Infiltrated Cells in Experimental Allergic Encephalomyelitis by Additional Intracerebral Injection in Myelin-Basic-Protein-SensitizedB6 Mice

We previously reported that murine experimental allergic encephalomyelitis can be induced by an additional intraperitoneal and intracerebral (i.c.) restimulation in resistant B6 mice after standard immunization with myelin antigens in complete Freund's adjuvant andBordetella pertussis coadjuvant. Neutrophils infiltrated into perivascular spaces at 12 h, followed by mononuclear cells 24 h after i.c. injection. In this study, we report that the i.c. injection induced the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The kinetic expression of ICAM-1 or VCAM-1 on brain endothelial cells paralleled the infiltration of neutrophils and mononuclear cells, respectively. The infiltrated lymphocytes also expressed very late antigen-4 (VLA-4) molecules. The microvascular endothelial cells were positive for VCAM-1, whereas the surrounding mononuclear cells were VLA-4 positive. Furthermore, we found a unique subpopulation of cells with characteristics of CD4-CD8-V8⁺ markers. The kinetic studies of this population showed that these cells were transiently depleted from 12 to 24 h after i.c. challenge (before the development of clinical symptoms) in cervical lymph nodes. These CD4-CD8-V8⁺ cells can be expanded by in vitro culture with myelin basic protein or IL-2. No significant changes of CD4⁺/CD8⁺ cells were noted. CD4⁺CD8^–CD3⁺ cells were also found in brain by double histochemical stains and were the major infiltrating cells at 24 or 48 h after i.c. challenge.     

Suppression of Th1-Mediated Autoimmunity by Embryonic Stem Cell-Derived Dendritic Cells

We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.     

The tick saliva immunosuppressor, Salp15, contributes to Th17-induced pathology during Experimental Autoimmune Encephalomyelitis

Salp15 is a tick saliva protein that inhibits CD4⁺ T cell differentiation through its interaction with CD4. The protein inhibits early signaling events during T cell activation and IL-2 production. Because murine Experimental Autoimmune Encephalomyelitis development is mediated by central nervous system-infiltrating CD4⁺ T cells that are specific for myelin-associated proteins, we sought to determine whether the treatment of mice with Salp15 during EAE induction would prevent the generation of proinflammatory T cell responses and the development of the disease. Surprisingly, Salp15-treated mice developed more severe EAE than control animals. The treatment of EAE-induced mice with the tick saliva protein did not result in increased infiltration of T cells to the central nervous system, indicating that Salp15 had not affected the permeability of the blood-brain barrier. Salp15 treatment did not affect the development of antibody responses against the eliciting peptide or the presence of IFNγ in the sera. The treatment with Salp15 resulted, however, in the increased differentiation of Th17 cells in vivo, as evidenced by higher IL-17 production from PLP_139-151-specific CD4⁺ T cells isolated from the central nervous system and the periphery. In vitro, Salp15 was able to induce the differentiation of Th17 cells in the presence of IL-6 and the absence of TGFβ These results suggest that a conductive milieu for the differentiation of Th17 cells can be achieved by restriction of the production of IL-2 during T cell differentiation, a role that may be performed by TGFβ and other immunosuppressive agents.     

Th1、Th2和Th17型细胞在类风湿性关节炎和系统性红斑狼疮中的活化特点

探讨Th1、Th2和Th17型细胞在类风湿性关节炎(RA)和系统性红斑狼疮(SLE)发病机制中的作用。收集37例RA患者、25例SLE患者和34例健康人的抗凝血,应用ELISA检测血清中IFN-γ、IL-10和IL-17的水平。与健康对照组比较,RA和SLE患者血清中IFN-γ的水平均具有统计学意义(P<0.05);SLE患者IL-10水平出现有意义的升高(P<0.05);而RA患者IL-17的升高具有统计学意义(P<0.05)。由此提示Th1、Th2和Th17细胞在自身免疫性疾病中均发挥不同的重要作用。     

Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region

Th17 cells have been reported to produce proinflammatory cytokines like Interleukin-17, IL-22, and regarded as important players in various inflammatory diseases. One of the IL-12 cytokine family cytokines, IL-23, composed of p19 and p40 subunit, is known for its potential to promote Th17 development and IL-17 producing, and the IL-23/IL-17 pathway is considered to be potential therapeutic target for autoimmune inflammation responses. Knockout mice deficient in either IL-23 or IL-17 related genes can suppress the allergic responses. Several IL-23 or IL-17 neutralizing agents are being evaluated in vitro or in vivo to disrupt the IL-23/IL-17 axis. Herein, we report that prokaryotically expressed soluble IL-23 receptor cytokine-binding homology region as an endogenous extracellular receptor analogue could be a natural antagonist against IL-23/IL-17 axis. We provide evidence that IL23R-CHR can bind to IL-23 in a dose-dependent manner in vitro, and block IL-23 signal by IL23R-CHR reducing the RORγt expression, which in turn lowers the expression of IL-17/IL-22, thus protecting naive CD4+ T cells against Th17 development. Together, this study indicates the importance of IL-23 pathway in Th17 development and the negative regulation of Th17 development by IL23R-CHR, and highlights the important roles of the soluble receptor extracellular region in the therapeutic strategy of neutralizing IL-23.     

The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation

CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4⁺, HLA-DR⁺, Ki67⁺, and IL-10⁺ Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.     

Prevalence and Clinical Relevance of T-Helper Cells,Th17 and Th1, in Hepatitis B Virus-Related Hepatocellular Carcinoma

Background and Aims

An immune imbalance in the cytokine profile exerts a profound influence on the progression of hepatitis B virus (HBV) infections and hepatocellular carcinoma (HCC). The present study evaluated the immune status of T helper (Th) 17 and Th1 cells in patients with HBV-related and non-HBV-related HCC.

Methods

We randomly enrolled 150 patients with HCC. Blood samples and tissue samples were obtained. The distributions and phenotypic features of Th17 and Th1 cells were determined by flow cytometry and/or immunohistochemistry.

Results

Compared to corresponding non-tumor regions, the levels of Th17 and Th1 cells were significantly increased in tumors of patients with HCC (P<0.001). The intratumoral densities of IL-17-producing cells and IFN-γ-producing cells were associated with overall survival (OS, P = 0.001) and disease-free survival (DFS, P = 0.001) of patients with HCC. The ratio of Th17 to Th1 in HBV-related HCC was higher than in non-HBV-related HCC. A multivariate Cox analysis revealed that the Th17 to Th1 ratio was an independent prognostic factor for OS (HR = 2.651, P = 0.007) and DFS (HR = 2.456, P = 0.002).

Conclusions

HBV infections can lead to an imbalance in immune status in patients with HCC. An elevated Th17 to Th1 ratio may promote tumor progression. The Th17 to Th1 ratio could serve as a potential prognostic marker for scoring the severity of HCC.     

Expansion of Pathogen-Specific Mono- and Multifunctional Th1 and Th17 Cells in Multi-Focal Tuberculous Lymphadenitis

Background

Th1 and Th17 responses are known to play an important role in immunity to pulmonary tuberculosis (PTB), although little is known about their role in extrapulmonary forms of tuberculosis (TB).

Methods

To identify the role of Th1, Th17, and Th22 cells in multi-focal TB lymphadenitis (TBL), we examined mycobacteria–specific immune responses in the whole blood of individuals with PTB (n = 20) and compared them with those with TBL (n = 25).

Results

Elevated frequencies of CD4⁺ T cells expressing IFN- γ, TNF-α, and IL-2 were present in individuals with TBL compared with those with PTB at baseline and in response to ESAT-6 and CFP-10. Similarly, increased frequencies of CD4⁺ T cells expressing IL-17A, IL-17F, and IFN-γ were also present in individuals with TBL at baseline and following ESAT-6 and CFP-10 stimulation although no significant difference in frequency of Th22 cells was observed. Finally, frequencies of Th1 (but not Th17) cells exhibited a significantly negative correlation with natural regulatory T cell frequencies at baseline.

Conclusions

Multi-focal TB lymphadenitis is therefore characterized by elevated frequencies of Th1 and Th17 cells, indicating that Th1 and Th17 responses in TB disease are probably correlates of disease severity rather than of protective immunity.     

Vitamin D Therapy in Experimental Allergic Encephalomyelitis Could be Limited by Opposing Effects of Sphingosine 1-Phosphate and Gelsolin Dysregulation

Several studies support a protective effect of vitamin D on multiple sclerosis and experimental allergic encephalomyelitis (EAE), but the mechanisms of these favorable effects are unclear. Our study demonstrates that sphingosine 1-phosphate (S1P) is upregulated in the serum and spinal cords of EAE rats, but that vitamin D reverses the upregulation to alleviate inflammation. Vitamin D, however, cannot prevent the disease process, suggesting that other factors may be involved. To identify additional factors that might limit vitamin D efficacy, we assessed the effects of vitamin D on plasma gelsolin (pGSN), a regulator of S1P that is downregulated in the CSF of MS patients. Our results show that pGSN is downregulated in the serum of EAE rats, whereas its cellular form, cytoplasmic gelsolin (cGSN), is upregulated in the spinal cord of EAE rats. Importantly, vitamin D causes a downregulation of both pGSN and cGSN, which may counteract the positive effects of S1P decrease. Furthermore, 48 and 42?kDa caspase-3 cleavage products of cGSN are detected in EAE spinal cords, suggesting enhanced apoptotic activity, but these cleaved products undergo a similar decrease upon vitamin D treatment. To directly test the role of cGSN in the apoptotic process, we performed RNA interference in PC-12, a rat sympathetic nerve cell line. Results verify that cGSN suppresses apoptosis induced by TNF-α. Collectively, these results support a therapeutic effect of vitamin D that is derived from its ability to reduce S1P, but is limited by its simultaneous effect in reducing pGSN and cGSN. Based on these observations, we postulate that combined therapy with recombinant human pGSN and vitamin D may produce more beneficial effect in treating multiple sclerosis.     

Phenotype switching by inflammation-inducing polarized Th17 cells, but not by Th1 cells

Th1 and Th17 cells are characterized by their expression of IFN-gamma or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-gamma suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-gamma was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-gamma or both IFN-gamma and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-gamma or IFN-gamma/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20-25% nonpolarized cells than in recipients of 35-40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-gamma or IFN-gamma/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-gamma t was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-gamma or IFN-gamma/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Partial Deficiency of Sphingosine-1-Phosphate Lyase Confers Protection in Experimental Autoimmune Encephalomyelitis

Background

Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span.

Methodology

We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation.

Principal Findings

The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS.

Significance

The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.