Protease-activated receptor 2 (PAR2) alleviates intestinal inflammation by upregulating autophagy. PAR2 also modulates tight junctions through β‑arrestin signaling. Therefore, we investigated the effect of PAR2-induced autophagy on intestinal epithelial tight junctions and permeability. RT-PCR, Western blot analysis, and immunoprecipitation were performed to investigate the underlying molecular mechanisms by which PAR2 regulates autophagy and intestinal epithelial tight junctions. Inhibition of PAR2 by GB83, a PAR2 antagonist, decreased the expression of autophagy-related and tight-junction-related factors in Caco-2 cells. Moreover, inhibition of PAR2 decreased intestinal transepithelial electrical resistance. When PAR2 was activated, intestinal permeability was maintained, but when autophagy was suppressed by chloroquine, intestinal permeability was significantly increased. In addition, the prolongation of ERK1/2 phosphorylation by PAR2–ERK1/2–β-arrestin assembly was reduced under autophagy inhibition conditions. Therefore, PAR2 induces autophagy to regulate intestinal epithelial permeability, suggesting that it is related to the β-arrestin–ERK1/2 pathway. In conclusion, regulating intestinal epithelial permeability through PAR2-induced autophagy can help maintain mucosal barrier integrity. Therefore, these findings suggest that the regulation of PAR2 can be a suitable strategy to treat intestinal diseases caused by permeability dysfunction.

Lactobacillus plantarum (L. plantarum) is a probiotic that has emerged as novel therapeutic agents for managing various diseases, such as cancer, atopic dermatitis, inflammatory bowel disease, and infections. In this study, we investigated the potential mechanisms underlying the anticancer effect of the metabolites of L. plantarum. We cultured L. plantarum cells to obtain their metabolites, created several dilutions, and used these solutions to treat human colonic Caco-2 cells. Our results showed a 10% dilution of L. plantarum metabolites decreased cell viability and reduced the expression of autophagy-related proteins. Moreover, we found co-treatment with L. plantarum metabolites and chloroquine, a known autophagy inhibitor, had a synergistic effect on cytotoxicity and downregulation of autophagy-related protein expression. In conclusion, we showed the metabolites from the probiotic, L. plantarum, work synergistically with chloroquine in killing Caco-2 cells and downregulating the expression of autophagy-related proteins, suggesting the involvement of autophagy, rather than apoptosis, in their cytotoxic effect. Hence, this study provides new insights into new therapeutic methods via inhibiting autophagy.

Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl‐phosphorylcholine into lysophosphatidic acid and sphingosine 1‐phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll‐like receptor 4 (TLR4) and its co‐receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4‐mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe‐induced intestinal inflammation. To study this hypothesis, we generated myeloid cell lineage‐restricted Atx‐knockout (ko) mice (Atx‐dME/dME) to investigate TLR4‐mediated immune and inflammatory responses and examined LPS‐receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Phagocytic activity of macrophages was assessed. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using Atx‐dME/dME;Il10‐/‐ mice that have both Atx‐deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients. With peritoneal macrophages from Atx‐dME/dME mice, we identified that Atx‐ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4‐mediated responses were substantially reduced in Atx‐ko macrophages. TLR4‐induced innate immunity such as phagocytosis was attenuated in Atx‐ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of Atx‐dME/dME mice compared to that of Atx‐wt littermates. Consequently, Atx‐dME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10‐/‐ mice, combining Atx‐dME/dME with Il10‐/‐ mice (Atx‐dME/dME;Il10‐/‐) did accelerate spontaneous colitis development. Atx‐dME/dME;Il10‐/‐ mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log‐rank P=0.0046), while Atx‐wt;Il10‐/‐ littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with intestinal inflammation. Collectively, we found that Atx deficiency suppresses TLR4‐mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe‐induced gut inflammation.