AutoresSumanta Mukherjee, Dennis M. Lindell, Aaron A. Berlin, Susan B. Morris, Thomas P. Shanley, Marc B. Hershenson, Nicholas W. Lukacs,
Tópico(s)Immune Cell Function and Interaction
ResumoSevere respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17−/− knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections. Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17−/− knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections. Nearly 98% of all infants become infected with respiratory syncytial virus (RSV) by the age of 2 years and experience severe bronchiolitis because their small airways easily become occluded.1Openshaw P.J. Dean G.S. Culley F.J. Links between respiratory syncytial virus bronchiolitis and childhood asthma: clinical and research approaches.Pediatr Infect Dis J. 2003; 22 (discussion S64–S65): S58-S64PubMed Google Scholar It is estimated by the U.S. Centers for Disease Control that up to 125,000 pediatric hospitalizations in the United States each year are due to RSV. In addition, RSV is pathogenic for elderly patients and for those with chronic lung disease and asthma, and further is associated with a mortality rate of 30% to 100% in immunosuppressed individuals.2Black C.P. Systematic review of the biology and medical management of respiratory syncytial virus infection.Respir Care. 2003; 48 (discussion 231–233): 209-231PubMed Google Scholar, 3Stensballe L.G. Devasundaram J.K. Simoes E.A. Respiratory syncytial virus epidemics: the ups and downs of a seasonal virus.Pediatr Infect Dis J. 2003; 22: S21-S32PubMed Google Scholar RSV also is associated with acute exacerbations of chronic obstructive pulmonary disease, causing prolonged episodes of illness. Recurrent infections with RSV are common, and the pulmonary pathology is known to persist long after the virus has been cleared efficiently. RSV disease pathology is clinically characterized by airway hyperreactivity (AHR), increased mucus production, and inflammation.4Jafri H.S. Chavez-Bueno S. Mejias A. Gomez A.M. Rios A.M. Nassi S.S. Yusuf M. Kapur P. Hardy R.D. Hatfield J. Rogers B.B. Krisher K. Ramilo O. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice.J Infect Dis. 2004; 189: 1856-1865Crossref PubMed Scopus (143) Google Scholar, 5Schwarze J. Cieslewicz G. Hamelmann E. Joetham A. Shultz L.D. Lamers M.C. Gelfand E.W. IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection.J Immunol. 1999; 162: 2997-3004PubMed Google Scholar, 6Tekkanat K.K. Maassab H. Miller A. Berlin A.A. Kunkel S.L. Lukacs N.W. RANTES (CCL5) production during primary respiratory syncytial virus infection exacerbates airway disease.Eur J Immunol. 2002; 32: 3276-3284Crossref PubMed Scopus (74) Google Scholar An altered immune environment due to an imbalance in the CD4 helper Th1 and Th2 responses is thought to underlie this disease phenotype. Recently it was reported that IL-17, produced by a subset of CD4 helper T cells (Th17 cells), was regulated by STAT-1 during RSV infections in rodents.7Hashimoto K. Durbin J.E. Zhou W. Collins R.D. Ho S.B. Kolls J.K. Dubin P.J. Sheller Jr, Goleniewska K. O'Neal J.F. Olson S.J. Mitchell D. Graham B.S. Peebles Jr, R.S. Respiratory syncytial virus infection in the absence of STAT 1 results in airway dysfunction, airway mucus, and augmented IL-17 levels.J Allergy Clin Immunol. 2005; 116: 550-557Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar The exact role of IL-17 in RSV disease pathogenicity is not known. Interleukin-17 belongs to a family of cytokines that has six members: IL-17 (also called IL-17A, the prototype) and IL-17B through IL-17F; IL-17E is also known as IL-25. IL-17F shares the strongest homology to IL-17.8Hymowitz S.G. Filvaroff E.H. Yin J.P. Lee J. Cai L. Risser P. Maruoka M. Mao W. Foster J. Kelley R.F. Pan G. Gurney A.L. de Vos A.M. Starovasnik M.A. IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding.EMBO J. 2001; 20: 5332-5341Crossref PubMed Scopus (436) Google Scholar, 9Kawaguchi M. Onuchic L.F. Li X.D. Essayan D.M. Schroeder J. Xiao H.Q. Liu M.C. Krishnaswamy G. Germino G. Huang S.K. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.J Immunol. 2001; 167: 4430-4435PubMed Google Scholar Both IL-17 and IL-17F are proinflammatory and have overlapping roles in the development of various autoimmune disorders, such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. However, IL-17 plays a critical role in host defense during bacterial and fungal infection, whereas IL-17F is largely involved in the development of asthma and airway inflammation.9Kawaguchi M. Onuchic L.F. Li X.D. Essayan D.M. Schroeder J. Xiao H.Q. Liu M.C. Krishnaswamy G. Germino G. Huang S.K. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.J Immunol. 2001; 167: 4430-4435PubMed Google Scholar, 10Hizawa N. Kawaguchi M. Huang S.K. Nishimura M. Role of interleukin-17F in chronic inflammatory and allergic lung disease.Clin Exp Allergy. 2006; 36: 1109-1114Crossref PubMed Scopus (98) Google Scholar, 11Ouyang W. Kolls J.K. Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation.Immunity. 2008; 28: 454-467Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar Moreover. IL-17F does not up-regulate proinflammatory molecules to the same degree as does IL-17.12Yang X.O. Chang S.H. Park H. Nurieva R. Shah B. Acero L. Wang Y.H. Schluns K.S. Broaddus R.R. Zhu Z. Dong C. Regulation of inflammatory responses by IL-17F.J Exp Med. 2008; 205: 1063-1075Crossref PubMed Scopus (602) Google Scholar The receptors for IL-17 and IL-17F are IL-17RA and IL-17RC, respectively; because these receptors have different tissue expression, the isoforms of IL-17 are tissue-specific. Expression of IL-17RC is limited to nonhematopoietic cells, but IL-17RA is expressed ubiquitously. IL-17 may promote Th2 responses in the lung through IL-17RA, whereas IL-17F has a regulatory role in limiting allergic asthma development.12Yang X.O. Chang S.H. Park H. Nurieva R. Shah B. Acero L. Wang Y.H. Schluns K.S. Broaddus R.R. Zhu Z. Dong C. Regulation of inflammatory responses by IL-17F.J Exp Med. 2008; 205: 1063-1075Crossref PubMed Scopus (602) Google Scholar, 13Nakae 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 (911) Google Scholar, 14Schnyder-Candrian S. Togbe D. Couillin I. Mercier I. Brombacher F. Quesniaux V. Fossiez F. Ryffel B. Schnyder B. Interleukin-17 is a negative regulator of established allergic asthma.J Exp Med. 2006; 203: 2715-2725Crossref PubMed Scopus (488) Google Scholar The combination of AHR and mucus production in the airways is a significant clinical outcome during viral infections. RSV infections induce significant AHR and mucus production in the airways of mice7Hashimoto K. Durbin J.E. Zhou W. Collins R.D. Ho S.B. Kolls J.K. Dubin P.J. Sheller Jr, Goleniewska K. O'Neal J.F. Olson S.J. Mitchell D. Graham B.S. Peebles Jr, R.S. Respiratory syncytial virus infection in the absence of STAT 1 results in airway dysfunction, airway mucus, and augmented IL-17 levels.J Allergy Clin Immunol. 2005; 116: 550-557Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Hashimoto K. Graham B.S. Ho S.B. Adler K.B. Collins R.D. Olson S.J. Zhou W. Suzutani T. Jones P.W. Goleniewska K. O'Neal J.F. Peebles Jr, R.S. Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and gob-5.Am J Respir Crit Care Med. 2004; 170: 306-312Crossref PubMed Google Scholar, 16Tekkanat K.K. Maassab H. Berlin A.A. Lincoln P.M. Evanoff H.L. Kaplan M.H. Lukacs N.W. Role of interleukin-12 and stat-4 in the regulation of airway inflammation and hyperreactivity in respiratory syncytial virus infection.Am J Pathol. 2001; 159: 631-638Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar and induce neutrophilia in the lung epithelium.11Ouyang W. Kolls J.K. Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation.Immunity. 2008; 28: 454-467Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 12Yang X.O. Chang S.H. Park H. Nurieva R. Shah B. Acero L. Wang Y.H. Schluns K.S. Broaddus R.R. Zhu Z. Dong C. Regulation of inflammatory responses by IL-17F.J Exp Med. 2008; 205: 1063-1075Crossref PubMed Scopus (602) Google Scholar, 17Al-Ramli W. Préfontaine D. Chouiali F. Martin J.G. Olivenstein R. Lemiere C. Hamid Q. T(H)17-associated cytokines (IL-17A and IL-17F) in severe asthma.J Allergy Clin Immunol. 2009; 123: 1185-1187Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar Although IL-17 has been reported to play a pathogenic role during the development of asthma by regulating mucin gene expression in the airways,11Ouyang W. Kolls J.K. Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation.Immunity. 2008; 28: 454-467Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar its specific role in pathogenic responses during RSV infection is not known. Here, we report increased IL-17 production in infants with RSV infection and identify a role of IL-17 in a mouse model of primary RSV infection, as well as during viral exacerbation of allergic lung disease. Using either IL-17-deficient mice or neutralization of IL-17 significantly inhibited mucus production during RSV infection. In addition, blocking IL-17 significantly decreased viral load and altered cytotoxic CD8 T-cell marker expression. These responses were also observed in RSV-induced allergic airway exacerbation, suggesting that IL-17 plays an important role in the pathogenesis of RSV-induced disease. Female BALB/c mice, 6 to 8 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME). The IL-17−/− mice, derived from breeder animals from the Jackson Laboratory, were a kind gift of Dr. Kathryn Eaton (University of Michigan).18Yang X.O. Nurieva R. Martinez G.J. Kang H.S. Chung Y. Pappu B.P. Shah B. Chang S.H. Schluns K.S. Watowich S.S. Feng X.H. Jetten A.M. Dong C. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs.Immunity. 2008; 29: 44-56Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar All mice were maintained in specific-pathogen-free facilities in the Unit for Laboratory Animal Medicine at the University of Michigan. The University Committee of Use and Care of Animals (UCUCA), University of Michigan, Ann Arbor, approved all animal experimental protocols, and experiments were conducted according to the guidelines provided by the UCUCA review committee. All human studies were performed in accordance with an approved University of Michigan institutional review board protocol after legal consent. The tracheal aspirate samples were diluted 50:50 with PBS containing complete anti-protease cocktail (Sigma-Aldrich, St. Louis, MO) and 0.5% Triton X-100 nonionic detergent to dissociate the mucus. Samples were aliquoted in 75 μL and stored at −80°C until analysis. IL-17 was analyzed using a Bio-Plex 200 System (Bio-Rad Laboratories, Hercules, CA) and IL-6 was analyzed using an enzyme-linked immunosorbent assay (human Quantikine ELISA kit; R&D Systems, Minneapolis, MN). Tracheal aspirate samples were obtained from RSV-infected infants who were hospitalized and given mechanical ventilation in a pediatric intensive care unit. Control uninfected tracheal infant samples were obtained from children undergoing surgery for reasons unrelated to RSV infection, after parental consent. Female BALB/c mice, 6 to 8 weeks old, were anesthetized and infected with RSV on day 0. Lungs were harvested on days 1, 2, 4, 6, 8, 10, and 12 for the time-course studies and on day 8 for all other studies. Cockroach (CRA) sensitization was performed as described previously.19Campbell E.M. Charo I.F. Kunkel S.L. Strieter R.M. Boring L. Gosling J. Lukacs N.W. Monocyte chemoattractant protein-1 mediates cockroach allergen-induced bronchial hyperreactivity in normal but not CCR2-/- mice: the role of mast cells.J Immunol. 1999; 163: 2160-2167PubMed Google Scholar, 20Campbell E.M. Kunkel S.L. Strieter R.M. Lukacs N.W. Temporal role of chemokines in a murine model of cockroach allergen-induced airway hyperreactivity and eosinophilia.J Immunol. 1998; 161: 7047-7053PubMed Google Scholar Briefly, mice were sensitized with a 1:1 mixture of clinical-grade CRA extract (Hollister-Stier Laboratories, Spokane, WA) and incomplete Freund's adjuvant (Sigma-Aldrich), administered subcutaneously and intraperitoneally on day 0. This cockroach allergen is a skin test/immunotherapy-grade preparation that has very little endotoxin contamination (<10 ng/mL). At day 14, mice were sensitized by intranasal challenges of CRA, which was followed by RSV infection on day 16. In animals treated with antibodies, control or anti-IL-17 purified IgG was given 2 hours before RSV infection on day 16. Two doses of control or anti-IL-17 purified IgG were also given on days 18 and 20. After a final intranasal challenge of CRA on day 21, the mice were euthanized and AHR studies were conducted on day 22. The lungs and draining lymph nodes (DLNs) of these mice were isolated for further characterization. RSV A strain (line 19) was derived from a clinical isolate at the University of Michigan.21Moore M.L. Chi M.H. Luongo C. Lukacs N.W. Polosukhin V.V. Huckabee M.M. Newcomb D.C. Buchholz U.J. Crowe Jr, J.E. Goleniewska K. Williams J.V. Collins P.L. Peebles Jr, R.S. A chimeric A2 strain respiratory syncytial virus (RSV) with the fusion protein of RSV strain line 19 exhibits enhanced viral load, mucus, and airway dysfunction.J Virol. 2009; 83: 4185-4194Crossref PubMed Scopus (125) Google Scholar, 22Herlocher M.L. Ewasyshyn M. Sambhara S. Gharaee-Kermani M. Cho D. Lai J. Klein M. Maassab H.F. Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine.Vaccine. 1999; 17: 172-181Crossref PubMed Scopus (27) Google Scholar The virus was administered to mice intratracheally by tongue pull at 1 × 105 plaque-forming units (PFU).23Lukacs N.W. Moore M.L. Rudd B.D. Berlin A.A. Collins R.D. Olson S.J. Ho S.B. Peebles Jr, R.S. Differential immune responses and pulmonary pathophysiology are induced by two different strains of respiratory syncytial virus.Am J Pathol. 2006; 169: 977-986Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Rudd B.D. Smit J.J. Flavell R.A. Alexopoulou L. Schaller M.A. Gruber A. Berlin A.A. Lukacs N.W. Deletion of TLR3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection.J Immunol. 2006; 176: 1937-1942PubMed Google Scholar, 25Smit J.J. Rudd B.D. Lukacs N.W. Plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus.J Exp Med. 2006; 203: 1153-1159Crossref PubMed Scopus (205) Google Scholar We have previously shown that the pathophysiology associated with UV-inactivated virus is comparable to that of naïve mice without RSV infection,26Miller A.L. Gerard C. Schaller M. Gruber A.D. Humbles A.A. Lukacs N.W. Deletion of CCR1 attenuates pathophysiologic responses during respiratory syncytial virus infection.J Immunol. 2006; 176: 2562-2567PubMed Google Scholar, 27Miller A.L. Bowlin T.L. Lukacs N.W. Respiratory syncytial virus-induced chemokine production: linking viral replication to chemokine production in vitro and in vivo.J Infect Dis. 2004; 189: 1419-1430Crossref PubMed Scopus (158) Google Scholar, 28John A.E. Gerard C.J. Schaller M. Miller A.L. Berlin A.A. Humbles A.A. Lukacs N.W. Respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon CCR1-associated immune responses.Eur J Immunol. 2005; 35 ([Erratum appeared in Eur J Immunol 2005;35:3083]): 108-116Crossref PubMed Scopus (29) Google Scholar and in the present study UV-inactivated virus failed to induce IL-17 in mice (data not shown). Plaque assays were performed on RSV-infected lungs. Whole lungs were harvested and ground with sand using a mortar and pestle. Supernatants were serially diluted and incubated with Vero cells for 4 days. Viral plaques were determined using a RSV-specific polyclonal antibody (Millipore, Billerica, MA). Rabbit anti-mouse IL-17 antibodies were prepared by multiple-site immunization of New Zealand White rabbits with recombinant mouse IL-17 (R&D Systems) in complete Freund's adjuvant and boosted with IL-17 in incomplete Freund's adjuvant, according to procedures from our laboratory as described previously (both purchased from Sigma-Aldrich).29Ito T. Schaller M. Hogaboam C.M. Standiford T.J. Sandor M. Lukacs N.W. Chensue S.W. Kunkel S.L. TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4.J Clin Invest. 2009; 119: 33-46PubMed Google Scholar Polyclonal antibodies were titered by direct ELISA against IL-17 coated onto 96-well plates. Specificity of the antibody was tested by direct ELISA against IL-17, IL-17E, and IL-17F, as described previously,29Ito T. Schaller M. Hogaboam C.M. Standiford T.J. Sandor M. Lukacs N.W. Chensue S.W. Kunkel S.L. TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4.J Clin Invest. 2009; 119: 33-46PubMed Google Scholar, 30Kallal L.E. Hartigan A.J. Hogaboam C.M. Schaller M.A. Lukacs N.W. Inefficient lymph node sensitization during respiratory viral infection promotes IL-17-mediated lung pathology.J Immunol. 2010; 185: 4137-4147Crossref PubMed Scopus (26) Google Scholar, 31Lukacs N.W. Smit J.J. Mukherjee S. Morris S.B. Nunez G. Lindell D.M. Respiratory virus-induced TLR7 activation controls IL-17-associated increased mucus via IL-23 regulation.J Immunol. 2010; 185: 2231-2239Crossref PubMed Scopus (90) Google Scholar and was found to react only with IL-17. The polyclonal antibody was further purified from the serum using Protein A columns (Pierce; Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's protocol. Control antibody (Cab) was purified from serum of unimmunized mice also using Protein A columns. Mice were pretreated intraperitoneally with 2.5 mg of purified polyclonal anti-mouse IL-17 antibody 2.5 hours before RSV infection on day 0 and then every other day until day 6. The control group similarly received 2.5 mg of anti-mouse IgG antibody (Cab). Airway hyperreactivity was assessed as described previously.19Campbell E.M. Charo I.F. Kunkel S.L. Strieter R.M. Boring L. Gosling J. Lukacs N.W. Monocyte chemoattractant protein-1 mediates cockroach allergen-induced bronchial hyperreactivity in normal but not CCR2-/- mice: the role of mast cells.J Immunol. 1999; 163: 2160-2167PubMed Google Scholar, 32Hogaboam C.M. Gallinat C.S. Taub D.D. Strieter R.M. Kunkel S.L. Lukacs N.W. Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis.J Immunol. 1999; 162: 6071-6079PubMed Google Scholar, 33Lukacs N.W. Tekkanat K.K. Berlin A. Hogaboam C.M. Miller A. Evanoff H. Lincoln P. Maassab H. Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms.J Immunol. 2001; 167 ([Erratum appeared in J Immunol 2005;175:8442]): 1060-1065PubMed Google Scholar, 34Tekkanat K.K. Maassab H.F. Cho D.S. Lai J.J. John A. Berlin A. Kaplan M.H. Lukacs N.W. IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent.J Immunol. 2001; 166 ([Erratum appeared in J Immunol 2005;175:8442]): 3542-3548PubMed Google Scholar Briefly, mice were anesthetized with sodium pentobarbital, intubated via cannulation of the trachea, and ventilated with a Harvard pump ventilator (0.3 mL tidal volume; 120 breaths/minute). Airway hyperreactivity was measured using a direct ventilation methodology with a sensitive mouse plethysmograph and software for calculation of the measurements (Buxco Research Systems, Wilmington, NC). After baseline measurements, mice were injected intravenously with 7.5 μg of methacholine (Sigma-Aldrich), and the peak airway resistance was recorded as a measure of AHR. Total RSV antigen levels in lung homogenates were measured by a specific ELISA as described previously.35Kumar M. Behera A.K. Matsuse H. Lockey R.F. Mohapatra S.S. Intranasal IFN-gamma gene transfer protects BALB/c mice against respiratory syncytial virus infection.Vaccine. 1999; 18: 558-567Crossref PubMed Scopus (58) Google Scholar, 36Matsuse H. Behera A.K. Kumar M. Lockey R.F. Mohapatra S.S. Differential cytokine mRNA expression in Dermatophagoides farinae allergen-sensitized and respiratory syncytial virus-infected mice.Microbes Infect. 2000; 2: 753-759Crossref PubMed Scopus (16) Google Scholar Briefly, the lung samples (100 μL) were incubated for 1 hour in 96-well plates coated with goat anti-RSV antibody (Millipore). After three washes, the plates were incubated with a mouse anti-RSV polyclonal secondary antibody (Vector Laboratories, Burlingame, CA). After a final incubation with a peroxidase-labeled goat anti-mouse IgG antibody (Millipore), positive wells were assessed by o-phenylenediamine dihydrochloride (Dako, Carpinteria, CA) substrate development. Optical density readings were at 450 nm. For histology studies, right-lung lobes from infected mice were removed, fixed in 10% formalin, and stained with H&E or PAS to detect mucus production. For RT-PCR, the lower left lobe of the freshly harvested lung was snap-frozen and stored at −80°C. Total RNA was extracted from the frozen tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) and was reverse transcribed to cDNA. Real-time PCR was performed with the cDNA using gene-specific primers. Murine primers for IL-4, IL-5, IL-13, IFN-γ, IL-17, IL-17F, IL-23p19, IL-6, RANTES, KC, eotaxin, MDC, MIP1a, MIP2a, T-bet, eomesodermin (Eomes), and GAPDH were purchased from Applied Biosystems (Carlsbad, CA). Primers and probes for Muc5ac, Gob5, RSV-F, RSV-N, and RSV-G were determined using primer/probe detection sets designed by PE Biosystems (Foster City, CA) and purchased from Sigma-Aldrich. All results were normalized to GAPDH expression. Single-cell suspensions of lymph nodes were seeded at a concentration of 5 × 106 cells/mL in a 96-well plate and were restimulated with RSV. At 48 hours, the supernatants were harvested and analyzed for cytokines, which were quantified using a Bio-Plex bead-based (Luminex) cytokine assay purchased from Bio-Rad Laboratories. Single-cell suspensions of lung (after collagenase dispersion) and lymph node cells were prepared as described previously.37Lundy S.K. Lira S.A. Smit J.J. Cook D.N. Berlin A.A. Lukacs N.W. Attenuation of allergen-induced responses in CCR6-/- mice is dependent upon altered pulmonary T lymphocyte activation.J Immunol. 2005; 174: 2054-2060PubMed Google Scholar To detect various surface markers (CD3, CD8, CD4, and CD69 from eBioscience, San Diego, CA; pan NK and γδ T cell from BioLegend, San Diego, CA; and IL-17RA from R&D Systems), cells were stained with the indicated antibodies after 10 minutes of preincubation with Fc block (BD Biosciences, San Jose, CA). For detection of RSV M protein-specific T-cell receptor-positive CD8 T cells (RSV tetramer, M82-90; H2-kd), staining antibody was added 30 minutes before the addition of any other antibody and the reaction took place at room temperature. The cells were fixed overnight with 4% formalin. For intracellular staining, the cells were fixed and permeabilized using a staining buffer kit (eBioscience) and then stained with antibodies against T-bet, granzyme B, IFN-γ, and Eomes (all from eBioscience). The samples were processed through a flow cytometer (LSRII; Becton-Dickinson, Franklin Lakes, NJ). The data were analyzed using FlowJo software version 8.8.4 (TreeStar, Ashland, OR). CD8 T cells were sorted from the spleen using magnetic beads and plated in 96-well plates at a concentration of 2 × 105 cells per well in RPMI-1640 complete medium (Lonza, Walkersville, MD). The cells were stimulated with plate-coated anti-CD3 and soluble anti-CD28 (eBioscience) with or without recombinant IL-17 (R&D Systems) at 100 ng/mL. RNA was extracted from the cells at 6 hours after stimulation and was quantified by real-time RT-PCR. After AHR measurement and cervical dislocation, bronchoalveolar lavage fluid was collected from each mouse. One milliliter sterile 0.9 N saline was instilled intratracheally and was suctioned out after a few seconds. Samples were placed in Eppendorf tubes and were centrifuged at 1500 rpm for 5 minutes (240 × g). The cells were centrifuged in a shandon cytospin 2, fixed, and a differential cell count was done after Diff-Quik staining (Dade Behring, Newark, DE). Data are reported as means ± SE. Statistical significance was determined by Student's t-test or one-way analysis of variance with Newman-Keuls post hoc test. P < 0.05 was considered significant. To investigate whether RSV infection induces IL-17 production in vivo, BALB/c mice were infected with RSV and a time-course analysis was performed of mRNA expression of two different IL-17 transcripts (IL-17 and IL-17F) and the cytokines involved in their production (IL-6) and maintenance (IL-23p19). RSV infection significantly induced IL-17 but not IL-17F in the lungs of infected mice compared with control mice, with expression levels peaking at day 8 after infection (Figure 1A). IL-6 and IL-23p19 mRNA expression increased threefold and twofold, respectively, on day 8 after RSV infection (Figure 1A). RSV infection also increased IL-17 protein production in the lungs, and peak IL-17 levels correlated with the transcript levels (Figure 1B). Furthermore, we observed a significant increase in antigen-specific IL-17 production in the DLNs taken at day 8 from infected animals when they were restimulated ex vivo with RSV (Figure 1C). To determine which lung cells were expressing IL-17 during peak IL-17 production, CD4 T cells, pan natural killer (NK) cells, and γδ T cells taken at day 8 from RSV-infected lungs were sorted by flow cytometry and intracellular IL-17 levels were measured. We observed significant increase in IL-17+CD4 T cells in the lungs of RSV-infected mice, compared with uninfected mice (Figure 1D). For NK and γδ T cells, however, no significant difference was observed between infected and uninfected groups. These data demonstrate that RSV infection induces IL-17 expression by CD4 T cells in the lungs during peak IL-17 production. To validate our data, we further analyzed IL-6 and IL-17 levels in tracheal aspirate samples from infants who had been hospitalized and given mechanical ventilation because of RSV infection. Both cytokines were significantly increased in infected samples (Figure 1, E and F). Thus, IL-17 appears to be induced during RSV infection in severely ill infants and correspondingly in our animal model of RSV
Referência(s)