Rationale: Glucocorticoid (GC) receptors (GR) are nuclear receptors that mediate both non-genomic and genomic effects. Upon ligand binding, the GC-GR complex translocates to the nucleus, binds specific DNA sequences, and modulates transcription. GR is a phosphoprotein, and its phosphorylation is crucial for receptor activation, affecting ligand binding, DNA interaction, subcellular localization, and biological activity. The N-terminal domain (NTD) of GR contains several phosphorylation sites, including Ser211 and Ser226. Our previous studies demonstrated that GR phosphorylation at Ser211 enhances transcriptional activity. Another antecedent related to the direct phosphorylation of Ser211 was the involvement of the cyclin-dependent kinase 5 (CDK5). However, the kinases involved in this process remain unclear. This study investigates the role of kinases in GR phosphorylation in airway smooth muscle (ASM) cells. Methods: We first sought to identify the kinases induced by glucocorticoids using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D) to measure 37 kinases in human ASM cells treated with cortisol (1 µg/mL) for 1, 3, and 5 minutes. Principal component analysis (PCA) was performed with R (version 4.4.1), and statistical analysis was conducted using GraphPad Prism (version 10.4.0) with one-way ANOVA followed by Dunnett's post-test (p<0.05). In separate experiments, ASM cells were treated with the pan-CDK inhibitor Roscovitine (1, 10, and 20 µM) for 2 hours, followed by cortisol stimulation (1 µg/mL) for 1 hour. GR phosphorylation at Ser211 and total GR expression were analyzed by Western blot, and cell viability was assessed using the MTT assay. Results: Kinase profiling showed significant changes in kinase activity following cortisol treatment. After 1 minute, beta-catenin increased by 31%, PLC-γ1 (pY783) by 13%, and Src (pY419) by 24%. At 3 minutes, STAT3 (pY705) increased by 42%, and STAT6 (pY641) peaked at 5 minutes. PCA of the first two components explained 91.5% of the variation and showed a positive correlation between beta-catenin and Src (pY419) and a negative correlation with HSP27 (pS78/pS82). In pharmacological inhibition studies, Roscovitine decreased GC-induced pSer211 phosphorylation by 60%, 72%, and 77% at 1, 10, and 20 µM, respectively, without affecting cell viability. Conclusions: Our results suggest that multiple kinases, particularly the CDK family, regulate GR phosphorylation at Ser211 in human ASM cells. These insights may guide the development of therapeutic strategies targeting these pathways to modulate glucocorticoid responses, especially in glucocorticoid-resistant diseases.

Rationale: Glucocorticoid (GC) insensitivity presents a significant treatment challenge in inflammatory diseases such as asthma, where reduced GC sensitivity is associated with increased disease severity. While GC insensitivity mechanisms in immune cells from severe asthma patients are well-studied, these mechanisms in airway smooth muscle cells (ASMCs) remain relatively poorly understood. ASMCs, key targets for GC therapy, contribute to bronchoconstriction, inflammation, and airway remodeling in asthma. The effects of GC are mediated through the glucocorticoid receptor (GR), which includes GRα and GRβ isoform produced by alternative splicing. These isoforms differentially influence GC cellular responses, with increased GRβ expression lowering the GRα:GRβ ratio, a shift associated with GC insensitivity. MicroRNAs (miRNAs), post-transcriptional regulators of gene expression, may modulate GRβ levels and thus impact GC sensitivity. This study investigates whether specific miRNAs regulate GRβ expression in ASMCs after stimulation with the mitogen EGF. Methods: Primary ASM (ASM) cells isolated from asthma and non-asthma patients were treated with the mitogen EGF (10 ng/mL) at intervals of 1, 2, 3, 6, 18, 24, and 48 hours. GRβ knockdown and miRNA inhibition were achieved by transfecting cells with specific siRNA targeting GRβ and inhibitors for miR-21, miR-33, and miR-144 using FuGENE 6. Cell proliferation was measured using the CyQUANT assay, while GRβ mRNA expression levels were quantified via qRT-PCR. Statistical analyses were performed with GraphPad Prism 10, utilizing two-way ANOVA followed by Tukey's multiple comparisons test. Significance was defined as p < 0.05. Results: Treatment with EGF significantly increased GRβ expression in ASM cells from both asthma and non-asthma patients, with the peak response at 3 hours (p < 0.01). Transfection with GRβ-targeting siRNA effectively reduced GRβ expression by approximately 60% compared to both non-transfected cells and scramble control-transfected cells. Interestingly, GRβ knockdown significantly decreased EGF-induced cell proliferation by 16.6% (33067 vs. 27560 cells) at 24 hours and 13.8% (26264 vs. 23823 cells) at 48 hours. Inhibition of miR-21 led to a significant reduction in GRβ expression in both in both untreated (p < 0.001) and EGF-treated (p < 0.001) cells; however, this effect was seen only in cells derived from non-asthma patients. Conversely, miR-144 inhibition significantly reduced GRβ expression in cells from both asthma and non-asthma patients. MiR-33 inhibition did not affect GRβ expression. Conclusions: These findings indicate that miR-144 may regulate GRβ expression in ASMCs from asthma patients, potentially affecting GC sensitivity and ASMC proliferation. Future studies using gain-of-function approaches are warranted to further these findings.

BACKGROUND:Novel specific therapy in chronic obstructive pulmonary disease (COPD) will require accessible targets for endotyping to identify responsive patients. It is therefore of interest that IL-26 in the bronchoalveolar space is enhanced and associates with bronchoalveolar pathology among long-term smokers (LTS) with and without COPD. OBJECTIVE:We determined whether IL-26 in the nasal cavity can be produced by T cells and associates with bronchoalveolar pathology and clinical symptoms in LTS with and without COPD. METHODS:We characterized LTS with and without COPD plus healthy nonsmokers by radiology, spirometry, modified Medical Research Council scale, and St George Respiratory Questionnaire. We determined extracellular IL-26 concentrations (via ELISA) in nasal (NAL) and bronchoalveolar lavage (BAL) samples, BAL neutrophil counts, and NAL IL-26+ T-cell expression (via flow cytometry). RESULTS:The NAL IL-26 concentrations were higher in LTS with COPD than in healthy nonsmokers. These enhanced IL-26 concentrations displayed a positive correlation with forced expiratory volume in 1 second/forced vital capacity ratio. The IL-26 protein was expressed in CD4+ and CD8+ T cells, but only a small portion of these cells coexpressed IL-15, IL-17A, or IL-22 in LTS with COPD. In this group, IL-26+ CD3+ T cells displayed a negative correlation with forced expiratory volume in 1 second, as did with extracellular NAL IL-26 concentrations. The relative mean fluorescence intensity for CD8+ T cells displayed a negative correlation with modified Medical Research Council and St George Respiratory Questionnaire score. CONCLUSION:In the nasal cavity, IL-26 can be produced by local T cells. This IL-26 reflects bronchoalveolar pathology and clinical symptoms, thereby constituting an accessible target with potential for clinically relevant endotyping in COPD.

Rationale Interleukin (IL)-38 is an anti-inflammatory cytokine in the IL-1 superfamily. Its production is upregulated in several chronic inflammatory disorders, acting as an endogenous antagonist of the neutrophil-mobilizing IL-36 cytokines. However, the role of IL-38 in the context of chronic obstructive pulmonary disease (COPD) remains largely unexplored. In this study, we aimed to determine whether systemic IL-38 concentrations are altered in COPD. Methods Patients with COPD (GOLD stages 2-4, groups A-E), as well as lung-healthy controls (LHC), were recruited at a tertiary clinical center. All subjects underwent pulmonary function tests (spirometry, plethysmography, and carbon monoxide diffusion capacity (DLCO)). Sarcopenia was assessed (hand grip strength test) and all COPD patients underwent chest imaging (X-ray or computed tomography (CT)). Emphysema was evaluated and graded in CT scans by a pulmonology specialist. IL-38 concentrations in serum were quantified by enzyme-linked immunosorbent assay (ELISA). Results Patients with COPD (n=25) comprised 60% females; a median age of 68 (64-73) years; 80% ex-smokers, 16% current smokers, and 4% never-smokers. Twenty percent were classified in GOLD stage 2, whereas 64% were classified in stage 3 and 8% in stage 4. Healthy subjects (n= 15) comprised 80% females; a median age of 42 (36-62) years; 68% never-smokers, and 33% ex-smokers. In the COPD group, 23 patients had a CT while two had an X-ray. Emphysema was present in a majority (78%), of which 30% were graded as severe. Serum IL-38 was markedly lower in the COPD group compared to the healthy group (Figure 1A). In contrast, within the COPD group, IL-38 concentrations were clearly higher in those with severe emphysema compared to those with mild or no emphysema (Figure 1B). IL-38 concentrations correlated with hand-grip strength in the entire (pooled) study population (Figure 1C), with a trend towards a similar correlation in the COPD group (n.s.). Conclusion The reduced systemic concentrations of IL-38 in the COPD group are suggestive of impaired systemic protection by this cytokine in COPD. Moreover, the positive correlation between IL-38 concentrations and hand-grip strength in the entire study population is compatible with a protective effect of IL-38 against sarcopenia. The enhanced concentrations of IL-38 among patients with severe emphysema in the COPD group are suggestive of a reactive but still protective role in this comorbidity. Taken together, our results motivate further study of a protective role IL-38 in COPD and emphysema to delineate its mechanism(s) of action.

To the Editor, Integrins are ubiquitous transmembrane glycoprotein receptors involved in bidirectional signaling across the cell membrane. Integrins are composed of a noncovalent complex that contains an α-subunit and a β-subunit, with 18 and 8 variations, respectively, forming a total of 24 distinct heterodimer.1 The integrin β1 subunit can interact with 12 α subunits, from α1 to α11 and αv. β2 integrins, along with α4β1, are cell adhesion ligands of leukocyte receptors.2 In asthma model, various polypeptides containing both cytosolic and extracellular β1 integrin subunits were detected in airway myocytes and connective tissue, suggesting the secretion of the β1 integrin subunit.3 In the context of asthma patients, studies have documented the presence of soluble extracellular domain of α1 and α2 integrin subunits in serum.4 However, it remains unclear whether cytosolic integrins domains are present in fluids, such as serum, within the context of the asthma. In patients with asthma (Supporting Information S1: Table S1), the levels of the extracellular domain of α1 and β1 integrin subunits, as well as the intracellular domain of α1 and β2 integrin subunits, were similar to those of healthy subjects (Figure 1). However, in asthma patients, the levels of the extracellular domain of α2 and β2 integrin subunits, as well as the intracellular domain of α2 and β1 integrin, were higher than those observed in healthy subjects (n = 23, p < 0.0001). This data is consistent with observations in persistent asthma patients, where an increase in serum soluble α2 integrin (extracellular domain) was observed compared to non-persistent asthma patients, while α1 integrin remained unchanged.4 Interestingly, the expression patterns of integrin β1, α1, and α2 subunits in asthma differed from those observed in scleroderma patients (Supporting Information S1: Figure S1). The integrins α1β1 and α2β1 act as receptors for various types of collagens (I, III, IV, and XIII), each with distinct functions.5 Additionally, integrin fragments have the ability to form heterodimers while retaining identical antigenic and ligand-binding properties as the complete transmembrane integrin.6 Therefore, the presence of functional soluble integrin heterodimers in serum is feasible. Levels of expression of the intracellular and extracellular domains of integrin α1, α2, β1, and β2 subunits in the serum of patients with asthma. Bars represent means ± standard error, n = 23 patients, *p < 0.0001, unpaired Student's t-test. The changes in the expression of the β1 integrin intracellular domain and both domains of the α2 integrin inversely correlate with FEV1 (Table 1), implying that the severity of asthma may be associated with an upregulation of these integrins. Conversely, the β2 integrin extracellular domain exhibited a direct correlation with FEV1. Supporting this finding, a recent study demonstrated that in asthma patients, this integrin subunit is predominantly released by metalloproteinase-9 (MMP-9) during asthma exacerbations, as this is when MMP-9 levels and activity increase.7 Interestingly, despite the lack of correlation between the β2 integrin subunit intracellular domain and FEV1, it does show a direct correlation with the percentage of blood neutrophils, as well as with the levels of IL-1β, IL-4, IL-5, IL-13, IL-17, and TNF-α. Conversely, it inversely correlates with lymphocytes and monocytes. This indicates the potential sensitivity of this subunit to inflammatory cytokines and T2 mediators. Finally, the association of neutrophil levels with the cytosolic domain suggests that these cells could likely be a significant source of cytosolic integrins, and that they might have been released by neutrophil degranulation during serum preparation. In guinea pigs modeling asthma, the levels of the soluble intracellular domain of β1 and β2 integrin subunits in both control and asthma-induced models (Supporting Information S1: Figure S2) were comparable in serum and bronchoalveolar lavage (BAL) samples from both young and aged animals. Conversely, the levels of the extracellular domain of these subunits were significantly lower in serum compared to BAL samples (n = 6, p < 0.05 and 0.01). It appears that the development of acute or chronic lung allergic processes in guinea pigs did not induce changes in serum or LBA β1 and β2 integrin patterns. In conclusion, our results indicate that integrin secretion occurs in humans and guinea pigs and can be identified in soluble fluids like serum. In humans, the secretion of the β2 integrin subunit may indicate inflammation, while the α2 integrin subunit secretion could signal worsening asthma. The disparities in integrin patterns between asthma and scleroderma underscore the potential of secreted integrin expression as a promising marker for asthma diagnostic. Rocío Chapela and Blanca Bazán-Perkins conceptualized the study design. Olivia Tellez-Jimenez, Christian Trejo-Jasso, Patricia Ramos-Ramirez, Maryana Tinoco-Cuellar, Diana García-Trejo, José Luis Miguel-Reyes, and Angélica Flores-Flores executed the experiments. Angélica Flores-Flores and Blanca Bazán-Perkins performed the interpretation and redaction of the manuscript. We thank Dr. Erasmo Martínez and Dr. Ignacio Páramo-Ramírez for providing samples from scleroderma and asthma patients. The authors declared that they have no conflict of interest. The study data is available upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Sex differences have been reported in the incidence, prevalence and severity of asthma. Previous findings from animal models have revealed sex-related differences in inflammatory pathways that may contribute to asthma pathogenesis, but human studies are limited. METHODS:Airway and blood samples (n = 55 and n = 85 respectively) were collected from adult females and males with asthma and healthy subjects. Type 2 innate lymphoid cells (ILC2s), T helper (Th)2 cells and their expression of IL-33R/ST2 (ST2L) were evaluated by flow cytometry. IL-13, thymic stromal lymphopoietin (TSLP), IL-33 and soluble IL-33R/ST2 (sST2) were measured by ELISA. Let-7 miRNA expression in bronchial biopsies was determined by qPCR. RESULTS:Females with asthma reported more exacerbations and had a higher number of airway eosinophils compared with males with asthma. Bronchial biopsy expression of Let-7f, Let-7g and miR-98 tended to be higher in males with asthma compared with females and inversely correlated with asthma exacerbations. In contrast, increased levels of IL-13, TSLP and sST2 were found in females with asthma compared with males. CONCLUSION:Our study demonstrates different inflammatory signatures between males and females with asthma. Let-7 miRNAs act as immune modulators by inhibiting the production of IL-13 and may be an important factor explaining the sex disparity seen in asthma.

The inflammation caused by asthma exacerbation can lead to permanent changes in the airways and loss of lung function. Integrins are membrane receptors that interact with components of the extracellular matrix and cell adhesion molecules. It is known that these receptors can be found in soluble form in some conditions such as asthma, but it is unknown if exacerbation during asthma leads to soluble integrins. Our results indicated that asthma patients showed higher levels of soluble alpha 1, alpha 2, and beta 2 integrin subunits in their serum compared to controls, as confirmed by both ELISA and western blot. During asthma exacerbation, the levels of alpha 2 and beta 2 integrin subunits increased even more compared to non-exacerbation and controls, while the alpha 1 integrin subunit decreased. Western blot analysis identified two beta 2 integrin subunits, one at 75 kDa and another at 120 kDa; the 120 kDa subunit increased during asthma exacerbation. The activity of matrix metalloproteinase 9 (MMP9) increased during exacerbation, while MMP2 remained unchanged. Lower forced expiratory volume in 1 second (FEV1) values were associated with higher expression levels of alpha 2, beta 1, and beta 2 integrin subunits. Active and latent MMP9 were correlated with the levels of the beta 2 integrin subunit, which means that at low levels of active and latent MMP9, there are lower levels of beta 2 integrin subunit. In conclusion, asthma exacerbation leads to the presence of soluble integrins, particularly the beta 2 subunit, most likely due to MMP9-induced proteolytic cleavage.

Background Asthma is a chronic inflammatory disease with structural changes in the lungs defined as airway remodelling. Mast cell responses are important in asthma as they, upon activation, release mediators inducing bronchoconstriction, inflammatory cell recruitment, and often remodelling of the airways. As guinea pigs exhibit anatomical, physiological, and pharmacological features resembling human airways, including mast cell distribution and mediator release, we evaluated the effect of extracts from two common allergens, house dust mite (HDM) and cat dander (CDE), on histopathological changes and the composition of tryptase- and chymase-positive mast cells in the guinea pig lungs. Methods Guinea pigs were exposed intranasally to HDM or CDE for 4, 8, and 12 weeks, and airway histology was examined at each time point. Hematoxylin and eosin, Picro-Sirius Red, and Periodic Acid-Schiff staining were performed to evaluate airway inflammation, collagen deposition, and mucus-producing cells. In addition, Astra blue and immunostaining against tryptase and chymase were used to visualize mast cells. Results Repetitive administration of HDM or CDE led to the accumulation of inflammatory cells into the proximal and distal airways as well as increased airway smooth muscle mass. HDM exposure caused subepithelial collagen deposition and mucus cell hyperplasia at all three time points, whereas CDE exposure only caused these effects at 8 and 12 weeks. Both HDM and CDE induced a substantial increase in mast cells after 8 and 12 weeks of challenges. This increase was primarily due to mast cells expressing tryptase, but not chymase, thus indicating mucosal mast cells. Conclusions We here show that exposure to HDM and CDE elicits asthma-like histopathology in guinea pigs with infiltration of inflammatory cells, airway remodelling, and accumulation of primarily mucosal mast cells. The results together encourage the use of HDM and CDE allergens for the stimulation of a clinically relevant asthma model in guinea pigs.

Background: Adiponectin is an anti-inflammatory adipokine that acts through adiponectin receptors 1 and 2 (AdipoR1/AdipoR2). We recently showed that under a T2 environment, adiponectin, via AdipoR1, promotes the IL-10 expression in cultured human circulating Tregs. We here sought to determine the effect of allergic inflammation on IL-10 production in lung AdipoR1+ Tregs in vivo. Method: BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA) on five consecutive days. Control mice were challenged with PBS. Lung Tregs, based on Foxp3 and Helios expression, were assessed for AdipoR1 and IL-10 expression by flow cytometry. Adiponectin levels were quantified in BALF and serum by ELISA. Results: We found a significant increase in the expression of AdipoR1 in CD4 T (P<0.001) and Treg (P<0.01) cells in the lung of OVA-challenged mice compared to PBS controls. While AdipoR1 expression in Heliosneg Tregs was more significant than in Heliospos Tregs (P<0.05), both subsets of AdipoR1+ Tregs were increased in OVA-challenged mice compared to PBS controls (P<0.01). Interestingly, IL-10 production in Heliosneg AdipoR1 Tregs was higher in response to OVA than that of PBS (4.8% and 1.6%, respectively, P<0.05), whereas no differences were found in Heliospos AdipoR1 Tregs. Adiponectin levels in both BALF (P<0.05) and serum (P<0.01) were significantly reduced in allergic mice compared to controls. Conclusion: Collectively, our findings show that allergic inflammation modulates the adiponectin/AdipoR1 axis in the lung, where IL-10 production by Heliosneg AdipoR1 Tregs might play an important role in their attempt to control the allergic response.

Although most patients with asthma symptoms are well controlled by inhaled glucocorticoids (GCs), a subgroup of patients suffering from severe asthma respond poorly to GC therapy. Such GC insensitivity (GCI) represents a profound challenge in managing patients with asthma. Even though GCI in patients with severe asthma has been investigated by several groups using immune cells (peripheral blood mononuclear cells and alveolar macrophages), uncertainty exists regarding the underlying molecular mechanisms in non-immune cells, such as airway smooth cells (ASM) cells. In asthma, ASM cells are among the targets of GC therapy and have emerged as key contributors not only to bronchoconstriction but also to airway inflammation and remodeling, as implied by experimental and clinical evidence. We here summarize the current understanding of the actions/signaling of GCs in asthma, and specifically, GC receptor (GR) “site-specific phosphorylation” and its role in regulating GC actions. We also review some common pitfalls associated with studies investigating GCI and the inflammatory mediators linked to asthma severity. Finally, we discuss and contrast potential molecular mechanisms underlying the impairment of GC actions in immune cells versus non-immune cells such as ASM cells.

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and "directly" regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.

Background Animal models are extensively used to study underlying mechanisms in asthma. Guinea pigs share anatomical, pharmacological and physiological features with human airways and may enable the development of a pre-clinical in vivo model that closely resembles asthma. Objectives To develop an asthma model in guinea pigs using the allergen house dust mite (HDM). Methods Guinea pigs were intranasally sensitized to HDM which was followed by HDM challenges once weekly for five weeks. Antigen-induced bronchoconstriction (AIB) was evaluated as alterations inR(n)(Newtonian resistance),G(tissue damping) andH(tissue elastance) at the first challenge with forced oscillation technique (FOT), and changes in respiratory pattern upon each HDM challenge were assessed as enhanced pause (Penh) using whole-body plethysmography. Airway responsiveness to methacholine was measured one day after the last challenge by FOT. Inflammatory cells and cytokines were quantified in bronchoalveolar lavage fluid, and HDM-specific immunoglobulins were measured in serum by ELISA. Airway pathology was evaluated by conventional histology. Results The first HDM challenge after the sensitization generated a marked increase inR(n)andG, which was abolished by pharmacological inhibition of histamine, leukotrienes and prostanoids. Repeated weekly challenges of HDM caused increase of Penh and a marked increase in airway hyperresponsiveness for all three lung parameters (R-n,GandH) and eosinophilia. Levels of IgE, IgG(1), IgG(2)and IL-13 were elevated in HDM-treated guinea pigs. HDM exposure induced infiltration of inflammatory cells into the airways with a pronounced increase of mast cells. Subepithelial collagen deposition, airway wall thickness and goblet cell hyperplasia were induced by repeated HDM challenge. Conclusion and Clinical Relevance Repeated intranasal HDM administration induces mast cell activation and hyperplasia together with an asthma-like pathophysiology in guinea pigs. This model may be suitable for mechanistic investigations of asthma, including evaluation of the role of mast cells.