Evodiae Fructus extract suppresses inflammatory response in HaCaT cells and improves house dust mite-induced atopic dermatitis in NC/Nga mice.

This study was conducted to assess the effect of Evodiae Fructus 70% ethanol extract (EFE) on the pathology of atopic dermatitis using in vitro and in vivo models. The major compounds in EFE were identified by ultra-performance liquid chromatography with tandem mass spectrometry as rutaecarpine, evodiamine, evodol, dehydroevodiamine, limonin, synephrine, evocarpine, dihydroevocarpine, and hydroxyevodiamine. EFE significantly decreased chemokine levels in tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells. In house dust mite-treated NC/Nga mice, topical application of EFE significantly decreased the dermatitis score, epidermal hyperplasia and thickening, mast cell infiltration, and plasma levels of histamine and corticosterone. Thymic stromal lymphopoietin, CD4+ T cells, interleukin-4, and intercellular adhesion molecule-1 expression in the lesioned skin was reduced in the treated mice. The mechanism of EFE was elucidated using transcriptome analysis, followed by experimental validation using Western blotting in HaCaT cells. EFE down-regulated the activation of Janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinases (MAPK) signaling pathways in HaCaT cells. EFE improves atopic dermatitis-like symptoms by suppressing inflammatory mediators, cytokines, and chemokines by regulating the JAK-STAT and MAPK signaling pathways, suggesting its use as a potential agent for the treatment of atopic dermatitis.

Tollip deficiency exaggerates airway type 2 inflammation in mice exposed to allergen and influenza A virus: role of the ATP/IL-33 signaling axis.

Toll-interacting protein (Tollip) is a negative regulator of the pro-inflammatory response to viruses, including influenza A virus (IAV). Genetic variation of Tollip has been associated with reduced airway epithelial Tollip expression and poor lung function in patients with asthma. Whether Tollip deficiency exaggerates type 2 inflammation (e.g., eosinophils) and viral infection in asthma remains unclear. We sought to address this critical, but unanswered question by using a Tollip deficient mouse asthma model with IAV infection. Further, we determined the underlying mechanisms by focusing on the role of the ATP/IL-33 signaling axis. Wild-type and Tollip KO mice were intranasally exposed to house dust mite (HDM) and IAV with or without inhibitors for IL-33 (i.e., soluble ST2, an IL-33 decoy receptor) and ATP signaling (i.e., an antagonist of the ATP receptor P2Y13). Tollip deficiency amplified airway type 2 inflammation (eosinophils, IL-5, IL-13 and mucins), and the release of ATP and IL-33. Blocking ATP receptor P2Y13 decreased IL-33 release during IAV infection in HDM-challenged Tollip KO mice. Furthermore, soluble ST2 attenuated airway eosinophilic inflammation in Tollip KO mice treated with HDM and IAV. HDM challenges decreased lung viral load in wild-type mice, but Tollip deficiency reduced the protective effects of HDM challenges on viral load. Our data suggests that during IAV infection, Tollip deficiency amplified type 2 inflammation and delayed viral clearance, in part by promoting ATP signaling and subsequent IL-33 release. Our findings may provide several therapeutic targets, including ATP and IL-33 signaling inhibition for attenuating excessive airway type 2 inflammation in human subjects with Tollip deficiency and IAV infection.

Dust mite component Analysis: Identifying key allergens components for effective immunotherapy in allergic rhinitis.

BACKGROUND: The aim of this study was to examine the frequency of sensitization to house dust mite (HDM) components among allergic rhinitis patients receiving subcutaneous immunotherapy (SCIT), and to assess the correlation between SCIT efficacy and specific IgE (sIgE) levels for allergenic HDM components. METHODS: Serum samples and clinical data were collected from 38 allergic rhinitis patients receiving HDM-SCIT at baseline and after 1 year of treatment. Effective treatment was defined as a therapeutic index (TI) of at least 50% after 1 year. Cytokine levels were analyzed using commercial ELISA kits, while serum total and specific IgE levels were determined by the fluoroenzymeimmunoassay technique. The ALLEOS 2000 magnetic particle chemiluminescence system was used to measure sIgE levels for Der f, Der p 1, Der p 2, Der p 10, and Der p 23. RESULTS: Allergic rhinitis patients undergoing HDM-SCIT had a high rate of allergic sensitization to the HDM major allergens Der p (100%), Der f (100%), Der p 1 (94.74%), Der p 2 (94.74%), and Der p 23 (36.84%). Patients who responded to SCIT had higher levels of IgE for HDM components at baseline, while those with ineffective treatment showed an opposite performance, particularly for Der p 1 (P＜0.05). After 1 year of treatment, effective and ineffective patients showed opposite trends in sIgE for dust mite components (decreased in effective patients, increased in ineffective patients). HDM-SCIT led to a significant reduction in IL-2, IL-4, IL-6, and EOS% (P＜0.05). IgE for Der p, Der f, Der p 1, Der p 2, and HDM sIgE were significantly positively correlated (P < 0.001). The correlation heatmap analysis based on changes in values reveals a negative correlation between CSMS score changes and sIgE for Der f and Der p 1, and a positive correlation with IL-2, IL-10, and TNF (P < 0.05). CONCLUSIONS: The molecular sensitization profiles during HDM-SCIT are variable and relate to treatment efficacy. Molecular diagnosis can assist allergists in identifying patients eligible for HDM-SCIT, thereby enhancing the treatment's clinical efficacy. Serum cytokine levels of IL-2, IL-4, IL-6，and EOS% may serve as useful biomarkers for monitoring HDM-SCIT efficacy.

Anti-atopic dermatitis effect of fish collagen on house dust mite-induced mice and HaCaT keratinocytes.

Collagen, a major structural protein in mammalian tissues, is effective against skin wounds and osteoarthritis. Although bovine and porcine collagens have mainly been used, several potential risks of mammalian collagen have led to the use of fish collagen (FC) as an alternative. FC and its peptides are used as common cosmeceutical products because of their antihypertensive, anti-bacterial, and antioxidant activities. Despite the effects of FC on wrinkle reduction, UV-protection, and wound healing, the relationship between FC and atopic dermatitis (AD) has not yet been reported. Therefore, we investigated the anti-AD effects of FC against house dust mite (Dermatophagoides farinae, HDM)-induced AD in NC/Nga mice and TNF-α/IFN-γ-stimulated HaCaT keratinocytes. FC alleviated AD apparent symptoms, such as dermatitis score, transepidermal water loss, epidermal thickness, and mast cell infiltration upon declining pro-inflammatory cytokines and mediators, IL-6, IL-5, IL-13, TSLP, and TNF-α. The skin barrier protein, filaggrin, was also recovered by FC administration in vivo and in vitro. Immune response and skin barrier dysfunction are both mitigated by three routes of FC administration: oral, topical, and both routes via the regulation of IκB, MAPKs, and STATs pathways. In summary, FC could be a potential therapeutic agent for AD by regulating immune balance and skin barrier function.

Dermatophagoides Pteronyssinus 1 (DerP1) May Trigger NLRP3-Mediated Corneal Epithelial Cell Pyroptosis by Elevating Interleukin-33 Expression Levels.

PURPOSE: To characterize the in vivo effects of Dermatophagoides pteronyssinus 1 (DerP1) in mice and determine the underlying NLRP3 inflammasome-mediated pyroptosis signaling mechanisms in the human corneal epithelial cells (HCECs). METHODS: DerP1 was used to induce allergic conjunctivitis in C57 mice. HCECs were sensitized with DerP1 in vitro to mimic their condition observed in allergic conjunctivitis in vivo. Transmission electron microscopy was used to evaluate pyroptosis in the HCECs, enzyme-linked immunosorbent assays to assess interleukin (IL)-33, IL-1ß and IL-4 levels, flow cytometry to detect the proportion of Th2 cells, MTT assays to assess cell metabolic activity, immunofluorescence to evaluate the effects of DerP1 on functional HCEC phenotypes, and Western blot assays to detect the expression of NOD-like receptor family pyrin domain-containing 3 (NLRP3), gasdermin D (GSDMD), N-terminal fragment of GSDMD (GSDMD-N), pro-caspase-1, cleaved caspase-1, IL-1ß, and IL-33. IL-33 expression in the HCECs was knocked down via lentivirus transfection. RESULTS: In vivo, DerP1 promotes pyroptosis, production of Th2 inflammatory cytokines and IL-33, and NLRP3 activation in mouse corneas. In vitro, pyroptotic bodies were found in the HCECs after sensitization with DerP1. Various concentrations of DerP1 increased the expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved caspase-1, and IL-1ß in the HCECs, with the largest increase observed after exposure to 20 µM DerP1. In vitro, recombinant human IL-33 mediated the expression of pyroptotic biomarkers in the HCECs, whereas IL-33 silencing diminished 20 µM DerP1-induced increase in their expression levels. CONCLUSIONS: DerP1 induces pyroptosis and allergic conjunctivitis, the expression of Th2 inflammatory cytokines, NLRP3 activation, and IL-33 in mouse corneas in our model. These effects would attribute to its activating NLRP3-GSDMD signaling pathway axis via enhancing IL-33 expression in HCECs.

Epithelium-derived cystatin SN inhibits house dust mite protease activity in allergic asthma.

BACKGROUND: Allergen source-derived proteases are a critical factor in the formation and development of asthma. The cysteine protease activity of house dust mite (HDM) disrupts the epithelial barrier function. The expression of cystatin SN (CST1) is elevated in asthma epithelium. CST1 inhibits the cysteine protease activity. We aimed to elucidate the role of epithelium-derived CST1 in the development of asthma caused by HDM. METHODS: CST1 protein levels in sputum supernatants and serum of patients with asthma and healthy volunteers were measured by ELISA. The ability of CST1 protein to suppress HDM-induced bronchial epithelial barrier function was examined in vitro. The effects of exogenous CST1 protein on abrogating HDM-induced epithelial barrier function and inflammation were examined in mice in vivo. RESULTS: CST1 protein levels were higher in sputum supernatants (142.4 ± 8.95 vs 38.87 ± 6.85 ng/mL, P < 0.0001) and serum (1129 ± 73.82 vs 703.1 ± 57.02 pg/mL, P = 0.0035) in patients with asthma than in healthy subjects. The levels were significantly higher in patients with not well- and very poorly controlled asthma than those with well-controlled asthma. Sputum and serum CST1 protein levels were negatively correlated with lung function in asthma. CST1 protein levels were significantly lower in the serum of HDM-specific IgE (sIgE)-positive asthmatics than in sIgE-negative asthmatics. The HDM-induced epithelial barrier function disruption was suppressed by recombinant human CST1 protein (rhCST1) in vitro and in vivo. CONCLUSION: Our data indicated that human CST1 protein suppresses asthma symptoms by protecting the asthmatic bronchial epithelial barrier through inhibiting allergenic protease activity. CST1 protein may serve as a potential biomarker for asthma control.

Secreted S100A4 causes asthmatic airway epithelial barrier dysfunction induced by house dust mite extracts via activating VEGFA/VEGFR2 pathway.

The airway epithelial barrier dysfunction plays a crucial role in pathogenesis of asthma and causes the amplification of downstream inflammatory signal pathway. S100 calcium binding protein A4 (S100A4), which promotes metastasis, have recently been discovered as an effective inflammatory factor and elevated in bronchoalveolar lavage fluid in asthmatic mice. Vascular endothelial growth factor-A (VEGFA), is considered as vital regulator in vascular physiological activities. Here, we explored the probably function of S100A4 and VEGFA in asthma model dealt with house dust mite (HDM) extracts. Our results showed that secreted S100A4 caused epithelial barrier dysfunction, airway inflammation and the release of T-helper 2 cytokines through the activation of VEGFA/VEGFR2 signaling pathway, which could be partial reversed by S100A4 polyclonal antibody, niclosamide and S100A4 knockdown, representing a potential therapeutic target for airway epithelial barrier dysfunction in asthma.

The HDM allergen orchestra and its cysteine protease maestro: Stimulators of kaleidoscopic innate immune responses.

House dust mite (HDM) encloses an explosive cocktail of allergenic proteins sensitizing hundreds of millions of people worldwide. To date, the innate cellular and molecular mechanism(s) orchestrating the HDM-induced allergic inflammation remains partially deciphered. Understanding the kaleidoscope of HDM-induced innate immune responses is hampered by (1) the large complexity of the HDM allergome with very diverse functional bioreactivities, (2) the perpetual presence of microbial compounds (at least LPS, ß-glucan, chitin) promoting as well pro-Th2 innate signaling pathways and (3) multiple cross-talks involving structural, neuronal and immune cells. The present review provides an update on the innate immune properties, identified so far, of multiple HDM allergen groups. Experimental evidence highlights the importance of HDM allergens displaying protease or lipid-binding activities on the initiation of the allergic responses. Specifically, group 1 HDM cysteine proteases are considered as the key initiators of the allergic response through their capacities to impair the epithelial barrier integrity, to stimulate the release of pro-Th2 danger-associated molecular patterns (DAMPs) in epithelial cells, to produce super-active forms of IL-33 alarmin and to mature thrombin leading to Toll-like receptor 4 (TLR4) activation. Remarkably, the recently evidenced primary sensing of cysteine protease allergens by nociceptive neurons confirms the critical role of this HDM allergen group in the early events leading to Th2 differentiation.

Physiological estrogen levels are dispensable for the sex difference in immune responses during allergen-induced airway inflammation.

Women show an increased prevalence of adult-onset asthma compared to men and previous studies have shown that testosterone inhibits while estrogen worsens allergen-induced airway inflammation. However, detailed knowledge about the aggravating effects of estrogen on immune responses remain unclear. Defining the effects of physiological levels of estrogen on immune responses in asthma would aid in the development of improved treatment strategies. In this study, the importance of estrogen for the sex difference in asthma was determined using a murine model of house dust mite (HDM)-induced airway inflammation on intact female and male mice, as well as on ovariectomized (OVX) female mice treated with a physiological dose of 17ß-estradiol (E2). Innate and adaptive immune responses were defined in bronchoalveolar lavage fluid, mediastinal lymph node (mLN) and lung tissue. The results reveal increased numbers of lung eosinophils, macrophages, and dendritic cells in female but not in male mice after HDM challenge. Females also exhibit higher numbers of Th17 cells in both mLN and lung in response to HDM. However, treatment of OVX mice with physiological levels of E2 does not influence any of the analyzed cell populations. Together, this study confirms the previously reported sex difference in allergen-induced airway inflammation and show that female mice mount stronger innate and adaptive immune responses to HDM challenge, but these effects are not mediated by physiological levels of E2.

Subendotyping of Dermatophagoides pteronyssinus-Induced Rhinitis and Its Impact on Respiratory Comorbidities.

BACKGROUND: The impact of delayed hypersensitivity to Dermatophagoides pteronyssinus (DP) on comorbidities of allergic rhinitis (AR) is unknown. OBJECTIVE: The primary end point was to test the hypothesis that DP-induced AR could be divided into 2 subendotypes on the basis of presence or absence of a delayed-type mite sensitization detected by the positive result of atopy patch test for DP (DP-APT). The second end point was to evaluate differences in the long-term risk of respiratory comorbidities and nasal airway response to mite exposure. METHODS: In a prospective observational study, we included 472 patients with DP-induced AR. A total of 343 patients had positive results of skin prick test/serum specific IgE and DP-APT and were assigned to a subendotype with both IgE- and T-cell-mediated mite sensitization (BMSS). The remaining 129 patients without delayed-type mite sensitization were included in the subendotype with only IgE-mediated mite sensitization. Nasal allergen provocation test with active anterior rhinomanometry, paranasal sinuses computed tomography scan, nasal endoscopy, and spirometry were performed. RESULTS: At baseline, BMSS showed a larger increase in nasal airway resistance, total nasal score, and visual analogue scale score to mite exposure. During a 15-year follow-up, 56 patients developed chronic rhinosinusitis with nasal polyps, with higher incidence in BMSS than in the subendotype with only IgE-mediated mite sensitization (50 patients, 14.6% vs 6 patients, 12.4%; P < .001). BMSS also showed a higher incidence of conjunctivitis (25.7% vs 12.4%; P < .01). The rate of adult-onset asthma did not differ between groups, but patients with BMSS showed a more frequent link to chronic rhinosinusitis with nasal polyps (6 of 29 patients, 20.7% vs 0 of 10 patients, 0%). DP-APT independently predicted chronic rhinosinusitis with nasal polyps and conjunctivitis. CONCLUSIONS: Two subendotypes with significantly different clinical outcome can be identified among patients with DP-induced AR according to the presence of delayed-type mite sensitization detected by positive DP-APT result.

Airway epithelial ITGB4 deficiency induces airway remodeling in a mouse model.

BACKGROUND: Airway epithelial cells (AECs) with impaired barrier function contribute to airway remodeling through the activation of epithelial-mesenchymal trophic units (EMTUs). Although the decreased expression of ITGB4 in AECs is implicated in the pathogenesis of asthma, how ITGB4 deficiency impacts airway remodeling remains obscure. OBJECTIVE: This study aims to determine the effect of epithelial ITGB4 deficiency on the barrier function of AECs, asthma susceptibility, airway remodeling, and EMTU activation. METHODS: AEC-specific ITGB4 conditional knockout mice (ITGB4-/-) were generated and an asthma model was employed by the sensitization and challenge of house dust mite (HDM). EMTU activation-related growth factors were examined in ITGB4-silenced primary human bronchial epithelial cells of healthy subjects after HDM stimulation. Dexamethasone, the inhibitors of JNK phosphorylation or FGF2 were administered for the identification of the molecular mechanisms of airway remodeling in HDM-exposed ITGB4-/- mice. RESULTS: ITGB4 deficiency in AECs enhanced asthma susceptibility and airway remodeling by disrupting airway epithelial barrier function. Aggravated airway remodeling in HDM-exposed ITGB4-/- mice was induced through the enhanced activation of EMTU mediated by Src homology domain 2-containing protein tyrosine phosphatase 2/c-Jun N-terminal kinase/Jun N-terminal kinase-dependent transcription factor/FGF2 (SHP2/JNK/c-Jun/FGF2) signaling pathway, which was partially independent of airway inflammation. Both JNK and FGF2 inhibitors significantly inhibited the aggravated airway remodeling and EMTU activation in HDM-exposed ITGB4-/- mice. CONCLUSIONS: Airway epithelial ITGB4 deficiency induces airway remodeling in a mouse model of asthma through enhanced EMTU activation that is regulated by the SHP2/JNK/c-Jun/FGF2 pathway.

Hedgehog Signaling as a Therapeutic Target for Airway Remodeling and Inflammation in Allergic Asthma.

Genome-wide association studies (GWAS) have shown that variants of patched homolog 1 (PTCH1) are associated with lung function abnormalities in the general population. It has also been shown that sonic hedgehog (SHH), an important ligand for PTCH1, is upregulated in the airway epithelium of patients with asthma and is suggested to be involved in airway remodeling. The contribution of hedgehog signaling to airway remodeling and inflammation in asthma is poorly described. To determine the biological role of hedgehog signaling-associated genes in asthma, gene silencing, over-expression, and pharmacologic inhibition studies were conducted after stimulating human airway epithelial cells or not with transforming growth factor ß1 (TGFß1), an important fibrotic mediator in asthmatic airway remodeling that also interacts with SHH pathway. TGFß1 increased hedgehog-signaling-related gene expression including SHH, GLI1 and GLI2. Knockdown of PTCH1 or SMO with siRNA, or use of hedgehog signaling inhibitors, consistently attenuated COL1A1 expression induced by TGFß1 stimulation. In contrast, Ptch1 over-expression augmented TGFß1-induced an increase in COL1A1 and MMP2 gene expression. We also showed an increase in hedgehog-signaling-related gene expression in primary airway epithelial cells from controls and asthmatics at different stages of cellular differentiation. GANT61, an inhibitor of GLI1/2, attenuated TGFß1-induced increase in COL1A1 protein expression in primary airway epithelial cells differentiated in air-liquid interface. Finally, to model airway tissue remodeling in vivo, C57BL/6 wildtype (WT) and Ptch1+/- mice were intranasally challenged with house dust mite (HDM) or phosphate-buffered saline (PBS) control. Ptch1+/- mice showed reduced sub-epithelial collagen expression and serum inflammatory proteins compared to WT mice in response to HDM challenge. In conclusion, TGFß1-induced airway remodeling is partially mediated through the hedgehog signaling pathway via the PTCH1-SMO-GLI axis. The Hedgehog signaling pathway is a promising new potential therapeutic target to alleviate airway tissue remodeling in patients with allergic airways disease.

EphA2 recognizes Dermatophagoidespteronyssinus to mediate airway inflammation in asthma.

Most of the asthma with low Th2 is severe steroid-resistant asthma, the exact pathogenesis of which has not yet been fully elucidated. We found that IL-6 and IL-8 were highly expressed in the sputum supernatant of severe asthma and ephrin type-A receptor 2 (EphA2) was highly expressed on bronchial epithelial cells. So, is there a connection between these two phenomena? To clarify this issue, we stimulated bronchial epithelial cells 16HBE with Dermatophagoides pteronyssinus and its compontents LPS, respectively, and detected the activation of EphA2, activation of downstream pathways and secretion of inflammatory cytokines. A mouse asthma model was established, and the therapeutic effects of inhibiting or blocking EphA2 on mouse asthma were investigated. The results showed that D. pteronyssinus and its component LPS phosphorylated EphA2 on 16HBE, activated downstream signaling pathways STAT3 and p38 MAPK, and promoted the secretion of IL-6 and IL-8. After knockout of EphA2 on 16HBE, the activation of inflammatory pathways was attenuated and the secretion of IL-6 and IL-8 was significantly reduced. Inhibition or blockade of EphA2 on mouse airways resulted in a significant reduction in airway hyperresponsiveness and airway inflammation, and a significant decrease in the expression levels of IL-6, IL-17F, IL-1α, IL-1ß and TNF in bronchoalveolar lavage fluid and lung tissue. Our study uncovers a novel role for EphA2 expressed on airway epithelial cells in the pathogenesis of asthma; EphA2 recognizes D. pteronyssinus or its component LPS and promotes the secretion of IL-6 and IL-8 by airway epithelial cell, thereby mediating airway inflammation. Thus, it is possible to provide a new molecular therapy for severe asthma.

First-in-human phase 2 trial with mite allergoids coupled to mannan in subcutaneous and sublingual immunotherapy.

BACKGROUND: Polymerized allergens conjugated to non-oxidized mannan (PM-allergoids) are novel vaccines targeting dendritic cells (DCs). Previous experimental data indicate that PM-allergoids are readily taken up by DCs and induce Treg cells. This first-in-human study was aimed to evaluate safety and to find the optimal dose of house dust mite PM-allergoid (PM-HDM) administered subcutaneously (SC) or sublingually (SL). METHODS: In a randomized, double-blind, double-dummy, placebo-controlled trial, 196 subjects received placebo or PM-HDM at 500, 1000, 3000, or 5000 mannan-conjugated therapeutic units (mTU)/mL in 9-arm groups for 4 months. All subjects received 5 SC doses (0.5 ml each) every 30 days plus 0.2 ml SL daily. The primary efficacy outcome was the improvement of titrated nasal provocation tests (NPT) with D. pteronyssinus at baseline and at the end of the study. All adverse events and reactions were recorded and assessed. Secondary outcomes were the combination of symptom and medication scores (CSMS) and serological markers. RESULTS: No moderate or severe adverse reactions were reported. Subjects improving the NPT after treatment ranged from 45% to 62% in active SC, 44% to 61% in active SL and 16% in placebo groups. Statistical differences between placebo and active groups were all significant above 500 mTU, being the highest with 3000 mTU SL (p = 0.004) and 5000 mTU SC (p = 0.011). CSMS improvement over placebo reached 70% (p < 0.001) in active 3000 mTU SC and 40% (p = 0.015) in 5000 mTU SL groups. CONCLUSIONS: PM-HDM immunotherapy was safe and successful in achieving primary and secondary clinical outcomes in SC and SL at either 3000 or 5000 mTU/ml.

ADAMTS7 Attenuates House Dust Mite-Induced Airway Inflammation and Th2 Immune Responses.

PURPOSE: ADAMTS7 is a secreted metalloproteinase enzyme and proteoglycan associated with the early progression of coronary artery disease. However, there is limited information regarding the role of ADAMTS7 in lung adaptive immunity and inflammation. Thus, we sought to assess whether ADAMTS7 expression in the lung modulates house dust mite (HDM)-induced airway inflammation and Th2 immune response. METHODS: The role of ADAMTS7 in HDM-induced airway disease was assessed in ADAMTS7-deficient (ADAMTS7-/-) mice and compared with the wild-type control mice by flow cytometry, ELISA, and histopathology. Furthermore, the antigen priming capability of dendritic cells (DC) was determined ex vivo by employing coculture with CD4+ OT-II cells. RESULTS: ADAMTS7-/- mice develop an augmented eosinophilic airway inflammation, mucous cell metaplasia, and increased Th2 immune response to inhaled HDM. In addition, allergen uptake by lung DC and migration to draining mediastinal lymph node were significantly increased in ADAMTS7-/- mice, which shows an enhanced capacity to mount allergen-specific T-cell proliferation and effector Th2 cytokine productions. We propose that the mechanism by which ADAMTS7 negatively regulates DC function involves attenuated antigen uptake and presentation capabilities, which reduces allergic sensitization and Th2 immune responses in the lung. CONCLUSION: In aggregate, we provide compelling evidence that ADAMTS7 plays a pivotal role in allergic airway disease and Th2 immunity and would be an attractive target for asthma.

Molecular Profile Sensitization to House Dust Mites as an Important Aspect for Predicting the Efficiency of Allergen Immunotherapy.

House dust mite (HDM) allergens are considered to be one of the most common causes of asthma and allergic rhinitis in the world. Cysteine proteases Der p 1 and Der f 1 (group 1) and also NPC 2 family proteins Der p 2 and Der f 2 (group 2) of D. pteronyssinus and D. farinae respectively are considered the main allergens of HDMs. The difference in the sensitivity of the population to these and other allergy causing components of HDM determines the treatment strategy. Thus, the purpose of this work was to determine the pattern of sensitization of the Ukrainian population to individual allergy causing molecular components of HDM in order to improve treatment strategies for the HDM allergy in various regions of Ukraine. To determine the molecular profile of sensitization to HDM, the data of multiplex allergy test Alex2 have been obtained from 10,651 patients. The sample included 57.86% children under the age of 18 and 42.14% adults. A Python language-based statistical analysis was performed, in order to group patients by sensitization to individual molecules and their combinations, regarding the age and geographical location of the patients. Simultaneous sensitization to Der f 2 and Der p 2 allergens was the most common among the entire group Simultaneous sensitization to 5 molecules-of group 1 (Der p 1 and Der f 1), group 2 (Der f 2 and Der p 2), and Der p 23-was the second most common for entire dataset and for the children group. This pattern differed in adults, where monosensitization to Der p 23 occupied the second position, suggesting that this molecule is an important factor of HDM allergy in Ukraine. Of the 16 analyzed regions, sensitization to Der p 23 prevailed in 2 Western regions of Ukraine. In the rest of the regions combination of Der p 2 and Der f 2 was the most prevalent. The established character of population sensitization to HDM in Ukraine is a good prognostic marker of allergen immunotherapy (AIT) efficacy.

Blocking function of allergen-specific immunoglobulin G, F(ab')2, and Fab antibodies prepared from patients undergoing Dermatophagoides pteronyssinus immunotherapy.

BACKGROUND: The blocking function of allergen-specific F(ab')2 [sF(ab')2] and Fab (sFab) fragment antibodies prepared from allergen immunotherapy-induced specific immunoglobulin G (sIgG) has not been fully investigated. OBJECTIVE: To investigate the inhibitory function of sIgG, sF(ab')2, and sFab antibodies in patients undergoing Dermatophagoides pteronyssinus (Der-p) subcutaneous immunotherapy (SCIT). METHODS: This study involved 10 subjects (aged 18-42 years) with house dust mite allergic rhinitis or asthma who received a 156-week course of Der-p SCIT. Total IgG levels were purified from the serum of the participants at weeks 0 and 156 by protein A affinity chromatography. Der-p sIgG was purified by affinity chromatography with Der-p as a ligand at week 156. The sF(ab')2 and sFab antibodies were prepared from Der-p sIgG by treatment with pepsin and papain, respectively. Furthermore, IgE-facilitated allergen binding assay, basophil activation inhibition test, and cytokine release inhibition assay were used to assess the inhibitory function of Der-p sIgG, sF(ab')2, and sFab antibodies. RESULTS: We found that the Der-p sIgG, sF(ab')2, and sFab antibodies markedly blocked Der-p-allergen sIgE complex binding to B cells, inhibited basophil activation, and markedly reduced the production of interleukin (IL)-5, IL-13, IL-17, and tumor necrosis factor-α by peripheral blood mononuclear cells. CONCLUSION: SCIT-induced Der-p sIgG, sF(ab')2, and sFab antibodies may block the formation of Der-p-sIgE complexes and may serve as a potential allergen-targeted biologics candidate for the treatment of allergic asthma. CLINICAL TRIAL REGISTRATION: This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and registered in the Chinese Clinical Trial Registry (ChiCTR-OOC-15006207, https://www.chictr.org.cn/enindex.aspx).

Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation.

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at KD 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD.

PlexinD1 Deficiency in Lung Interstitial Macrophages Exacerbates House Dust Mite-Induced Allergic Asthma.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.