Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3.

Purpose: Blood vessels distribute cells, oxygen, and nutrients throughout the body to support tissue growth and balance. Pericytes and endothelial cells form the inner wall of blood vessels, crucial for organ development and tissue homeostasis by producing paracrine signaling molecules. In the skeletal system, pericyte-derived vascular factors along with angiogenic factors released by bone cells regulate angiogenesis and bone formation. Although the involvement of angiogenic factors and skeletal blood vessels in bone homeostasis is relatively clear, the role of pericytes and the underlying mechanisms remain unknown. Here, our objective was to elucidate the significance of pericytes in regulating osteoclast differentiation. Methods: We used tissue staining to detect the coverage of pericytes and osteoclasts in femoral tissues of osteoporotic mice and mice of different ages, analyzing their correlation. We developed mice with conditionally deleted pericytes, observing changes in bone mass and osteoclast activity using micro-computer tomography and tissue staining to detect the regulatory effect of pericytes on osteoclasts. Pericytes-derived exosomes (PC-EVs) were collected and co-cultured with monocytes that induce osteoclast differentiation to detect the effect of the former on the exosomes. Finally, the specific mechanism of PC-EVs regulating osteoclast differentiation was verified using RNA sequencing and Western blotting. Results: Our study indicates a significant correlation between pericytes and age-related bone resorption. Conditional deletion of pericytes activated bone resorption and led to osteopenia in vivo. We discovered that PC-EVs inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which is mediated by tumor necrosis factor receptor-associated factor 3 (Traf3), negatively regulating osteoclast development and bone resorption. Silencing Traf3 in PC-EVs canceled their inhibitory effect on osteoclast differentiation. Conclusion: Our study provides a novel perspective into the regulatory role of pericytes on bone resorption and may provide potential strategies for developing novel anti-bone resorption therapies.

TRAF3/STAT6 axis regulates macrophage polarization and tumor progression.

Converting tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype is considered an effective strategy for cancer therapy. TRAF3 is known to regulate NF-κB signaling. However, the role of TRAF3 in TAM polarization has not yet been completely elucidated. Here, we found that ablation of TRAF3 increased M1 markers, iNOS, FGR and SLC4A7, while down-regulated M2 markers, CD206, CD36 and ABCC3, expression levels in macrophages. Moreover, TRAF3 deficiency enhanced LPS-induced M1 and abolished IL-4-induced macrophage polarization. Next, quantitative ubiquitomics assays demonstrated that among the quantitative 7618 ubiquitination modification sites on 2598 proteins, ubiquitination modification of IL-4 responding proteins was the most prominently reduced according to enrichment analysis. STAT6, a key factor of IL-4 responding protein, K450 and K129 residue ubiquitination levels were dramatically decreased in TRAF3-deficient macrophages. Ubiquitination assay and luciferase assay demonstrated that TRAF3 promotes STAT6 ubiquitination and transcriptional activity. Site mutation analysis revealed STAT6 K450 site ubiquitination played a vital role in TRAF3-mediated STAT6 activation. Finally, B16 melanoma mouse model demonstrated that myeloid TRAF3 deficiency suppressed tumor growth and lung metastasis in vivo. Taken together, TRAF3 plays a vital role in M2 polarization via regulating STAT6 K450 ubiquitination in macrophages.

Analysis of RANK-c interaction partners identifies TRAF3 as a critical regulator of breast cancer aggressiveness.

Breast cancer is a highly heterogeneous disease both at the histological and molecular levels. We have previously shown that RANK-c is a regulator of NF-κB signaling and exerts a suppressive effect on aggressive properties of ER negative breast cancer cells, while there is an opposite effect on ER positive cell lines. In order to identify molecular determinants that govern the opposing function of RANK-c in breast cancer cells we employed the two cell lines with the highest degree of phenotypic divergence upon RANK-c-expression (SKBR3 and BT474) and identified proteins that interact with RANK-c by affinity-enrichment mass spectrometry (AE-MS) analysis. Annotating enriched proteins with NF-κB signaling pathway revealed TRAF3 as an interacting partner of RANK-c in SKBR3 cell protein lysates, but not in BT474 breast cancer cells in which RANK-c induces cell aggressiveness. To determine the role of TRAF3 in the phenotype of BT474-RANK-c cells, we reconstructed the TRAF3/RANK-c interaction both in parental BT474 and RANK-c expressing cells and tested for aggressive properties through colony formation, migration and invasion assays. TRAF3 forced expression was able to reverse BT474 phenotypic changes imposed by RANK-c, rendering cells less aggressive. Finally, TRAF3 gene expression data and TRAF3 immunohistochemical (IHC) analysis on breast cancer samples indicated that TRAF3 expression correlates with Overall Survival (OS), Recurrence Free Survival (RFS) and several clinicopathological parameters (histological grade, proliferation index) of breast cancer disease.

[Anti-herpes simplex virus type â of tectorigenin derivative and effect on Toll-like receptors in vitro].

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 µg·mL~(-1) and 251.78 µg·mL~(-1), respectively, and TC_(50) was 1 749.98 µg·mL~(-1) and 2 977.50 µg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 µg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1ß(IL-1ß) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.

CircTRHDE knockdown protects WI-38 cells against LPS-induced inflammatory injury.

BACKGROUND: Circular RNAs (circRNAs) have been reported to be involved in the progression of infantile pneumonia. Here, we investigated the function of circTRHDE in lipopolysaccharide (LPS)-induced cell inflammatory injury to evaluate its role in infantile pneumonia progression. METHODS: The circTRHDE, microRNA (miR)-381-3p and TNF-receptor associated factor 3 (TRAF3) expression were detected by quantitative real-time PCR. LPS-induced WI-38 cells were used to construct an inflammatory injury model. Cell viability, inflammation and apoptosis were measured by cell counting kit assay, ELISA assay and flow cytometry. Caspase3 activity, MDA level and SOD activity were analysed to assess cell apoptosis and oxidative stress. Protein levels were determined using western blot analysis. The interaction between miR-381-3p and circTRHDE or TRAF3 was confirmed by dual-luciferase activity assay and RNA pull-down assay. RESULTS: CircTRHDE had increased expression in infantile pneumonia patients and LPS-induced WI-38 cells. LPS treatment inhibited WI-38 cell viability while promoting inflammation, apoptosis and oxidative stress. However, knockdown of circTRHDE remitted LPS-triggered WI-38 cell injury. CircTRHDE could sponge miR-381-3p to positively regulate TRAF3 expression. MiR-381-3p suppressed LPS-induced WI-38 cell inflammatory injury, and this effect was revoked by TRAF3 overexpression. Also, LPS-induced WI-38 cell inflammatory injury restrained by circTRHDE knockdown also were reversed by miR-318-3p inhibitor or TRAF3 overexpression. CONCLUSION: Our findings demonstrated that circTRHDE might be a target for infantile pneumonia treatment, which relieved LPS-induced cell inflammatory injury by the regulation of the miR-318-3p/TRAF3 axis.

Variations within Toll-like receptor (TLR) and TLR signaling pathway-related genes and their synergistic effects on the risk of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is the commonest post-infectious polyradiculopathy. Although genetic background of the host seems to play an important role in the susceptibility to GBS, genes conferring major risk are not yet known. Dysregulation of Toll-like receptor (TLR) molecules exacerbates immune-inflammatory responses and the genetic variations within TLR pathway-related genes contribute to differential risk to infection. The aim of this study was to delineate the impact of genetic variations within TLR2, TLR3, and TLR4 genes as well as TLR signaling pathway-related genes such as MyD88, TRIF, TRAF3, TRAF6, IRF3, NFκß1, and IκBα on risk of developing GBS. Fourteen polymorphisms located within TLR2 (rs3804099, rs111200466), TLR3 (rs3775290, rs3775291), TLR4 (rs1927911, rs11536891), MyD88 (rs7744, rs4988453), TRIF (rs8120), TRAF3 (rs12147254), TRAF6 (rs4755453), IRF3 (rs2304204), NFκß1 (rs28362491), and IκBα (rs696) genes were genotyped in 150 GBS patients and 150 healthy subjects either by PCR-RFLP or TaqMan Allelic Discrimination Assay. Genotypes of two polymorphic variants, Del/Del of rs111200466 insertion and deletion (INDEL) polymorphism of TLR2 gene and TT of rs3775290 single nucleotide polymorphism (SNP) of TLR3 gene had significantly higher frequencies among GBS patients, while the frequencies of TT genotype of rs3804099 SNP of TLR2 gene and TT genotype of rs11536891 SNP of TLR4 gene were significantly higher in controls. Gene-gene interaction study by Multifactor Dimensionality Reduction analysis also suggested a significant combined effect of TLR2, and NFκß1 genes on the risk of GBS. The SNPs in the IκBα and IRF3 genes correlated with severity of GBS. The genes encoding TLRs and TLR signaling pathway-related molecules could serve as crucial genetic markers of susceptibility and severity of GBS.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.