Project description:Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi.
Project description:Asthma, chronic rhinosinusitis, and related incurable allergic afflictions of the upper and lower airways are medically important because of their association with the disabling symptom of dyspnea and, at least for asthma, the potential to cause fatal asphyxiation. Extensive research over the past two decades has uncovered both the physiological basis of airway obstruction in asthma and key governing molecular pathways. Exaggerated airway constriction in response to diverse provocative stimuli, termed airway hyperresponsiveness, is mediated through the cytokines interleukin 4 (IL-4) and IL-13 and the transcription factor signal transducer and activator of transcription 6 (STAT6). Overproduction of mucus has long been known to be an essential second component of airway obstruction and is also mediated in part through the IL-4/IL-13/STAT6 pathway. In this review, we discuss a second major signaling pathway which underlies mucus production that is mediated through proteinase-cleaved fibrinogen signaling through Toll-like receptor 4. Unexpectedly, our analysis of human sputum and paranasal sinus fluid indicates that in most cases of severe allergic airway disease, a unique type of airway fungal infection, termed airway mycosis, is pathogenically linked to these conditions. We further discuss how fungal and endogenous proteinases mediate the fibrinogenolysis that is essential to both Toll-like receptor 4 signaling and fibrin deposition that, together with mucus, contribute to airway obstruction.
Project description:Phospholipase A2 (sPLA2), pivotal for allergic and inflammatory response, hydrolyses phosphatidylcholine (PC) to lysophosphatidylcholine (LPC). In present study, the role of LPC in allergic airway disease manifestation was studied using mouse model. Balb/c mice were immunized using cockroach extract (CE) and LPC release was blocked by sPLA2 inhibitor. Airway hyperresponse (AHR), lung-histology, total and differential leukocyte count (TLC&DLC), Th2 type cytokines, sPLA2 activity and LPC levels in bronchoalveolar lavage fluid (BALF) were measured. Exogenous LPC was given to the mice with or without CE sensitization, to demonstrate its role in allergic airway disease manifestation. Anti-CD1d antibody was given to study the involvement of natural killer T (NKT) cells in LPC induced response. AHR, lung-inflammation, TLC, DLC, Th2 type cytokines, sPLA2 activity and LPC levels were increased on CE challenge. sPLA2 activity and LPC release was blocked by sPLA2-inhibitor, which decreased AHR, and inflammatory parameters. Exogenous LPC with or without CE sensitization increased above parameters. CE challenge or LPC exposure increased LY49C(+)TCR?(+) NKT cells in BALF and spleen, which was reduced by anti-CD1d antibody, accompanied with reduction in AHR and allergic airway inflammation parameters. Conclusively, LPC induces allergic airway disease manifestation and it does so probably via CD1d-restricted LY49C(+)TCR?(+) NKT cells.
Project description:The innate immune response of airway epithelial cells to airborne allergens initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25, we show here that epithelial matrix metalloproteinase 7 (MMP7) was expressed during asthma and was required for the maximum activity of interleukin 25 in promoting the differentiation of T helper type 2 cells. Allergen-challenged Mmp7(-/-) mice had less airway hyper-reactivity and production of allergic inflammatory cytokines and higher expression of retinal dehydrogenase 1. Inhibition of retinal dehydrogenase 1 restored the asthma phenotype of Mmp7(-/-) mice and inhibited the responses of lung regulatory T cells, whereas exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating interleukin 25 while simultaneously inhibiting retinoid-dependent development of regulatory T cells.
Project description:In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant.We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum.Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR).Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17-dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR.Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma.
Project description:The formylpeptide receptor-like 1, now officially termed FPR2, in human and its mouse homolog mFPR2 mediate leukocyte migration in response to agonists associated with inflammation and immune responses. To clarify the in vivo role of the receptor, we generated mice deficient in mFPR2. mFPR2(-/-) mice showed markedly reduced severity in OVA/alum-induced allergic airway inflammation. This was associated with diminished recruitment of CD11c(+) dendritic cells into the airway mucosa and secondary lymphoid organs, as well as reduced production of Type 2 cytokines and Igs. We also found that the bronchoalveolar lavage fluid from wild type mice with airway inflammation contained mFPR2 agonist activity. This study reveals a critical role for mFPR2 in the progression of allergic airway inflammation and immune responses.
Project description:To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8(-/-) mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite-treated Adam8(-/-) mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8's anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8(-/-) mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8(-/-) macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.
Project description:Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the ?-smooth muscle actin (?-SMA) promoter drives HAS2 expression were generated. Mixed male and female ?-SMA-HAS2 mice (HAS2+ mice, n?=?16; HAS2- mice, n?=?13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and ?-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, ?-SMA, and collagen deposition in OVA-challenged ?-SMA-HAS2+ mice compared with ?-SMA-HAS2- mice. Unexpectedly, OVA-challenged ?-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated ?-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged ?-SMA-HAS2+ mice and ?-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.
Project description:Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:?-6-D-mannoside ?1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNF?, IFN?)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.
Project description:Proteinases and the innate immune receptor Toll-like receptor 4 (TLR4) are essential for expression of allergic inflammation and diseases such as asthma. A mechanism that links these inflammatory mediators is essential for explaining the fundamental basis of allergic disease but has been elusive. Here, we demonstrate that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen, yielding fibrinogen cleavage products that acted as TLR4 ligands on airway epithelial cells and macrophages. Thus, allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation. These findings clarify the molecular basis of allergic disease and suggest new therapeutic strategies.