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.

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.

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.

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.

Chitosan Oligosaccharide Reduces Intestinal Inflammation That Involves Calcium-Sensing Receptor (CaSR) Activation in Lipopolysaccharide (LPS)-Challenged Piglets.

Chitosan oligosaccharide (COS) is a degradation product of chitosan with antioxidative, anti-inflammatory, and antibacterial effects. This study was conducted to investigate the effects of dietary COS on the intestinal inflammatory response and the calcium-sensing receptor (CaSR) and nuclear transcription factor kappa B (NF-κB) signaling pathways that may be involved using a lipopolysaccharide (LPS)-challenged piglet model. A total of 40 weaned piglets were used in a 2 × 2 factorial design; the main factors were dietary treatment (basal or 300 µg/kg COS) and inflammatory challenge (LPS or saline). On the morning of days 14 and 21 after the initiation of treatment, the piglets were injected intraperitoneally with Escherichia coli LPS at 60 and 80 µg/kg body weight or the same amount of sterilized saline, respectively. Blood and small intestine samples were collected on day 14 or 21, respectively. The results showed that piglets challenged with LPS have a significant decrease in average daily gain and gain:feed and histopathological injury in the jejunum and ileum, whereas dietary supplementation with COS significantly alleviated intestinal injury induced by LPS. Piglets fed the COS diet had lower serum concentrations of tumor necrosis factor alpha (TNF-α), interleukin (IL) 6, and IL-8 as well as lower intestinal abundances of pro-inflammatory cytokine mRNA but higher anti-inflammatory cytokine mRNA compared with piglets fed the basal diet among LPS-challenged piglets (p < 0.05). Dietary COS increased intestinal CaSR and PLCß2 protein expressions in both saline- and LPS-treated piglets, but decreased p-NF-κB p65, IKKα/ß, and IκB protein expressions in LPS-challenged piglets (p < 0.05). These findings indicate that COS has the potential to reduce the intestinal inflammatory response, which is concomitant with the activation of CaSR and the inhibition of NF-κB signaling pathways under an inflammatory stimulus.

Dietary arginine supplementation of mice alters the microbial population and activates intestinal innate immunity.

Currently, little is known about the function of arginine in the homeostasis of the intestinal immune system. This study was conducted to test the hypothesis that dietary arginine supplementation may alter intestinal microbiota and innate immunity in mice. Mice were fed a basal diet (containing 0.93% l-arginine; grams per gram) or the basal diet supplemented with 0.5% l-arginine for 14 d. We studied the composition of intestinal microbiota, the activation of innate immunity, and the expression of toll-like receptors (Tlrs), proinflammatory cytokines, and antimicrobials in the jejunum, ileum, or colon of mice. Signal transduction pathway activation in the jejunum and ileum, including TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide-3 kinase (PI3K)/PI3K-protein kinase B (Akt), was analyzed by Western blotting. Quantitative polymerase chain reaction analysis revealed that arginine supplementation induced (P < 0.05) a shift in the Firmicutes-to-Bacteroidetes ratio to favor Bacteroidetes in the jejunum (0.33 ± 0.04 vs. 1.0 ± 0.22) and ileum (0.20 ± 0.08 vs. 1.0 ± 0.27) compared with the control group. This finding coincided with greater (P < 0.05) activation of the innate immune system, including TLR signaling, as well as expression of proinflammatory cytokines, ​secretory immunoglobulin A, mucins, and Paneth antimicrobials in the jejunum and ileum. Finally, arginine supplementation reduced (P < 0.05) expression of the proteins for NF-κB, MAPK, and PI3K-Akt signaling pathways but activated (P < 0.05) p38 and c-Jun N-terminal protein kinase in the jejunum and the ileum, respectively. Collectively, dietary arginine supplementation of mice changes the intestinal microbiota, contributing to the activation of intestinal innate immunity through NF-κB, MAPK, and PI3K-phosphorylated Akt signaling pathways.

Serum amino acids profile and the beneficial effects of L-arginine or L-glutamine supplementation in dextran sulfate sodium colitis.

This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS)-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment). Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5%) or glutamine (0.5%, 1.0% and 2.0%) supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px)], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1ß), IL-6, IL-17 and tumor necrosis factor alpha (TNF-α)] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt), and myosin light chain kinase (MLCK)-myosin light chain (MLC20), were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (P<0.05). Dietary arginine or glutamine supplementation had significant (P<0.05) influence on the clinical and biochemical parameters (T-SOD, IL-17 and TNF-α) in colitis model. These results were associated with colonic NF-κB, PI3K-Akt and MLCK signaling pathways. In conclusion, arginine or glutamine could be a potential therapy for intestinal inflammatory diseases.