Activated Leukocyte Cell Adhesion Molecule Regulates the Expression of Interleukin-33 in RSV Induced Airway Inflammation by Regulating MAPK Signaling Pathways.

PURPOSE: The respiratory syncytial virus (RSV) is a common respiratory virus that causes acute lower respiratory tract infectious diseases, particularly in young children and older individuals. Activated leukocyte cell adhesion molecule (ALCAM) is a membrane glycoprotein expressed in various cell types, including epithelial cells, and is associated with inflammatory responses and various cancers. However, the precise role of ALCAM in RSV-induced airway inflammation remains unclear, and our study aimed to explore this gap in the literature. METHODS: C57BL/6 wild-type, ALCAM knockout mice and airway epithelial cells were infected with RSV and the expression of ALCAM and inflammatory cytokines were measured. We also conducted further experiments using Anti-ALCAM antibody and recombinant ALCAM in airway epithelial cells. RESULTS: The expression levels of ALCAM and inflammatory cytokines increased in both RSV-infected mice and airway epithelial cells. Interestingly, IL-33 expression was significantly reduced in ALCAM-knockdown cells compared to control cells following RSV infection. Anti-ALCAM antibody treatment also reduced IL-33 expression following RSV infection. Furthermore, the phosphorylation of ERK1/2, p38, and JNK was diminished in ALCAM-knockdown cells compared to control cells following RSV infection. Notably, in the control cells, inhibition of these pathways significantly decreased the expression of IL-33. In vivo study also confirmed a reduction in inflammation induced by RSV infection in ALCAM deficient mice compared to wild-type mice. CONCLUSION: These findings demonstrate that ALCAM contributes to RSV-induced airway inflammation at least partly by influencing IL-33 expression through mitogen-activated protein kinase signaling pathways. These results suggest that targeting ALCAM could be a potential therapeutic strategy for alleviating IL-33-associated lung diseases.

NLRX1 knockdown attenuates pro-apoptotic signaling and cell death in pulmonary hyperoxic acute injury.

Hyperoxia is frequently used for treating acute respiratory failure, but it can cause acute lung injury. Nucleotide-binding domain and leucine-rich-repeat-containing family member X1 (NLRX1) is localized in mitochondria and involved in production of reactive oxygen species, inflammation, and apoptosis, which are the features of hyperoxic acute lung injury (HALI). The contribution of NLRX1 to HALI has not previously been addressed. Thus, to investigate the role of NLRX1 in hyperoxia, we generated a murine model of HALI in wild-type (WT) and NLRX1-/- mice by exposure to > 95% oxygen for 72 h. As a result, NLRX1 expression was elevated in mice exposed to hyperoxia. In acute lung injury, levels of inflammatory cells, protein leakage, cell cytotoxicity, and pro-inflammatory cytokines were diminished in NLRX1-/- mice compared to WT mice. In a survival test, NLRX1-/- mice showed reduced mortality under hyperoxic conditions, and apoptotic cell death and caspase expression and activity were also lower in NLRX1-/- mice. Furthermore, levels of the MAPK signaling proteins ERK 1/2, JNK, and p38 were decreased in NLRX1-deficient mice than in WT mice exposed to hyperoxia. The study shows that a genetic deficit in NLRX1 can suppress hyperoxia-induced apoptosis, suggesting that NLRX1 acts as a pivotal regulator of HALI.

Interleukin-18 Receptor α Modulates the T Cell Response in Food Allergy.

PURPOSE: The prevalence of food allergy, triggered by T-helper type 2 (Th2) cell-mediated inflammation, is increasing worldwide. Interleukin (IL)-18 plays an important role in inflammatory diseases by binding with the IL-18 receptor. IL-18/IL-18 receptor α (IL-18Rα) is a cofactor for immunoglobulin E (IgE) production and Th2 cell development. Studies have not investigated the association between the IL-18/IL-18Rα signaling pathway and food allergy. Here, we investigated the role of IL-18Rα in food allergy induction and development. METHODS: Wild-type (WT) and IL-18Rα-null mutant (IL-18Rα-/-) C57BL/6 mice were sensitized and challenged using ovalbumin (OVA) for food allergy induction. Food allergy symptoms, T cell-mediated immune responses, and signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways were analyzed in mice. RESULTS: IL-18Rα expression was increased in WT mouse intestines after OVA treatment. Food allergy-induced IL-18Rα-/- mice showed attenuated systemic food allergic reactions, OVA-specific IgE and mouse mast cell protease-1 production, inflammatory cell infiltration, and T cell activation. Ex vivo experiments showed that cell proliferation and Th2 cytokine production were lower in IL-18Rα-/- mouse splenocytes than in WT mouse splenocytes. IL-18Rα blockade in WT splenocytes attenuated cell proliferation and Th2 cytokine production. Moreover, STAT3 phosphorylation was reduced in IL-18Rα-/- mice, and SOCS3 and SOCS1 activation were diminished in IL-18Rα-/- intestinal T cells. CONCLUSIONS: IL-18Rα regulates allergic reactions and immune responses by regulating T cell responses in food allergies. Moreover, IL-18Rα is involved in the STAT/SOCS signaling pathways. Targeting IL-18Rα signaling might be a novel therapeutic strategy for food allergy.

A Novel Regulatory Role of Activated Leukocyte Cell-Adhesion Molecule in the Pathogenesis of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by fibroproliferative matrix molecule accumulation, collagen deposition, and apoptosis. Activated leukocyte cell-adhesion molecule (ALCAM; CD166) is a cell-adhesion molecule that has been implicated in adhesive and migratory attribution, including leukocyte homing and trafficking and cancer metastasis. We investigated the role of ALCAM on pulmonary fibrosis development in murine models. Thus, a bleomycin-induced pulmonary fibrosis model was established with wild-type and ALCAM-/- mice. Pulmonary fibrosis was also induced in transforming growth factor-ß1 (TGF-ß1)-transgenic mice that conditionally overexpress TGF-ß1 upon doxycycline administration. In both models, observed reduced ALCAM levels in lung tissue and BAL fluid in pulmonary fibrosis-induced wild-type mice compared with control mice. We also observed reduced ALCAM expression in the lung tissue of patients with pulmonary fibrosis compared with normal lung tissue. ALCAM-/- mice showed an exacerbated lung fibrosis response compared with wild-type mice, and this was accompanied by increased cell death. Further investigation for identification of the signaling pathway revealed that PI3K and ERK signaling pathways are involved in bleomycin-induced fibrosis. Collectively, these results highlight that ALCAM plays a protective role in the pathogenesis of pulmonary fibrosis that inhibits epithelial cell apoptosis through the PI3K-Akt signaling pathway. Our findings demonstrate the potential of ALCAM as a therapeutic target for IPF and may aid the development of new strategies for the management and treatment of patients with IPF.

Chitinase 3-like 1 is involved in the induction of IL-8 expression by double-stranded RNA in airway epithelial cells.

Viral respiratory infection causes inflammatory lung disease. Chitinase 3-like 1 (CHI3L1) contributes to airway inflammation, but its role in human airway epithelial cells following viral infection is unclear. Thus, we investigated whether CHI3L1 regulates inflammatory responses caused by viral infections in airway epithelial cells. Human bronchial epithelial cells, BEAS-2B, were stimulated with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)). To confirm the specific role of CHI3L1, CHI3L1 was knocked down in BEAS-2B cells using shRNA lentivirus. The expression of CHI3L1 and proinflammatory cytokines such as IL-8 and phosphorylation of mitogen-activated protein kinase (MAPK) pathways were analyzed. In addition to poly(I:C), BEAS-2B cells were infected with the human respiratory syncytial virus (RSV) A2 strain, and CHI3L1 and IL-8 expression was analyzed. Stimulating the cells with poly(I:C) increased CHI3L1 and IL-8 expression, whereas IL-8 expression was abrogated in CHI3L1 knockdown BEAS-2B cells. Poly(I:C) stimulation of BEAS-2B cells resulted in phosphorylation of MAPK pathways, and inhibition of MAPK pathways significantly abolished IL-8 secretion. Phosphorylation of MAPK pathways was diminished in CHI3L1 knockdown BEAS-2B cells. Infection with RSV increased CHI3L1 and IL-8 expression. IL-8 expression induced by RSV infection was abrogated in CHI3L1 knockdown cells. In conclusion, CHI3L1 may be involved in IL-8 secretion by regulating MAPK pathways during respiratory viral infections in airway epithelial cells.

Metabolomic profiling revealed altered lipid metabolite levels in childhood food allergy.

BACKGROUND: The pathophysiology of childhood food allergy (FA) and its natural history are poorly understood. Clarification of the underlying mechanism may help identify novel biomarkers and strategies for clinical intervention in children with FA. OBJECTIVE: This study aimed to identify metabolites associated with the development and resolution of FA. METHODS: The metabolomic profiles of 20 children with FA and 20 healthy controls were assessed by liquid chromatography-tandem mass spectrometry. Comparative analysis was performed to identify metabolites associated with FA and FA resolution. For subjects with FA, serum samples were collected at the time of diagnosis and after resolution to identify the changes in metabolite levels. The selected metabolites were then quantified in a quantification cohort to validate the results. Finally, genome-wide association analysis of the metabolite levels was performed. RESULTS: The study demonstrated a significantly higher level of sphingolipid metabolites and a lower level of acylcarnitine metabolites in children with FA than those in healthy controls. At diagnosis, subjects with resolving FA had a significantly high level of omega-3 metabolites and a low level of platelet-activating factors compared to persistent FA. However, the level of omega-3 metabolites decreased in children with resolving FA but increased in children with persistent FA during the same time. The quantification data of omega-3-derived resolvins, platelet-activating factor, and platelet-activating factor acetylhydrolase activity further supported these results. CONCLUSION: The lipid metabolite profile is closely related to childhood FA and FA resolution. This study suggests potential predictive biomarkers and provides insight into the mechanisms underlying childhood FA.

Genome-wide association study identifies TNFSF15 associated with childhood asthma.

BACKGROUND: Genome-wide association studies (GWASs) of asthma have identified several risk alleles and loci, but most have been conducted in individuals with European-ancestry. Studies in Asians, especially children, are still lacking. We aimed to identify susceptibility loci by performing the first GWAS of asthma in Korean children with persistent asthma. METHODS: We used a discovery set of 741 children with persistent asthma as cases and 589 healthy children and 551 healthy adults as controls to perform a GWAS. We validated our GWAS findings using UK Biobank data. We then used the Genotype-Tissue Expression database to identify expression quantitative trait loci of candidate variants. Finally, we quantified proteins of genes associated with asthma. RESULTS: Variants at the 17q12-21 locus and SNPs in CYBRD1 and TNFSF15 genes were associated with persistent childhood asthma at genome-wide thresholds of significance. Four SNPs in the TNFSF15 gene were also associated with childhood-onset asthma in British white participants in the UK Biobank data. The asthma-associated rs7856856-C allele, the lead SNP, was associated with decreased TNFSF15 expression in whole blood and in arteries. Korean children with asthma had lower serum TNFSF15 levels than controls, and those with the asthma risk rs7856856-CC genotype exhibited the lowest serum TNFSF15 levels overall, especially asthmatic children. CONCLUSIONS: Our GWAS of persistent childhood asthma with allergic sensitization identified a new susceptibility gene, TNFSF15, and replicated associations at the 17q12-21 childhood-onset asthma locus. This novel association may be mediated by reduced expression of serum TNFSF15 and loss of suppression of angiogenesis.

Genome-wide association study identifies BTNL2 associated with atopic asthma in children.

ABSTRACT: Asthma is a heterogeneous disease characterized by chronic airway inflammation with a genetic predisposition. Butyrophilin-like 2 (BTNL2) is a member of the immunoglobulin superfamily that plays an important role in regulating T cell activation and immune homeostasis. Here, we aimed to investigate the association of the genetic variants of BTNL2 with childhood asthma and asthma-related traits by utilizing extreme asthma phenotypes and employing a genome-wide association study. Our study included 243 children with well-defined moderate to severe atopic asthma and 134 healthy children with no history of allergic diseases and allergic sensitization. DNA from these subjects was genotyped using AxiomTM Genome-Wide Array Plates. Although no single nucleotide polymorphisms (SNPs) reached a genome-wide threshold of significance, 3 SNPs, rs3817971, rs41355746, and rs41441651, at BTNL2 were significantly associated with moderate to severe atopic asthma after performing Bonferroni correction. These SNPs were also associated with the risk of allergic sensitization toward house dust mites and the presence and degree of bronchial hyperresponsiveness. Thus, we identified that BTNL2 was associated with atopic moderate to severe persistent asthma in Korean children, and this may play an important role in disease development and susceptibility.

Gram-negative microbiota is related to acute exacerbation in children with asthma.

BACKGROUND: The upper-airway microbiota may be associated with the pathogenesis of asthma and useful for predicting acute exacerbation. However, the relationship between the lower-airway microbiota and acute exacerbation in children with asthma is not well understood. We evaluated the characteristics of the airway microbiome using induced sputum from children with asthma exacerbation and compared the microbiota-related differences of inflammatory cytokines with those in children with asthma. METHODS: We analysed the microbiome using induced sputum during acute exacerbation of asthma in children. We identified microbial candidates that were prominent in children with asthma exacerbation and compared them with those in children with stable asthma using various analytical methods. The microbial candidates were analysed to determine their association with inflammatory cytokines. We also developed a predictive functional profile using PICRUSt. RESULTS: A total of 95 children with allergic sensitisation including 22 with asthma exacerbation, 67 with stable asthma, and 6 controls were evaluated. We selected 26 microbial candidates whose abundances were significantly increased, decreased, or correlated during acute exacerbation in children with asthma. Among the microbial candidates, Campylobacter, Capnocytophaga, Haemophilus, and Porphyromonas were associated with inflammatory cytokines including macrophage inflammatory protein (MIP)-1ß, programmed death-ligand 1, and granzyme B. Both Campylobacter and MIP-1ß levels were correlated with sputum eosinophils. Increased lipopolysaccharide biosynthesis and decreased glycan degradation were observed in children with asthma exacerbation. CONCLUSION: Gram-negative microbes in the lower airway were related to acute exacerbation in children with asthma. These microbes and associated cytokines may play a role in exacerbating asthma in children.

Clusterin Deficiency Exacerbates Hyperoxia-Induced Acute Lung Injury.

Exposure to high oxygen concentrations leads to generation of excessive reactive oxygen species, causing cellular injury and multiple organ dysfunctions and is associated with a high mortality rate. Clusterin (CLU) is a heterodimeric glycoprotein that mediates several intracellular signaling pathways, including cell death and inflammation. However, the role of CLU in the pathogenesis of hyperoxic acute lung injury (HALI) is unknown. Wild-type (WT) and CLU-deficient mice and cultured human airway epithelial cells were used. Changes in cell death- and inflammation-related molecules with or without hyperoxia exposure in cells and animals were determined. Hyperoxia induced an increase in CLU expression in mouse lungs and human airway epithelial cells. Mice lacking CLU had increased HALI and mortality rate compared with WT mice. In vitro, CLU-disrupted cells showed enhanced release of cytochrome c, Bax translocation, cell death and inflammatory cytokine expression. However, treatment with recombinant CLU attenuated hyperoxia-induced apoptosis. Moreover, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed metabolic pathways, hematopoietic cell lineage, response to stress and localization and regulation of immune system that were differentially regulated between WT and CLU-/- mice. These results demonstrate that prolonged hyperoxia-induced lung injury is associated with CLU expression and that CLU replenishment may alleviate hyperoxia-induced cell death.

Integrated genetic and epigenetic analyses uncover MSI2 association with allergic inflammation.

BACKGROUND: The relationship between allergic and eosinophilic inflammation, either systemic or local, in allergic diseases remains unclear. OBJECTIVE: We performed combined genome-wide association study (GWAS) and epigenome-wide (EWAS) for atopy and tissue eosinophilia to identify both genetic and epigenetic signatures between systemic and local allergic inflammation, and to capture global patterns of gene regulation. METHODS: We included 126 subjects for atopy analysis and 147 for tissue eosinophilia analysis, as well as 18 normal nasal tissue samples. We identified differentially methylated positions (DMPs) and genes associated with atopy and tissue eosinophilia. Furthermore, we performed mendelian randomization analysis and penalized regression along with replication in an independent cohort. RESULTS: EWAS identified genes, including Musashi RNA binding protein 2 (MSI2), associated with atopy, which contained enriched DMPs that genetically affect atopy. A direct association was observed between MSI2 single-nucleotide polymorphisms and atopy, as was a causal effect of changes in MSI2 expression and methylation on atopy, which was replicated in a Costa Rican population. Regarding tissue eosinophilia, EWAS identified genes with enriched DMPs directly contributing to tissue eosinophilia at the gene level, including CAMK1D. The gene ontology terms of the identified genes for both phenotypes encompassed immune-related terms. CONCLUSION: EWAS combined with GWAS identified novel candidate genes, especially the methylation of MSI2, contributing to systemic allergic inflammation. Certain genes displayed a greater association with either systemic or local allergic inflammation; however, it is expected that a harmonized effect of these genes influences immune responses.

Chitinase 3-Like 1 Contributes to Food Allergy via M2 Macrophage Polarization.

PURPOSE: Food allergy is a hypersensitive immune response to specific food proteins. Chitinase 3-like 1 (CHI3L1, also known as YKL-40 in humans or BRP-39 in mice) is associated with various chronic diseases, such as cancer, rheumatoid arthritis, and allergic disease. CHI3L1 is involved in allergen sensitization and type 2 helper T (Th2) inflammation, but the role of CHI3L1 in food allergy remains unclear. In this study, we sought to investigate the role of CHI3L1 in the development of food allergy. METHODS: We measured serum levels of CHI3L1 in food allergic patients. Food allergy was induced in wild-type (WT) and CHI3L1 null mutant (CHI3L1-/-) BALB/c mice with ovalbumin (OVA). We investigated Th2 immune responses, M2 macrophage polarization, and mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) signaling pathways, and also performed transcriptome analysis. RESULTS: Serum levels of CHI3L1 were significantly higher in children with food allergy compared with those in healthy controls. Furthermore, CHI3L1 expression levels were elevated in WT mice after OVA treatment. Food allergy symptoms, immunoglobulin E levels, Th2 cytokine production, and histological injury were attenuated in food allergy-induced CHI3L1-/- mice compared with those in food allergy-induced WT mice. CHI3L1 expression was increased in OVA-treated WT intestinal macrophages and caused M2 macrophage polarization. Furthermore, CHI3L1 was involved in the extracellular signal-regulated kinases (ERK) and AKT signaling pathways and was associated with immune response and lipid metabolism as determined through transcriptome analysis. CONCLUSIONS: CHI3L1 plays a pivotal role in Th2 inflammation and M2 macrophage polarization through MAPK/ERK and PI3K/AKT phosphorylation in food allergy.

Activated Leukocyte Cell Adhesion Molecule Modulates Th2 Immune Response in Atopic Dermatitis.

PURPOSE: Activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily, is highly expressed on dendritic cells. ALCAM and its receptor CD6 are co-stimulatory molecules in the immunological synapse; their interaction is required for T cell activation. While atopic dermatitis (AD) is recognized as a T helper 2 (Th2)-mediated allergic disease, the role of ALCAM in its pathogenesis is unclear. METHODS: ALCAM levels were measured in the serum of AD patients and AD-induced murine model by ovalbumin treatment. We next investigated transepidermal water loss, clinical score, Th2-immune responses, skin barrier gene expression and T-cell activation using wild-type (WT) and ALCAM deficiency mice. An oxazolone-induced AD-like model was also established and analyzed using WT- and ALCAM-deficient mice. RESULTS: We found that serum ALCAM levels were elevated in pediatric AD patients as well as WT AD mice, whereas Th2-type cytokine production and AD symptoms were suppressed in ALCAM-deficient mice. In addition, CD4⁺ effector T-cell counts in murine skin and skin-draining lymph nodes were lower in ALCAM-deficient mice than in their WT counterparts. ALCAM deficiency was also linked to higher expression of skin barrier genes and number of lamellar bodies. CONCLUSIONS: These findings indicate that ALCAM may contribute to AD pathogenesis by meditating a Th2-dominant immune response and disrupting the barrier function of the skin.