In vitro induced human regulatory T cells (iTregs) have in vivo therapeutic utility. MicroRNAs (miRNAs) are a family of approximately 22-nucleotide non-coding RNAs that are processed from longer precursors by the RNases Drosha and Dicer. miRNAs regulate post-transcriptional protein expression through messenger RNA destabilization or translational silencing; miR-142-3p regulates natural Treg function through autophagy. We hypothesized that this miRNA may also have an iTreg regulation function. Antagomir-mediated knockdown of miR-142-3p improved Foxp3 (forkhead box P3) expression, regulatory function, cytokine expression, and apoptosis of iTregs in vitro, with or without inflammatory cytokine stimulation. miR-142-3p knockdown increased autophagy-related protein 16-1-mediated autophagy. Target prediction and luciferase assay results indicated that miR-142-3p binds directly to lysine demethylase 6A (KDM6A), which resulted in demethylation of H3K27me3 and in turn upregulated expression of the anti-apoptotic protein Bcl-2. Based on these results, we propose a novel strategy that uses knockdown of miR-142-3p to enhance anti-apoptotic ability and function of iTregs by increasing KDM6A and Bcl-2 expression. This approach might be used as a treatment to control established chronic immune-mediated autoimmune and inflammatory diseases.

Although diabetes mellitus/hyperglycemia is a risk factor for acute liver injury, the underlying mechanism remains largely unknown. Liver-resident macrophages (Kupffer cells, KCs) and oxidative stress play critical roles in the pathogenesis of toxin-induced liver injury. Here, we evaluated the role of oxidative stress in regulating KC polarization against acetaminophen (APAP)-mediated acute liver injury in a streptozotocin-induced hyperglycemic murine model. Compared to the controls, hyperglycemic mice exhibited a significant increase in liver injury and intrahepatic inflammation. KCs obtained from hyperglycemic mice secreted higher levels of the proinflammatory factors, such as TNF-α and IL-6, lower levels of the anti-inflammatory factor IL-10. Furthermore, enhanced oxidative stress was revealed by increased levels of reactive oxygen species (ROS) in KCs from hyperglycemic mice post APAP treatment. In addition, ROS inhibitor NAC resulted in a significant decrease of ROS production in hyperglycemic KCs from mice posttreated with APAP. We also analyzed the role of hyperglycemia in macrophage M1/M2 polarization. Interestingly, we found that hyperglycemia promoted M1 polarization, but inhibited M2 polarization of KCs obtained from APAP-exposed livers, as evidenced by increased MCP-1 and inducible NO synthase (iNOS) gene induction but decreased Arg-1 and CD206 gene induction accompanied by increased STAT1 activation and decreased STAT6 activation. NAC restored Arg-1, CD206 gene induction, and STAT6 activation. To explore the mechanism how hyperglycemia regulates KCs polarization against APAP-induced acute liver injury, we examined the AMPK/PI3K/AKT signaling pathway and found decreased AMPK activation and increased AKT activation in liver and KCs from hyperglycemic mice post APAP treatment. AMPK activation by its agonist AICAR or PI3K inhibition by its antagonist LY294002 inhibited ROS production in KCs from hyperglycemic mice post APAP treatment and significantly attenuated APAP-induced liver injury in the hyperglycemic mice, compared to the control mice. Our results demonstrated that hyperglycemia exacerbated APAP-induced acute liver injury by promoting liver-resident macrophage proinflammatory response via AMPK/PI3K/AKT-mediated oxidative stress.

Background: Cancer-associated fibroblasts (CAFs) are a major component of hepatocellular carcinoma (HCC) stroma that are critically involved in HCC cancer chemoresistance, but the mechanism has not been elucidated. Previous studies have reported CD73 exerted an immunosuppressive function in cancer. Here, we explored the mechanism by which CAFs regulates CD73 + HCC cells and clarified whether CAFs promote chemoresistance of CD73 + cells.Methods: We used the co-culture method to study the relationship between CAFs and HCC cells.Immunohistochemistry was applied to evaluate the correlation between α-smooth-muscle actin (α-SMA) and CD73. CD73 mRNA and protein were determined by real-time polymerase chain reaction (RT-PCR) and western blotting, and hepatocyte growth factor (HGF) was assayed by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to explore the regulated pathway of CD73 + HCC. We then knocked down CD73 in cells, and then assessed the effect of CD73 on the apoptosis by flow cytometry. Finally, a sphere formation assay was applied to investigate the stemness of cancer cells, and xenograft tumors in nude mice were built to investigate the tumorigenicity.Results: We found that the proportion of CAFs was positively correlated with CD73 expression in HCC cells. Mechanistically, c-Met and the MEK-ERK1/2 pathway were activated by HGF from CAFs which upregulated CD73 expression in HCC cells. Also, we found that CD73 promote sorafenib and cisplatin resistance in HCC, and CD73 + HCC cells indicated the higher capability of tumorigenicity compared to CD73 − HCC cells in vivo. Furthermore, HGF further enhanced the chemoresistant characteristics of CD73 + tumor cells.Conclusions: Our findings collectively suggest that CD73 is a vital HCC-chemoresistance force controlled by cross-talking between CAFs and HCC cells, thereby establishing CD73 as a potential new therapeutic target for HCC.