髓样分化因子88对姜黄素诱导肝星状细胞凋亡的影响

目的观察髓样分化因子88在姜黄素促进肝星状细胞(HSC)凋亡中的作用。方法体外培养大鼠肝星状细胞株HSCT6,并分为空白对照组、Control siRNA组、MyD88 siRNA干扰组、姜黄素组、姜黄素+Control siRNA组、姜黄素+MyD88 siRNA干扰组,siRNA处理组给予siRNA干扰48 h后,姜黄素组加入姜黄素作用24 h,各组均在收集细胞前12 h给予LPS诱导,收集各组细胞,Western blotting法检测MyD88蛋白表达;流式细胞术检测细胞凋亡。结果 MyD88 siRNA干扰、姜黄素均可降低MyD88蛋白的表达(P0.05),同时给予MyD88 siRNA干扰和姜黄素作用时与单独给予姜黄素比较,MyD88蛋白下降更明显(P0.05)。MyD88siRNA干扰后HSCs凋亡率无明显增加(P0.05),给予姜黄素处理HSCs凋亡率增加(P0.05),且姜黄素+MyD88 siRNA干扰组的凋亡率升高更明显。结论降低MyD88表达可加强姜黄素促进HSCs凋亡的作用。     

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

Background: In a previous report, it was shown that Toll‐like receptor (TLR) 2 knockdown modulates interleukin (IL)‐6 and IL‐8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF‐related adaptor molecule (TRAM), could affect mRNA expression of IL‐6, IL‐8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA‐mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL‐6, IL‐8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL‐8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL‐6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non‐stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL‐6 and IL‐8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.     

Role of myeloid differentiation factor 88 in Rhesus rotavirus-induced biliary atresia

Background

Biliary atresia (BA) is a unique neonatal disease resulting from inflammatory and fibrosing obstruction of the extrahepatic biliary tree. Previous studies have demonstrated the critical role of innate immunity and the Th1 response to activated inflammatory cells and overexpressed cytokines in the pathogenesis of BA. Myeloid differentiation factor 88 (MyD88) is a critical adaptor molecule that has been shown to play a crucial role in immunity. We investigated the role of MyD88 in the inflammatory response and development of cholangiopathy in murine BA.

Methods

MyD88 knockout (MyD88^−/−) and wild-type (WT) BALB/c pups were injected with Rhesus rotavirus or saline on day 1 of life. The mice were monitored for clinical symptoms of BA, including jaundice, acholic stools, bilirubinuria, and death. The liver and extrahepatic bile ducts were harvested for histologic evaluation and the quantification of viral content, determination of cytokine expression, and detection of inflammatory cells.

Results

Rhesus rotavirus infection produced symptoms in 100% of both MyD88^−/− and WT pups, with survival of 18% of WT and 0% of MyD88^−/− mice. Histologic analysis demonstrated bile duct obstruction in both MyD88^−/− and WT mice. Viral titers obtained 7 d after infection and expression of interferon-γ and tumor necrosis factor-α at day 3, 5, 8, and 12 after infection revealed no significant differences between the WT and MyD88^−/− mice. Flow cytometry demonstrated similar levels of activated CD8+ T cells and natural killer cells.

Conclusions

The pathogenesis of murine BA is independent of the MyD88 signaling inflammatory pathway, suggesting alternative mechanisms are crucial in the induction of the model.     

髓样分化因子88的研究进展：把握TLR信号通路中的瓶颈

目的总结髓样分化因子88（myeloid differentiation factor 88,MyD88）在Toll样受体（toll like receptor,TLR）信号通路中的作用、MyD88与相关疾病的关系及其潜在的应用价值。方法收集近年来国内外有关MyD88在TLR信号通路中的作用及其与相关疾病关系的文献并作综述。结果 MyD88是一种重要的接头蛋白,在TLR信号通路中处于节点位置,起到承上启下的作用,是通路中的＂瓶颈＂。其传导的信号可导致下游多种转录因子的激活,从而启动先天性免疫反应,并且其与多种疾病的发生均有关。结论 MyD88是TLR信号通路的核心接头蛋白,在先天性免疫、获得性免疫和多种疾病的发生中均起着重要作用,是一个潜在的临床疾病的治疗靶点。     

MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans (_AaLPS), in the oral environment is made mainly by surface receptors known as Toll‐like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary‐response protein 88 (MyD88) ‐dependent and ‐independent pathways, which may involve the adaptor Toll/interleukin‐1 receptor‐domain‐containing adaptor inducing interferon‐β (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by _AaLPS in mice. C57BL6/J wild‐type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of _AaLPS strain FDC Y4 (5 μg in 3 μl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate‐buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of _AaLPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by _AaLPS was observed. In contrast, responses in TRIF^?/? mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP‐positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor‐α production in MyD88^?/? mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88^?/? mice after _AaLPS stimulation. Hence, the signaling through MyD88 is pivotal for _AaLPS‐induced osteoclast formation and alveolar bone loss.     

Sustained IL-1β expression impairs adult hippocampal neurogenesis independent of IL-1 signaling in nestin+ neural precursor cells

Alterations in adult hippocampal neurogenesis have been observed in numerous neurological diseases that contain a neuroinflammatory component. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that contributes to neuroinflammation in many CNS disorders. Our previous results reveal a severe reduction in adult hippocampal neurogenesis due to focal and chronic expression of IL-1β in a transgenic mouse model, IL-1β^XAT, that evokes a complex neuroinflammatory response. Other investigators have shown that IL-1β can bind directly to neural precursors to cause cell cycle arrest in vitro. In order to observe if IL-1 signaling is necessary in vivo, we conditionally knocked out MyD88, an adapter protein essential for IL-1 signaling, in nestin⁺ neural precursor cells (NPCs) in the presence of IL-1β-dependent inflammation. Our results show that conditional knockout of MyD88 does not prevent IL-1β-induced reduction in neuroblasts using a genetic fate mapping model. Interestingly, MyD88 deficiency in nestin⁺ NPCs causes an increase in the number of astrocytes in the presence of IL-1β, suggesting that MyD88-dependent signaling is important in limiting astroglial differentiation due to inflammation. MyD88 deficiency does not alter the fate of NPCs in the absence of inflammation. Furthermore, the inflammatory milieu due to IL-1β is not affected by the absence of MyD88 in nestin⁺ NPCs. These results show that sustained IL-1β causes a reduction in adult hippocampal neurogenesis that is independent of MyD88-dependent signaling in nestin⁺ NPCs, suggesting an indirect negative effect of IL-1β on neurogenesis.     

Short‐term MyD88 inhibition ameliorates cardiac graft rejection and promotes donor‐specific hyporesponsiveness of skin grafts in mice

Recognition of evolutionarily conserved ligands by Toll‐like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T‐cell proliferation in vitro. A short‐course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 ± 0.92 days vs. 7.25 ± 0.46 days). ST2825‐treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti‐CD154 induced long‐term skin allograft acceptance in about one‐third of recipients (>100 days). ‘Skin‐tolerant’ recipients showed attenuated donor‐specific IFN‐γ responses, intact IL‐4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor‐specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection.     

髓样分化因子88在妊娠期糖尿病发病机制中的作用研究

目的:通过调控孕妇单核细胞中髓样分化因子88(My D88)的表达水平,研究My D88在妊娠期糖尿病(GDM)发病机制中的作用。方法:30例正常妊娠孕妇为正常组,30例GDM患者为GDM组,两组每个样本处理均相同,每个样本各分为4个组,分别为未处理组、LPS组、ST2825组、LPS+ST2825组。采用Western blot法检测并比较各组单核细胞中My D88及核转录因子-κB(NF-κB)/p65的表达量,分析各组中My D88与NF-κB/p65的相关性;采用ELISA法检测并比较各组培养液中肿瘤坏死因子-α(TNF-α)、白介素-1(IL-1)、白介素-10(IL-10)水平。结果:①正常组和GDM组组内的比较:LPS组、ST2825组的My D88及NF-κB/p65的表达水平与同组未处理组相比,差异有统计学意义(P0.05);LPS+ST2825组的My D88及NF-κB/p65的表达水平明显低于同组的LPS组(P0.05)。正常组与GDM组组间比较:GDM组各处理组My D88及NF-κB/p65的表达水平均较正常组相同处理组明显升高(P0.05)。②两组中除未处理组外,其他处理组的My D88与NF-κB/p65的表达水平均呈正相关关系(P0.05)。③GDM组中未处理组和LPS组中的细胞因子(TNF-α、IL-1、IL-10)的表达水平均高于正常组中未处理组及LPS组(P0.05);GDM组中LPS组3个细胞因子的表达水平高于其未处理组(P0.05),ST2825组3个细胞因子的表达水平低于其未处理组(P0.05)。GDM组中LPS+ST2825组3个细胞因子的表达水平低于其LPS组(P0.05)。结论:调控GDM孕妇单核细胞中上游My D88的表达水平,下游NF-κB/p65及细胞因子(TNF-α、IL-1、IL-10)水平发生相应变化,提示My D88可能参与了GDM的发生并在其发病机制中起着重要作用。     

MYD88 L265P mutation in intraocular lymphoma: A potential diagnostic marker

Purpose:Vitreoretinal lymphoma (VRL) is the most common intraocular lymphoma (IOL). This can be either primary or secondary to the central nervous system lymphoma. The diagnosis of primary intraocular lymphoma (PIOL) currently relies on clinical diagnosis and cytological analysis of the vitreous or subretinal biopsy. Although most cases are diagnosed without much issue, the limited amount of vitreous fluid, subjectivity in cytological reporting, and special expertise in ocular pathology make the diagnosis challenging. MYD88 L265P mutation has been implicated to have diagnostic utility in PIOL. In this study, we screened consecutive vitreous biopsies for the presence of MYD88 L265P mutation to understand its diagnostic utility compared to conventional cytological analysis.Methods:Cytological analysis and MYD88 L265P mutation by PCR-based sequencing and restriction fragment length polymorphism (RFLP) were carried out on consecutive vitreous and subretinal biopsies collected from 21 patients. The diagnostic utility of the cytology and MYD88 L265P mutation analysis were compared.Results:Out of the 21 patients, 15 had clinical suspicion of having PIOL. Out of these suspected cases of PIOL, nine were confirmed on follow-up, while six were diagnosed as other intraocular pathologies. Diagnostic utility of MYD88 L265P mutation analysis revealed a sensitivity of 88.9%, specificity of 91.6%, positive and negative predictive value of 88.9% and 91.7%, respectively. Diagnostic accuracy of 90.5% was achieved with the mutation analysis that shows the superiority of MYD88 in both ruling in and ruling out PIOL. The diagnostic utility of MYD88 L265P mutation was superior to conventional cytological analysis.Conclusion:The analysis of MYD88 L265P mutation is reliable and efficient in the diagnosis of PIOL.