High throughput screening of airway constriction in mouse lung slices.

The level of airway constriction in thin slices of lung tissue is highly variable. Owing to the labor-intensive nature of these experiments, determining the number of airways to be analyzed in order to allocate a reliable value of constriction in one mouse is challenging. Herein, a new automated device for physiology and image analysis was used to facilitate high throughput screening of airway constriction in lung slices. Airway constriction was first quantified in slices of lungs from male BALB/c mice with and without experimental asthma that were inflated with agarose through the trachea or trans-parenchymal injections. Random sampling simulations were then conducted to determine the number of airways required per mouse to quantify maximal constriction. The constriction of 45 ± 12 airways per mouse in 32 mice were analyzed. Mean maximal constriction was 37.4 ± 32.0%. The agarose inflating technique did not affect the methacholine response. However, the methacholine constriction was affected by experimental asthma (p = 0.003), shifting the methacholine concentration-response curve to the right, indicating a decreased sensitivity. Simulations then predicted that approximately 35, 16 and 29 airways per mouse are needed to quantify the maximal constriction mean, standard deviation and coefficient of variation, respectively; these numbers varying between mice and with experimental asthma.

Lung Volumes in a Mouse Model of Pulmonary Allergic Inflammation.

PURPOSE: Air trapping, often attested in humans by elevated residual volume (RV) and ratio of RV on total lung capacity (RV/TLC), is frequently observed in asthma. Confirming these alterations in experimental asthma would be important for translational purposes. Herein, lung volumes were investigated in a mouse model of pulmonary allergic inflammation. METHODS: Eight- to 10-week-old male C57BL/6 and BALB/c mice were exposed once daily to intranasal house dust mite (HDM) for 10 consecutive days. All readouts were measured 24 h after the last exposure. Lung volumes were assessed with the flexiVent using a new automated method consisting of degassing the lungs followed by a full-range pressure-volume maneuver. The weight and the volume of the lungs were also measured ex vivo and a lobe was further processed for histological analyses. RESULTS: HDM exposure led to tissue infiltration with inflammatory cells, goblet cell hyperplasia, thickening of the airway epithelium, and elevated ex vivo lung weight and volume. It also decreased TLC and vital capacity but without affecting RV and RV/TLC. These observations were similar between the two mouse strains. CONCLUSION: Alterations of lung volumes in a murine model of pulmonary allergic inflammation are inconsistent with observations made in human asthma. These discrepancies reflect the different means whereby lung volumes are measured between species. The invasive method used herein enables RV to be measured more precisely and without the confounding effect of air trapping, suggesting that changes in RV and RV/TLC using this method in mice should be interpreted differently than in humans.

Sensitivity of the airway smooth muscle in terms of force, shortening and stiffness.

Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10-8 to 10-5 M. For each strip, the concentration-response was repeated four times in a randomized order to measure isometric force, isotonic shortening against a load corresponding to either 5 or 10 % of a reference force, and average force, stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5 % at 0.2 Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5 % load had the lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was followed by isotonic shortening with a 10 % load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38), isometric force (-6.32), and average force (-6.30). Some of these differences were significant. For example, the EC50 with the average force was 44 % greater than with the elastance (p=0.001). The methacholine sensitivity is thus affected by the contractile readout being measured.

A Quick Method to Assess Airway Distensibility in Mice.

Airway distensibility is defined as the ease whereby airways are dilating in response to inflating lung pressure. If measured swiftly and accurately, airway distensibility would be a useful readout to parse the various elements contributing to airway wall stiffening, such as smooth muscle contraction, surface tension, and airway remodeling. The goal of the present study was to develop a method for measuring airway distensibility in mice. Lungs of BALB/c and C57BL/6 mice from either sex were subjected to stepwise changes in pressure. At each pressure step, an oscillometric perturbation was used to measure the impedance spectrum, on which the constant-phase model was fitted to deduce a surrogate for airway caliber called Newtonian conductance (GN). The change in GN over the change in pressure was subsequently used as an index of airway distensibility. An additional group of mice was infused with methacholine to confirm that smooth muscle contraction changes airway distensibility. GN increased with increasing steps in pressure, suggesting that the extent to which this occurs can be used as an index of airway distensibility. Airway distensibility was greater in BALB/c than C57BL/6 mice, and its variation by sex was mouse strain dependent, being greater in female than male in BALB/c mice with an inverse trend in C57BL/6 mice. Airway distensibility was also decreased by methacholine. This novel method swiftly measures airway distensibility in mice. Airway distensibility was also shown to vary with sex and mouse strain and to be sensitive to the contraction of smooth muscle.

Lung stiffness of C57BL/6 versus BALB/c mice.

This study was undertaken to determine whether a smaller lung volume or a stiffer lung tissue accounts for the greater lung elastance of C57BL/6 than BALB/c mice. The mechanical properties of the respiratory system and lung volumes were measured with the flexiVent and compared between male C57BL/6 and BALB/c mice (n = 9). The size of the excised lung was also measured by volume liquid displacement. One lobe was then subjected to sinusoidal strains in vitro to directly assess the mechanical properties of the lung tissue, and another one was used to quantify the content of hydroxyproline. In vivo elastance was markedly greater in C57BL/6 than BALB/c mice based on 5 different readouts. For example, respiratory system elastance was 24.5 ± 1.7 vs. 21.5 ± 2.4 cmH2O/mL in C57BL/6 and BALB/c mice, respectively (p = 0.007). This was not due to a different lung volume measured by displaced liquid volume. On the isolated lobes, both elastance and the hydroxyproline content were significantly greater in C57BL/6 than BALB/c mice. These results suggest that the lung elastance of C57BL/6 mice is greater than BALB/c mice not because of a smaller lung volume but because of a stiffer lung tissue due to a greater content of collagen.

Bronchial thermoplasty attenuates bronchodilator responsiveness.

INTRODUCTION: Bronchial thermoplasty is an effective intervention to improve respiratory symptoms and to reduce the rate of exacerbations in uncontrolled severe asthma. A reduction in airway smooth muscle is arguably the most widely discussed mechanisms accounting for these clinical benefits. Yet, this smooth muscle reduction should also translate into an impaired response to bronchodilator drugs. This study was designed to address this question. METHODS: Eight patients with clinical indication for thermoplasty were studied. They were uncontrolled severe asthmatics despite optimal environmental control, treatment of comorbidities, and the use of high-dose inhaled corticosteroids and long-acting ß2-agonists. Lung function measured by spirometry and respiratory mechanics measured by oscillometry were examined pre- and post-bronchodilator (salbutamol, 400 µg), both before and at least 1 year after thermoplasty. RESULTS: Consistent with previous studies, thermoplasty yielded no benefits in terms of baseline lung function and respiratory mechanics, despite improving symptoms based on two asthma questionnaires (ACQ-5 and ACT-5). The response to salbutamol was also not affected by thermoplasty based on spirometric readouts, including forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio. However, a significant interaction was observed between thermoplasty and salbutamol for two oscillometric readouts, namely reactance at 5 Hz (Xrs5) and reactance area (Ax), showing an attenuated response to salbutamol after thermoplasty. CONCLUSIONS: Thermoplasty attenuates the response to a bronchodilator. We argue that this result is a physiological proof of therapeutic efficacy, consistent with the well-described effect of thermoplasty in reducing the amount of airway smooth muscle.

Force adaptation through the intravenous route in naïve mice.

Aim of the study: Force adaptation is a process whereby the contractile capacity of the airway smooth muscle increases during a sustained contraction (aka tone). Tone also increases the response to a nebulized challenge with methacholine in vivo, presumably through force adaptation. Yet, due to its patchy pattern of deposition, nebulized methacholine often spurs small airway narrowing heterogeneity and closure, two important enhancers of the methacholine response. This raises the possibility that the potentiating effect of tone on the methacholine response is not due to force adaptation but by furthering heterogeneity and closure. Herein, methacholine was delivered homogenously through the intravenous (i.v.) route. Materials and Methods: Female and male BALB/c mice were subjected to one of two i.v. methacholine challenges, each of the same cumulative dose but starting by a 20-min period either with or without tone induced by serial i.v. boluses. Changes in respiratory mechanics were monitored throughout by oscillometry, and the response after the final dose was compared between the two challenges to assess the effect of tone. Results: For the elastance of the respiratory system (Ers), tone potentiated the methacholine response by 64 and 405% in females (37.4 ± 10.7 vs. 61.5 ± 15.1 cmH2O/mL; p = 0.01) and males (33.0 ± 14.3 vs. 166.7 ± 60.6 cmH2O/mL; p = 0.0004), respectively. For the resistance of the respiratory system (Rrs), tone potentiated the methacholine response by 129 and 225% in females (9.7 ± 3.5 vs. 22.2 ± 4.3 cmH2O·s/mL; p = 0.0003) and males (10.7 ± 3.1 vs. 34.7 ± 7.9 cmH2O·s/mL; p < 0.0001), respectively. Conclusions: As previously reported with nebulized challenges, tone increases the response to i.v. methacholine in both sexes; albeit sexual dimorphisms were obvious regarding the relative resistive versus elastic nature of this potentiation. This represents further support that tone increases the lung response to methacholine through force adaptation.

More airway smooth muscle in males versus females in a mouse model of asthma: A blessing in disguise?

NEW FINDINGS: What is the central question of this study? The lung response to inhaled methacholine is reputed to be greater in male than in female mice. The underpinnings of this sex disparity are ill defined. What is the main finding and its importance? We demonstrated that male airways exhibit a greater content of airway smooth muscle than female airways. We also found that, although a more muscular airway tree in males might contribute to their greater responsiveness to inhaled methacholine than females, it might also curb the heterogeneity in small airway narrowing. ABSTRACT: Mouse models are helpful in unveiling the mechanisms underlying sex disparities in asthma. In comparison to their female counterparts, male mice are hyperresponsive to inhaled methacholine, a cardinal feature of asthma that contributes to its symptoms. The physiological details and the structural underpinnings of this hyperresponsiveness in males are currently unknown. Herein, BALB/c mice were exposed intranasally to either saline or house dust mite once daily for 10 consecutive days to induce experimental asthma. Twenty-four hours after the last exposure, respiratory mechanics were measured at baseline and after a single dose of inhaled methacholine that was adjusted to trigger the same degree of bronchoconstriction in both sexes (it was twice as high in females). Bronchoalveolar lavages were then collected, and the lungs were processed for histology. House dust mite increased the number of inflammatory cells in bronchoalveolar lavages to the same extent in both sexes (asthma, P = 0.0005; sex, P = 0.96). The methacholine response was also markedly increased by asthma in both sexes (e.g., P = 0.0002 for asthma on the methacholine-induced bronchoconstriction). However, for a well-matched bronchoconstriction between sexes, the increase in hysteresivity, an indicator of airway narrowing heterogeneity, was attenuated in males for both control and asthmatic mice (sex, P = 0.002). The content of airway smooth muscle was not affected by asthma but was greater in males (asthma, P = 0.31; sex, P < 0.0001). These results provide further insights regarding an important sex disparity in mouse models of asthma. The increased amount of airway smooth muscle in males might contribute functionally to their greater methacholine response and, possibly, to their decreased propensity for airway narrowing heterogeneity.

The cumulative effect of methacholine on large and small airways when deep inspirations are avoided.

BACKGROUND AND OBJECTIVE: The effect of serial incremental concentrations of methacholine is only slightly cumulative when assessed by spirometry. This limited cumulative effect may be attributed to the bronchodilator effect of deep inspirations that are required between concentrations to measure lung function. Using oscillometry, the response to methacholine can be measured without deep inspirations. Conveniently, oscillometry can also dissociate the contribution of large versus small airways. Herein, oscillometry was used to assess the cumulative effect of methacholine in the absence of deep inspirations on large and small airways. METHODS: Healthy and asthmatic volunteers underwent a multiple-concentration methacholine challenge on visit 1 and a single-concentration challenge on visit 2 using the highest concentration of visit 1. The maximal response was compared between visits to assess the cumulative effect of methacholine. The lung volume was also measured after the final concentration to assess hyperinflation. RESULTS: In both healthy and asthmatic subjects, increases in resistance at 19 Hz (Rrs19 ), reflecting large airway narrowing, did not differ between the multiple- and the single-concentration challenge. However, increases in resistance at 5 Hz (Rrs5 ) minus Rrs19 , reflecting small airway narrowing, were 117 and 270% greater in the multiple- than the single-concentration challenge in healthy (p = 0.006) and asthmatic (p < 0.0001) subjects, respectively. Hyperinflation occurred with both challenges and was greater in the multiple- than the single-concentration challenge in both groups. CONCLUSION: Without deep inspirations, the effect of methacholine is cumulative on small airways but not on large airways. Lung hyperinflation and derecruitment may partially explain these different responses.

In mice of both sexes, repeated contractions of smooth muscle in vivo greatly enhance the response of peripheral airways to methacholine.

BALB/c mice from both sexes underwent one of two nebulized methacholine challenges that were preceded by a period of 20 min either with or without tone induced by repeated contractions of the airway smooth muscle. Impedance was monitored throughout and the constant phase model was used to dissociate the impact of tone on conducting airways (RN - Newtonian resistance) versus the lung periphery (G and H - tissue resistance and elastance). The effect of tone on smooth muscle contractility was also tested on excised tracheas. While tone markedly potentiated the methacholine-induced gains in H and G in both sexes, the gain in RN was only potentiated in males. The contractility of female and male tracheas was also potentiated by tone. Inversely, the methacholine-induced gain in hysteresivity (G/H) was mitigated by tone in both sexes. Therefore, the tone-induced muscle hypercontractility impacts predominantly the lung periphery in vivo, but also promotes further airway narrowing in males while protecting against narrowing heterogeneity in both sexes.

Sensitive physiological readouts to evaluate countermeasures for lipopolysaccharide-induced lung alterations in mice.

Despite decades of research, studies investigating the physiological alterations caused by an acute bout of inflammation induced by exposing the lung to lipopolysaccharide have yielded inconsistent results. This can be attributed to small effects and/or a lack of fitted physiological testing. Herein, a comprehensive investigation of lung mechanics was conducted on 270 male C57BL/6 mice at 24, 48, or 96 h after an intranasal exposure to saline or lipopolysaccharide at either 1 or 3 mg/kg (30 mice per group). Traditional techniques that probe the lung using small-amplitude perturbations (i.e., oscillometry) were used, together with less conventional and new techniques that probe the lung using maneuvers of large amplitudes. The latter include a partial and a full-range pressure-volume maneuvers to measure quasi-static elastance, compliance, total lung volume, vital capacity, and residual volume. The results demonstrate that lung mechanics assessed by oscillometry was only slightly affected by lipopolysaccharide, confirming previous findings. In contradistinction, lipopolysaccharide markedly altered mechanics when the lung was probed with maneuvers of large amplitudes. With the dose of 3 mg/kg at the peak of inflammation (48 h postexposure), lipopolysaccharide increased quasi-static elastance by 26.7% (P < 0.0001) and decreased compliance by 34.5% (P < 0.0001). It also decreased lung volumes, including total lung capacity, vital capacity, and residual volume by 33.3%, 30.5%, and 43.3%, respectively (all P < 0.0001). These newly reported physiological alterations represent sensitive outcomes to efficiently evaluate countermeasures (e.g., drugs) in the context of several lung diseases.

Effects of airway smooth muscle contraction and inflammation on lung tissue compliance.

There are renewed interests in using the parameter K of Salazar-Knowles' equation to assess lung tissue compliance. K either decreases or increases when the lung's parenchyma stiffens or loosens, respectively. However, whether K is affected by other common features of respiratory diseases, such as inflammation and airway smooth muscle (ASM) contraction, is unknown. Herein, male C57BL/6 mice were treated intranasally with either saline or lipopolysaccharide (LPS) at 1 mg/kg to induce pulmonary inflammation. They were then subjected to either a multiple or a single-dose challenge with methacholine to activate ASM to different degrees. A quasi-static pressure-driven partial pressure-volume (P-V) maneuver was performed before and after methacholine. The Salazar-Knowles' equation was then fitted to the deflation limb of the P-V loop to obtain K, as well as the parameter A, an estimate of lung volume (inspiratory capacity). The fitted curve was also used to derive the quasi-static elastance (Est) at 5 cmH2O. The results demonstrate that LPS and both methacholine challenges increased Est. LPS also decreased A, but did not affect K. In contradistinction, methacholine decreased both A and K in the multiple-dose challenge, whereas it decreased K but not A in the single-dose challenge. These results suggest that LPS increases Est by reducing the open lung volume (A) and without affecting tissue compliance (K), whereas methacholine increases Est by decreasing tissue compliance with or without affecting lung volume. We conclude that lung tissue compliance, assessed using the parameter K of Salazar-Knowles' equation, is insensitive to inflammation but sensitive to ASM contraction.

Double-chamber plethysmography versus oscillometry to detect baseline airflow obstruction in a model of asthma in two mouse strains.

AIM OF THE STUDY: The current gold standard to assess respiratory mechanics in mice is oscillometry, a technique from which several readouts of the respiratory system can be deduced, such as resistance and elastance. However, these readouts are often not altered in mouse models of asthma. This is in stark contrast with humans, where asthma is generally associated with alterations when assessed by either oscillometry or other techniques. In the present study, we have used double-chamber plethysmography (DCP) to evaluate the breathing pattern and the degree of airflow obstruction in a mouse model of asthma. MATERIALS AND METHODS: Female C57BL/6 and BALB/c mice were studied at day 1 using DCP, as well as at day 11 using both DCP and oscillometry following a once-daily exposure to either house-dust mite (HDM) or saline for 10 consecutive days. RESULTS: All DCP readouts used to describe either the breathing pattern (e.g., tidal volume and breathing frequency) or the degree of airflow obstruction (e.g., specific airway resistance) were different between mouse strains at day 1. Most of these strain differences persisted at day 11. Most oscillometric readouts (e.g., respiratory system resistance and elastance) were also different between strains. Changes caused by HDM were obvious with DCP, including decreases in tidal volume, minute ventilation, inspiratory time and mid-tidal expiratory flow and an increase in specific airway resistance. HDM also caused some strain specific alterations in breathing pattern, including increases in expiratory time and end inspiratory pause, which were only observed in C57BL/6 mice. Oscillometry also detected a small but significant increase in tissue elastance in HDM versus saline-exposed mice. CONCLUSIONS: DCP successfully identified differences between C57BL/6 and BALB/c mice, as well as alterations in mice from both strains exposed to HDM. We conclude that, depending on the study purpose, DCP may sometimes outweigh oscillometry.

Smooth Muscle Hypocontractility and Airway Normoresponsiveness in a Mouse Model of Pulmonary Allergic Inflammation.

The contractility of airway smooth muscle (ASM) is labile. Although this feature can greatly modulate the degree of airway responsiveness in vivo, the extent by which ASM's contractility is affected by pulmonary allergic inflammation has never been compared between strains of mice exhibiting a different susceptibility to develop airway hyperresponsiveness (AHR). Herein, female C57BL/6 and BALB/c mice were treated intranasally with either saline or house dust mite (HDM) once daily for 10 consecutive days to induce pulmonary allergic inflammation. The doses of HDM were twice greater in the less susceptible C57BL/6 strain. All outcomes, including ASM contractility, were measured 24 h after the last HDM exposure. As expected, while BALB/c mice exposed to HDM became hyperresponsive to a nebulized challenge with methacholine in vivo, C57BL/6 mice remained normoresponsive. The lack of AHR in C57BL/6 mice occurred despite exhibiting more than twice as much inflammation than BALB/c mice in bronchoalveolar lavages, as well as similar degrees of inflammatory cell infiltrates within the lung tissue, goblet cell hyperplasia and thickening of the epithelium. There was no enlargement of ASM caused by HDM exposure in either strain. Unexpectedly, however, excised tracheas derived from C57BL/6 mice exposed to HDM demonstrated a decreased contractility in response to both methacholine and potassium chloride, while tracheas from BALB/c mice remained normocontractile following HDM exposure. These results suggest that the lack of AHR in C57BL/6 mice, at least in an acute model of HDM-induced pulmonary allergic inflammation, is due to an acquired ASM hypocontractility.

Flexibility of microstructural adaptations in airway smooth muscle.

The airway smooth muscle undergoes an elastic transition during a sustained contraction, characterized by a gradual decrease in hysteresivity caused by a relatively greater rate of increase in elastance than resistance. We recently demonstrated that these mechanical changes are more likely to persist after a large strain when they are acquired in dynamic versus static conditions; as if the microstructural adaptations liable for the elastic transition are more flexible when they evolve in dynamic conditions. The extent of this flexibility is undefined. Herein, contracted ovine tracheal smooth muscle strips were kept in dynamic conditions simulating tidal breathing (sinusoidal length oscillations at 5% amplitude) and then subjected to simulated deep inspirations (DI). Each DI was straining the muscle by either 10%, 20%, or 30% and was imposed at either 2, 5, 10, or 30 min after the preceding DI. The goal was to assess whether and the extent by which the time-dependent decrease in hysteresivity is preserved following the DI. The results show that the time-dependent decrease in hysteresivity seen pre-DI was preserved after a strain of 10%, but not after a strain of 20% or 30%. This suggests that the microstructural adaptations liable for the elastic transition withstood a strain at least twofold greater than the oscillating strain that pertained during their evolution (10% vs. 5%). We propose that a muscle adapting in dynamic conditions forges microstructures exhibiting a substantial degree of flexibility.NEW & NOTEWORTHY This study confirms that airway smooth muscle undergoes an elastic transition during a sustained contraction even when it operates in dynamic conditions simulating breathing at tidal volume. It also demonstrates that the microstructural adaptations liable for this elastic transition withstand a strain that is at least twice as large as the oscillating strain that pertains during their evolution. This degree of flexibility might be an asset with major significant impact for a tissue such as the airway smooth muscle that displays an everchanging shape due to breathing.

Intranasal versus intratracheal exposure to lipopolysaccharides in a murine model of acute respiratory distress syndrome.

Due to frequent and often severe lung affections caused by COVID-19, murine models of acute respiratory distress syndrome (ARDS) are increasingly used in experimental lung research. The one induced by a single lipopolysaccharide (LPS) exposure is practical. However, whether it is preferable to administer LPS intranasally or intratracheally remains an open question. Herein, female C57Bl/6 J mice were exposed intranasally or intratracheally to one dose of either saline or 3 mg/kg of LPS. They were studied 24 h later. The groups treated with LPS, either intranasally or intratracheally, exhibited a pronounced neutrophilic inflammation, signs of lung tissue damage and protein extravasation into the alveoli, and mild lung dysfunction. The magnitude of the response was generally not different between groups exposed intranasally versus intratracheally. However, the variability of some the responses was smaller in the LPS-treated groups exposed intranasally versus intratracheally. Notably, the saline-treated mice exposed intratracheally demonstrated a mild neutrophilic inflammation and alterations of the airway epithelium. We conclude that an intranasal exposure is as effective as an intratracheal exposure in a murine model of ARDS induced by LPS. Additionally, the groups exposed intranasally demonstrated less variability in the responses to LPS and less complications associated with the sham procedure.

Organic components of airborne dust influence the magnitude and kinetics of dendritic cell activation.

Bioaerosol exposure in highly contaminated occupational settings is associated with an increased risk of disease. Yet, few determinants allow for accurate prediction of the immunopathogenic potential of complex bioaerosols. Since dendritic cells are instrumental to the initiation of immunopathological reactions, we studied how dendritic cell activation was modified in response to individual agents, combined microbial agents, or air sample eluates from highly contaminated environmental settings. We found that combinations of agents accelerated and enhanced the activation of in vitro-generated murine bone marrow-derived dendritic cell cultures, when compared to individual agents. We also determined that endotoxins are not sufficient to predict the potential of air samples to induce bone marrow-derived dendritic cell activation, especially when endotoxin levels are low. Importantly, bone marrow-derived dendritic cell activation stratified samples from three environmental settings (swine barns, dairy barns, and wastewater treatment plants) according to their air quality status. As a whole, these results support the notion that the interplay between bioaerosol components impacts on their ability to activate dendritic cells and that bone marrow-derived dendritic cell cultures are promising tools to study the immunomodulatory impact of air samples and their components.

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

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.