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Introduction: Rheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. Objectives: To evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. Methods: The effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. Results: We found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. Conclusion: Our results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

Abstract The aberrant regulation of PD‐L1 in tumor cells remains poorly understood. Here, the authors systematically investigate the endosomal trafficking of plasma membrane PD‐L1 in tumor cells. They show that plasma membrane PD‐L1 is continuously internalized, and then trafficked from early endosomes to multivesicular bodies/late endosomes, recycling endosomes, lysosomes, and/or extracellular vesicles (EVs). This constitutive endocytic trafficking of PD‐L1 is Rab5‐ and clathrin‐dependent. Triazine compound 6J1 blocks the endosomal trafficking of PD‐L1 and induces its accumulation in endocytic vesicles by activating Rab5. 6J1 also promotes exosomal PD‐L1 secretion by activating Rab27. Together, these effects result in a decrease in the membrane level of PD‐L1 in 6J1‐treated tumor cells and enables tumor cells to be more susceptible to the tumor‐killing activity of T cells in vitro. 6J1 also increases tumor‐infiltrating cytotoxic T cells and promotes chemokines secretion in the tumor microenvironment. Rab27 knockdown abolishes 6J1‐induced PD‐L1 secretion in EVs and revokes the exhausted tumor‐infiltrating T cells in tumors, thereby improving the anticancer efficacy of 6J1. Furthermore, a combination of 6J1 and an anti‐PD‐1 antibody significantly improves the anticancer immune response. Therefore, manipulating PD‐L1 endosomal trafficking provides a promising means to promote an anticancer immune response in addition to the immune checkpoint‐blocking antibody therapy.