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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23790号 / 医博第4836号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山本 洋介, 教授 佐藤 俊哉, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects at least five million people worldwide. An increased expression of type I interferon (IFN) regulated genes is a hallmark of SLE, but the precise etiology of SLE initiation and flares is poorly understood. Because plasmacytoid dendritic cells (pDCs) are the primary type I IFN producers, their role in SLE has long been suspected, with murine pDC depletion models successfully delaying the progression of murine lupus-like disease. However, the mechanism behind how exactly how pDCs contribute to lupus autoimmunity is unknown, contributing to the current dearth lack of disease modifying treatments; current treatments only succeed in suppressing symptoms, and do not halt disease progression. In this study, we take a multifactorial approach to understanding the biology of pDCs in the context of lupus autoimmunity. Although the exact etiology of lupus is unknown, infections are an important environmental trigger for / Infectious Disease & Immunity

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24994号 / 医博第5028号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 椛島 健治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. The crosstalk between the gut microbiota and the immune system plays an important role in the tolerance induction to self-antigens both in the intestinal mucosa and at the systemic level. The MRL/lpr mouse model exhibits lupus-like symptoms early in life due to multiple SLE susceptible loci of the MRL background, plus the Faslpr mutation that offers an accelerated model. Recently, we experienced a loss of disease phenotype in our in-house colony compared to the previous published phenotype of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX) with our in-house MRL/lpr mice and found that the phenotypic drift, most significantly the attenuation of glomerulonephritis, was present in both colonies. In addition, while JAX mice and mice in our colony are genetically identical, there were minor differences in disease that might be due to differences in splenic microRNAs and the gut microbiota. Once confirming that our MRL/lpr mouse model was as good as that from JAX, we continued our investigation of the role of Lactobacilli in the pathogenesis of lupus-like disease in MRL/lpr mice. We previously published that the mixture of Lactobacillus reuteri (L. reuteri), L. oris, L. johnsonii, L. gasseri, and L. rhamnosus significantly attenuated disease in MRL/lpr mice by restoring the imbalance between regulatory T cells and T helper-17 cells. To further understand the role of Lactobacillus spp., we treated MRL/lpr mice with the combined culture supernatant of the 5 strains containing secreted metabolites, given that the metabolites may induce an immunosuppressive response. The results showed significant attenuation of the inflammation of the spleen and renal lymph nodes similar to the effect of the bacteria themselves. There was also a trending decrease of double-stranded DNA autoantibodies with the combined supernatant. We thus tested the strains individually but none was able to recapitulate the effect of the bacterial mixture. This suggests cell-to-cell contact among different strains of lactobacilli may be required in ameliorating the disease. With these results, we now have a better understanding of the role of probiotic Lactobacillus spp. against SLE. Future investigations will focus on the potential therapeutic effect of Lactobacillus spp. as a combination. Additionally, our group generated a Cx3cr1-deficient MRL/lpr mouse which exhibits a distinct phenotype of exacerbated glomerulonephritis with concurrent change of the gut microbiota composition compared to Cx3cr1+/+ MRL/lpr littermates. Interestingly, upon correction of the gut microbiota with Lactobacillus administration, the phenotype of exacerbated glomerulonephritis was reversed, suggesting that CX3CR1 controls glomerulonephritis in MRL/lpr mice through a gut microbiota-dependent mechanism. In addition, a collaborative project revealed that Cx3cr1 deficiency-mediated pathogenic mechanisms also contributed to SLE-associated cardiovascular disease in MRL/lpr mice. The results of these studies will lead to the identification of new therapeutic targets for the treatment of two severe manifestations, glomerulonephritis and cardiovascular disease, that together account for most of the morbidity and mortality in SLE. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is an autoimmune disease with no known cure. Commensal microbiota, mostly bacteria living in our gut, and the immune system have a strong relationship in maintaining a healthy state of the gut as well as the whole body. Alterations in the gut microbiota, known as dysbiosis, can facilitate SLE in human and animal models. Current treatments for SLE are primarily focused on using immunosuppressants, but the side effects are still a concern. The use of long-term nonselective immunosuppressant conducts a higher incidence of severe infections in SLE patients. It is thus necessary to develop new approaches and treatments against SLE. My dissertation research is focused on understanding how commensal bacteria influence in the pathogenesis of SLE. My studies have shown that environmental factors can manipulate the gut microbiota leading to different disease outcomes. In addition, following upon previously published studies from our laboratory, I have delineated the mechanism how a mixture of probiotic Lactobacilli can exert a beneficial effect against lupus. Finally, I have revealed a new, CX3CR1-mediated mechanism through which the gut microbiota controls kidney disease in the MRL/lpr lupus-prone mouse model.

Systemic lupus erythematosus (SLE) is a chronic multifactorial inflammatory autoimmune disease with heterogeneous clinical manifestations. Among different manifestations, lupus nephritis (LN) remains a major cause of morbidity and mortality. There are few FDA approved treatments for LN. In general, they are non-selective and lead to global immunosuppression with significant side effects including an increased risk of infection. In the past 60 years, only one new drug, belimumab was approved for lupus disease with modest efficacy in clinic and not approved for patients suffering for nephritis. Therefore, it is urgent to develop new treatments to replace or reduce the use of current ones. Histone deacetylase 6 (HDAC6) plays a variety of biologic functions in a number of important molecular pathways in diverse immune cells. Both innate and adaptive immune cells contribute to pathogenesis of lupus. Among those cells, B cells play a central role in pathogenesis of lupus nephritis in an anti-body dependent manner through differentiation into plasma cells (PCs). As a result, HDAC6 inhibitors represent an entirely new class of agents that could have potent effects in SLE. Importantly, the available toxicity profile suggests that HDAC6 inhibitors could be advanced into SLE safely. We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. ACY-738 is a hydroxamic acid HDAC6 inhibitor that is highly selective for HDAC6. In our current studies, we tested if an orally selective HDAC6 inhibitor, ACY-738, would decrease disease pathogenesis in a lupus mouse model with established early disease. Moreover, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. In order to define the mechanism by which HDAC6 inhibition decreases disease pathogenesis in NZB/W mice by using RNAseq to evaluate the transcriptomic signatures of splenocytes from treated and untreated mice coupled with applied computational cellular and pathway analysis. In addition, we sought to bridge between the transcriptomic data obtained from the HDAC6 treated mice and human gene expression information to determine the relevance to this target in possibly controlling human lupus. We treated 20-week-old (early-disease) NZB/W F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 4~5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by ELISA, and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells, and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. Our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. RNA sequence data showed that PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was in part due to inhibition of B cell development and response. RNA sequence data analysis show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggests that a critical event might be modulation of cellular metabolism. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease by which immune cells mistakenly attacks healthy self-cells in different organs. Kidney inflammation occurs in nearly 50% of patients with lupus resulting in kidney damage leading to end stage renal disease. Lupus nephritis (LN) is major cause of morbidity and mortality associated with SLE. Current treatments for LN consist primarily of immunosuppressants that block the immune response and leave the patients with unwanted side effects including an increased risk of infection. To circumvent the unwanted side effects, we explored a novel mechanism to target the immune response. My project was to determine whether histone deacetylase 6 (HDAC6) inhibition would suppress the autoimmune inflammatory response in lupus. We found that inhibition of HDAC6 was effective at attenuating early LN, probably by down-regulating innate immune response, which suppressed subsequent adaptive immune responses downstream. HDAC6 inhibition affected the innate immune response by inhibiting type I interferon production by plasmacytoid dendritic cells. HDAC6 inhibition affected the cell mediated immune response by decreasing T helper cell and B cell activation. To determine the mechanism by which HDAC6 inhibits immune cells activation, we used RNAseq to reveal HDAC6 inhibition on multiple signaling events associated with the induction of lupus disease. These results suggest that HDAC6 could be a potential therapeutic target in the early stage of LN.