Background Calcitonin gene-related peptide (CGRP), an immunomodulatory neuropeptide, is important for regulating pain transmission, vasodilation, and the inflammatory response. However, the molecular mechanisms of the CGRP-mediated immune response remain unknown. Methods The effects of CGRP on bacterial meningitis (BM) and its underlying mechanisms were investigated in BM mice in vivo and macrophages in vitro. Results Peripheral injection of CGRP attenuated cytokine storms and protected mice from fatal pneumococcal meningitis, marked by increased bacterial clearance, improved neuroethology, and reduced mortality. When the underlying mechanisms were investigated, we found that CGRP induces proteasome-dependent degradation of major histocompatibility complex class II (MHC-II) in macrophages and then inhibits CD4+ T-cell activation. MARCH1 was identified as an E3 ligase that can be induced by CGRP engagement and promote K48-linked ubiquitination and degradation of MHC-II in macrophages. These results provide new insights into neuropeptide CGRP-mediated immune regulation mechanisms. Conclusions We conclude that targeting the nervous system and manipulating neuroimmune communication is a promising strategy for treating intracranial infections like BM.

目的 探讨间皮素(Mesothelin)对三阴性乳腺癌(TNBC)细胞增殖、凋亡、迁移及侵袭能力的影响.方法免疫化学染色检测TNBC组织、癌旁组织及乳腺癌细胞系中Mesothelin的表达.将过表达Mesothelin慢病毒质粒(Mesothelin组)及对照Scramble质粒(Scramble组)转染TNBC细胞系MDA-MB-231细胞,未感染细胞作为Control组.流式细胞术检测病毒感染效率;实时荧光定量PCR、流式细胞术及Western blot检测Mesothelin表达;CCK-8检测细胞增殖情况;利用Annexin V-APC/PI检测细胞凋亡;Western blot检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)表达;划痕实验检测细胞迁移能力;Transwell实验检测细胞的侵袭能力.结果 Mesothelin在TNBC肿瘤组织中表达高于癌旁组织(P＜0.05),在乳腺癌细胞系中有表达.流式细胞术验证发现慢病毒感染效率达到90%以上.与Scramble组相比,Mesothelin组Mesothelin mRNA和蛋白表达水平升高,细胞增殖率升高,细胞凋亡率降低,Bax、E-cadherin蛋白表达减少,Bcl-2、N-cadherin和Vimentin蛋白表达增加,细胞的迁移及侵袭能力增强(P＜0.05).结论 Mesothelin能够促进乳腺癌细胞增殖,抑制细胞凋亡,增加细胞的迁移及侵袭能力.

The mechanisms by which retinoic acid-inducible gene I (RIG-I), a critical RNA virus sensor, is regulated in many biological and pathological processes remain to be determined. Here, we demonstrate that T cell immunoglobulin and mucin protein-3 (Tim-3), an immune checkpoint inhibitor, mediates infection tolerance by suppressing RIG-I-type I interferon pathway. Overexpression or blockade of Tim-3 affects type I interferon expression, virus replication, and tissue damage in mice following H1N1 infection. Tim-3 signaling decreases RIG-I transcription via STAT1 in macrophages and promotes the proteasomal dependent degradation of RIG-I by enhancing K-48-linked ubiquitination via the E3 ligase RNF-122. Silencing RIG-I reversed Tim-3 blockage-mediated upregulation of type I interferon in macrophages. We thus identified a new mechanism through which Tim-3 mediates the immune evasion of H1N1, which may have clinical implications for the treatment of viral diseases.

Tim-3, an immune checkpoint inhibitor, is widely expressed on the immune cells and contributes to immune tolerance. However, the mechanisms by which Tim-3 induces immune tolerance remain to be determined. Major histocompatibility complex II (MHC-II) plays a key role in antigen presentation and CD4+T cell activation. Dysregulated expressions of Tim-3 and MHC-II are associated with the pathogenesis of many autoimmune diseases including multiple sclerosis. Here we demonstrated that, by suppressing MHC-II expression in macrophages via the STAT1/CIITA pathway, Tim-3 inhibits MHC-II-mediated autoantigen presentation and CD4+T cell activation. As a result, overexpression or blockade of Tim-3 signaling in mice with experimental autoimmune encephalomyelitis (EAE) inhibited or increased MHC-II expression respectively and finally altered clinical outcomes. We thus identified a new mechanism by which Tim-3 induces immune tolerance in vivo and regulating the Tim-3-MHC-II signaling pathway is expected to provide a new solution for multiple sclerosis treatment.

Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.

目的 探讨T细胞免疫球蛋白和黏蛋白结构域分子-3(Tim-3)在李斯特菌(Listeria)感染中通过核因子相关因子2(Nrf2)/环氧化酶2(COX-2)通路对巨噬细胞的影响及可能机制.方法 提取Tim-3转基因小鼠和野生小鼠腹腔巨噬细胞,利用RT-PCR检测COX-2的表达;Tim-3融合蛋白阻断RAW264.7巨噬细胞系Tim-3信号,利用RT-PCR检测COX-2表达;李斯特菌感染RAW264.7细胞并用流式细胞术检测吞噬变化.结果 经检测发现,Tim-3通过抑制COX-2的表达减弱了巨噬细胞对李斯特菌的吞噬作用.结论 阻断巨噬细胞Tim-3信号,可以上调COX-2的表达,进而增强巨噬细胞等免疫细胞的抗感染功能.

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

The receptor for activated C kinase 1 (RACK1) adaptor protein has been implicated in viral infection. However, whether RACK1 promotes in vivo viral infection in mammals remains unknown. Moreover, it remains elusive how RACK1 is engaged in antiviral innate immune signaling. In this study, we report that myeloid RACK1 deficiency does not affect the development and survival of myeloid cells under resting conditions but renders mice less susceptible to viral infection. RACK1-deficient macrophages produce more IFN-α and IFN-β in response to both RNA and DNA virus infection. In line with this, RACK1 suppresses transcriptional activation of type 1 IFN gene promoters in response to virus infection. Analysis of virus-mediated signaling indicates that RACK1 inhibits the phosphorylation of IRF3/7. Indeed, RACK1 interacts with IRF3/7, which is enhanced after virus infection. Further exploration indicates that virus infection triggers AMPK activation, which in turn phosphorylates RACK1 at Thr50. RACK1 phosphorylation at Thr50 enhances its interaction with IRF3/7 and thereby limits IRF3/7 phosphorylation. Thus, our results confirm that myeloid RACK1 promotes in vivo viral infection and provide insight into the control of type 1 IFN production in response to virus infection.

Nuclear factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering stress granule (SG) formation. However, the regulation of the NF90-SG pathway remains largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SG-mediated antiviral immunity. Vesicular stomatitis virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages, which in turn recruit the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiate a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of Tim-3 enhances the NF90 downstream SG formation by selectively increasing the phosphorylation of protein kinase R and eukaryotic translation initiation factor 2α, the expression of SG markers G3BP1 and TIA-1, and protecting mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

目的 构建李斯特菌脑膜炎小鼠模型,以评价T细胞免疫球蛋白和黏蛋白结构域分子3(Tim-3)在李斯特菌脑膜炎小鼠中的可能作用.方法 体内实验通过延髓池注射李斯特菌的方法构建半数致死量的李斯特菌脑膜炎小鼠模型,应用Tim-3敲除(knock out,KO)模式小鼠和Tim-3抗体评价Tim-3对李斯特菌脑膜炎的作用.体外实验通过小胶质细胞BV2与Tim-3抗体共培养,检测阻断Tim-3对BV2细胞因子表达的影响.结果 成功构建了半数致死剂量的脑膜炎小鼠模型,与对照组相比,KO小鼠及应用Tim-3抗体小鼠临床评分较高.小胶质细胞阻断Tim-3后,肿瘤坏死因子α(TNF-α)水平升高.结论 敲除及阻断Tim-3引起小鼠李斯特菌脑膜炎病情加重.

T cell immunoglobulin and mucin domain‐3 (Tim‐3), an immune checkpoint molecule, plays critical roles in maintaining innate immune homeostasis; however, the mechanisms underlying these roles remain to be determined. Here, we determined that Tim‐3 controls glycolysis in macrophages and thus contributes to phenotype shifting. Tim‐3 signal blockade significantly increases lactate production by macrophages, but does not influence cell proliferation or apoptosis. Tim‐3 attenuates glucose uptake by inhibiting hexokinase 2 (HK2) expression in macrophages. Tim‐3‐mediated inhibition of macrophage glycolysis and the expression of proinflammatory cytokines, tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β are reversed by HK2 silencing. Finally, we demonstrated that Tim‐3 inhibits HK2 expression via the STAT1 pathway. We have thus discovered a new way by which Tim‐3 modulates macrophage function.

The generation of large numbers of plasma cells (PCs) is a main factor in systemic lupus erythematosus (SLE). We hypothesize that Hspa13, a member of the heat shock protein family, plays a critical role in the control of PC differentiation. To test the hypothesis, we used lipopolysaccharide (LPS)-activated B cells and a newly established mouse line with a CD19cre-mediated, B cell–specific deletion of Hspa13: Hspa13 cKO mice. We found that Hspa13 mRNA was increased in PCs from atacicept-treated lupus-prone mice and in LPS-stimulated plasmablasts (PBs) and PCs. A critical finding was that PBs and PCs [but not naïve B cells and germinal center (GC) B cells] expressed high levels of Hspa13. In contrast, the Hspa13 cKO mice had a reduction in BPs, PCs, and antibodies induced in vitro by LPS and in vivo by sheep red blood cells (SRCs)- or 4-hydroxy-3-nitrophenylacetyl (NP)-immunization. Accordingly, the Hspa13 cKO mice had reduced class-switched and somatically hypermutated antibodies with defective affinity maturation. Our work also showed that Hspa13 interacts with proteins (e.g., Bcap31) in the endoplasmic reticulum (ER) to positively regulate protein transport from the ER to the cytosol. Importantly, Hspa13 mRNA was increased in B220+ cells from patients with multiple myeloma (MM) or SLE, whereas Hspa13 cKO led to reduced autoantibodies and proteinuria in both pristane-induced lupus and lupus-prone MRL/lpr mouse models. Collectively, our data suggest that Hspa13 is critical for PC development and may be a new target for eliminating pathologic PCs.

Background: T-cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint inhibitor that has therapeutic implications for many tumors and infectious diseases. However, the mechanisms by which Tim-3 promotes immune evasion remain unclear. Methods: In this study, we demonstrated that Tim-3 inhibits the expression of major histocompatibility complex class I (MHC-I) in macrophages at both the messenger ribonucleic acid and protein levels by inhibiting the STAT1-NLRC5 signaling pathway. Results: As a result, MHC-I-restricted antigen presentation by macrophages was inhibited by Tim-3 both in vitro and in a Listeria monocytogenes infection model in vivo. Systemic overexpression of Tim-3 or specific knockout of Tim-3 in macrophages significantly attenuated or enhanced CD8(+) T-cell activation and infection damage in L monocytogenes-infected mice, respectively. Conclusions: Thus, we identified a new mechanism by which Tim-3 promotes L monocytogenes immune evasion. Further studies on this pathway might shed new light on the physio-pathological roles of Tim-3 and suggest new approaches for intervention.

目的:研究慢性心理应激以及起主要作用的应激激素对于γδT细胞的影响.方法:采用连续5次16 h/次束缚模型诱导慢性心理应激,取小鼠胸腺、脾脏进行称重和白细胞计数,取肠系膜淋巴结进行白细胞计数,流式细胞术检测CD3+γδTCR+细胞的百分比,检测糖皮质激素受体抑制剂和儿茶酚胺β肾上腺素能受体抑制剂的可能逆转作用,分析体外糖皮质激素对纯化的γδT细胞凋亡的影响.结果:连续5次16 h/次束缚导致小鼠免疫器官的重量和细胞数量减少,γδT细胞数量减少;阻断糖皮质激素的外周作用可逆转γδT细胞数量减少,糖皮质激素可在体外直接诱导纯化的γδT细胞凋亡.结论:慢性心理应激是通过糖皮质激素的刺激作用导致γδT细胞的凋亡和减少.

Alternative splicing (AS) of mRNA is known to be involved in regulation of immune cell differentiation and activation. Elongation factor Tu GTP binding domain containing 2 (Eftud2) is an AS factor to potentially modulate innate immune response in macrophages. In this study, we investigate its involvement in the pathogenesis of colitis-associated cancer (CAC). Using an established mouse model of CAC, we show that Eftud2 is constantly overexpressed in the colonic tissues as well as infiltrating macrophages. Myeloid-specific knockout of Eftud2 remarkably suppresses chronic intestinal inflammation and tumorigenesis, which is associated with decreased production of inflammatory cytokines and tumorigenic factors. Repression of colonic inflammation and colorectal tumor development in Eftud2-deficient mice is due to the impaired activation of NF-κB signaling in LPS-challenged macrophages. Furthermore, the alteration of Eftud2-mediated AS involving the components of TLR4-NF-κB cascades underlies the impairment of NF-κB activation. Overall, these findings provide new insights into the tight link between inflammation and cancer and modulation of AS in innate immune signals may be a potentially therapeutic avenue for CAC treatment.

目的 探究T细胞免疫球蛋白黏蛋白-3(Tim-3)对白细胞介素-2(IL-2)的调控作用及机制.方法 通过酶联免疫吸附实验(ELISA)、流式细胞术(FACS)以及实时定量荧光聚合酶链反应(RT-PCR)方法,检测T细胞活化后,Jurkat高表达Tim-3(JurkatTim-3-high)与低表达Tim-3(JurkatTim-3-low)细胞系中IL-2的基因转录及蛋白表达水平;使用中和性抗人Tim-3抗体阻断Jurkat细胞系中Tim-3功能,ELISA及RT-PCR方法检测IL-2蛋白分泌及转录水平变化;通过Western蛋白免疫印迹法检测Tim-3对IL-2表达调控相关分子[包括蛋白激酶C(PKC)β1及核因子(NF)-κB亚基P65]的磷酸化的影响.结果 ELISA、FACS和RT-PCR结果均表明,Tim-3抑制IL-2的表达,使用抗人Tim-3抗体阻断后,IL-2的表达上调;Western印迹结果显示,与JurkatTim-3-low细胞相比,JurkatTim-3-high细胞中蛋白激酶PKCβ1及NF-κB亚基P65的磷酸化受到明显抑制.使用中和性抗人Tim-3抗体阻断后,PKCβ1及P65的磷酸化明显增强.结论 Tim-3对IL-2的基因转录及蛋白表达起负性调控作用,并且可能是通过抑制NF-κB(NF-κB/p65)及PKCβ1的磷酸化,从而抑制IL-2的产生.

BackgroundMultiple myeloma (MM), characterized by cancerous proliferation of plasmablasts (PB) and plasma cells (PC), remains incurable in many patients. Differentially expressed molecules between MM PCs and healthy PCs have been explored in order to identify novel targets for treating MM. In the present study, we searched for novel MM therapeutic targets by comparing mRNA expression patterns between the Mus musculus myeloma plasmablast-like SP 2/0 cell line and LPS-induced PB/PC.MethodsGene expression profiles of LPS-induced PB/PC and SP 2/0 cells were determined using RNA-sequencing. A predicted gene (Gm40600) was found to be expressed at a low level in SP 2/0 cells. To study the role of Gm40600 in malignant PC, Gm40600 cDNA was cloned into a lentiviral vector (LV201) containing a puromycin selectable marker that was then transfected into SP 2/0 cells. Stable Gm40600-expressing SP 2/0 cells were selected using puromycin. The effect of Gm40600 on SP 2/0 cell proliferation, cell cycle/apoptosis, and tumor progression was assessed by cell counting kit-8 (CCK8), flow cytometry (FACS), and the SP 2/0 isograft mouse model, respectively. The effect of Gm40600 on mRNA and protein expression was evaluated by RNA-sequencing and western blotting, respectively.ResultsWe found that SP 2/0 cells expressed lower level of Gm40600 mRNA as compared to LPS-induced PB/PC. Overexpression of Gm40600 significantly suppressed SP 2/0 cell proliferation and isograft tumor progression in an isograft mouse model by promoting apoptosis. In addition, Gm40600 overexpression suppressed transcription of the gene encoding Bcl2. Gm40600 overexpression also reduced the expression of PC-associated transcription factors Blimp1 and Xbp1, which promote transcription of the gene that encodes Bcl2.ConclusionsGm40600 reduced SP 2/0 cell proliferation and isograft tumor growth and progression by suppressing Blimp1 and Xbp1-mediated Bcl2 transcription to induce apoptosis. Thus, regulation of a human homolog of Gm40600, or associated factors, may be a potential therapeutic approach for treating MM.

Although sharing the same subunit Ebi3, IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3) have different biological functions, suggesting that Ebi3 subunit may functions as a carrier. Our data demonstrated that activated T cells and B cells effectively up-regulated Ebi3 expression. In addition, Ebi3 effectively promoted T-cell activation and the differentiation of helper T 1 (Th1), Th17, and Foxp3+ regulatory T (Treg) cells induced by Th1, Th17, and Treg polarizing condition, respectively. Naturally, Ebi3 could promote B-cell activation and the production of CD138+ plasma cells (PC) induced by LPS. Conversely, neutralizing anti-Ebi3 antibody could significantly suppress T/B-cell activation and production of Th1, Th17, Tregs, and PC induced by Th1, Th17, Treg polarizing condition, and LPS, respectively. Furthermore, we found that Ebi3 time-dependently induced STAT3 activation in CD4+T cells and B cells. Conversely, STAT3-/- effectively reduced Ebi3 expression and the production of Th1, Th17, Tregs, and plasma cells. Finally, we showed that gp130 but not IL-27Rα mediates Ebi3-induced STAT3 activation. These results suggest that Ebi3 promotes Th- and B-cell differentiation via gp130-STAT3 signaling pathway. Thus, autocrine Ebi3 may play an important role in the differentiation of Th and B cells and thus in infection, inflammation, and autoimmune disorders.