BACKGROUND & AIMS: :The mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes. The three main MAPK cascades are the extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases. A typical MAPK cascade is composed of MAP3K-MAP2K-MAPK kinases that are held by scaffold proteins. Scaffolds function to assemble the protein tier and contribute to the specificity and efficacy of signal transmission. WD repeat domain 62 (WDR62) is a JNK scaffold protein, interacting with JNK, MKK7, and several MAP3Ks. The loss of WDR62 in human leads to microcephaly and pachygyria. Yet the role of WDR62 in cellular function is not fully studied. We used the CRISPR/Cas9 and short hairpin RNA approaches to establish a human breast cancer cell line MDA-MB-231 with WDR62 loss of function and studied the consequence to JNK signaling. In growing cells, WDR62 is responsible for the basal expression of c-Jun. In stressed cells, WDR62 specifically mediates TNFα-dependent JNK activation through the association with both the adaptor protein, TNF receptor-associated factor 2 (TRAF2), and the MAP3K protein, mixed lineage kinase 3. TNFα-dependent JNK activation is mediated by WDR62 in HCT116 and HeLa cell lines as well. MDA-MB-231 WDR62-knockout cells display increased resistance to TNFα-induced cell death. Collectively, WDR62 coordinates the TNFα receptor signaling pathway to JNK activation through association with multiple kinases and the adaptor protein TRAF2.

背景与目标： : 丝裂原活化蛋白激酶 (MAPKs) 调节多种细胞过程。三个主要的MAPK级联是细胞外信号调节激酶 (ERK)，c 6月N端激酶 (JNK) 和p38激酶。典型的MAPK级联反应由支架蛋白持有的MAP3K-MAP2K-MAPK激酶组成。支架的功能是组装蛋白质层，并有助于信号传递的特异性和有效性。WD重复结构域62 (WDR62) 是一种JNK支架蛋白，与JNK、MKK7和几个map3k相互作用。人类中WDR62的丢失会导致小头畸形和粗大症。然而，WDR62在细胞功能中的作用尚未得到充分研究。我们使用CRISPR/Cas9和短发夹RNA方法建立了具有WDR62功能丧失的人乳腺癌细胞系MDA-MB-231，并研究了JNK信号传导的后果。在生长的细胞中，WDR62负责c-6月的基础表达在应激细胞中，WDR62通过与衔接蛋白，TNF受体相关因子2 (TRAF2) 和MAP3K蛋白混合谱系激酶3结合，特异性介导TNF α 依赖性JNK激活。Tnf α 依赖性JNK激活也由HCT116和HeLa细胞系中的WDR62介导。MDA-MB-231 WDR62-knockout细胞显示对tnf α 诱导的细胞死亡的抗性增加。总的来说，WDR62通过与多种激酶和衔接蛋白traf2结合，协调tnf α 受体信号通路与JNK激活。

BACKGROUND & AIMS: :Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood-brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke.

背景与目标： : 肿瘤坏死因子 (TNF)-α 在大脑中产生，以响应急性脑缺血，并促进神经元凋亡。生物肿瘤坏死因子抑制剂 (TNFIs)，如依那西普，不能作为新的中风治疗方法开发，因为这些大分子药物不会穿过血脑屏障 (BBB)。通过将II型人TNF受体 (TNFR) 融合到针对小鼠转铁蛋白受体 (TfR) 的基因工程嵌合单克隆抗体 (MAb) 的每个重链上，工程化了BBB穿透性生物TNFI，称为cTfRMAb-TNFR融合蛋白。融合蛋白的cTfRMAb结构域充当分子特洛伊木马，将融合的TNFR传递穿过BBB。依那西普或cTfRMAb-TNFR融合蛋白 (1  mg/kg) 在成年小鼠中静脉注射，该成年小鼠在闭塞后长达90  分钟的可逆性大脑中动脉闭塞1小时。在闭塞后24小时或7天评估神经保护作用。cTfRMAb-TNFR融合蛋白处理分别导致半球，皮质和皮质下中风体积以及神经缺陷的显着45%，48%，42% 和54% 减少。静脉注射依那西普无疗效。可以对生物TNFIs进行重组以进行BBB渗透，并且在实验性中风中延迟静脉内给药后，IgG-TNFR融合蛋白可以治疗。

BACKGROUND & AIMS: :The deprivation of zinc, caused by malnutrition or as a consequence of aging or disease, strongly affects immune cell functions, causing higher frequency of infections. Among other effects, an increased production of reactive oxygen species (ROS) and proinflammatory cytokines has been observed in zinc-deficient patients, but the underlying mechanisms were unknown. The aim of the current study was to define mechanisms explaining the increase in proinflammatory cytokine production during zinc deficiency, focusing on the role of epigenetic and redox-mediated mechanisms. Interleukin (IL)-1β and tumor necrosis factor (TNF)α production was increased in HL-60 cells under zinc deficiency. Analyses of the chromatin structure demonstrated that the elevated cytokine production was due to increased accessibilities of IL-1β and TNFα promoters in zinc-deficient cells. Moreover, the level of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase-produced ROS was elevated under zinc deficiency, subsequently leading to p38 mitogen-activated protein kinase (MAPK) phosphorylation. The increased activation of p38 MAPK appeared to be necessary for posttranscriptional processes in IL-1β and TNFα synthesis. These data demonstrate that IL-1β and TNFα expression under zinc deficiency is regulated via epigenetic and redox-mediated mechanisms. Assuming an important role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases.

背景与目标： : 由于营养不良或由于衰老或疾病而导致的锌剥夺强烈影响免疫细胞功能，导致更高的感染频率。除其他影响外，在缺锌患者中观察到活性氧 (ROS) 和促炎细胞因子的产生增加，但其潜在机制尚不清楚。当前研究的目的是定义解释锌缺乏期间促炎细胞因子产生增加的机制，重点是表观遗传和氧化还原介导机制的作用。缺锌条件下HL-60细胞中白细胞介素 (IL)-1β 和肿瘤坏死因子 (TNF)α 的产生增加。对染色质结构的分析表明，细胞因子产生的增加是由于缺锌细胞中IL-1β 和TNF α 启动子的可及性增加。此外，缺锌条件下烟酰胺腺嘌呤二核苷酸磷酸氧化酶 (NADPH) 氧化酶产生的ROS水平升高，随后导致p38丝裂原活化蛋白激酶 (MAPK) 磷酸化。p38 MAPK的激活增加似乎是IL-1β 和tnf α 合成中的转录后过程所必需的。这些数据证明了在锌缺乏下IL-1β 和tnf α 的表达是通过表观遗传和氧化还原介导的机制来调节的。假设锌的重要作用在促炎细胞因子调节中，这应该鼓励研究使用锌补充剂治疗炎症性疾病。

BACKGROUND & AIMS: OBJECTIVE:We previously demonstrated that the activation of transient receptor potential vanilloid 1 (TRPV1), a nociceptive ion channel receptor, by capsaicin led to the up-regulation of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) ratio in human periodontal ligament (HPDL) cells. Since TRPV1 is recognised as one of the thermo-sensitive cation channels, this study investigated the response of TRPV1 to thermal stimulation in HPDL cells. METHODS:HPDL cells were incubated at 45°C for thermal stimulation. The mRNA expression of OPG, RANKL, tumour necrosis factor α (TNFα), and interleukin-1 β (IL-1β) was determined by using RT-PCR. OPG secretion and RANKL protein expression were analysed by ELISA and Western blot analysis, respectively. The mechanisms of heat-induced TNFα expression were studied using several TRPV1 inhibitors. RESULTS:In contrast to capsaicin, thermal stimulation had no effect on OPG or RANKL expression. Interestingly, the mRNA expression of TNFα, but not IL-1β, was increased by heat. Using TRPV1 antagonists, we confirmed that TNFα up-regulation was mediated by TRPV1. Phospholipase C (PLC) was previously shown to be involved in capsaicin-induced OPG expression. However, we found that protein kinase C, not PLC, was required for heat-induced TNFα expression. Additionally, the use of cytochalasin D, an inhibitor of actin polymerisation, revealed that cytoskeleton rearrangement might be an important mechanism for cellular sensing of thermal stimuli. CONCLUSION:Our results indicate that TRPV1 plays a multi-functional role in HPDL cells depending on the stimuli. In response to heat, TRPV1 activation leads to the induction of TNFα expression.

背景与目标：

BACKGROUND & AIMS: :Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

背景与目标： : 据报道，瞬时受体电位Melastatin-8 (TRPM8) 在各种过程中起着重要作用，包括冷感，体温调节，疼痛转导和肿瘤发生。然而，TRPM8在寒冷条件下炎症中的作用尚不清楚。由于冷却可以使原发性损伤和损伤引起的炎症趋于一致，因此我们假设冷却的保护作用机制可能与trpm8有关。因此，我们研究了TRPM8激活参与炎症细胞因子的调节。结果表明，当环境温度降低时，TRPM8在小鼠下丘脑中的表达上调; 同时，肿瘤坏死因子 α (tnf α) 下调。TRPM8对tnf α 的抑制作用由核因子 κ B (nf κ B) 介导。具体来说，冷应激刺激TRPM8的表达，从而促进TRPM8和nf κ b的相互作用，从而抑制nf κ b核定位。因此，这种抑制导致tnf α 基因转录的抑制。目前的数据为TRPM8激活的抗炎作用提供了可能的理论基础，为创伤患者的冷却疗法的发展提供了实验基础。

BACKGROUND & AIMS: :In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr(-/-), tlr2(-/-), trif(-/-), and myd88(-/-) mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor-knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.-Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ.

背景与目标： : 在皮肤利什曼病中，亚马逊利什曼原虫激活巨噬细胞双链RNA激活的蛋白激酶R (PKR) 以促进寄生虫的生长。在我们的研究中，利什曼原虫major在原始细胞，原始表达的显性负PKR (PKR-DN) 细胞和巨噬细胞中正常生长PKR-敲除小鼠，表明PKR对于巨噬细胞的L. major生长是必不可少的。poly I:C感染的巨噬细胞中的PKR激活导致寄生虫死亡。作为PKR激活的结果，50% 的ecotin样丝氨酸肽酶抑制剂 (ISP2; Δisp2/isp3) (一种中性粒细胞弹性蛋白酶 (NE) 的抑制剂) 的L. major敲除品系死于129Sv小鼠的原始细胞或巨噬细胞。抑制PKR或NE或中和Toll样受体4或2(TLR4或TLR2) 可防止 Δisp2/isp3死亡。Δisp2/isp3在来自129Sv PKR (-/-)，tlr2(-/-)，trif(-/-) 和myd88(-/-) 小鼠的RAW-pkr-DN细胞或巨噬细胞中正常生长，使NE活性，PKR和TLR反应与寄生虫死亡。Δ isp2/isp3以PKR依赖性方式使TNF-α 的mRNA表达增加2倍，干扰素 β (ifn β) 的mRNA表达增加2倍。TNF-α 抗体逆转了 Δ isp2/isp3对95% 的杀伤，而它们在来自IFN受体敲除小鼠的巨噬细胞中正常生长。我们建议ISP2通过NE-TLR4-TLR2轴阻止PKR的激活，以控制有助于杀死L的先天反应。少校。-法里亚，男S、，卡莱加里-席尔瓦，T。C.，德卡瓦略·维瓦里尼，A.，莫特拉姆，JC.，洛佩斯，美国。G.，利马，A。P。C.A。蛋白激酶R在巨噬细胞通过tnf α 和ifn β 响应中性粒细胞弹性蛋白酶和TLR4杀死利什曼原虫中的作用。

BACKGROUND & AIMS: :While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNFα or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNFα-induced cell death and ROS accumulation in NF-κB activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNFα stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNFα-induced necroptosis and ROS accumulation in NF-κB activation-deficient cells. However, Nec-1, but not BHA, inhibited TNFα-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNFα-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation.

背景与目标： : 虽然细胞凋亡被认为与程序性细胞死亡相同，但在形态上与坏死有关的坏死性坏死已成为一种新型的程序性细胞死亡。坏死性依赖于两种结构相关的激酶，受体相互作用的丝氨酸-苏氨酸激酶 (RIPK)1和ripk3。RIPK1通过由TNF α 或Fas配体触发的上游衔接子分子 (例如具有死亡结构域 (FADD) 和TNF受体相关死亡结构域 (TRADD) 的Fas相关蛋白) 的寡聚而被激活。激活的RIPK1随后与RIPK3相互作用并激活，导致坏死性。然而，氧化应激对坏死性坏死的影响仍然存在争议。我们发现RIPK1，necrostatin-1 (Nec-1) 的选择性抑制剂可显着阻止tnf α 诱导的NF-κ b活化缺陷细胞中的细胞死亡和ROS积累。这表明这些细胞在tnf α 刺激下大多死于坏死性。有趣的是，抗氧化剂丁基羟基茴香醚 (BHA) 阻止了tnf α 诱导的坏死和ROS在NF-κ b活化缺陷细胞中的积累。然而，Nec-1 (而不是BHA) 抑制了tnf α 诱导的这些细胞中RIPK1的磷酸化，这表明ROS在RIPK1激活下游的坏死性坏死中起着至关重要的作用。使用BHA相关化合物进行的结构和功能分析表明，BHA的叔丁基和羟基对于其抗坏死功能至关重要。总之，这些结果表明，tnf α 诱导的坏死性坏死与氧化应激密切相关，而氧化应激是在RIPK1激活的下游诱导的。

BACKGROUND & AIMS: :Pro-inflammatory cytokines secreted by macrophages and other cell types are implicated as intraovarian factors affecting different aspects of ovarian function including follicle and corpus luteum 'turnover', steroidogenesis and angiogenesis. Here, we compared granulosal (GC) and thecal (TC) expression of TNF, IL6 and their receptors (TNFRSF1A, TNFRSF1B and IL6R) during bovine antral follicle development; all five mRNA transcripts were detected in both GC and TC and statistically significant cell-type and follicle stage-related differences were evident. Since few studies have examined cytokine actions on TC steroidogenesis, we cultured TC under conditions that retain a non-luteinized 'follicular' phenotype and treated them with TNFα and IL6 under basal and LH-stimulated conditions. Both TNFα and IL6 suppressed androgen secretion concomitantly with CYP17A1 and LHCGR mRNA expression. In addition, TNFα reduced INSL3, HSD3B1 and NOS3 expression but increased NOS2 expression. IL6 also reduced LHCGR and STAR expression but did not affect HSD3B1, INSL3, NOS2 or NOS3 expression. As macrophages are a prominent source of these cytokines in vivo, we next co-cultured TC with macrophages and observed an abolition of LH-induced androgen production accompanied by a reduction in CYP17A1, INSL3, LHCGR, STAR, CYP11A1 and HSD3B1 expression. Exposure of TC to bacterial lipopolysaccharide also blocked LH-induced androgen secretion, an effect reduced by a toll-like receptor blocker (TAK242). Collectively, the results support an inhibitory action of macrophages on thecal androgen production, likely mediated by their secretion of pro-inflammatory cytokines that downregulate the expression of LHCGR, CYP17A1 and INSL3. Bovine theca interna cells can also detect and respond directly to lipopolysaccharide.

背景与目标： : 巨噬细胞和其他细胞类型分泌的促炎细胞因子被认为是影响卵巢功能不同方面的卵巢内因素，包括卵泡和黄体 “周转”，类固醇生成和血管生成。在这里，我们比较了牛窦卵泡发育过程中TNF，IL6及其受体 (TNFRSF1A，TNFRSF1B和IL6R) 的颗粒 (GC) 和thecal (TC) 表达; 在GC和TC中均检测到所有五个mRNA转录本，并且具有统计学意义的细胞类型和卵泡阶段相关差异。由于很少有研究检查细胞因子对TC类固醇生成的作用，因此我们在保留非黄体化 “卵泡” 表型的条件下培养TC，并在基础和LH刺激的条件下用tnf α 和IL6处理它们。Tnf α 和IL6均与CYP17A1和LHCGR mRNA表达同时抑制雄激素分泌。此外，tnf α 降低了INSL3，HSD3B1和NOS3的表达，但增加了NOS2的表达。IL6还降低了LHCGR和STAR的表达，但不影响HSD3B1，INSL3，NOS2或NOS3的表达。由于巨噬细胞是体内这些细胞因子的主要来源，我们接下来将TC与巨噬细胞共培养，并观察到LH诱导的雄激素产生的消除，伴随着CYP17A1，INSL3，LHCGR，STAR，CYP11A1和HSD3B1表达的降低。将TC暴露于细菌脂多糖中也会阻止LH诱导的雄激素分泌，这种作用被toll样受体阻滞剂 (TAK242) 降低。总的来说，结果支持巨噬细胞对鞘雄激素产生的抑制作用，可能是由其分泌促炎细胞因子介导的，这些细胞因子下调了LHCGR，CYP17A1和insl3的表达。牛卵泡膜细胞也可以检测脂多糖并直接响应。

BACKGROUND & AIMS: :TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.

背景与目标： : TLR4信号通过MyD88和TRIF依赖途径启动转录因子NF-κ b向细胞核的转运。在使用数学建模和功能分析的细胞群体研究中，Cheng等人。建议在没有TRIF的情况下，LPS驱动的MyD88激活会损害NF-κ b易位。我们测试了Cheng等人提出的模型。在表达EGFP标记的p65和tnf α 启动子驱动的mCherry的巨噬细胞中使用实时单细胞分析。LPS刺激后，缺乏TRIF的细胞显示出NF-κ b动力学模式，该模式与野生型细胞没有改变，但tnf α 启动子的激活受到损害。在缺乏MyD88的巨噬细胞中，响应LPS刺激，NF-κ b向细胞核的易位很小，并且tnf α 启动子没有激活。这些发现证实，通过MyD88的信号传导是LPS依赖性NF-κ b易位到细胞核的主要驱动因素。但是，TRIF缺陷细胞中NF-κ b动力学的模式并不直接反映tnf α 启动子激活的动力学，从而支持TRIF依赖性信号传导在该细胞因子的转录中起重要作用的概念。

BACKGROUND & AIMS: :TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5-7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.

背景与目标： : tnf α 在许多损伤和疾病情况下持续升高。先前关于tnf α 对少突胶质细胞及其祖细胞的细胞毒性的报道表明，创伤性损伤或多发性硬化症患者的内源性髓鞘再生不良可能部分归因于持续的炎症。因此，了解炎性细胞因子对潜在细胞治疗候选者的影响对于评估其使用的可行性非常重要。在这项研究中，我们评估了长期暴露于tnf α 对培养的大鼠嗅鞘细胞 (OECs) 和雪旺细胞 (SCs) 的活力，增殖，迁移和tnf α 受体表达的影响。尽管移植到CNS中的OECs和SCs产生相似的髓鞘表型，并且可能具有相似的治疗用途，但我们报告它们对tnf α 的敏感性非常不同。OECs在比SCs更宽的浓度范围内对tnf α 表现出阳性增殖反应。低tnf α 浓度增加了OECs和SCs的增殖和迁移，但在100 ng/ml或更高浓度的tnf α 存在下SC数下降。相反，即使在高TNF α 浓度 (高达1 µ g/ml) 下，OECs也显示出增强的增殖，并且即使在治疗后4周也没有显示出TNF细胞毒性的证据。此外，尽管OECs和SCs均表达tnf αr1和tnf αr2，但暴露于100 ng/ml tnf α 5-7天后，OECs中的tnf α 受体水平下调，但SCs中的tnf α 受体水平升高或保持不变。这些结果表明，如果将OECs移植到具有持续炎症的区域中，则OECs可能是更合适的细胞治疗候选者。

BACKGROUND & AIMS: BACKGROUND:The role of peripheral tumor necrosis factor alpha (TNFα) in inflammatory bowel disease (IBD) is well established, but its central nervous system (CNS) effects are not understood. Thrombin, another mediator of inflammation in IBD, has been implicated in CNS vagal neuron apoptosis in the dorsal motor nucleus of the vagus (DMV). This study evaluates DMV TNFα exposure, characterizes effects of TNFα on DMV neurons, and identifies a relationship between DMV TNFα and thrombin in IBD. METHODS:2,4,6-Trinitrobenzene sulfonic acid was administered via enema to induce colonic inflammation in rats. TNFα in serum, cerebrospinal fluid (CSF), and DMV tissues were determined by ELISA and DMV TNFα expression by quantitative reverse transcription PCR (RT-PCR). TNFα was administered into the fourth intracerebral ventricle (4 V) adjacent to the DMV, with and without blockade of TNF receptor 1 (TNFR1) and the thrombin receptor proteinase-activated receptor 1 (PAR1). Immunofluorescence was used to evaluate microglial activation (Cd11b) and prothrombin presence in DMV sections. Apoptosis was examined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and activated caspase-3 immunofluorescence. RESULTS:IBD is associated with increased TNFα protein in serum, CSF, and DMV tissue; DMV TNFα transcription is also increased. TNFα (4 V) caused a 54 % increase in microglial activation, a 27 % increase in DMV prothrombin protein, and a 31 % increase in vagal neuron apoptosis by TUNEL. There was a 52 % increase in activated caspase-3 immunofluorescence in TNFα-treated animals (p < 0.05). All effects of 4 V TNFα were prevented by TNFR1 blockade. TNFα-induced apoptosis was prevented by PAR1 blockade. CONCLUSIONS:IBD is associated with DMV exposure to TNFα, causing excess DMV prothrombin and vagal apoptosis.

背景与目标：

BACKGROUND & AIMS: :Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), may contribute to hepatic steatosis in the situation of excess lipid accumulation in farmed fish. Pigment epithelium-derived factor (PEDF) is an endogenous anti-inflammatory factor and promotes lipolysis. Accordingly, we isolated PEDF from grass carp and investigated its role in TNFα-induced hepatic steatosis. Sequence analysis showed that PEDF gene, which possesses 8 exons and 7 introns, encodes a protein with 409 amino acids. PEDF was a critical determinant of the transcriptional response to nutrient availability in grass carp. Endogenous PEDF was an intracellular protein with cytoplasmic distribution and directly interacts with adipose triglyceride lipase (ATGL), which might mediate PEDF-induced lipolysis. TNFα significantly promoted lipid accumulation in vivo and in vitro, accompanied with a decrease in mRNA levels of PEDF and peroxisome proliferator-activated receptor alpha (PPARα). Recombinant PEDF and PPARα agonist diminished the TNFα-induced hepatic steatosis. Meanwhile, PPARα agonist caused an increase in PEDF expression, suggesting that TNFα antagonizes the actions of PEDF possibly in a PPARα-dependent manner. These findings suggest that PEDF is an important protective factor against hepatic steatosis induced by TNFα, which provided a new therapeutic target for inflammation-associated hepatic steatosis.

背景与目标： : 促炎性细胞因子，例如肿瘤坏死因子 α (tnf α)，可能在养殖鱼类中脂质积累过多的情况下导致肝脂肪变性。色素上皮衍生因子 (PEDF) 是一种内源性抗炎因子，可促进脂肪分解。因此，我们从草鱼中分离出PEDF，并研究了其在tnf α 诱导的肝脂肪变性中的作用。序列分析表明，PEDF基因具有8个外显子和7个内含子，编码一个409个氨基酸的蛋白质。PEDF是草鱼对养分利用率的转录反应的关键决定因素。内源性PEDF是一种具有细胞质分布的细胞内蛋白，与脂肪甘油三酯脂肪酶 (ATGL) 直接相互作用，可能介导PEDF诱导的脂肪分解。Tnf α 显着促进体内和体外脂质积累，并伴有PEDF和过氧化物酶体增殖物激活受体 α (ppar α) 的mRNA水平降低。重组PEDF和ppar α 激动剂减少了tnf α 诱导的肝脂肪变性。同时，ppar α 激动剂引起PEDF表达增加，表明tnf α 可能以ppar α 依赖性方式拮抗PEDF的作用。提示PEDF是抑制tnf α 诱导的肝脂肪变性的重要保护因子，为炎症相关肝脂肪变性提供了新的治疗靶点。

BACKGROUND & AIMS: :Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal-entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X(7) receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2'-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X(7) receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X(7) receptors with the specific antagonists oxATP, BBG, and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X(7) signaling. Furthermore, TNFα activation of P2X(7) receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X(7) signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα.

背景与目标： 神经胶质细胞及其祖细胞的激活和增殖是与许多神经退行性疾病相关的神经炎症的关键过程。在神经胶质细胞活化和增殖明显的神经病理条件下，控制神经胶质细胞的数量可能具有治疗重要性。细胞因子肿瘤坏死因子 α (tnf α) 对小胶质细胞的增殖作用已有报道，但tnf α 调节胶质细胞增殖的分子机制尚不清楚。使用器官型海马-内嗅皮层 (HEC) 切片培养模型，我们研究了tnf α 在ATP-P2X(7) 受体信号传导在神经胶质增殖中的作用。用5-溴-2 '-脱氧尿苷 (BrdU) 标记HEC培养物中的增殖细胞群体。Tnf α 处理可诱导BrdU细胞中P2X(7) 受体mRNA的强烈表达和免疫反应性，同时显着增加BrdU细胞的增殖。此外，tnf α 增加了水通道蛋白4 (AQP4) 的表达，这是一种参与神经胶质增殖的离子通道。Tnf α 的增殖作用通过用特异性拮抗剂oxATP，BBG和KN62阻断P2X(7) 受体或通过用ATP水解apyrase降低细胞外ATP而减弱。BrdU + 细胞的基础增殖也对ATP-P2X(7) 信号传导的阻断敏感。此外，P2X(7) 受体的tnf α 激活似乎通过蛋白激酶C级联调节AQP4的表达，而AQP4表达的下调可以减少tnf α 刺激的BrdU细胞增殖。总之，这些新发现证明了在与tnf α 增加相关的病理条件下，ATP-P2X(7) 信号在控制神经胶质祖细胞增殖中的重要性。

BACKGROUND & AIMS: BACKGROUND:Mesenchymal stem/stromal cells (MSCs) have been widely used to treat various inflammatory diseases. The immunomodulatory capabilities of MSCs are usually licensed by inflammatory cytokines and may vary depending on the levels and the types of inflammatory cytokines. However, how the inflammatory microenvironment affects the fate of MSCs remains elusive. Here we characterized the molecular mechanism underlying the apoptosis of mouse MSCs triggered by the synergistic action of IFNγ and TNFα. METHODS:We isolated and expanded MSCs by flushing the femoral and tibial bone marrow of wild-type, iNOS-/-, and Fas-/- mice. BM-MSCs were treated with IFNγ and TNFα in vitro, and cell viability was evaluated by a CCK-8 kit. Apoptosis was assessed by Annexin V/propidium iodide-stained flow cytometry. Expression of genes related to apoptosis and endoplasmic reticulum (ER) stress was measured by reverse transcription-polymerase chain reaction (RT-PCR). Apoptosis and autophagy-related proteins were examined by Western blot analysis. RESULTS:IFNγ and TNFα synergistically trigger apoptosis of mouse BM-MSCs. The two cytokines were shown to stimulate the expression of inducible nitric oxide synthase (iNOS) and consequently the generation of nitric oxide (NO), which is required for the apoptosis of mouse BM-MSCs. The two cytokines similarly induced apoptosis in Fas-/- BM-MSCs. iNOS and NO were shown to upregulate Fas in mouse MSCs and sensitize them to Fas agonist-induced apoptosis. Moreover, NO stimulated by IFNγ/TNFα impairs autophagy, which aggravates ER stress and promotes apoptosis. CONCLUSIONS:IFNγ/TNFα-induced apoptosis in mouse MSCs is mediated by NO. Our findings shed new light on cytokine-induced apoptosis of MSCs and have implications in MSC-based therapy of inflammatory diseases.

背景与目标：

BACKGROUND & AIMS: :We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein-Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.

背景与目标： : 我们以前报道过，HSV-2 R1，单纯疱疹病毒2型核糖核苷酸还原酶的R1亚基 (ICP10; UL39)，通过在caspase-8激活处或上游中断DR介导的信号传导，保护细胞免受死亡受体 (DR) 配体肿瘤坏死因子-α-(tnf α) 和Fas配体 (FasL) 诱导的凋亡。对HSV-2 R1保护的分子机制的进一步研究表明，细胞外调节激酶1/2 (ERK1/2)，磷脂酰肌醇3-激酶 (PI3-K)/Akt，NF-κ b和JNK存活途径在这种抗凋亡功能中不发挥主要作用。相互作用研究表明，HSV-2 R1与caspase-8组成性相互作用。HSV-2 R1缺失突变体R1(1-834)-GFP和爱泼斯坦-巴尔病毒 (EBV) R1，其不保护免受DR配体诱导的凋亡，不与caspase-8相互作用，表明相互作用是保护所必需的。HSV-2 R1损害了caspase-8过表达诱导的caspase-8活化，表明两种蛋白质之间的相互作用阻止了caspase-8二聚化/活化。HSV-2 R1通过其前结构域直接与caspase-8结合，但不与其半胱天冬酶结构域或Fas相关死亡结构域蛋白 (FADD) 相互作用。HSV-2 R1和caspase-8之间的相互作用破坏了FADD-caspase-8结合。我们进一步证明，单独表达的HSV-1 R1 (ICP6) 与HSV-2 R1共享结合caspase-8和保护细胞免受DR诱导的凋亡的能力。最后，由于长寿命的Fas蛋白在感染的早期保持稳定，因此使用HSV-1 UL39缺失突变体ICP6 ∆ 进行的实验表明，HSV-1 R1对于保护HSV-1-infected细胞免受FasL侵害可能至关重要。