IL-25 regulates Th17 function in autoimmune inflammation

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17-producing T cells. We have generated IL-25-deficient (il25-/-) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25-/- mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17-, IFNgamma-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNgamma in il25-/- mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.     

Cytosolic phospholipase A2 alpha-deficient mice are resistant to experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.     

IL-21R signaling is critical for induction of spontaneous experimental autoimmune encephalomyelitis

IL-17–producing CD4⁺ T cells (Th17 cells) have well-described pathogenic roles in tissue inflammation and autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE); however, the involvement of IL-21 in these processes has remained controversial. While IL-21 is an essential autocrine amplification factor for differentiation of Th17 cells, the loss of IL-21 or IL-21 receptor (IL-21R) does not protect mice from actively induced EAE. Here, we utilized a transgenic EAE mouse model, in which T and B cells overexpress receptors for myelin oligodendrocyte glycoprotein (MOG) (referred to as 2D2xTH mice), and demonstrated that IL-21 is critical for the development of a variant form of spontaneous EAE in these animals. Il21r deletion in 2D2xTH mice reduced the incidence and severity of spontaneous EAE, which was associated with a defect in Th17 cell generation. Moreover, IL-21R deficiency limited IL-23R expression on Th17 cells and inhibited expression of key molecules involved in the generation of pathogenic Th17 cells. Conversely, loss of IL-23R in 2D2xTH mice resulted in complete resistance to the development of spontaneous EAE. Our data identify a previously unappreciated role for IL-21 in EAE and reveal that IL-21–mediated signaling supports generation and stabilization of pathogenic Th17 cells and development of spontaneous autoimmunity.     

Dual phase regulation of experimental allergic encephalomyelitis by platelet-activating factor

Experimental allergic encephalomyelitis (EAE) serves as a model for multiple sclerosis and is considered to be a CD4+ Th1 cell-mediated autoimmune disease. To investigate the role of platelet-activating factor (PAF) in this disease, PAF receptor (PAFR) KO (PAFR-KO) and wild-type (WT) mice, on a C57BL/6 genetic background, were immunized with myelin oligodendrocyte glycoprotein 35-55. The levels of PAF production and PAFR mRNA expression in the spinal cord (SC) correlated with the EAE symptoms. PAFR-KO mice showed lower incidence and less severe symptoms in the chronic phase of EAE than WT mice. However, no difference was observed in T cell proliferation, Th1-cytokine production, or titer of IgG2a between both genotypes. Before onset, as revealed by microarray analysis, mRNAs of inflammatory mediators and their receptors-including IL-6 and CC chemokine receptor 2-were down-regulated in the SC of PAFR-KO mice compared with WT mice. Moreover, in the chronic phase, the severity of inflammation and demyelination in the SC was substantially reduced in PAFR-KO mice. PAFR-KO macrophages reduced phagocytic activity and subsequent production of TNF-alpha. These results suggest that PAF plays a dual role in EAE pathology in the induction and chronic phases through the T cell-independent pathways.     

Th17 lymphocytes traffic to the central nervous system independently of α4 integrin expression during EAE

The integrin α4β1 (VLA-4) is used by encephalitogenic T cells to enter the central nervous system (CNS). However, both Th1 and Th17 cells are capable of inducing experimental autoimmune encephalomyelitis (EAE), and the molecular cues mediating the infiltration of Th1 versus Th17 cells into the CNS have not yet been defined. We investigated how blocking of α4 integrins affected trafficking of Th1 and Th17 cells into the CNS during EAE. Although antibody-mediated inhibition of α4 integrins prevented EAE when MOG(35-55)-specific Th1 cells were adoptively transferred, Th17 cells entered the brain, but not the spinal cord parenchyma, irrespective of α4 blockade. Accordingly, T cell-conditional α4-deficient mice were not resistant to actively induced EAE but showed an ataxic syndrome with predominantly supraspinal infiltrates of IL-23R(+)CCR6(+)CD4(+) T cells. The entry of α4-deficient Th17 cells into the CNS was abolished by blockade of LFA-1 (αLβ2 integrin). Thus, Th1 cells preferentially infiltrate the spinal cord via an α4 integrin-mediated mechanism, whereas the entry of Th17 cells into the brain parenchyma occurs in the absence of α4 integrins but is dependent on the expression of αLβ2. These observations have implications for the understanding of lesion localization, immunosurveillance, and drug design in multiple sclerosis.     

Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis

Monocyte chemoattractant protein (MCP)-1 plays a critical role in innate immunity by directing the migration of monocytes into inflammatory sites. Recent data indicated a function for this chemokine in adaptive immunity as a regulator of T cell commitment to T helper cell type 2 (Th2) effector function. Studies in a Th1-dependent animal model, experimental autoimmune encephalomyelitis (EAE), showed that MCP-1 was highly expressed in the central nervous system (CNS) of affected rodents, and MCP-1 antibodies could block relapses of the disease. Mice deficient for the major MCP-1 receptor, CC chemokine receptor (CCR)2, did not develop EAE after active immunization but generated effector cells that could transfer the disease to naive wild-type recipients. We analyzed EAE in mice deficient for MCP-1 to define the relevant ligand for CCR2, which responds to murine MCP-1, MCP-2, MCP-3, and MCP-5. We found that C57BL/6 MCP-1-null mice were markedly resistant to EAE after active immunization, with drastically impaired recruitment of macrophages to the CNS, yet able to generate effector T cells that transferred severe disease to naive wild-type recipients. By contrast, adoptive transfer of primed T cells from wild-type mice into naive MCP-1-null recipients did not mediate clinical EAE. On the SJL background, disruption of the MCP-1 gene produced a milder EAE phenotype with diminished relapses that mimicked previous findings using anti-MCP-1 antibodies. There was no compensatory upregulation of MCP-2, MCP-3, or MCP-5 in MCP-1-null mice with EAE. These results indicated that MCP-1 is the major CCR2 ligand in mice with EAE, and provided an opportunity to define the role of MCP-1 in EAE. Compared with wild-type littermates, MCP-1-/- mice exhibited reduced expression of interferon gamma in draining lymph node and CNS and increased antigen-specific immunoglobulin G1 antibody production. Taken together, these data demonstrate that MCP-1 is crucial for Th1 immune responses in EAE induction and that macrophage recruitment to the inflamed CNS target organ is required for primed T cells to execute a Th1 effector program in EAE.     

Studies in B7-deficient mice reveal a critical role for B7 costimulation in both induction and effector phases of experimental autoimmune encephalomyelitis.

The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7-CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2-deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2(-/-) mice show reduced proliferative responses, but greater interferon gamma production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55-specific T lines were adoptively transferred into the B7-1/B7-2(-/-) and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2(-/-) compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system.     

基因工程Th17细胞的研制及其功能探讨

目的 探讨并优化制备基因工程Th17细胞的方法,评估制备的工程Th17细胞是否具有同天然Th17细胞类似的表型和功能.方法 包装含RORγt基因的重组慢病毒pXZ9-RORγt和空载体对照质粒pXZ9.C57BL6小鼠CD4+ CD25 - na(i)ve T细胞体外活化36 h后依据感染慢病毒颗粒的不同及是否添加TGF-β和IL-6,分为五组:pXZ9-RORγt组、TGF-β+ IL-6组(pXZ9+ TGF-β+ IL-6组)、联合组(pXZ9-RORγt+ TGF-β+ IL-6组)、空载体对照组(pXZ9组)及空白对照组.流式细胞术检测各组IL-17A阳性细胞比例,荧光定量PCR检测各组细胞IL-17A、IL-17F、IL-21和IFN-γ表达水平.通过小鼠实验性变应性脑脊髓炎(EAE)模型评估各组细胞是否具有加重EAE的功能.结果 ①pXZ9-RORγt组、TGF-β +IL-6组和联合组均制备出工程Th17细胞,其中联合组IL-17A阳性率达(60.59±8.15)％,明显高于pXZ9-RORγt组[(40.25±5.46)％]和TGF-β+IL-6组[(14.36±5.27)％].②各组工程Th17细胞均表达IL-17A与IL-17F,而IL-21仅在给予TGF-β和IL-6时明显升高.③各组工程Th17细胞均可加重小鼠EAE.结论 成功建立了制备高效工程Th17细胞的方法,其中过表达RORγt基因与联合TGF-β和IL-6不仅可提高Th17细胞的制备效率,而且制备的工程Th17细胞在表型和功能上与天然Th17细胞相类似.     

MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis

Accumulation of IL-17–producing Th17 cells is associated with the development of multiple autoimmune diseases; however, the contribution of microRNA (miRNA) pathways to the intrinsic control of Th17 development remains unclear. Here, we demonstrated that miR-21 expression is elevated in Th17 cells and that mice lacking miR-21 have a defect in Th17 differentiation and are resistant to experimental autoimmune encephalomyelitis (EAE). Furthermore, we determined that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Moreover, the decreases in Th17 differentiation in miR-21–deficient T cells were associated with defects in SMAD-2/3 activation and IL-2 suppression. Finally, we found that treatment of WT mice with an anti–miR-21 oligonucleotide reduced the clinical severity of EAE, which was associated with a decrease in Th17 cells. Thus, we have characterized a T cell–intrinsic miRNA pathway that enhances TGF-β signaling, limits the autocrine inhibitory effects of IL-2, and thereby promotes Th17 differentiation and autoimmunity.     

Chronological changes of CD4(+) and CD8(+) T cell subsets in the experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). The etiology of MS remains unclear, but T cells specific for myelin components, such as myelin oligodendrocyte glycoprotein (MOG), are thought to play a critical role in the onset of MS. Experimental autoimmune encephalomyelitis (EAE) has been used as an animal model of MS, and T helper type 1 (Th1) cells play an essential role for the pathogenesis of EAE through the production of Th1 cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). We examined CD4(+) and CD8(+) T cell responses in the spleen and CNS of EAE mice, generated by immunization with a peptide (35-55 amino acid residues) of MOG. The number of both CD4(+) and CD8(+) T cells and their MOG-reactivity in the CNS were associated with increasing disease severity but not those in the spleen, suggesting that the MOG-specific CD4(+) and CD8(+) T cells in the CNS are involved in the development of EAE. Polymerase chain reaction analysis suggested that both CD4(+) and CD8(+) T cells produced IFN-gamma and TNF-alpha, while CD4(+) T cells also produced interleukin-17 (IL-17), an important factor in the development of EAE. Thus, CD4(+) T cells may contribute to the induction of EAE by producing IL-17. Furthermore, CD8(+) T cells express higher levels of a suppressive cytokine, IL-10. Taking together, our data suggest that CD4(+) T cells are involved in the early phase of EAE, whereas CD8(+) T cells have a regulatory role in the later stage of EAE.     

IL-17A and IL-17F do not contribute vitally to autoimmune neuro-inflammation in mice

The clear association of Th17 cells with autoimmune pathogenicity implicates Th17 cytokines as critical mediators of chronic autoimmune diseases such as EAE. To study the impact of IL-17A on CNS inflammation, we generated transgenic mice in which high levels of expression of IL-17A could be initiated after Cre-mediated recombination. Although ubiquitous overexpression of IL-17A led to skin inflammation and granulocytosis, T cell–specific IL-17A overexpression did not have a perceptible impact on the development and health of the mice. In the context of EAE, neither the T cell–driven overexpression of IL-17A nor its complete loss had a major impact on the development of clinical disease. Since IL-17F may be able to compensate for the loss of IL-17A, we also generated IL-17F–deficient mice. This strain was fully susceptible to EAE and displayed unaltered emergence and expansion of autoreactive T cells during disease. To eliminate potential compensatory effects of either cytokine, we treated IL-17F–deficient mice with antagonistic monoclonal antibodies specific for IL-17A and found again only a minimal beneficial impact on disease development. We conclude therefore that both IL-17A and IL-17F, while prominently expressed by an encephalitogenic T cell population, may only marginally contribute to the development of autoimmune CNS disease.     

Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon gamma-producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.     

骨髓基质干细胞治疗实验性自身免疫性脑脊髓炎的效果及免疫调节机制

目的:探讨骨髓基质干细胞(BMSCs)对实验性自身免疫性脑脊髓炎(EAE)的治疗作用及相关免疫调节机制。方法:分离纯化培养BMSCs,建立EAE动物模型,随机分为CFA组、BMSCs回输CFA组、MBP组及BMSCs回输MBP组。BMSCs回输CFA组和BMSCs回输MBP组于免疫后第6天尾静脉回输BMSCs,发病高峰期取材,全部大鼠予临床评价,检测淋巴细胞和IL-17+细胞浸润及T淋巴细胞增殖情况。建立BMSCs与淋巴细胞的共培养体系,观察BMSCs在封闭IL-27后对T淋巴细胞中Th17的影响。用ELISA检测大鼠血清或细胞培养上清中相关细胞因子表达。结果:BMSCs回输MBP组与MBP组相比,发病率降低,发病时间延后,临床评分显著降低(P<0.05),炎性细胞浸润明显减轻(P<0.01),IL-17+T细胞表达显著减少(P<0.01),血清中IFN-γ、TNF-α、IL-17、IL-23表达水平降低(P<0.05),IL-4、TGF-β和IL-27表达水平升高(P<0.05)。IL-27中和的BMSCs与病理性T细胞共培养后,细胞培养上清中TNF-α、IFN-γ和IL-23的分泌水平明显升高(P<0.05),IL-4的分泌水平降低(P<0.01),IL-17的分泌水平显著升高(P<0.01)。结论:BMSCs经静脉移植治疗可以降低EAE发病程度,其作用机制是通过降低T淋巴细胞的反应性,通过IL-27的信号传导通路抑制EAE的靶细胞Th17产生生物学效应的。     

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.