Site Of Autoimmune Inflammation Research Articles

Pathogenic T cell subset Th17 is regulated by chromatin remodeling dependent manner to switch Th1 phenotype

Abstract In many autoimmune disease such as type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease (IBD), it has been reported that pathogenic Th17 cells exhibit remarkable plasticity to classical Th1 phenotype at the site of autoimmune inflammation. This effecter Th17 cells is also called non-classical Th1 cells because they show the ability to produce IFNγ, similar to conventional Th1 cells, and also continue to produce IL-17 cytokine. The precise mechanism of transcriptional regulation in Th17/IFNγ cells is still not clear yet. By using the splenic naive CD4 T cells, cells were cultured under the Th17 condition including IL-6 and TGFβ for 3 days to 7 days and then cells were added IL-12 to provide Th1-like differentiation condition. In vitro induced Th17 cells were stimulated with anti TCR antibody and assessed the ratio of IL-17 protein production, RORγt and T-bet expression as protein level by flow cytometer. Then, induced Th17 cells were performed to the secondary culture under the several concentration of IL-12 and TGFβ to be able to detect the production of IFNγ, and T-bet. Also we analyzed the specific gene expressions of transcription factor by RT-PCR or quantitative PCR on each day. In this study, we focused on the plasticity of Th17 to Th1 and also compared to the effecter T cell function. We focused on the function of chromatin remodeling in Th17 cells producing both Il-17 and IFNγ by performing ChIP analysis of double producing Th subset and detected that this type plasticity is unique in pathogenic Th17 cell.

B cells in the formation of tertiary lymphoid organs in autoimmunity, transplantation and tumorigenesis

Tertiary lymphoid organs named also tertiary lymphoid structures (TLS) often occur at sites of autoimmune inflammation, organ transplantation and cancer. Although the mechanisms for their formation/function are not entirely understood, it is known that TLS can display features of active germinal centres supporting the proliferation and differentiation of (auto)-reactive B cells. In this Review, we discuss current knowledge on TLS-associated B cells with particular reference on how within diseased tissues these structures are linked to either deleterious or protective outcomes in patients and the potential for therapeutic targeting of TLS through novel drugs.

A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo.

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

Ex-Th17 (Nonclassical Th1) Cells Are Functionally Distinct from Classical Th1 and Th17 Cells and Are Not Constrained by Regulatory T Cells.

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly resistant to suppression of proliferation and cytokines by regulatory T cells. Finally, we showed that ex-Th17 cells accumulated in the joints of rheumatoid arthritis patients. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the rheumatoid arthritis joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Self-Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon-γ Blockade.

Resistance of Teff cells to Treg cell-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self-sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically. CD8+ or CD4+ Teff cells were cultured with or without antigen-presenting cells (APCs) in Treg cell-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff cells were crosscultured with peripheral blood (PB) Treg cells from JIA patients or healthy controls. Tumor necrosis factor (TNF) or interferon-γ (IFNγ) blocking agents were used to restore Teff cell responsiveness to suppression. Suppression of cell proliferation and cytokine production in CD8+ Teff cells from the SF of JIA patients was severely impaired compared to that in CD8+ Teff cells from the PB of JIA patients, regardless of the presence of APCs and CD4+ Teff cells. Similar to CD4+ Teff cells, impaired suppression of CD8+ Teff cells was shown to be an intrinsic feature of this cell population. While TNF blockade restored both CD8+ and CD4+ Teff cell susceptibility to suppression, autocrine release of IFNγ selectively sustained CD8+ Teff cell resistance, which could be relieved by IFNγ blockade. Unlike CD4+ Teff cells, resistance of CD8+ Teff cells to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFNγ, and blockade of IFNγ restores CD8+ Teff cell responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFNγ to restore immune regulation in JIA.

T‐bet is essential for Th1‐mediated, but not Th17‐mediated, CNS autoimmune disease

T cells that produce both IL-17 and IFN-γ, and co-express ROR-γt and T-bet, are often found at sites of autoimmune inflammation. However, it is unknown whether this co-expression of T-bet with ROR-γt is a prerequisite for immunopathology. We show here that T-bet is not required for the development of Th17-driven experimental autoimmune encephalomyelitis (EAE). The disease was not impaired in T-bet−/− mice and was associated with low IFN-γ production and elevated IL-17 production among central nervous system (CNS) infiltrating CD4+ T cells. T-bet−/− Th17 cells generated in the presence of IL-6/TGF-β/IL-1 and IL-23 produced GM-CSF and high levels of IL-17 and induced disease upon transfer to naïve mice. Unlike their WT counterparts, these T-bet−/– Th17 cells did not exhibit an IL-17→IFN-γ switch upon reencounter with antigen in the CNS, indicating that this functional change is not critical to disease development. In contrast, T-bet was absolutely required for the pathogenicity of myelin-responsive Th1 cells. T-bet-deficient Th1 cells failed to accumulate in the CNS upon transfer, despite being able to produce GM-CSF. Therefore, T-bet is essential for establishing Th1-mediated inflammation but is not required to drive IL-23-induced GM-CSF production, or Th17-mediated autoimmune inflammation.

Foxp3+Treg cells in the inflamed CNS are insensitive to IL‐6‐driven IL‐17 production

Foxp3(+) T regulatory (Treg) cells can be induced to produce interleukin (IL)-17 by in vitro exposure to proinflammatory cytokines, drawing into question their functional stability at sites of inflammation. Unlike their splenic counterparts, Treg cells from the inflamed central nervous system (CNS-Treg cells) during EAE resisted conversion to IL-17 production when exposed to IL-6. We show that the highly activated phenotype of CNS-Treg cells includes elevated expression of the Th1-associated molecules CXCR3 and T-bet, but reduced expression of the IL-6 receptor α chain (CD126) and the signaling chain gp130. We found a lack of IL-6 receptor on all CNS CD4(+) T cells, which was reflected by an absence of both classical and trans-IL-6 signaling in CNS CD4(+) cells, compared with their splenic counterparts. We propose that extinguished responsiveness to IL-6 (via down-regulation of CD126 and gp130) stabilizes the regulatory phenotype of activated Treg cells at sites of autoimmune inflammation.

Increased stimulatory capacity of antigen presenting cells at the site of autoimmune inflammation interferes with regulatory T cell function

Background FOXP3+ regulatory T cells (Treg) are critical in maintaining self tolerance and are therefore considered important targets for the treatment of autoimmune disease. However, environmental factors at the site of autoimmune inflammation, such as enhanced costimulatory potential of antigen presenting cells (APC) and increased proinflammatory cytokine production, can negatively affect Treg function, thereby limiting effectiveness of these Treg targeted approaches. Aim Here we studied the phenotype of APC present at the site of inflammation in patients with Juvenile Idiopathic Arthritis (JIA) and investigated whether these cells can interfere with Treg mediated suppression. Methods Mononuclear cells were isolated from peripheral blood (PB) of healthy controls (HC) and from paired PB and synovial fluid (SF) of JIA patients. The phenotype of APC was analysed using flow cytometry. In vitro suppression assays were performed to study T cell activation and Treg mediated suppression in the presence of SF and PB derived APC. Results Monocytes from the site of inflammation displayed a more pro-inflammatory phenotype, with significantly increased costimulatory molecule expression, compared to monocytes from PB. In line with this pro-inflammatory phenotype, SF APC induced enhanced proliferation of effector cells and decreased suppression of effector cell proliferation in the presence of Treg. Conclusions APC from the site of inflammation have an enhanced stimulatory capacity that interferes with Treg mediated suppression. Therefore, this increased stimulatory potential should be targeted as well, in order for a Treg enhancing approach to be fully effective in the treatment of autoimmune inflammation.

B7‐H1 and CD8+ Treg: The enigmatic role of B7‐H1 in peripheral tolerance

The interaction between B7-H1 (PD-L1) expressed on APC with PD-1 expressed by T cells was shown previously to result in inhibition of T-cell activation and autoimmune diseases. A paper in this issue of the European Journal of Immunology demonstrates that DC B7-H1 expression can in fact enhance autoimmunity, rather than suppress it. Using a model of direct injection of self antigen-loaded DC into the CNS, the authors demonstrate that DC with intact B7-H1 expression exacerbate CNS autoimmune disease. Importantly, the improved disease outcome in animals treated with B7-H1(-/-) DC is a result of a population of CD8(+) Treg cells that expand at the site of autoimmune inflammation.

Neutrophil-derived leukotriene B4 is required for inflammatory arthritis

Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.