Toll-like receptor signaling pathway involved in pathogenesis of thromboangiitis obliterans through activating of NF-κB

Abstract Objectives The pathogenic mechanisms of Thromboangiitis Obliterans (TAO) are not entirely known and autoimmune inflammation plays a vital role in the initiation and continuance of TAO activity. The authors investigated in this study the role of the TLR signaling pathway in the pathogenesis of TAO. Methods First, the authors detected the expressions of MyD88, TRIF and NF-κB in vascular walls of 46 patients with TAO and 32 patients with trauma and osteosarcoma by western blot assay. Second, the authors detected the cellular localization of MyD88, TRIF and NF-κB in vascular walls of patients with TAO by immunofluorescent assay. Results The protein expressions of MyD88, TRIF and NF-κB were much higher in vascular walls of TAO patients (p < 0.05). Higher expressions of MyD88 and NF-κB were detected both on vascular endothelial and vascular smooth muscle cells of TAO patients. However, higher expression of TRIF was just detected on vascular smooth muscle cells of TAO patients. Conclusions These dates suggest that the TLR signaling pathway might play an important role in the pathogenesis of TAO, it might induce vasospasm, vasculitis and thrombogenesis to lead to the pathogenesis and progression of TAO.

Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Toll-like receptor 3 (TLR3) is a member of the TLR family, mediating the transcriptional induction of type I interferons (IFNs), proinflammatory cytokines, and chemokines, thereby collectively establishing an antiviral host response. Studies have shown that unlike other TLR family members, TLR3 is the only RNA sensor that is utterly dependent on the Toll-interleukin-1 receptor (TIR)‍-domain-containing adaptor-inducing IFN-‍β (TRIF). However, the details of how the TLR3-TRIF signaling pathway works in an antiviral response and how it is regulated are unclear. In this review, we focus on recent advances in understanding the antiviral mechanism of the TRIF pathway and describe the essential characteristics of TLR3 and its antiviral effects. Advancing our understanding of TLR3 may contribute to disease diagnosis and could foster the development of novel treatments for viral diseases.

Cardamine komarovii flower extract reduces lipopolysaccharide-induced acute lung injury by inhibiting MyD88/TRIF signaling pathways / 中国天然药物

In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.

MRTF-A alleviates myocardial ischemia reperfusion injury via inhibiting TLR4/TRIF signaling pathways / 中华急诊医学杂志

Objective To observe the effect of myocardial transcription factor MRTF-A on myocardium inflammation and its mechanism.Methods Totally 30 rats were randomly divided into the sham,ischemia-reperfusion (myocardial ischemia 30 min and reperfusion 2 h),and MRTF-A groups(myocardial ischemia 30 min and reperfusion 2 h & Lentivirus infection MRTF-A) (n=10 each group).Serum myocardial enzyme activity was detected by biochemical analysis,myocardial infarct size detected by TTC,and degree of myocardial injury was measured by HE staining.The TLR4 and TRIF expression was analyzed by immunohistochemistry and qPCR.Results Compared with the sham group,the MRTF-A group significantly increased the activity of serum myocardial enzymes CK-MB and LDH (P＜0.05).The infarct area of myocardial tissue was gray-white,and the infarct area was (54.31±3.07)％ (P ＜ 0.05).Myocardial fibrosis was disorder,myocardial cell was swollen and burst,and inflammatory cell infiltration was obvious.Protein and mRNA expressions of TRL4 and TRIF were significantly up-regulated (P＜0.05).Compared with the ischemia-reperfusion group,the levels of CK-MB and LDH were significantly reduced after myocardial infection with MRTF-A (P＜0.05).The myocardial infarction area was significantly reduced to (16.74±4.26)％ (P＜ 0.05).The myocardial structure was nearly normal with mild edema.Protein and mRNA expression of TRL4 and TRIF decreased significantly (P＜0.05).Conclusions The overexpression of transcription factor MRTF-A in myocardial cells alleviates the myocardial ischemia reperfusion injury by inhibiting the TLR4/TRIF signaling pathway and reducing the serum myocardial enzyme activity and myocardial damage.

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells. MATERIALS AND METHODS: First, siRNA targeted autophagy–related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo. RESULTS: Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p < 0.05). LC3 and TRIF colocation was observed in HepG2 cells. Decreased cell viability, increased apoptosis, decrease in xenograft tumor volume, and angiogenesis potential were also observed in order (p < 0.05). CONCLUSION: Suppression of intracellular autophagy resulted in decreased degradation of TRIF protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.

Roles of MyD88 and TRIF in cardiac dysfunction during sepsis / 中华危重病急救医学

Objective To investigate the roles of myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing interferon-β (TRIF) in sepsis-induced myocardial dysfunction, and to analyze whether strain rate (SR) can be early sensitive evaluation for septic heart failure.Methods Sixty-four healthy male C57BL/6 mice were divided into four groups by random number table (n = 16 in each group): sham group, cecum ligation and puncture (CLP)-induced sepsis model group, anti-MyD88 group and anti-TRIF group. The anti-MyD88 group and anti-TRIF group were injected with 5μL/g of anti-MyD88 antibody or anti-TRIF antibody through the tail veins 2 hours before CLP. Eight animals in each group were used to observe the survival of 24 hours, and the other 8 myocardial tissues were harvested for examination. The cardiac function was evaluated by echocardiography before and 6 hours and 12 hours after operation. The mRNA expressions of MyD88, TRIF and inflammatory factors in myocardium were measured by polymerase chain reaction (PCR) at 24 hours after operation, and the degree of neutrophils infiltration was detected by myeloperoxidase (MPO) activity.Results The number of 24-hour survive in anti-MyD88 group and anti-TRIF group were higher than that in CLP group (number: 4, 3 vs. 2,P = 0.044,P = 0.047). Compared with sham group, the cardiac function was significantly decreased, the mRNA expressions of myocardial tissues MyD88, TRIF, interleukin (IL-1, IL-6) and tumor necrosis factor-α (TNF-α) were significantly increased, and the infiltration of neutrophils were obvious in CLP group. Compared with CLP group, the left ventricular short axis fractional shortening rate (FS) and SR were significantly increased after 12 hours in anti-MyD88 group and anti-TRIF group [FS: (49.52±1.78)%, (49.89±1.49)%vs. (41.11±1.63)%, SR (s-1): 17.63±2.16, 17.85±1.64 vs. 12.55±1.84]; the mRNA expressions of MyD88, TRIF and inflammatory factors were significantly decreased [MyD88 mRNA (A value): 0.463±0.046, 0.505±0.048 vs. 0.638±0.102, TRIF mRNA (A value): 0.413±0.031, 0.410±0.021 vs. 0.625±0.057, IL-1 mRNA (A value):0.569±0.101, 0.570±0.091 vs. 0.946±0.171, IL-6 mRNA (A value): 0.551±0.143, 0.431±0.157 vs. 0.850±0.194, TNF-α mRNA (A value): 0.471±0.082, 0.444±0.093 vs. 0.707±0.094]; and the infiltration of neutrophils were significantly decreased [MPO (U/L): 62.34±2.60, 60.87±2.40 vs. 73.83±4.90], with statistically significant differences (allP 0.05).Conclusions Blocking MyD88 and TRIF expression play significant and similar roles in protecting cardiac deterioration from sepsis by attenuating cytokine release, reducing neutrophil infiltration. SR can sensitively assess septic cardiac dysfunction.

The regulation of TLR3 on NF-κB signaling pathway and its significance in the pathogenesis of tumor / 实用肿瘤学杂志

NF-κBs is a member of Rel protein family of transcription factors ,which is expressed in all cells and is involved in important physiological and pathological regulatory mechanisms ,including the regulation of TLR3 on NF-κB signaling pathway ,which has been attracted widespread attention .This paper reviews the mo-lecular mechanisms of the regulation .We summarizes the role of host immune responses in tumor development , which is mediated by the related signal molecules .It reveals that some key proteins in the regulation have a great significance to the treatment of diseases ,as used as the therapeutic targets ,and is conducive to the understanding of function and diversity in tumor tissue .

TRIF Deficiency does not Affect Severity of Ovalbumin-induced Airway Inflammation in Mice

Allergic asthma is a chronic pulmonary inflammatory disease characterized by reversible airway obstruction, hyperresponsiveness and eosinophils infiltration. Toll-like receptors (TLRs) signaling are closely associated with asthma and have emerged as a novel therapeutic target in allergic disease. The functions of TLR3 and TLR4 in allergic airway inflammation have been studied; however, the precise role of TIR-domain-containing adapter-inducing interferon-beta (TRIF), the adaptor molecule for both TLR3 and TLR4, is not yet fully understood. To investigate this, we developed a mouse model of OVA-induced allergic airway inflammation and compared the severity of allergic airway inflammation in WT and TRIF-/- mice. Histopathological assessment revealed that the severity of inflammation in airway inflammation in TRIF-deficient mice was comparable to that in WT mice. The total number of cells recovered from bronchoalveolar lavage fluid did not differ between WT and TRIF-deficient mice. Moreover, TRIF deficiency did not affect Th1 and Th2 cytokine production in lung tissue nor the level of serum OVA-specific IgE, IgG1 and IgG2c. These findings suggest that TRIF-mediated signaling may not be critical for the development of allergic airway inflammation.

Effects of MyD88 and Trif on production of donor-specific antibodies and memory T cells in mouse cardiac transplant model / 中华器官移植杂志

Objective To determine the roles of MyD88 and Trif,critical adaptor proteins for TLR signaling,in production of donor-specific antibodies (DSA) and memory T cells in a presensitized mouse cardiac transplant model.Methods Skin grafts from Balb/c mice were transplanted into either wild type B6 mice or B6 Myd88 and Trif double knockout mice (Myd88/Trif DKO).The recipients were subsequently transplanted heterotopically with cardiac grafts from the same donors two weeks after skin transplantation.Plasma DSA levels and spleen phenotypical analysis were performed prior to heart transplant or at time of cardiac rejection by using flow cytometry.Results Recipients presensitized with skin grafts developed accelerated cardiac allograft rejection in the absence of Myd88 and Trif.However,plasma DSA,especially IgG2,was significantly decreased (P＜0.05) in Myd88/Trif DKO mice,compared to that in Wild Type mice at 2nd week after skin transplantation.The production of DSAs including all IgG subtypes was further reduced 3 days following heart transplantation in the Myd88/Trif DKO.In addition,MyD88/Trif DKO mice had impaired ability to generate memory T cells,as percentages of both CD44hi CD4+ and CD44hi CD8+ were significantly lower in the DKO than in Wild Type mice (P＜0.01,P＜0.05).Conclusion Simultaneous ablation of MyD88 and Trif in recipients significantly decreases the production of serum DSAs and spleen memory T cells following allogeneic skin and heart transplantation,supporting a crucial role of TLR signaling in adaptive immune responses in organ transplantation.

Relationship of the Arl8a with TLR4-TRIF pathway in dendritic cells / 中华微生物学和免疫学杂志

Objective To explore the relationship of the ADP-ribosylation factor-like 8A (Arl8a)with TLR4-TRIF-GEFH1 -RhoB pathway in dendritic cells(DCs). Methods DCs were prepared from wildtype and TRIF-knockout (TRIFKO) mice. After LPS stimulation, the cells were collected for cDNA amplification. Real-time PCR method was used to detect Arl8a mRNA levels. DCs from wild-type mice were transfected with guanine nucleotide-exchange factors H1 ( GEFH1 ) small interference RNA ( siRNA ), Arl8a mRNA levels were examined with or without LPS stimulation. Then RhoB mRNA expression was analyzed in DCs transfected with the siRNA of GEFH1 and Arl8a gene, respectively. Results LPS induced the up-regulation of Arl8a mRNA in DCs from control mice but not in DCs from TRIFKO, indicating that LPS-mediated up-regulation of Arl8a was suppressed in TRIFKO DCs. In addition, siRNA of GEFH1 significantly suppressed the LPS-mediated up-regulation of Arl8a mRNA, RNAi of Arl8a and GEFH1 significantly decreased RhoB mRNA level in DCs after LPS stimulation ( P ＜ 0. 01 ). Conclusion The expression of Arl8a is involved in the TLR4-TRIF pathway in DCs, and Arl8a is closely associated with GEFH1 and RhoB at transcriptional level.