Targeted inhibition of m6A demethylase FTO by FB23 attenuates allergic inflammation in the airway epithelium.

Epithelial cells play a crucial role in asthma, contributing to chronic inflammation and airway hyperresponsiveness. m6A modification, which involves key proteins such as the demethylase fat mass and obesity-associated protein (FTO), is crucial in the regulation of various diseases, including asthma. However, the role of FTO in epithelial cells and the development of asthma remains unclear. In this study, we investigated the demethylase activity of FTO using a small-molecule inhibitor FB23 in epithelial cells and allergic inflammation in vivo and in vitro. We examined the FTO-regulated transcriptome-wide m6A profiling by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq under FB23 treatment and allergic inflammation conditions. Immunofluorescence staining was performed to assess the tissue-specific expression of FTO in asthmatic bronchial mucosa. We demonstrated that FB23 alleviated allergic inflammation in IL-4/IL-13-treated epithelial cells and house dust mite (HDM)-induced allergic airway inflammation mouse model. The demethylase activity of FTO contributed to the regulation of TNF-α signaling via NF-κB and epithelial-mesenchymal transition-related pathways under allergic inflammation conditions in epithelial cells. FTO was expressed in epithelial, submucosal gland, and smooth muscle cells in human bronchial mucosa. In conclusion, FB23-induced inhibition of FTO alleviates allergic inflammation in epithelial cells and HDM-induced mice, potentially through diverse cellular processes and epithelial-mesenchymal transition signaling pathways, suggesting that FTO is a potential therapeutic target in asthma management.

Microbiological characteristics of the lower airway in adults with bronchiectasis: a prospective cohort study.

BACKGROUND: Microbial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis. METHODS: We conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up. RESULTS: We recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis. CONCLUSION: We have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.

Heterozygous CARD9 mutation favors the development of allergic bronchopulmonary aspergillosis.

BACKGROUND: Previous research demonstrated that a homozygous mutation of g.136372044G>A (S12N) in caspase recruitment domain family member 9 ( CARD9 ) is critical for producing Aspergillus fumigatus -induced ( Af -induced) T helper 2 (T H 2)-mediated responses in allergic bronchopulmonary aspergillosis (ABPA). However, it remains unclear whether the CARD9S12N mutation, especially the heterozygous occurrence, predisposes the host to ABPA. METHODS: A total of 61 ABPA patients and 264 controls (including 156 healthy controls and 108 asthma patients) were recruited for sequencing the CARD9 locus to clarify whether patients with this heterozygous single-nucleotide polymorphisms are predisposed to the development of ABPA. A series of in vivo and in vitro experiments, such as quantitative real-time polymerase chain reaction, flow cytometry, and RNA isolation and quantification, were used to illuminate the involved mechanism of the disease. RESULTS: The presence of the p.S12N mutation was associated with a significant risk of ABPA in ABPA patients when compared with healthy controls and asthma patients, regardless of Aspergillus sensitivity. Relative to healthy controls without relevant allergies, the mutation of p.S12N was associated with a significant risk of ABPA (OR: 2.69 and 4.17 for GA and AA genotypes, P = 0.003 and 0.029, respectively). Compared with patients with asthma, ABPA patients had a significantly higher heterozygous mutation (GA genotype), indicating that p.S12N might be a significant ABPA-susceptibility locus ( aspergillus sensitized asthma: OR: 3.02, P = 0.009; aspergillus unsensitized asthma: OR: 2.94, P = 0.005). The mutant allele was preferentially expressed in ABPA patients with heterozygous CARD9S12N , which contributes to its functional alterations to facilitate Af -induced T H 2-mediated ABPA development. In terms of mechanism, Card9 wild-type ( Card9WT ) expression levels decreased significantly due to Af -induced decay of its messenger RNA compared to the heterozygous Card9S12N . In addition, ABPA patients with heterozygous CARD9S12N had increased Af -induced interleukin-5 production. CONCLUSION: Our study provides the genetic evidence showing that the heterozygous mutation of CARD9S12N , followed by allele expression imbalance of CARD9S12N , facilitates the development of ABPA.

The Role of Myeloid-Derived Suppressor Cells in Multiple Sclerosis and Its Animal Model.

Myeloid-derived suppressor cells (MDSCs), a heterogeneous cell population that consists of mostly immature myeloid cells, are immunoregulatory cells mainly characterized by their suppressive functions. Emerging findings have revealed the involvement of MDSCs in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). MS is an autoimmune and degenerative disease of the central nervous system characterized by demyelination, axon loss, and inflammation. Studies have reported accumulation of MDSCs in inflamed tissues and lymphoid organs of MS patients and EAE mice, and these cells display dual functions in EAE. However, the contribution of MDSCs to MS/EAE pathogenesis remains unclear. This review aims to summarize our current understanding of MDSC subsets and their possible roles in MS/EAE pathogenesis. We also discuss the potential utility and associated obstacles in employing MDSCs as biomarkers and cell-based therapies for MS.

GABA signaling enforces intestinal germinal center B cell differentiation.

Recent compelling results indicate possible links between neurotransmitters, intestinal mucosal IgA+ B cell responses, and immunoglobulin A nephropathy (IgAN) pathogenesis. Here, we demonstrated that γ-amino butyric acid (GABA) transporter-2 (GAT-2) deficiency induces intestinal germinal center (GC) B cell differentiation and worsens the symptoms of IgAN in a mouse model. Mechanistically, GAT-2 deficiency enhances GC B cell differentiation through activation of GABA-mammalian target of rapamycin complex 1 (mTORC1) signaling. In addition, IgAN patients have lower GAT-2 expression but higher activation of mTORC1 in blood B cells, and both are correlated with kidney function in IgAN patients. Collectively, this study describes GABA signaling-mediated intestinal mucosal immunity as a previously unstudied pathogenesis mechanism of IgAN and challenges the current paradigms of IgAN.

Oral administration of Lactobacillus delbrueckii enhances intestinal immunity through inducing dendritic cell activation in suckling piglets.

Lactobacillus delbrueckii (LAB) has been demonstrated to exert versatile beneficial effects on modulating intestinal immunity, increasing gut microbial diversity, promoting growth performance, and even preventing disease onset in pigs. However, the underlying mechanism of LAB-mediated gut immunity regulation in piglets remains unclear. In this study, we found that supplementation of LAB significantly increases serum TNF-α, ileum IL-4, and IL-10 levels compared with the control group. Meanwhile, oral supplementation of LAB-modified gut microbial communities was evidenced by the increased abundance of the Lactobacillus genus in the colon. Mechanistically, LAB induced dendritic cell (DC) maturation and activation, which may be relevant to the activation of NF-κB and MAPK signaling pathways. Moreover, we found that oral administration of LAB during the suckling period shows long-lasting immunomodulatory impacts on intestinal immunity after weaning. Collectively, this study uncovers the mechanism of LAB in regulating the intestinal immunity of piglets, suggesting that LAB can be developed as an immunoenhancing biological agent during the suckling period.

CD69 mediates the protective role of adipose tissue-derived mesenchymal stem cells against Pseudomonas aeruginosa pulmonary infection.

BACKGROUND: Our previous study shows that Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising strategy for cell-based therapy against pulmonary infection with Pseudomonas aeruginosa (P. aeruginosa), but the underlying mechanisms remain unclear. METHODS: cDNA microarray assay was performed to explore the transcriptome of ASCs primed by P. aeruginosa. Small interfering RNA (siRNA) was constructed to select the receptor candidates for P. aeruginosa recognition and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in ASCs. The soluble protein chimeras containing the extracellular domain of human CD69 fused to the Fc region of human immunoglobulin IgG1 were used as a probe to validate the recognition of P. aeruginosa. The association between CD69 and extracellular regulated protein kinases 1/2 (ERK1/2) was explored via co-immunoprecipitation, siRNA, and inhibitor. The murine models of P. aeruginosa pneumonia treated with WT-ASCs, GM-CSF-/- -ASCs Cd69-/- -ASCs or Erk1-/- -ASCs were used to determine the role of GM-CSF, CD69, and ERK1 in ASCs against P. aeruginosa infection. RESULTS: We showed that C-type lectin receptor CD69 mediated the protective effects of ASCs partly through GM-CSF. CD69 could specifically recognize P. aeruginosa and regulate GM-CSF secretion of ASCs. CD69 regulated the production of GM-CSF via ERK1 in ASCs after P. aeruginosa infection. Moreover, the Administration of ASCs with deficiency of CD69 or ERK1 completely blocked its protective effects in a murine model of P. aeruginosa pneumonia. CONCLUSIONS: CD69 recognizes P. aeruginosa and further facilitates ERK1 activation, which plays a crucial role in ASCs-based therapy against P. aeruginosa pneumonia. CD69 may be a novel target molecule to improve ASCs-based therapy against P. aeruginosa infection.

Low-protein diets supplemented with glutamic acid or aspartic acid ameliorate intestinal damage in weaned piglets challenged with hydrogen peroxide.

Glutamic acid (Glu) and aspartic acid (Asp) are acidic amino acids with regulatory roles in nutrition, energy metabolism, and oxidative stress. This study aimed to evaluate the effects of low-protein diets supplemented with Glu and Asp on the intestinal barrier function and energy metabolism in weaned piglets challenged with hydrogen peroxide (H2O2). Forty piglets were randomly divided into 5 groups: NC, PC, PGA, PG, and PA (n = 8 for each group). Pigs in the NC and PC groups were fed a low-protein diet, while pigs in the PGA, PG, or PA groups were fed the low-protein diet supplemented with 2.0% Glu +1.0% Asp, 2.0% Glu, or 1.0% Asp, respectively. On day 8 and 11, pigs in the NC group were intraperitoneally injected with saline (1 mL/kg BW), while pigs in the other groups were intraperitoneally administered 10% H2O2 (1 mL/kg BW). On day 14, all pigs were sacrificed to collect jejunum and ileum following the blood sample collection in the morning. Notably, low-protein diets supplemented with Glu or Asp ameliorated the intestinal oxidative stress response in H2O2-challenged piglets by decreasing intestinal expression of genes (P < 0.05) (e.g., manganese superoxide dismutase [MnSOD], glutathione peroxidase [Gpx]-1, and Gpx-4) encoding oxidative stress-associated proteins, reducing the serum concentration of diamine oxidase (P < 0.05), and inhibiting apoptosis of the intestinal epithelium. Glu and Asp supplementation attenuated the upregulated expression of energy metabolism-associated genes (such as hexokinase and carnitine palmitoyltransferase-1) and the H2O2-induced activation of acetyl-coenzyme A carboxylase (ACC) in the jejunum and adenosine monophosphate-activated protein kinase-acetyl-ACC signaling in the ileum. Dietary Glu and Asp also ameliorated intestinal barrier damage as indicated by restored intestinal histology and morphology. In conclusion, low-protein diets supplemented with Glu and Asp protected against oxidative stress-induced intestinal dysfunction in piglets, suggesting that this approach could be used as a nutritional regulatory protectant against oxidative stress.

E3 ligase c-Cbl regulates intestinal inflammation through suppressing fungi-induced noncanonical NF-κB activation.

Intestinal fungi are critical for modulating host immune homeostasis and underlying mechanisms remain unclear. We show that dendritic cell (DC)-specific deficiency of casitas B-lineage lymphoma (c-Cbl) renders mice susceptible to dextran sodium sulfate (DSS)-induced colitis. Mechanistically, we identify that c-Cbl functions downstream of Dectin-2 and Dectin-3 to mediate the ubiquitination and degradation of noncanonical nuclear factor κB subunit RelB. Thus, c-Cbl deficiency in DCs promotes α-mannan-induced activation of RelB, which suppresses p65-mediated transcription of an anti-inflammatory cytokine gene, il10, thereby aggravating DSS-induced colitis. Moreover, suppressing fungal growth with fluconazole or inhibition of RelB activation in vivo attenuates colitis in mice with DC-specific deletion of c-Cbl. We also demonstrate an interaction between c-Cbl and c-Abl tyrosine kinase and find that treatment with DPH, a c-Abl agonist, synergistically increases fungi-induced c-Cbl activation to restrict colitis. Together, these findings unravel a previously unidentified fungi-induced c-Cbl/RelB axis that sustains intestinal homeostasis and protects against intestinal inflammation.

Priming with FLO8-deficient Candida albicans induces Th1-biased protective immunity against lethal polymicrobial sepsis.

The morphological switch between yeast and hyphae of Candida albicans is essential for its interaction with the host defense system. However, the lack of understanding of host-pathogen interactions during C. albicans infection greatly hampers the development of effective immunotherapies. Here, we found that priming with the C. albicans FLO8-deficient (flo8) mutant, locked in yeast form, protected mice from subsequent lethal C. albicans infection. Deficiency of Dectin-2, a fungus-derived α-mannan recognition receptor, completely blocked flo8 mutant-induced protection. Mechanistically, the flo8 mutant-induced Dectin-2/CARD9-mediated IL-10 production in DCs and macrophages to block thymus atrophy by inhibiting the C. albicans-induced apoptosis of thymic T cells, which facilitated the continuous output of naive T cells from the thymus to the spleen. Continuous recruitment of naive T cells to the spleen enhanced Th1-biased antifungal immune responses. Consequently, depletion of CD4+ T cells or blockade of IL-10 receptor function using specific antibodies in mice completely blocked the protective effects of flo8 mutant priming against C. albicans infection. Moreover, mannans exposed on the surface of the flo8 mutant were responsible for eliciting protective immunity by inhibiting the C. albicans-induced apoptosis of thymic T cells to sustain the number of naive T cells in the spleen. Importantly, priming with the flo8 mutant extensively protected mice from polymicrobial infection caused by cecal ligation and puncture (CLP) by enhancing Th1-biased immune responses. Together, our findings imply that targeting FLO8 in C. albicans elicits protective immune responses against polymicrobial infections and that mannans extracted from the flo8 mutant are potential immunotherapeutic candidate(s) for controlling infectious diseases.

LL37 Inhibits Aspergillus fumigatus Infection via Directly Binding to the Fungus and Preventing Excessive Inflammation.

The incidence of Aspergillus fumigatus infection and the rate of resistance to antifungal drugs have sharply increased in recent years. LL37 has been reported as a host defense peptide with broad-spectrum antibacterial activities. However, the role of LL37 during A. fumigatus infection remains unclear. Here, we examined the interaction between LL37 and A. fumigatus and found that synthetic LL37 could directly bind to the surface of A. fumigatus, disrupting the integrity of the cell wall in vitro. LL37 inhibited mycelial growth in a concentration-dependent manner, rather than fungicidal effect even at high concentration (e.g., 20 µM). Interestingly, low concentrations of LL37 (e.g., 4 µM) significantly attenuated mycelial adhesion and prevented the invasion and destruction of epithelial cells. Following LL37 treatment, the levels of proinflammatory cytokines released by A. fumigatus-stimulated macrophages decreased significantly, accompanied by downregulation of M1 type markers. In a mouse model of pulmonary A. fumigatus infection, LL37-treated mice showed lower amounts of fungi load, moderate pathological damage, and reduced proinflammatory cytokines. Further, LL37 transgenic mice (LL37+/+) were examined to investigate the effects of endogenous LL37 in an A. fumigatus infection model and showed lower susceptibility to A. fumigatus infection in comparison with wild-type mice. In addition, LL37 also played a protective role in an immunosuppressed mouse model of A. fumigatus infection. Thus, LL37 inhibits A. fumigatus infection via directly binding to mycelia and reducing excessive inflammation. LL37 or its analogs may therefore constitute potential drug components for A. fumigatus infection.

Slc6a13 deficiency promotes Th17 responses during intestinal bacterial infection.

The Î³-amino butyric acid (GABA)ergic system shapes the activation and function of immune cells. The present study was conducted to explore the regulation of GABA transporter (GAT)-2 on the differentiation of Th17 cells. Here we found that Th17 cells show higher abundance of GAT-2, and have distinct cellular metabolic signatures, such as the GABA shunt pathway, as compared to naïve T cells. GAT-2 deficiency had little effect on the metabolic signature in naïve T cells, but impaired the GABA uptake and GABA shunt pathway in Th17 cells. GAT-2 deficiency had little effect on T cell development and peripheral T cell homeostasis; however, its deficiency promoted Th17 cell differentiation in vitro. Mechanistically, GAT-2 deficiency promoted differentiation of Th17 cells through activation of GABA-mTOR signaling. In a mouse model of intestinal infection and inflammation, GAT-2 deficiency promoted Th17 responses. Collectively, GAT-2 deficiency promotes Th17 cell responses through activation of GABA-mTOR signaling.

CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses.

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell-mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell-mediated pathologic responses.

Alpha-ketoglutarate (AKG) lowers body weight and affects intestinal innate immunity through influencing intestinal microbiota.

Alpha-ketoglutarate (AKG), a precursor of glutamate and a critical intermediate in the tricarboxylic acid cycle, shows beneficial effects on intestinal function. However, the influence of AKG on the intestinal innate immune system and intestinal microbiota is unknown. This study explores the effect of oral AKG administration in drinking water (10 g/L) on intestinal innate immunity and intestinal microbiota in a mouse model. Mouse water intake, feed intake and body weight were recorded throughout the entire experiment. The ileum was collected for detecting the expression of intestinal proinflammatory cytokines and innate immune factors by Real-time Polymerase Chain Reaction. Additionally, the ileal luminal contents and feces were collected for 16S rDNA sequencing to analyze the microbial composition. The intestinal microbiota in mice was disrupted with an antibiotic cocktail. The results revealed that AKG supplementation lowered body weight, promoted ileal expression of mammalian defensins of the alpha subfamily (such as cryptdins-1, cryptdins-4, and cryptdins-5) while influencing the intestinal microbial composition (i.e., lowering the Firmicutes to Bacteroidetes ratio). In the antibiotic-treated mouse model, AKG supplementation failed to affect mouse body weight and inhibited the expression of cryptdins-1 and cryptdins-5 in the ileum. We concluded that AKG might affect body weight and intestinal innate immunity through influencing intestinal microbiota.

Interferon Tau Affects Mouse Intestinal Microbiota and Expression of IL-17.

This study was conducted to explore the effects of interferon tau (IFNT) on the intestinal microbiota and expression of interleukin 17 (IL-17) in the intestine of mice. IFNT supplementation increased microbial diversity in the jejunum and ileum but decreased microbial diversity in the feces. IFNT supplementation influenced the composition of the intestinal microbiota as follows: (1) decreasing the percentage of Firmicutes and increasing Bacteroidetes in the jejunum and ileum; (2) enhancing the percentage of Firmicutes but decreasing Bacteroidetes in the colon and feces; (3) decreasing Lactobacillus in the jejunum and ileum; (4) increasing the percentage of Blautia, Bacteroides, Alloprevotella, and Lactobacillus in the colon; and (5) increasing the percentage of Lactobacillus, Bacteroides, and Allobaculum, while decreasing Blautia in the feces. Also, IFNT supplementation decreased the expression of IL-17 in the intestines of normal mice and of an intestinal pathogen infected mice. In conclusion, IFNT supplementation modulates the intestinal microbiota and intestinal IL-17 expression, indicating the applicability of IFNT to treat the intestinal diseases involving IL-17 expression and microbiota.

Proteome analysis for the global proteins in the jejunum tissues of enterotoxigenic Escherichia coli -infected piglets.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in humans and livestock. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) combined with multidimensional liquid chromatography (LC) and MS analysis was used for screening the differentially expressed proteins in piglet jejunum after ETEC infection. Totally 1,897 proteins were identified with quantitative information in piglet jejunum. We identified 92 differentially expressed proteins in ETEC-induced diarrhea, of which 30 were up regulated and 62 down regulated. Most of the differentially expressed proteins were involved in intestinal function of binding, metabolic process, catalytic activity and immune responses. The inhibition of intestinal immune responses in the jejunum in ETEC-induced diarrhea was also validated by immunobloting and RT-PCR. Our study is the first attempt to analyze the protein profile of ETEC-infected piglets by quantitative proteomics, and our findings could provide valuable information with respect to better understanding the host response to ETEC infection.

L-Cysteine metabolism and its nutritional implications.

L-Cysteine is a nutritionally semiessential amino acid and is present mainly in the form of L-cystine in the extracellular space. With the help of a transport system, extracellular L-cystine crosses the plasma membrane and is reduced to L-cysteine within cells by thioredoxin and reduced glutathione (GSH). Intracellular L-cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for production of GSH, hydrogen sulfide (H(2)S), and taurine. L-Cysteine-dependent synthesis of GSH has been investigated in many pathological conditions, while the pathway for L-cysteine metabolism to form H(2)S has received little attention with regard to prevention and treatment of disease in humans. The main objective of this review is to highlight the metabolic pathways of L-cysteine catabolism to GSH, H(2)S, and taurine, with special emphasis on therapeutic and nutritional use of L-cysteine to improve the health and well-being of animals and humans.