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Methanosphaera stadtmanae induces a type IV hypersensitivity response in a mouse model of airway inflammation

2017; Wiley; Volume: 5; Issue: 7 Linguagem: Inglês

10.14814/phy2.13163

2051-817X

Émilie Bernatchez, Matthew J. Gold, Anick Langlois, Pascale Blais‐Lecours, Magali Boucher, Caroline Duchaine, David Marsolais, Kelly M. McNagny, Marie-Renée Blanchet,

Asthma and respiratory diseases

Physiological ReportsVolume 5, Issue 7 e13163 Original ResearchOpen Access Methanosphaera stadtmanae induces a type IV hypersensitivity response in a mouse model of airway inflammation Emilie Bernatchez, Emilie Bernatchez Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorMatthew J. Gold, Matthew J. Gold The Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, CanadaSearch for more papers by this authorAnick Langlois, Anick Langlois Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorPascale Blais-Lecours, Pascale Blais-Lecours Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorMagali Boucher, Magali Boucher Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorCaroline Duchaine, Caroline Duchaine Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorDavid Marsolais, David Marsolais Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorKelly M. McNagny, Kelly M. McNagny The Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, CanadaSearch for more papers by this authorMarie-Renée Blanchet, Corresponding Author Marie-Renée Blanchet marie-renee.blanchet@criucpq.ulaval.ca Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, Canada Correspondence Marie-Renée Blanchet, Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec City, QC, Canada. Tel: 1-418-656-8711 ext. 3247 Fax: 418-656-4509 E-mail: marie-renee.blanchet@criucpq.ulaval.caSearch for more papers by this author Emilie Bernatchez, Emilie Bernatchez Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorMatthew J. Gold, Matthew J. Gold The Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, CanadaSearch for more papers by this authorAnick Langlois, Anick Langlois Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorPascale Blais-Lecours, Pascale Blais-Lecours Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorMagali Boucher, Magali Boucher Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorCaroline Duchaine, Caroline Duchaine Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorDavid Marsolais, David Marsolais Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, CanadaSearch for more papers by this authorKelly M. McNagny, Kelly M. McNagny The Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, CanadaSearch for more papers by this authorMarie-Renée Blanchet, Corresponding Author Marie-Renée Blanchet marie-renee.blanchet@criucpq.ulaval.ca Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Quebec City, Quebec, Canada Correspondence Marie-Renée Blanchet, Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec City, QC, Canada. Tel: 1-418-656-8711 ext. 3247 Fax: 418-656-4509 E-mail: marie-renee.blanchet@criucpq.ulaval.caSearch for more papers by this author First published: 31 March 2017 https://doi.org/10.14814/phy2.13163Citations: 11 Funding Information: This work was supported by AllerGen NCE, E.B., D.M. and M.R.B. are Fonds de Recherche du Québec en Santé scholars and E.B. is a Institut de recherche Robert-Sauvé en santé et sécurité du travail scholar. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Despite improved awareness of work-related diseases and preventive measures, many workers are still at high risk of developing occupational hypersensitivity airway diseases. This stems from a lack of knowledge of bioaerosol composition and their potential effects on human health. Recently, archaea species were identified in bioaerosols, raising the possibility that they play a major role in exposure-related pathology. Specifically, Methanosphaera stadtmanae (MSS) and Methanobrevibacter smithii (MBS) are found in high concentrations in agricultural environments and respiratory exposure to crude extract demonstrates immunomodulatory activity in mice. Nevertheless, our knowledge of the specific impact of methanogens exposure on airway immunity and their potential to induce airway hypersensitivity responses in workers remains scant. Analysis of the lung mucosal response to methanogen crude extracts in mice demonstrated that MSS and MBS predominantly induced TH17 airway inflammation, typical of a type IV hypersensitivity response. Furthermore, the response to MSS was associated with antigen-specific IgG1 and IgG2a production. However, despite the presence of eosinophils after MSS exposure, only a weak TH2 response and no airway hyperresponsiveness were observed. Finally, using eosinophil and mast cell-deficient mice, we confirmed that these cells are dispensable for the TH17 response to MSS, although eosinophils likely contribute to the exacerbation of inflammatory processes induced by MSS crude extract exposure. We conclude that, as MSS induces a clear type IV hypersensitivity lung response, it has the potential to be harmful to workers frequently exposed to this methanogen, and that preventive measures should be taken to avoid chronic hypersensitivity disease development in workers. Background To date, despite treatments and avoidance/prevention measures (Jolly et al. 2015; Cano-Jimenez et al. 2016; Suojalehto et al. 2016), workers in various environments are still regularly diagnosed with occupational lung hypersensitivity diseases such as asthma, obstructive pulmonary disease and hypersensitivity pneumonitis (HP) (Baldassarre et al. 2016; Burge 2016; Cushen et al. 2016; Gao and Li 2016; Kraim-Leleu et al. 2016). The struggle in preventing these occupational diseases stems from the presence of unknown antigens, including microorganisms, in environmental bioaerosols and their unknown potential to elicit an inflammatory response in the lung. A better understanding of the nature and immunogenicity of these antigens would lead to improved prevention and a better quality of life for workers. Interestingly, non-viable microorganisms from the archaea domain, namely methanogens from the Methanobacteriacaea group (Methanobrevibacter or Methanosphaera genus), were found in high concentrations (up to 108 archaea/m3) in bioaerosols from poultries, dairy farms and swine confinement buildings (Nehme et al. 2009; Blais-Lecours et al. 2012; Just et al. 2013). Furthermore, a mouse model of lung inflammation induced by crude extracts of these methanogens was recently developed and demonstrated species-dependent lung immune responses to Methanosphaera stadtmanae (MSS) and Methanobrevibacter smithii (MBS), with MSS being the more potent inducer (Blais-Lecours et al. 2011). This study demonstrated that archaeal crude extracts induce the recruitment of CD4+ and CD19+ cells in the lung along with a strong production of serum IgG (Blais-Lecours et al. 2011). Importantly, human endogenous viable methanogens species are associated with oral diseases (Lepp et al. 2004; Vianna et al. 2006, 2008, 2009; Vickerman et al. 2007; Jiang et al. 2009; Efenberger et al. 2015), intestinal diseases (Scanlan et al. 2008; Lee et al. 2013; Blais-Lecours et al. 2014; Mira-Pascual et al. 2015) and obesity (Zhang et al. 2009; Mbakwa et al. 2015). Methanogens activate human peripheral blood cells to release the important immune mediator TNF (Blais-Lecours et al. 2014), and methanogen-specific IgGs are detectable in periodontic and inflammatory bowel disease (IBD) patients, documenting their potential as activators of the human immune system in environments where the strict methanogen conditions allow their survival. Nevertheless, because of a lack of detailed information on the specific immune mechanisms induced by these microorganisms (alive or dead), the role of methanogen-laden bioaerosols in human inflammatory responses remains unclear. Hypersensitivity responses are defined as a pathogenic immune response to non-harmful antigens, and can lead to the development of various occupational hypersensitivity diseases such as occupational asthma and HP. These responses are classically separated in four types. The type 1 hypersensitivity response, also known as the allergic response, is, for example, involved in allergic asthma (Bogaert et al. 2009). It is mainly characterized by the recruitment and activation of eosinophils and mast cells through release of cytokines, such as IL-4, 5, 13, 33, and eotaxins, by type 2 innate lymphoid cells (ILC2s) and CD4+ T cells (TH2 CD4+ cells) (Hammad and Lambrecht 2015). These also drive isotype switching of B cells and the production of IgE and IgG1 immunoglobulins (Snapper et al. 1988). In the lung, chronic activation of this pathway normally leads to the development of airway hyperresponsiveness (AHR) (Lauzon and Martin 2016). Type II and III hypersensitivity responses (the latter being involved in HP (Bogaert et al. 2009)), lead to antibody production (IgG) resulting in either the killing of host cells by induction of apoptosis (type II) or in the formation of precipitates that drive a strong local immune response and tissue injury (type III) (Descotes and Choquet-Kastylevsky 2001; Rajan 2003; Warrington et al. 2011). Finally, type IV hypersensitivity responses can be found in diseases such as HP (Bogaert et al. 2009). This response is mainly cell-mediated, either by secretion of inflammatory mediators by type 1 (TH1) and type 17 (TH17) effector CD4+ T cells (interferon-gamma; IFNγ by TH1, and IL-17A by TH17 cells) or by the cytotoxic activity of CD8+ T cells (Descotes and Choquet-Kastylevsky 2001; Rajan 2003; Warrington et al. 2011). Using a mouse model of lung exposure to archaeal crude extracts and an array of genetically modified mice, we set out to characterize the type of hypersensitivity response induced by methanogen exposure to resolve their potential to induce hypersensitivity disease in highly exposed workers (Nehme et al. 2009; Blais-Lecours et al. 2012; Just et al. 2013). We demonstrate that MSS crude extracts induce a strong eosinophilic response at low dose that is associated with a mixed TH2/TH17 and a IgG1 lung mucosal response, while exposure to high doses result solely in a TH17 and a strong IgG1/IgG2a response typical of a type IV hypersensitivity response. Furthermore, although eosinophils are present in high quantity and contribute to the inflammatory response to MSS, we show they are dispensable for the development of the type IV hypersensitivity response and, accordingly, that MSS fails to induce AHR. We find that MBS crude extract also induces a TH17 lung response. We conclude that airborne, non-infectious exposure to methanogen bioaerosols, especially MSS, initiates a type IV hypersensitivity airway response, and that exposure of workers to these bioaerosols should be limited, if not prevented. Methods Animals C57BL/6J and C.129S1(B6)-Gata1tm6Sho/J (ΔdblGATA) were obtained from Jackson Laboratories and Cpa3-Cre mice were kindly provided by Feyerabend et al. (2011). Mice were kept in a pathogen-free animal unit (BRC, University of British Columbia, Vancouver, BC, Canada; CRIUCPQ, Laval University, Québec, QC, Canada) for the duration of the experiments. Ethics statement Experiments were approved by local ethics committees and followed Canadian Animal Care guidelines for the use of experimental mice. The study was approved by the Laval University Committee for Animal Care (protocol 2013-124-2). Mice were euthanized by an overdose of ketamine/xylazine according to the committee guidelines on rodent euthanasia. Cell culture and preparation of archaeal crude extract Methanosphaera stadtmanae (DSMZ-11975; MSS) and Methanobrevibacter smithii (DSMZ-3091, MBS) (DSMZ, Germany) were grown according to DSMZ's cultivation conditions. Cells were lyophilized and reconstituted as described by Blais-Lecours et al. (2011). Endotoxins were quantified in MSS and MBS crude extracts, revealing 124 and 22 EU/mg, respectively, using the Kinetic Chromogenic LAL Assay according to the manufacturer's instructions (Lonza Walkersville, Inc., Walkersville, MD). Induction and assessment of the airway inflammation The timeline used for the chronic model is presented in Figure 1A. Mice were anesthetized with isoflurane and received intranasal instillations (i.n.) of either 3 μg or 100 μg of MSS crude extract or 6.25 μg of MBS crude extract on three consecutive days/week for 3 weeks. Mice were euthanized 4 days after the last exposure. Upon euthanasia, mice were tracheotomized with an 18G catheter, and a broncho-alveolar lavage (BAL) was performed via three separate injections/aspirations of 1 mL saline. Total BAL cells were counted and differential counts obtained using Giemsa stain (HemaStain Set, Fisher Scientific, Kalamazoo, MI). For the administration of anti-IL-17A, mice received 50 μg of anti-IL-17A or isotype control antibodies (Bio X Cell, West Lebanon, NH) intra-peritoneally (i.p.) 1 h prior to each MSS i.n. exposure. Figure 1Open in figure viewerPowerPoint MSS induces a mixed TH2/TH17 immune lung response. (A) Timeline of the model of exposure to methanogens. Full line represents intranasal instillation of either PBS, MSS or MBS crude extract while dashed line represents day of euthanasia. (B) Flow cytometry gating strategy for the polarity of the effector lung response after ex-vivo stimulation of lung leukocytes isolated from methanogen-exposed mice. CD4+ T cells were gated from total lung cells and cytokine-positive cells were analyzed using Fluorescence Minus One (FMO) controls. (C) Severity of the inflammatory lung response after 3 μg MSS exposure was measured using total broncho-alveolar lavage (BAL) count and differential count. (D) Polarity of the effector response evaluated as number and the % of CD4+ cells expressing IFNg, IL-13 or IL-17A. (E) Il13 and Il17a expression measured by qRT-PCR on lung tissue of mice exposed to MSS compared with PBS. (F) MSS-specific IgG1 and IgG2a and total IgE production was measured from serum using ELISA. Results are representative of at least three separate experiments; n = 3–6 mice/group. * = P ˂ 0.05. Assessment of the airway hyperresponsiveness Four days after the final challenge, WT mice were anesthetized with ketamine/xylazine, tracheotomized and intubated with an 18G catheter. The airway resistance (Rrs) was measured with a Flexivent apparatus (SCIREQ, Montreal, Qc, Canada). Respiratory frequency was set at 150 breaths/min with a tidal volume of 0.2 mL, and a positive end-expiratory pressure of 3 mL H2O was applied. 50uL of increasing concentrations of methacholine (MCh) (0–64 mg/mL) was administered by nebulization. Detection of antigen-specific IgG1 and IgG2a and total IgE Upon euthanasia, blood was harvested through a cardiac puncture and serum was collected. For IgGs, ELISA plates were coated with 50 μg of MSS, blocked with 10% FBS in PBS and incubated with serum dilutions. Samples were then incubated with either anti-mouse IgG1-HRP or IgG2a-HRP (BD Biosciences, San Diego, CA). Total IgE were measured using a Mouse IgE ELISA Set (BD OptEIA) according to the manufacturer's instructions. The reactions were revealed with BD OptEIA TMB (BD Biosciences) and stopped with 1N HCl. Isolation of lung leukocytes Lung leukocytes were obtained by digestion of lung tissue with Collagenase IV (Sigma, Oakville, ON, Canada) for 0.5 h at 37°C. Digested tissue was pressed through a 70 μm cell strainer and leukocytes were enriched using a 30% Percoll gradient (GE Healthcare, Uppsala, Sweden). Red blood cells were lysed using ammonium chloride. T cell cytokine production assays CD4+ T cell cytokine production was induced by stimulating 1 × 106 lung-isolated leukocytes with PMA (50 ng/mL), ionomycin (750 ng/mL) and brefeldin A (3 μg/mL) in RPMI 1640 supplemented with 10% FBS and 1% antibiotic/antimycotic in a 96-well plate for 4 h. Intracellular cytokine production was analyzed by flow cytometry as described below. Flow cytometry analysis of isolated leukocytes Cells were suspended in PBS supplemented with 10% FBS and 0.02% sodium azide for cell surface staining. Cells were stained with CD4 (eBioscience). For intracellular staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience). Antibodies used were: IL-13, IL-17A and IFNg (BioLegend). We measured the polarity of the inflammatory response by gating on CD4+ T cells from whole lung cells and using fluorescence minus one (FMO) controls (gating presented on Fig. 1B). Lung ILC2s were gated as described in (Gold et al. 2014) and stained with: CD25, ST2, Sca-1 (eBioscience), CD3e, CD11b, CD11c, CD19, Gr1, NK1.1, Ter119, CD45, and CD90.2 (Ablab, Vancouver, B.C., Canada). RNA isolation and quantitative PCR Lung tissue was homogenized in Trizol and RNA samples were prepared using EZ-10 DNAaway RNA Mini-Preps kit (BioBasic Canana INC., Markham, CA). Using iScript cDNA Synthesis Kit (Bio-rad, Mississauga, CA), cDNA was obtained and used for quantitative RT-PCR using SsoAdvenced Universal SYBR Green Supermix (Bio-rad) and a rotor gene 6000 (QIAGEN, Valencia, Calif). Validated primers (efficiency between 90% and 105%) used for quantitative RT-PCR: Ccl11 5′-GAATCACCAACAACAGATGCAC-3′ (fwr) and 5′-ATCCTGGACCCACTTCTTCTT-3′ (rev); Ccl24 5′-ATTCTGTGACCATCCCCTCAT-3′ (fwr) and 5′-TGTATGTGCCTCTGAACCCAC-3′ (rev); Il13 5′-CCTGGCTCTTGCTTGCCT T-3′ (fwr) and 5′-GGTCTTGTGTGATGTTGCTCA-3′ (rev); Il17a 5′-AGCAGCGATCATCCCTCAAAG-3′ (fwr) and 5′-TCACAGAGGATATCTATCAGGGTC-3′ (rev); and Il33 5′-GGGAAGAAGGTGATGGTGAA-3′ (fwr) and 5′-CCGAAGGACTTTTTGTGAAGG-3′ (rev). Relative quantification was calculated with 2−ΔCT and using GAPDH and GNB as reference genes (Pfaffl 2001; Vandesompele et al. 2002). Statistics Data are presented as mean ± SEM. Data were tested for normality and homogeneity of variance. When appropriate, variables were log-transformed. Statistical analysis for multiple comparisons was performed using an ANOVA table followed by Tukey's multiple comparison test or by a Kruskal-Wallis rank sum test followed by Nemenyi-Tests. Non-multiple comparisons were analyzed using unpaired T-tests. Statistical significance was determined at P < 0.05. Results To characterize the immune response induced by MSS and its potential to induce hypersensitivity responses, we first evaluated the polarity of the effector response following exposure to MSS crude extract. MSS crude extract quantities used in our protocol were similar to the ones used by Blais-Lecours et al. (2011), which are based on the well-described model of hypersensitivity pneumonitis to Saccharopolyspora rectivirgula (SR) antigen and widely accepted to mimic human pathology onset, severity and inflammatory processes (Denis et al. 1991; Gudmundsson and Hunninghake 1997; Nance et al. 2004). To insure the response to MSS in our study was similar to what was previously described (Blais-Lecours et al. 2011), the broncho-alveolar lavage (BAL) inflammatory response was analyzed. As described by Blais-Lecours et al., exposure to low dose of MSS (3 μg) increased the number of immune cells in the BAL (Fig. 1C), and these were characterized by the presence of lymphocytes, neutrophils and eosinophils (Fig. 1C). To complement the analysis previously published by Blais-Lecours et al., the polarity of the response to MSS was thoroughly studied. The analysis of cytokine polarity production by CD4+ T cells revealed a predominant TH17 response, as determined by the high frequency of CD4/IL-17A+ cells (Fig. 1D). Interestingly, exposure to 3 μg of MSS also induced weak TH2 responses, as denoted by an increased number and % of the CD4/IL-13+ cells (Fig. 1D). Polarization towards TH17 was further confirmed by increased expression of Il17a, but not Il13, via qRT-PCR (Fig. 1E). Analyses of serum immunoglobulins revealed a strong production of MSS-specific IgG1, a small production of IgG2a, and no IgE production (Fig. 1F). These data suggest that a type IV hypersensitivity response is induced after exposure to low dose of MSS, although we also observed a low level of allergic, type I hypersensitivity response. Similar to previous studies (Blais-Lecours et al. 2015), an exacerbated total response was observed at 100 μg MSS compared with 3 μg, characterized mainly by macrophages and lymphocytes (Fig. 2A). Of important note is the inversely proportional presence of eosinophils to the quantity of instilled MSS, as denoted by the higher % of eosinophils response at 3 μg MSS compared with 100 μg. This is very interesting as the total number of cells is similar after exposure to low or high quantities of MSS (Fig. 2A). This suggests that different types of hypersensitivity responses can be developed with different levels of MSS exposure, or that the higher dose shifts the timing of the response to include more macrophages at the time of euthanasia. Independently of the explanation, this is an important observation and a significant factor in determining the potential impact of MSS bioaerosols exposure in humans. Additionally, when looking at the effector response, we found that the cell number and the % of CD4/IL-13+ T cells are decreased in mice exposed to 100 μg compared with 3 μg, while the CD4/IL-17A+ T cells population remains unchanged (Fig. 2B). Furthermore, IgG1 and IgG2a levels are increased in mice exposed to 100 μg MSS (Fig. 2C), while the IgG2a production in response to 3 μg MSS was very weak. Thus, MSS exposure mainly promotes a TH17-dominated and strong IgG1/IgG2a response, indicative of a type IV hypersensitivity response. Figure 2Open in figure viewerPowerPoint MSS mainly induces a TH17 polarized immune response. (A) Severity of the inflammatory response following exposure to 3 μg versus 100 μg of MSS was quantified using total broncho-alveolar lavage (BAL) count and differential count. Results are representative of at least three separate experiments; n = 3–6 mice/group. * = P ˂ 0.05. (B) Polarity of the effector response evaluated as number and the % of CD4+ cells expressing IFNg, IL-13 or IL-17A. (C) MSS-specific IgG1 and IgG2a production was measured from serum using ELISA. Results were pooled from two experiments; n = 6–10 mice/group. † = P ˂ 0.05 with multi-comparison test. Using the Flexivent Apparatus, (D) airway resistance (Rrs) was evaluated in mice exposed to 3 μg or 100 μg MSS. Results were pooled from two experiments; n = 6–12 mice/group. * = P ˂ 0.05. As mentioned, the lung TH2 response and the presence of eosinophils in BAL are associated with the development of AHR (Gauvreau et al. 1999; Wynn 2015). Using a Flexivent apparatus, we set out to verify whether exposure to MSS crude extract induces an exaggerated increase in the airway resistance (Rrs), which is a measure of AHR, compared with the negative control PBS (Blanchet et al. 2007; Bernatchez et al. 2015). Our group has extensively described that an increase in Rrs in the area of 500% over baseline is considered a positive response for AHR in C57Bl/6J background (Blanchet et al. 2007; Bernatchez et al. 2015). We find that neither 3 μg nor 100 μg of MSS induce an exaggerated increase in Rrs compared with the controls. Indeed, the response observed in mice exposed to MSS is similar to mice exposed to the negative control PBS, and in the area of 200% increase in Rrs compared with the baseline (Fig. 2D). Taking into account the absence of AHR, in addition to the weak presence of CD4/IL-13+ cells at 3 μg of MSS and the absence of IgE, these results indicate that MSS does not induce a type I hypersensitivity allergic reaction in the lung. To test whether other methanogens found in bioaerosols have the potential to induce a type IV hypersensitivity response, we exposed mice to MBS crude extract, which has also been shown to cause an immune response in the lung (Blais-Lecours et al. 2011). As described before (Blais-Lecours et al. 2011), BAL counts from mice exposed to 6.25 μg of MBS showed a weaker inflammatory response than 3 μg MSS and this was characterized by fewer granulocytes (Fig. 3A). When looking at the polarity of the effector response, we found that MBS also induces mainly CD4/IL-17A+ cells (Fig. 3B). Furthermore, neither IgG1 nor IgG2a production could be observed (Fig. 3C). Low-dose exposure to MBS therefore induces a TH17 response, highlighting its potential to induce a type IV hypersensitivity response, although the overall inflammatory response is weaker than MSS and with less inflammatory cells in the lung. Figure 3Open in figure viewerPowerPoint MBS induces a weak TH17 immune lung response. (A) Severity of the inflammatory lung response after 6.25 μg MBS exposure was measured using total broncho-alveolar lavage (BAL) count and differential count. (B) Polarity of the effector response evaluated as number and the % of CD4+ cells expressing IFNg, IL-13 or IL-17A. (C) MBS-specific IgG1 and IgG2a production was measured from serum using ELISA. Results are representative of at least three separate experiments; n = 3–6 mice/group. * = P ˂ 0.05. Eosinophils are present in response to low doses of MSS and the presence of eosinophils in the lung after exposure to non-infectious agents is anticipated to be associated to allergic, asthma-like symptoms. Importantly, eosinophils can also contribute to resolution processes in asthma (Takeda et al. 2015), but their significance and role in MSS-induced airway hypersensitivity remains misunderstood. Therefore, we set out to further evaluate the importance of this cell population in the response to MSS crude extract exposure. However, we found that low-dose exposure to MSS induces only a weak CD4/IL-13+ response and recruitment of eosinophils (Nakajima et al. 1992; Pope et al. 2001), and no devolopment of AHR, a response associated with the presence of eosinophils (Cockcroft and Davis 2006). Accordingly, we examined the functional significance of eosinophils in the airway inflammatory response to MSS, and on their potential harmful impact in disease. We first assessed the activation of eosinophil recruitment pathways in response to MSS. We analyzed eotaxins and IL-33 production, which are well-known chemokines involved in eosinophil recruitment. qRT-PCR analyses revealed no increase in eotaxins Ccl11 (Eotaxin-1) and Ccl24 (Eotaxin-2) following MSS crude extract exposure, but an increase in Il33 mRNA expression (Fig. 4A). IL-33 is a potent activator of lung ILC2s (Neill et al. 2010), which, in turn, recruit eosinophils in allergy development (Gold et al. 2014). To assess whether MSS-induced expression of IL-33 promotes the expansion of lung ILC2s, we quantified lung ILC2s following antigen challenge by flow cytometry (Lineage−CD45+Sca1+CD90.2+CD25+ST2+). The frequency of lung ILC2s was unchanged following exposure to MSS (Fig. 4B). Thus, these results suggest that MSS-induced eosinophil recruitment to the lungs is not induced by type I hypersensitivity mechanisms. Figure 4Open in figure viewerPowerPoint IL-17A blockade leads to reduced total inflammation and eosinophil influx in the airways. (A) Expression of Ccl11, Ccl24, and Il33 measured by qRT-PCR. (B) The % of ILC2s (Lineage−CD45+Sca1+CD90.2+CD25+ST2+) in lung of mice exposed to MSS compared with PBS. (C) The severity of the inflammatory lung response in mice that received isotype or anti-IL-17A 1 h before each exposure to 3 μg MSS was verified using broncho-alveolar lavage (BAL) count. Results were pooled from two experiments; n = 6–12 mice/group. * = P ˂ 0.05. One of the major cytokine involved in type IV hypersensitivity responses, IL-17A, is recognized for its role in neutrophil recruitment (Hellings et al. 2003) and stromal cell activation (Molet et al. 2001; Hasan et al. 2013), which can lead to development of fibrosis, such as in HP (Hasan et al. 2013). However, IL-17A also plays a role in eosinophil recruitment to the lung (Schnyder-Candrian et al. 2006; Murdock et al. 2012). Thus, we verified whether this cytokine is involved in eosinophil accumulation in response to MSS. To do so, mice were given 50 μg i.p. of anti-IL-17A antibodies 1 h prior to 3 μg MSS exposure, and the airway inflammatory response was evaluated. We find that blockade of IL-17A reduced the total inflammatory response, characterized by fewer numbers of granulocytes, compared with the mice that received the isotype