Essential role of NK cells in IgG therapy for experimental autoimmune encephalomyelitis.
ABSTRACT: Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fc? receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fc? receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon ? and interleukin 17 production. The percentage of CD4(+)Foxp3(+) regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4(+) T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4(+)Foxp3(+) T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.
Project description:Interleukin-15 (IL-15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL-15 signaling by TM?-1 mAb treatment aggravated EAE severity. The key mechanism was not NK-cell depletion but depletion of CD8+ CD122+ T cells. Adoptive transfer of exogenous CD8+ CD122+ T cells to TM?-1-treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8+ CD122+ T cells prevented EAE development and significantly reduced IL-17 secretion. Naïve effector CD4+ CD25- T cells cultured with either CD8+ CD122+ T cells from wild-type mice or IL-15 transgenic mice displayed lower frequencies of IL-17A production with lower amounts of IL-17 in the supernatants when compared with production by effector CD4+ CD25- T cells cultured alone. Addition of a neutralizing antibody to IL-10 led to recovery of IL-17A production in Th17 cultures. Furthermore, coculture of CD8+ CD122+ T cells with effector CD4+ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL-15 dependent CD8+ CD122+ T cells. Taken together, these observations suggest that IL-15, acting through CD8+ CD122+ T cells, has a negative regulatory role in reducing IL-17 production and Th17-mediated EAE inflammation.
Project description:CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4(+)Foxp3(+) regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4(+)Foxp3(+) Treg homing has not been investigated.Infected wild-type (Ccr5(+/+)) and CCR5-deficient (Ccr5(-/-)) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs was used to evaluate the role of Tregs in JE progression.CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Compared to Ccr5(+/+) mice, Ccr5(-/-) mice unexpectedly showed increased responses of IFN-?(+)NK and CD8(+) T cells in the spleen, but not CD4(+) T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17(+)CD4(+) Th17 cells and correspondingly reduced CD4(+)Foxp3(+) Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17(+)CD4(+) Th17 cells, myeloid-derived Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Instead, adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-?) in the spleen and brain, and transferred CCR5(+) Tregs were found to produce IL-10.CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4(+)Foxp3(+) Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.
Project description:The transcription factor STAT1 plays a critical role in modulating the differentiation of CD4+ T cells producing IL-17 and GM-CSF, which promote the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The protective role of STAT1 in MS and EAE has been largely attributed to its ability to limit pathogenic Th cells and promote Tregs. Using mice with selective deletion of STAT1 in T cells (STAT1CD4-Cre), we identified a potentially novel mechanism by which STAT1 regulates neuroinflammation independently of Foxp3+ Tregs. STAT1-deficient effector T cells became the target of NK cell-mediated killing, limiting their capacity to induce EAE. STAT1-deficient T cells promoted their own killing by producing more IL-2 that, in return, activated NK cells. Elimination of NK cells restored EAE susceptibility in STAT1CD4-Cre mice. Therefore, our study suggests that the STAT1 pathway can be manipulated to limit autoreactive T cells during autoimmunity directed against the CNS.
Project description:In this study, we have evaluated our recently developed method for antigen-cell coupling using sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) heterobifunctional crosslinker in prevention and reversal of experimental autoimmune encephalomyelitis (EAE). We demonstrate that infusion of MOG35-55-coupled spleen cells (MOG-SP) significantly prevents and reverses EAE. Further studies show that the protected animals exhibit significantly delayed EAE upon EAE reinduction. Moreover, adoptive transfer of CD4+ T cells from the protected mice to naïve syngeneic mice renders the recipient mice resistant to EAE induction. Unexpectedly, CD4+ T cell proliferation is similar upon ex vivo stimulation by MOG35-55 amongst all groups. However, further analysis of those proliferating CD4+ T cells shows remarkable differences in Foxp3+ regulatory T cells (70% in MOG-SP groups versus 10-25% in control groups) and in IL-17+ cells (2-3% in MOG-SP groups versus 6-9% in control groups). In addition, we discover that MOG-SP treatment also significantly attenuates MOG35-55-responding IFN-?-producing Th1 cells. These findings suggest that MOG-SP treatment induces EAE protective MOG35-55-specific regulatory T cells and suppresses EAE pathogenic Th17 and Th1 cells. Our study provides a novel approach for antigen-based EAE immunotherapy, which can potentially be translated into clinical application for immunotherapy of multiple sclerosis.
Project description:<h4>Objective</h4>To evaluate the influence of oral laquinimod, a candidate multiple sclerosis (MS) treatment, on induction of T follicular helper cells, development of meningeal B cell aggregates, and clinical disease in a spontaneous B cell-dependent MS model.<h4>Methods</h4>Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with recombinant myelin oligodendrocyte glycoprotein (rMOG) protein. Spontaneous EAE was evaluated in C57BL/6 MOG p35-55-specific T cell receptor transgenic (2D2) × MOG-specific immunoglobulin (Ig)H-chain knock-in (IgH<sup>MOG-ki</sup> [Th]) mice. Laquinimod was administered orally. T cell and B cell populations were examined by flow cytometry and immunohistochemistry.<h4>Results</h4>Oral laquinimod treatment (1) reduced CD11c<sup>+</sup>CD4<sup>+</sup> dendritic cells, (2) inhibited expansion of PD-1<sup>+</sup>CXCR5<sup>+</sup>BCL6<sup>+</sup> T follicular helper and interleukin (IL)-21-producing activated CD4<sup>+</sup>CD44<sup>+</sup> T cells, (3) suppressed B cell CD40 expression, (4) diminished formation of Fas<sup>+</sup>GL7<sup>+</sup> germinal center B cells, and (5) inhibited development of MOG-specific IgG. Laquinimod treatment not only prevented rMOG-induced EAE, but also inhibited development of spontaneous EAE and the formation of meningeal B cell aggregates. Disability progression was prevented when laquinimod treatment was initiated after mice developed paralysis. Treatment of spontaneous EAE with laquinimod was also associated with increases in CD4<sup>+</sup>CD25<sup>hi</sup>Foxp3<sup>+</sup> and CD4<sup>+</sup>CD25<sup>+</sup>IL-10<sup>+</sup> regulatory T cells.<h4>Conclusions</h4>Our observations that laquinimod modulates myelin antigen-specific B cell immune responses and suppresses both development of meningeal B cell aggregates and disability progression in spontaneous EAE should provide insight regarding the potential application of laquinimod to MS treatment. Results of this investigation demonstrate how the 2D2 × Th spontaneous EAE model can be used successfully for preclinical evaluation of a candidate MS treatment.
Project description:Forkhead box P3 (Foxp3) is the major transcription factor controlling the development and function of regulatory T (Treg) cells. Previous studies have indicated epigenetic regulation of Foxp3 expression. Here, we investigated whether the deoxyribonucleic acid (DNA) methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) applied peripherally could modulate central nervous system (CNS) inflammation, by using a mouse experimental autoimmune encephalomyelitis (EAE) model. We found that disease activity was inhibited in a myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE mouse briefly pretreated with low-dose (0.15 mg/kg) 5-Aza, ameliorating significant CNS inflammatory responses, as indicated by greatly decreased proinflammatory cytokines. On the contrary, control EAE mice expressed high levels of IFN-? and interleukin (IL)-17. In addition, 5-Aza treatment in vitro increased GFP expression in CD4(+)GFP(-) T cells isolated from GFP knock-in Foxp3 transgenic mice. Importantly, 5-Aza treatment increased Treg cell numbers, in EAE mice, at both disease onset and peak. However, Treg inhibition assays showed 5-Aza treatment did not enhance per-cell Treg inhibitory function, but did maintain a lower activation threshold for effector cells in EAE mice. In conclusion, 5-Aza treatment prevented EAE development and suppressed CNS inflammation, by increasing the number of Treg cells and inhibiting effector cells in the periphery.
Project description:Experimental autoimmune encephalomyelitis (EAE) is a CD4? T cell mediated inflammatory demyelinating disease that is induced in mice by administration of peptides derived from myelin proteins. We developed EAE in SJL mice by administration of PLP139-151 peptide. The effect of treating these mice with 1?,25-Dihydroxyvitamin D? (vitamin D?), or with monomethyl fumarate (MMF) was then examined. We observed that both vitamin D? and MMF inhibited and/or prevented EAE in these mice. These findings were corroborated with isolating natural killer (NK) cells from vitamin D?-treated or MMF-treated EAE mice that lysed immature or mature dendritic cells. The results support and extend other findings indicating that an important mechanism of action for drugs used to treat multiple sclerosis (MS) is to enhance NK cell lysis of dendritic cells.
Project description:Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, demyelinating disease of the CNS that mimics human multiple sclerosis (MS), and it is thought to be driven by Th1 and Th17 myelin-reactive cells. Although adaptive immunity is clearly pivotal in the pathogenesis of EAE, with an essential role of CD4<sup>+</sup> T cells, little is known of early, innate responses in this experimental setting. CpG-rich oligodeoxynucleotides (ODNs), typically found in microbial genomes, are potent activators of TLR9 in plasmacytoid dendritic cells (pDCs). In this study, we compared the effects of two types of CpG, namely, type A and type B, on EAE. We found that treatment with CpG type A ODN (CpG-A), known to induce high amounts of IFN-<i>?</i> in pDCs, significantly reduced disease severity in EAE, relative to controls (12.63 ± 1.86 versus 23.49 ± 1.46, resp.; <i>p</i> = 0.001). Treatment also delayed onset of neurological deficits and reduced spinal cord demyelination, while increasing the percentage of splenic regulatory (Foxp3<sup>+</sup> CD4<sup>+</sup>) T cells. CpG-A likewise reduced the levels of IL-17 and IFN-<i>?</i> in the CNS. Mechanistic insight into those events showed that CpG-A promoted a regulatory phenotype in pDCs. Moreover, adoptive transfer of pDCs isolated from CpG-A-treated mice inhibited CNS inflammation and induced disease remission in acute-phase EAE. Our data thus identify a link between TLR9 activation by specific ligands and the induction of tolerance via innate immunity mechanisms.
Project description:CD4 T cells are involved in the pathogenesis of atherosclerosis, but atherosclerosis-specific CD4 T cells have not been described. Moreover, the chemokine(s) that regulates T-cell trafficking to the atherosclerotic lesions is also unknown.In Apoe(-/-) mice with mature atherosclerotic lesions (5 months of high fat diet), we find that most aortic T cells express CCR5 and interferon-? with a unique combination of cell surface markers (CD4(+)CD25(-)CD44(hi)CD62L(lo)) and transcription factors (FoxP3(+)T-bet(+)). We call these cells CCR5Teff. We investigated the role of CCR5 in regulating T-cell homing to the atherosclerotic aorta and the functionality of the CCR5Teff cells.CCR5Teff cells are exclusively found in the aorta and para-aortic lymph nodes of Apoe(-/-) mice. They do not suppress T-cell proliferation in vitro and are less potent than regulatory T cells at inhibiting cytokine secretion. Blocking or knocking out CCR5 or its ligand CCL5 significantly blocks T-cell homing to atherosclerotic aortas. Transcriptomic analysis shows that CCR5Teff cells are more similar to effector T cells than to regulatory T cells. They secrete interferon-?, interleukin-2, interleukin-10, and tumor necrosis factor. Adoptive transfer of these CCR5Teff cells significantly increases atherosclerosis.CCR5 is specifically needed for CD4 T-cell homing to the atherosclerotic plaques. CCR5(+)CD4 T cells express an unusual combination of transcription factors, FoxP3 and T-bet. Although CCR5Teff express FoxP3, we showed that they are not regulatory and adoptive transfer of these cells exacerbates atherosclerosis.
Project description:<h4>Background</h4>A growing body of evidence supports the hypothesis that vitamin D is an important environmental factor in the etiology of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS).<h4>Aim</h4>The purpose of this study was exploring the mechanisms underlying the beneficial effect of vitamin D3 in encephalomyelitis (EAE).<h4>Methods</h4>We treated monophasic experimental autoimmune EAE, induced in Lewis rat, with vitamin D3 and adoptively transfer tolerogenic bone marrow-derived DCs generated in the presence of vitamin D3.<h4>Results</h4>This study provides evidence that the in vivo administration of vitamin D3, as well as the adoptive transfer of vitamin D3 -induced IDO(+) immature/tolerogenic dendritic cells, leads to a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) regulatory T cells in the lymph nodes in a rat model of MS, experimental autoimmune EAE. Concomitant with the increase in this cell population, there is a significant decrease in the number of autoreactive T cells in the central nervous system. Bone marrow-derived DCs cultivated in the presence of vitamin D3 present a tolerogenic profile with high IL-10, TNF?, and IDO expression and decreased MHC-II and CD80 expression. The adoptive transfer of IDO (+) DCs induces a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) T cells in the lymph nodes, comparable with vitamin D3 treatment.<h4>Conclusion</h4>These mechanisms contribute actively to the generation of a microenvironment in the lymph nodes that suppresses the activation of encephalitogenic T cells, resulting in the downregulation of the inflammatory response in the central nervous system.