The protective effect of Wnt3a on inflammatory response in oxygen-glucose deprivation/reoxygenation (OGD/R) astrocyte model.

Involving in the immune response after cerebral infarction, astrocytes could secrete large amounts of pro- and anti-inflammatory factors. The aim of this study is to investigate the effect of Wnt3a intervention on the inflammatory response of oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R) astrocyte model, and to provide a new target for immunoprotective treatment of cerebral infarction. We constructed the OGD/R rat astrocyte model, the astrocytes were treated by different concentrations of glucose (25, 50, 100 mM) intervened with/without Wnt3a (25 µg/ml). Microscope was used to observe the cell survival in rat astrocytes. The relative expression of inflammatory factors (TNF-a, IL-6, HIF-a) in rat astrocytes was detected by qRT-PCR. The expression of inflammatory factors such as TNF-a, IL-6 and HIF-a in rat astrocytes was increased after OGD/R treatment. The Wnt3a intervention promoted cell survival and decreased the expression of inflammatory factors in rat astrocytes induced by OGD/R. There is a neuroprotective effect that Wnt3a intervention could reduce inflammatory response in the OGD/R rat astrocyte model.

TRIM11 attenuates Treg cell differentiation by p62-selective autophagic degradation of AIM2.

Ubiquitination is an important protein modification that regulates diverse biological processes, including CD4+ T cell differentiation and functions. However, the function of most E3 ubiquitin ligases in CD4+ T cell differentiation and CD4+ T cell-mediated pathological diseases remains unclear. In this study, we find that tripartite motif-containing motif 11 (TRIM11) specifically negatively regulates regulatory T (Treg) cell differentiation in CD4+ T cells and promotes autoimmune disease development in an AIM2-dependent manner. Mechanistically, TRIM11 interacts with absent in melanoma 2 (AIM2) and promotes the selective autophagic degradation of AIM2 by inducing AIM2 ubiquitination and binding to p62 in CD4+ T cells. AIM2 attenuates AKT and FOXO1 phosphorylation, MYC signaling, and glycolysis, thereby promoting the stability of Treg cells during experimental autoimmune encephalomyelitis (EAE). Our findings suggest that TRIM11 serves as a potential target for immunotherapeutic intervention for dysregulated immune responses that lead to autoimmunity and cancers.

Egr-1 transactivates WNT5A gene expression to inhibit glucose-induced ß-cell proliferation.

Selective ß-cell loss is a characteristic of type 2 diabetes mellitus (T2DM). Inhibition of glucose-stimulated ß-cell proliferation is one of the in vivo results of the lipotoxicity of saturated fatty acids (SFAs). However, the mechanism by which lipotoxicity inhibits ß-cell proliferation is still unclear. In this study, we found palmitate, a saturated fatty acid, inhibited the ß-cell proliferation induced by high glucose through the induction of Wnt5a expression in vitro and in vivo. We also found that Wnt5a was both sufficient and necessary for inhibition of ß-cell proliferation. Additionally, Egr-1, but not NF-κB, FOXO1, Smad2, Smad3, SP1 or SP3 mediated the expression of Wnt5a. Deletion and site-directed mutagenesis of the WNT5A promoter revealed that activation of WNT5A gene transcription depends primarily on a putative Egr-binding sequence between nucleotides -52 to -44, upstream of the transcription start site. Furthermore, Egr-1 bound directly to this sequence in response to palmitate treatment, both in vitro and in vivo. Moreover, after mice islets were treated with Egr inhibitors, the expression of Wnt5a decreased significantly and the glucose-induced ß-cell proliferation inhibited by palmitate was resumed. These findings establish Wnt5a as an Egr-1 target gene in ß-cells, uncovering a novel Egr-1/Wnt5a pathway by which saturated free fatty acids block glucose-induced ß-cell proliferation. Our study lends support for the potential of Egr-1 inhibitors or Wnt5a antibodies as therapeutics for the treatment of T2DM.

Stimulator of Interferon Genes Promotes Host Resistance Against Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa (PA) is the leading cause of bacterial keratitis, especially in those who wear contact lens and who are immunocompromised. Once the invading pathogens are recognized by pattern recognition receptors expressed on the innate immune cells, the innate immune response is stimulated to exert host defense function, which is the first line to fight against PA infection. As a converging point of cytosolic DNA sense signaling, stimulator of interferon genes (STING) was reported to participate in host-pathogen interaction. However, the role of STING in regulating PA-induced corneal inflammation and bacterial clearance remains unknown. Our data demonstrated that STING was activated in murine model of PA keratitis and in in vitro-cultured macrophages, indicated by Western blot, immunostaining, and flow cytometry. To explore the role of STING in PA keratitis, we used siRNA to silence STING and 2',3'-cGAMP to activate STING in vivo and in vitro, and the in vivo data found out that STING promoted host resistance against PA infection. To investigate the reason why STING played a protective role in PA keratitis, the inflammatory cytokine secretion and bacterial load were measured by using real-time PCR and bacterial plate count, respectively. Our data demonstrated that STING suppressed the production of inflammatory cytokines and enhanced bacterial elimination in murine model of PA keratitis and in PA-infected macrophages. To further investigate the mechanism beneath, the phosphorylation of mitogen-activated protein kinase, the nuclear translocation of nuclear factor-κB (NF-κB) and the bactericidal mechanism were measured by western-blot, immunofluorescence, and real-time PCR, respectively. Our data indicated that STING suppressed inflammatory cytokine expressing via restraining NF-κB activity and enhanced inducible NO synthase expression, an oxygen-dependent bactericidal mechanism. In conclusion, this study demonstrated that STING promoted host resistance against PA keratitis and played a protective role in PA-infected corneal disease, via inhibiting corneal inflammation and enhancing bacterial killing.

ß-catenin promotes intracellular bacterial killing via suppression of Pseudomonas aeruginosa-triggered macrophage autophagy.

Objective To investigate ß-catenin-mediated bacterial elimination during Pseudomonas aeruginosa infection of macrophage-like RAW264.7 cells. Methods Cell viability and catenin beta 1 ( CTNNB1) expression in RAW264.7 cells following P. aeruginosa infection versus uninfected cells, were detected by cell counting kit-8 assay and ß-catenin Western blots. RAW264.7 cells with CTNNB1 overexpression were established with ß-catenin lentivirus using flow cytometry and clonogenic limiting dilution assays. Bacterial killing was measured by plate counts; phagocytosis and nitric oxide (NO) were measured by flow cytometry; and reactive oxygen species (ROS) were measured using Griess reaction. Autophagy was determined by microtubule-associated protein 1 light chain 3 alpha-phosphatidylethanolamine conjugate (LC3-II) protein levels and formation of LC3 puncta, using Western blot and immunofluorescence staining. Results Following P. aeruginosa infection, RAW264.7 cell ß-catenin levels were reduced in a time- and multiplicity of infection-dependent manner. CTNNB1 overexpression was associated with increased P. aeruginosa elimination, but had no effect on RAW264.7 cell phagocytosis, ROS and NO. CTNNB1 overexpression reduced LC3-II levels and formation of LC3 puncta, suggesting autophagy inhibition. Rapamycin/starvation-induced autophagy resulted in reduced bacterial killing following P. aeruginosa infection. Conclusion ß-catenin may promote bacterial killing via suppression of P. aeruginosa-induced macrophage autophagy.

Exendin-4 promotes pancreatic ß-cell proliferation via inhibiting the expression of Wnt5a.

Exendin-4, a glucagon-like peptide-1 receptor agonist, is currently regarded as an effective therapeutic strategy for type-2 diabetes. Previous studies indicated that exendin-4 promoted ß cell proliferation. However, the underlying mechanisms remain largely unknown. Recently it was reported that exendin-4 promoted pancreatic ß cell proliferation by regulating the expression level of Wnt4. The present study was designed to investigate whether other Wnt isoforms take part in accommodation of ß-cell proliferation. We found that exendin-4 promotes the proliferation and suppresses the expression of Wnt5a in INS-1 cell line and C57Bl/6 mouse pancreatic ß-cells. Further mechanistic study demonstrated that exendin-4 promoted INS-1 cell proliferation partly through down-regulating the expression of Wnt5a. Furthermore, Wnt5a could induce the activation of calmodulin-dependent protein kinase II in INS-1 cells, thereby decreasing the cellular stable ß-catenin and its nuclear translocation, and finally reduce the expression of cyclin D1. In addition, we also found that both of the receptors (Frz-2 and Ror-2) mediated the effect of Wnt5a on ß cell line INS-1 proliferation. Taken together, this study suggests that Wnt5a plays a critical role in exendin-4-induced ß-cell proliferation, indicating that Wnt5a might be a novel regulator in counterbalance of ß cell mass.