Granulysin-mediated reduction of PDZRN3 induces Cx43 gap junctions activity exacerbating skin damage in trichloroethylene hypersensitivity syndrome.

Trichloroethylene (TCE)-induced hypersensitivity syndrome (THS) has been a concern for many researchers in the field of environmental and occupational health. Currently, there is no specific treatment for THS, leaving patients to contend with severe infections arising from extensive skin lesions, consequently leading to serious adverse effects. However, the pathogenesis of severe skin damage in THS remains unclear. This study aims to investigate the specific danger signals and mechanisms underlying skin damage in THS through in vivo and in vitro experiments. We identified that cell supernatant containing 15 kDa granulysin (GNLY), released from activated CD3-CD56+NK cells or CD3+CD56+NKT cells in PBMC induced by TCE or its metabolite, promoted apoptosis in HaCaT cells. The apoptosis level decreased upon neutralization of GNLY in the supernatant by a GNLY-neutralizing antibody in HaCaT cells. Subcutaneous injection of recombinant 15 kDa GNLY exacerbated skin damage in the THS mouse model and better mimicked patients' disease states. Recombinant 15 kDa GNLY could directly induce cellular communication disorders, inflammation, and apoptosis in HaCaT cells. In addition to its cytotoxic effects, GNLY released from TCE-activated NK cells and NKT cells or synthesized GNLY alone could induce aberrant expression of the E3 ubiquitin ligase PDZRN3, causing dysregulation of the ubiquitination of the cell itself. Consequently, this resulted in the persistent opening of gap junctions composed of connexin43, thereby intensifying cellular inflammation and apoptosis through the "bystander effect". This study provides experimental evidence elucidating the mechanisms of THS skin damage and offers a novel theoretical foundation for the development of effective therapies targeting severe dermatitis induced by chemicals or drugs.

Involvement of autologous myeloid dendritic cells in the evaluation of immediate hypersensitivity reactions to betalactams.

BACKGROUND: Amoxicillin (AX) and clavulanic acid (CLV) are the betalactam antibiotics (BLs) most used to treat bacterial infections, although they can trigger immediate hypersensitivity reactions (IDHRs). The maturation analysis of monocyte-derived dendritic cells (moDCs) and their capacity to induce proliferative response of lymphocytes are useful to test the sensitisation to a drug, although without optimal sensitivity. Nevertheless, this can be improved using directly isolated DCs such as myeloid DCs (mDCs). METHODS: mDCs and moDCs were obtained from 28 allergic patients (AP), 14 to AX, 14 to CLV and from 10 healthy controls (HC). The expression of CCR7, CD40, CD80, CD83, and CD86 was analysed after stimulation with both BLs. We measured the capacity of these pre-primed DCs to induce drug-specific activation of different lymphocyte subpopulations, CD3+, CD4+, CD8+, CD4+Th1, and CD4+Th2, by flow cytometry. RESULTS: Higher expression of CCR7, CD40, CD80, CD83, and CD86 was observed on mDCs compared to moDCs from AP after stimulating with the culprit BL. Similarly, mDCs induced higher proliferative response, mainly of CD4+Th2 cells, compared to moDCs, reaching up to 67% of positive results with AX, whereas of only 25% with CLV. CONCLUSIONS: mDCs from selective AP efficiently recognise the culprit drug which trigger the IDHR. mDCs also trigger proliferation of lymphocytes, mainly those with a Th2 cytokine pattern, although these responses depend on the nature of the drug, mimicking the patient's reaction.

Beyond Allergies-Updates on The Role of Mas-Related G-Protein-Coupled Receptor X2 in Chronic Urticaria and Atopic Dermatitis.

Mast cells (MCs) are an important part of the immune system, responding both to pathogens and toxins, but they also play an important role in allergic diseases, where recent data show that non-IgE-mediated activation is also of relevance, especially in chronic urticaria (CU) and atopic dermatitis (AD). Skin MCs express Mas-related G-protein-coupled receptor X2 (MRGPRX2), a key protein in non-IgE-dependent MC degranulation, and its overactivity is one of the triggering factors for the above-mentioned diseases, making MRGPRX2 a potential therapeutic target. Reviewing the latest literature revealed our need to focus on the discovery of MRGPRX2 activators as well as the ongoing vast research towards finding specific MRGPRX2 inhibitors for potential therapeutic approaches. Most of these studies are in their preliminary stages, with one drug currently being investigated in a clinical trial. Future studies and improved model systems are needed to verify whether any of these inhibitors may have the potential to be the next therapeutic treatment for CU, AD, and other pseudo-allergic reactions.

[Enhancement of mast cells activation by ATP via P2X4 receptor].

Adenosine-5'-triphosphate (ATP) is an important intracellular energy currency, but it is released extracellularly in response to various stimuli and acts as an intercellular signaling molecule by stimulating various P2 receptors. ATP and ADP are stored in synaptic vesicles and secretory granules, and are released extracellularly upon stimulation, playing important roles in neurotransmission and platelet aggregation. Furthermore, considerable amount of ATP is released by mechanical stimuli such as skin scraping or by cell damage, which in turn activates immune cells to promote inflammatory responses. Mast cells (MCs) are derived from hematopoietic stem cells and play a central role in type I allergic reactions. MCs are activated by IgE-mediated antigen recognition, leading to type I allergic reactions. MCs express P2X7 receptors that are activated by high concentrations of ATP (>0.5 |mM), and reported to aggravate inflammatory bowel disease and dermatitis. In contrast, role of MC P2 receptors that respond to lower concentrations of ATP remains to be investigated. We investigated in detail the effects of ATP in mouse bone marrow-derived MCs, and found that lower concentrations of ATP (<100 |µM) promotes IgE-dependent and GPCR-mediated degranulation via the ionotropic P2X4 receptor. In mouse allergic models, P2X4 receptor signal promote MC-mediated allergic responses through comprehensively increasing the sensitivity of MCs to different stimuli. Since ATP is known to be released from various cells upon mechanical stimuli such as cell damage or scratching, inhibition of P2X4 receptor signaling may represent a novel strategy to abrogate allergic reaction.

TGFß prevents IgE-mediated allergic disease by restraining T follicular helper 2 differentiation.

Allergic diseases are common, affecting more than 20% of the population. Genetic variants in the TGFß pathway are strongly associated with atopy. To interrogate the mechanisms underlying this association, we examined patients and mice with Loeys-Dietz syndrome (LDS) who harbor missense mutations in the kinase domain of TGFΒR1/2. We demonstrate that LDS mutations lead to reduced TGFß signaling and elevated total and allergen-specific IgE, despite the presence of wild-type T regulatory cells in a chimera model. Germinal center activity was enhanced in LDS and characterized by a selective increase in type 2 follicular helper T cells (TFH2). Expression of Pik3cg was increased in LDS TFH cells and associated with reduced levels of the transcriptional repressor SnoN. PI3Kγ/mTOR signaling in LDS naïve CD4+ T cells was elevated after T cell receptor cross-linking, and pharmacologic inhibition of PI3Kγ or mTOR prevented exaggerated TFH2 and antigen-specific IgE responses after oral antigen exposure in an adoptive transfer model. Naïve CD4+ T cells from nonsyndromic allergic individuals also displayed decreased TGFß signaling, suggesting that our mechanistic discoveries may be broadly relevant to allergic patients in general. Thus, TGFß plays a conserved, T cell-intrinsic, and nonredundant role in restraining TFH2 development via the PI3Kγ/mTOR pathway and thereby protects against allergic disease.

12-(S)-Hydroxyeicosatetraenoic Acid and GPR31 Signaling in Spinal Cord in Neuropathic Pain.

Neuropathic pain is a pressing unmet medical need requiring novel nonopioid-based therapeutic approaches. Using unbiased transcriptomic analysis, we found that the expression of Gpr31, a G protein-coupled receptor, increased in the dorsal horn of the spinal cord in rats with traumatic nerve injury-induced neuropathic pain. Daily intrathecal injections of siGpr31 reversed behavioral hypersensitivities in a time-dependent manner. GPR31, a Gα i protein-coupled receptor, has recently been cloned and is a receptor for 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. The lack of commercially available GPR31 antagonists has hampered the understanding of this receptor in pathophysiological states, including pain. To investigate this, our first approach was to identify novel GPR31 antagonists. Using a multidisciplinary approach, including in silico modeling, we identified the first highly potent and selective small-molecule GPR31 antagonist, SAH2. Here, we characterize the pharmacological activity in well-described models of neuropathic pain in rodents and provide evidence that 12-(S)-HETE/GPR31-dependent behavioral hypersensitivities are mediated through mitogen-activated protein kinase (MAPK) activation in the spinal cord. Our studies provide the pharmacological rationale for investigating contributions of GPR31 along the pain neuroaxis and the development of nonopioid GPR31-targeted strategies. SIGNIFICANCE STATEMENT: We have identified the first highly selective GPR31 antagonist. Using this antagonist, we have demonstrated that GPR31 signaling in the spinal cord is pronociceptive and MAPK pathways provided signaling mechanisms downstream of GPR31 activation in these processes.

Screening of Prospective Antiallergic Compound as FcεRI Inhibitors and Its Antiallergic Efficacy Through Immunoinformatics Approaches.

The occurrence of allergy, a type I hypersensitivity reaction, is rising exponentially all over the world. Sometimes, allergy proves to be fatal for atopic patients, due to the occurrence of anaphylaxis. This study is aimed to find an anti-allergic agent that can inhibit the binding of IgE to Human High Affinity IgE Receptor (FCεRI), thereby preventing the degranulation of mast cells. A considerable number of potential anti-allergic compounds were assessed for their inhibitory strength through ADMET studies. AUTODOCK was used for estimating the binding energy between anti-allergic compounds and FCεRI, along with the interacting amino acids. The docked pose showing favorable binding energy was subjected to molecular dynamics simulation study. Marrubiin, a diterpenoid lactone from Lamiaceae, and epicatechin-3-gallate appears to be effective in blocking the Human High Affinity IgE Receptor (FCεRI). This in-silico study proposes the use of marrubiin and epicatechin-3-gallate, in the downregulation of allergic responses. Due to the better inhibition constant, future direction of this study is to analyze the safety and efficacy of marrubiin in anti-allergic activities through in-vivo clinical human trials.

Chemokine CCL19 promotes type 2 T-cell differentiation and allergic airway inflammation.

BACKGROUND: Allergic asthma is driven largely by allergen-specific TH2 cells, which develop in regional lymph nodes on the interaction of naive CD4+ T cells with allergen-bearing dendritic cells that migrate from the lung. This migration event is dependent on CCR7 and its chemokine ligand, CCL21. However, is has been unclear whether the other CCR7 ligand, CCL19, has a role in allergic airway disease. OBJECTIVE: This study sought to define the role of CCL19 in TH2 differentiation and allergic airway disease. METHODS: Ccl19-deficient mice were studied in an animal model of allergic asthma. Dendritic cells or fibroblastic reticular cells from wild-type and Ccl19-deficient mice were cultured with naive CD4+ T cells, and cytokine production was measured by ELISA. Recombinant CCL19 was added to CD4+ T-cell cultures, and gene expression was assessed by RNA-sequencing and quantitative PCR. Transcription factor activation was assessed by flow cytometry. RESULTS: Lungs of Ccl19-deficient mice had less allergic airway inflammation, reduced airway hyperresponsiveness, and less IL-4 and IL-13 production compared with lungs of Ccl19-sufficient animals. Naive CD4+ T cells cocultured with Ccl19-deficient dendritic cells or fibroblastic reticular cells produced lower amounts of type 2 cytokines than did T cells cocultured with their wild-type counterparts. Recombinant CCL19 increased phosphorylation of STAT5 and induced expression of genes associated with TH2 cell and IL-2 signaling pathways. CONCLUSIONS: These results reveal a novel, TH2 cell-inducing function of CCL19 in allergic airway disease and suggest that strategies to block this pathway might help to reduce the incidence or severity of allergic asthma.

The role of glycosylation in clinical allergy and immunology.

While glycans are among the most abundant macromolecules on the cell with widespread functions, their role in immunity has historically been challenging to study. This is in part due to difficulties assimilating glycan analysis into routine approaches used to interrogate immune cell function. Despite this, recent developments have illuminated fundamental roles for glycans in host immunity. The growing field of glycoimmunology continues to leverage new tools and approaches to uncover the function of glycans and glycan-binding proteins in immunity. Here we utilize clinical vignettes to examine key roles of glycosylation in allergy, inborn errors of immunity, and autoimmunity. We will discuss the diverse functions of glycans as epitopes, as modulators of antibody function, and as regulators of immune cell function. Finally, we will highlight immune modulatory therapies that harness the critical role of glycans in the immune system.

The transcription factor NFIL3/E4BP4 regulates the developmental stage-specific acquisition of basophil function.

BACKGROUND: Basophils are rare but important effector cells in many allergic disorders. Contrary to their early progenitors, the terminal developmental processes of basophils in which they gain their unique functional properties are unknown. OBJECTIVE: We sought to identify a novel late-stage basophil precursor and a transcription factor regulating the terminal maturation of basophils. METHODS: Using flow cytometry, transcriptome analysis, and functional assays, we investigated the identification and functionality of the basophil precursors as well as basophil development. We generated mice with basophil-specific deletion of nuclear factor IL-3 (NFIL3)/E4BP4 and analyzed the functional impairment of NFIL3/E4BP4-deficient basophils in vitro and in vivo using an oxazolone-induced murine model of allergic dermatitis. RESULTS: We report a new mitotic transitional basophil precursor population (referred to as transitional basophils) that expresses the FcεRIα chain at higher levels than mature basophils. Transitional basophils are less responsive to IgE-linked degranulation but produce more cytokines in response to IL-3, IL-33, or IgE cross-linking than mature basophils. In particular, we found that the expression of NFIL3/E4BP4 gradually rises as cells mature from the basophil progenitor stage. Basophil-specific deletion of NFIL3/E4BP4 reduces the expression of genes necessary for basophil function and impairs IgE receptor signaling, cytokine secretion, and degranulation in the context of murine atopic dermatitis. CONCLUSIONS: We discovered transitional basophils, a novel late-stage mitotic basophil precursor cell population that exists between basophil progenitors and postmitotic mature basophils. We demonstrated that NFIL3/E4BP4 augments the IgE-mediated functions of basophils, pointing to a potential therapeutic regulator for allergic diseases.

The dichotomy of regulatory B cells in cancer versus allergic disease.

Regulatory B cells (Bregs) are an immunosuppressive cell phenotype that affects the immune system by limiting the inflammatory cascade. Dysregulation of Bregs can interestingly play a dichotomous role in the pathophysiology of many diseases and is especially highlighted when examining cancer pathology compared to allergic disease. This study reviews the existing literature on Bregs and compares their role in allergic disease in contrast to cancer development. Upregulation of Bregs in cancer states has been associated with poor prognostic outcomes across various cancer types, and Breg proliferation was associated with chronic interferon signaling, activation of the BCR-BTK (B cell receptor-Bruton's tyrosine kinase) pathway, and release of C-X-C motif ligand 13. In contrast, Breg dysfunction has been identified as a key mechanism in many allergic diseases, such as allergic asthma, allergic rhinitis, atopic dermatitis, and contact dermatitis. Development of Breg-targeted immunotherapies is currently at the preclinical level, but strategies differentially focus on Breg depletion in cancer versus Breg stimulation in allergy. Our review highlights the divergent functions that Bregs play in cancer compared to allergy. We conclude that natural homeostasis hinges on a fine balance between the dichotomous role of Bregs-over or underactivation can result in a pathological state.

Alveolar macrophage modulation via the gut-lung axis in lung diseases.

Several studies have demonstrated great potential implications for the gut-lung axis in lung disease etiology and treatment. The gut environment can be influenced by diet, metabolites, microbiotal composition, primary diseases, and medical interventions. These changes modulate the functions of alveolar macrophages (AMs) to shape the pulmonary immune response, which greatly impacts lung health. The immune modulation of AMs is implicated in the pathogenesis of various lung diseases. However, the mechanism of the gut-lung axis in lung diseases has not yet been determined. This mini-review aimed to shed light on the critical nature of communication between the gut and AMs during the development of pulmonary infection, injury, allergy, and malignancy. A better understanding of their crosstalk may provide new insights into future therapeutic strategies targeting the gut-AM interaction.

Mechanism underlying polyvalent IgG-induced regulatory T cell activation and its clinical application: Anti-idiotypic regulatory T cell theory for immune tolerance.

The regulatory T (Treg) cells constitute a functionally defined subpopulation of T cells that modulate the immune system and maintain immune tolerance through suppression of the development of autoimmune responses to self-antigens and allergic reactions to external antigens. Reduction in the number or function of Treg cells has been suggested as a key immune abnormality underlying the development of autoimmune and allergic diseases. In vitro studies have demonstrated that purified polyvalent immunoglobulin G (IgG) from multiple healthy blood donors can exert immunomodulatory effects on Treg cells. Incubation of polyvalent human IgG with purified CD4+CD25high T cells increased the intracellular expression of interleukin (IL)-10. Intravenous administration of polyvalent human IgG induced significant expansions of CD4+ Foxp3+ Treg cells and clinical improvements in patients with autoimmune diseases. In human clinical trials, intramuscular administration of autologous total IgG significantly increased the percentage of IL-10-producing CD4+ Treg cells in the peripheral blood of healthy subjects and provided significant clinical improvements in patients with atopic dermatitis. These results suggest a clinical usefulness of polyvalent IgG-induced activation of Treg cells in human subjects. This review proposes a new hypothesis for immune tolerance mechanism by integrating the pre-existing "idiotypic network theory" and "Treg cell theory" into an "anti-idiotypic Treg cell theory." Based on this hypothesis, an "active anti-idiotypic therapy" for allergic and autoimmune diseases using autologous polyvalent IgG (as immunizing antigens) is suggested as follows: (1) Intramuscular or subcutaneous administration of autologous polyvalent IgG produces numerous immunogenic peptides derived from idiotypes of autologous IgG through processing of dendritic cells, and these peptides activate anti-idiotypic Treg cells in the same subject. (2) Activated anti-idiotypic Treg cells secrete IL-10 and suppress Th2 cell response to allergens and autoimmune T cell response to self-antigens. (3) These events can induce a long-term clinical improvements in patients with allergic and autoimmune diseases. Further studies are needed to evaluate the detailed molecular mechanism underlying polyvalent IgG-induced Treg cell activation and the clinical usefulness of this immunomodulatory therapy for autoimmune and allergic diseases.

Dermatophagoides farinae microRNAs released to external environments via exosomes regulate inflammation-related gene expression in human bronchial epithelial cells.

Background: Dermatophagoides farinae (DFA) is an important species of house dust mites (HDMs) that causes allergic diseases. Previous studies have focused on allergens with protein components to explain the allergic effect of HDMs; however, there is little knowledge on the role of microRNAs (miRNAs) in the allergic effect of HDMs. This study aimed to unravel the new mechanism of dust mite sensitization from the perspective of cross-species transport of extracellular vesicles-encapsulated miRNAs from HDMs. Methods: Small RNA (sRNA) sequencing was performed to detect miRNAs expression profiles from DFA, DFA-derived exosomes and DFA culture supernatants. A quantitative fluorescent real-time PCR (qPCR) assay was used to detect miRNAs expression in dust specimens. BEAS-2B cells endocytosed exosomes were modeled in vitro to detect miRNAs from DFA and the expression of related inflammatory factors. Representative dfa-miR-276-3p and dfa-novel-miR2 were transfected into BEAS-2B cells, and then differentially expressed genes (DEGs) were analyzed by RNA sequencing. Protein-protein interaction (PPI) network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment analyses were performed on the first 300 nodes of DEGs. Results: sRNA sequencing identified 42 conserved miRNAs and 66 novel miRNAs in DFA, DFA-derived exosomes, and DFA culture supernatants. A homology analysis was performed on the top 18 conserved miRNAs with high expression levels. The presence of dust mites and miRNAs from HDMs in living environment were also validated. Following uptake of DFA-derived exosomes by BEAS-2B cells, exosomes transported miRNAs from DFA to target cells and produced pro-inflammatory effects in corresponding cells. RNA sequencing identified DEGs in dfa-miR-276-3p and dfa-novel-miR2 transfected BEAS-2B cells. GO and KEGG enrichment analyses revealed the role of exosomes with cross-species transporting of DFA miRNAs in inflammatory signaling pathways, such as JAK-STAT signaling pathway, PI3K/AKT signaling pathway and IL-6-mediated signaling pathway. Conclusion: Our findings demonstrate the miRNAs expression profiles in DFA for the first time. The DFA miRNAs are delivered into living environments via exosomes, and engulfed by human bronchial epithelial cells, and cross-species regulation may contribute to inflammation-related processes.

[Regulación de la expresión de IL-33 e IL-17 por la modulación farmacológica de HIF-1 en un modelo murino de inflamación alérgica pulmonar].

Objective: To evaluate the effect of pharmacological modulation of HIF-1 on the expression of IL-33 and IL-17 in a murine model of allergic pulmonary inflam- mation (API) with different degrees of severity. Methods: 5 mice/group received ovalbumin (OVA) 1(mild), 2(moderate) or 3(severe) challenges via i.t. prior to allergen sensitization, in addition to the HIF-1 induction or inhibition groups, received EDHB (OVA+EDHB) i.p. or 2ME (OVA+2ME) i.t. respectively. Control groups received saline solution (SS) in the same way. HE (inflammatory infiltrate), PAS (mucus production) and immunohistochemical staining for HIF-1a, IL-33, IL-17 were performed, quantitatively analyzing by digital pathology. Results: We obtained different degrees of severity with a greater number of challenges, increasing the expression of HIF-1, correlating with the expression of IL-33/IL-17. Increasing or decreasing, respectively by pharmacological modulation. Conclusions: The above suggests that the high expression of HIF-1 favors the production of IL-33 and IL-17 contributing to the damage in lung tissue and the severity of the disease and these can be regulated through the modulation of HIF- 1.

Objetivo: Evaluar el efecto de la modulación farmacológica de HIF-1 en la expresión de IL-33 e IL-17 en un modelo murino de inflamación alérgica pulmonar (IAP) con diferentes grados de severidad. Métodos: 5 ratones/grupo recibieron ovoalbúmina (OVA) 1(leve), 2(moderada) o 3(severa) retos vía i.t. previa sensibilización como alergeno, además los grupos de inducción o inhibición de HIF-1a, recibieron EDHB (OVA+EDHB) i.p. o 2ME (OVA+2ME) i.t. respectivamente. Los grupos controles recibieron solución salina (SS) de igual forma. Se realizaron tinciones de HE (infiltrado inflamatorio), PAS (producción de moco) e inmunohistoquímicas de HIF-1a, IL-33, IL-17, analizando cuantitativamente por patología digital. Resultados: Obtuvimos diferentes grados de severidad a mayor número de retos, incrementando la expresión de HIF-1, correlacionando con la expresión de IL- 33/IL-17. Aumentando o disminuyendo, respectivamente por la modulación farmacológica. Conclusiones: Lo anterior sugiere que la alta expresión de HIF-1 favorece la producción de IL-33 e IL-17 contribuyendo al daño en el tejido pulmonar y la severi- dad de la enfermedad y estas pueden ser reguladas a través de la modulación de HIF-1.

[Research progress on eosinophils in lung cancer].

Eosinophils are important immune cells that contain eosinophilic particles and play a key role in allergic diseases such as asthma and helminth infections. An increasing number of studies have confirmed that eosinophils infiltrate a variety of tumor tissues, which can synthesize and secrete a large number of bioactive substances under certain circumstances, such as cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes and so on, which may affect angiogenesis and matrix remodeling or change the tumor microenvironment, thereby affecting tumor progression. This review focused on the role of eosinophils in lung cancer and provided an outlook on the issues in clinical and basic research.

Isthmin-1 attenuates allergic Asthma by stimulating adiponectin expression and alveolar macrophage efferocytosis in mice.

BACKGROUND: Allergic asthma is a common respiratory disease that significantly impacts human health. Through in silico analysis of human lung RNASeq, we found that asthmatic lungs display lower levels of Isthmin-1 (ISM1) expression than healthy lungs. ISM1 is an endogenous anti-inflammatory protein that is highly expressed in mouse lungs and bronchial epithelial cells, playing a crucial role in maintaining lung homeostasis. However, how ISM1 influences asthma remains unclear. This study aims to investigate the potential involvement of ISM1 in allergic airway inflammation and uncover the underlying mechanisms. METHODS: We investigated the pivotal role of ISM1 in airway inflammation using an ISM1 knockout mouse line (ISM1-/-) and challenged them with house dust mite (HDM) extract to induce allergic-like airway/lung inflammation. To examine the impact of ISM1 deficiency, we analyzed the infiltration of immune cells into the lungs and cytokine levels in bronchoalveolar lavage fluid (BALF) using flow cytometry and multiplex ELISA, respectively. Furthermore, we examined the therapeutic potential of ISM1 by administering recombinant ISM1 (rISM1) via the intratracheal route to rescue the effects of ISM1 reduction in HDM-challenged mice. RNA-Seq, western blot, and fluorescence microscopy techniques were subsequently used to elucidate the underlying mechanisms. RESULTS: ISM1-/- mice showed a pronounced worsening of allergic airway inflammation and hyperresponsiveness upon HDM challenge. The heightened inflammation in ISM1-/- mice correlated with enhanced lung cell necroptosis, as indicated by higher pMLKL expression. Intratracheal delivery of rISM1 significantly reduced the number of eosinophils in BALF and goblet cell hyperplasia. Mechanistically, ISM1 stimulates adiponectin secretion by type 2 alveolar epithelial cells partially through the GRP78 receptor and enhances adiponectin-facilitated apoptotic cell clearance via alveolar macrophage efferocytosis. Reduced adiponectin expression under ISM1 deficiency also contributed to intensified necroptosis, prolonged inflammation, and heightened severity of airway hyperresponsiveness. CONCLUSIONS: This study revealed for the first time that ISM1 functions to restrain airway hyperresponsiveness to HDM-triggered allergic-like airway/lung inflammation in mice, consistent with its persistent downregulation in human asthma. Direct administration of rISM1 into the airway alleviates airway inflammation and promotes immune cell clearance, likely by stimulating airway adiponectin production. These findings suggest that ISM1 has therapeutic potential for allergic asthma.

Stimulus-responsive proteins involved in multi-process regulation of storage substance accumulation during rice grain filling under elevated temperature.

BACKGROUND: The intensified global warming during grain filling deteriorated rice quality, in particular increasing the frequency of chalky grains which markedly impact market value. The formation of rice quality is a complex process influenced by multiple genes, proteins and physiological metabolic processes. Proteins responsive to stimulus can adjust the ability of plants to respond to unfavorable environments, which may be an important protein involved in the regulation of quality formation under elevated temperature. However, relatively few studies have hindered our further understanding of rice quality formation under elevated temperature. RESULTS: We conducted the actual field elevated temperature experiment and performed proteomic analysis of rice grains at the early stage of grain filling. Starting with the response to stimulus in GO annotation, 22 key proteins responsive to stimulus were identified in the regulation of grain filling and response to elevated temperature. Among the proteins responsive to stimulus, during grain filling, an increased abundance of signal transduction and other stress response proteins, a decreased abundance of reactive oxygen species-related proteins, and an increased accumulation of storage substance metabolism proteins consistently contributed to grain filling. However, the abundance of probable indole-3-acetic acid-amido synthetase GH3.4, probable indole-3-acetic acid-amido synthetase GH3.8 and CBL-interacting protein kinase 9 belonged to signal transduction were inhibited under elevated temperature. In the reactive oxygen species-related protein, elevated temperature increased the accumulation of cationic peroxidase SPC4 and persulfide dioxygenase ETHE1 homolog to maintain normal physiological homeostasis. The increased abundance of alpha-amylase isozyme 3E and seed allergy protein RA5 was related to the storage substance metabolism, which regulated starch and protein accumulation under elevated temperature. CONCLUSION: Auxin synthesis and calcium signal associated with signal transduction, other stress responses, protein transport and modification, and reactive oxygen species-related proteins may be key proteins responsive to stimulus in response to elevated temperature. Alpha-amylase isozyme 3E and seed allergy protein RA5 may be the key proteins to regulate grain storage substance accumulation and further influence quality under elevated temperature. This study enriched the regulatory factors involved in the response to elevated temperature and provided a new idea for a better understanding of grain response to temperature.

Extracellular vesicles of the probiotic bacteria E. coli O83 activate innate immunity and prevent allergy in mice.

BACKGROUND: E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. METHODS: EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. CONCLUSIONS: We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Video Abstract.

Degranulation of human mast cells: modulation by P2 receptors' agonists.

Since the late 1970s, there has been an alarming increase in the incidence of asthma and its morbidity and mortality. Acute obstruction and inflammation of allergic asthmatic airways are frequently caused by inhalation of exogenous substances such as allergens cross-linking IgE receptors expressed on the surface of the human lung mast cells (HLMC). The degree of constriction of human airways produced by identical amounts of inhaled allergens may vary from day to day and even hour to hour. Endogenous factors in the human mast cell (HMC)'s microenvironment during allergen exposure may markedly modulate the degranulation response. An increase in allergic responsiveness may significantly enhance bronchoconstriction and breathlessness. This review focuses on the role that the ubiquitous endogenous purine nucleotide, extracellular adenosine 5'-triphosphate (ATP), which is a component of the damage-associated molecular patterns, plays in mast cells' physiology. ATP activates P2 purinergic cell-surface receptors (P2R) to trigger signaling cascades resulting in heightened inflammatory responses. ATP is the most potent enhancer of IgE-mediated HLMC degranulation described to date. Current knowledge of ATP as it relates to targeted receptor(s) on HMC along with most recent studies exploring HMC post-receptor activation pathways are discussed. In addition, the reviewed studies may explain why brief, minimal exposures to allergens (e.g., dust, cat, mouse, and grass) can unpredictably lead to intense clinical reactions. Furthermore, potential therapeutic approaches targeting ATP-related enhancement of allergic reactions are presented.