The aim of this study was to investigate the anti-inflammatory effect of IL-21 on LPS-induced mouse peritoneal macrophages. The results showed that IL-21 significantly inhibited LPS-induced mRNA expression of IL-1β, TNF-α, and IL-6 in macrophages, but not of IFN-γ, IL-10, CCL5, or CXCL2. ELISA analysis showed that IL-21 also suppressed LPS-induced production of TNF-αand IL-6 in culture supernatants. Western blot analysis showed that IL-21 clearly inhibited ERK and IκBαphosphorylation and NF-κB translocation in LPS-stimulated macrophages, but it increased STAT3 phosphorylation. Flow cytometric and Western blot analysis showed that IL-21 decreased M1 macrophages surface markers expression of CD86, iNOS, and TLR4 in LPS-stimulated cells. All results suggested that IL-21 decreases IL-6 and TNF-αproduction via inhibiting the phosphorylation of ERK and translocation of NF-κB and promotes a shift from the M1 to M2 macrophage phenotype by decreasing the expression of CD86, iNOS, and TLR4 and by increasing STAT3 phosphorylation in LPS-stimulated cells.

The rise of antibiotic resistance poses a significant public health crisis, particularly due to limited antimicrobial options for the treatment of infections with Gram‐negative pathogens. Here, an antimicrobial peptide (AMP) SR25 is characterized, which effectively kills both Gram‐negative and Gram‐positive bacteria through a unique dual‐targeting mechanism without detectable resistance. Meanwhile, an SR25‐functionalized hydrogel is developed for the efficient treatment of infected diabetic wounds. SR25 is obtained through genome mining from an uncultured bovine enteric actinomycete named Nonomuraea Jilinensis sp. nov. Investigations reveal that SR25 has two independent cellular targets, disrupting bacterial membrane integrity and restraining the activity of succinate:quinone oxidoreductase (SQR). In a diabetic mice wound infection model, the SR25‐incorporated hydrogel exhibits high efficacy against mixed infections of Escherichia coli (E. coli) and methicillin‐resistant Staphylococcus aureus (MRSA), accelerating wound healing. Overall, these findings demonstrate the therapeutic potential of SR25 and highlight the value of mining drugs with multiple mechanisms from uncultured animal commensals for combating challenging bacterial pathogens.

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes. High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes, Blautia, Coprococcus_1, and Ruminococcus_1. Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes, indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Obesity is one of the prevalent chronic diseases in human and companion animals usually associated with several metabolic disorders. The gut commensal bacterium Akkermansia muciniphila (A. muciniphila) is known for its therapeutic effects on metabolic disorders and inflammations. Here, we isolated the A. muciniphila AKK2 strain from the feces of interferon-inducible protein 204^−/− (IFI204^−/−) mice and further evaluated its anti-obesity effects on high-fat diet (HFD)-fed C57BL/6J mice and beagles. The results showed that it effectively controlled weight gain. Microbiome analysis using 16S rRNA gene sequencing revealed that HFD alters gut microbiota composition and A. muciniphila AKK2 increases the Firmicutes/Bacteroidetes (F/B) ratio in beagles. Furthermore, we prepared microcapsules containing A. muciniphila AKK2, and tolerance tests showed the encapsulation maintained high viability and stability in an aerobic environment and simulated the secretion of gastrointestinal fluids. Overall, this study widens the spectrum of A. muciniphila applications to prevent obesity.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.