Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Dispensable roles of Gsdmd and Ripk3 in sustaining IL-1ß production and chronic inflammation in Th17-mediated autoimmune arthritis.

Programmed necrosis, such as necroptosis and pyroptosis, is a highly pro-inflammatory cellular event that is associated with chronic inflammation. Although there are various triggers of pyroptosis and necroptosis in autoimmune tissue inflammation and subsequent lytic forms of cell death release abundant inflammatory mediators, including damage-associated molecular patterns and IL-1ß, capable of amplifying autoimmune Th17 effector functions, it remains largely unclear whether the programs play a crucial role in the pathogenesis of autoimmune arthritis. We herein report that Gasdermin D (Gsdmd) and receptor interacting serine/threonine kinase 3 (Ripk3)-key molecules of pyroptosis and necroptosis, respectively-are upregulated in inflamed synovial tissues, but dispensable for IL-1ß production and the development of IL-17-producing T helper (Th17) cell-mediated autoimmune arthritis in SKG mice. Gsdmd-/-, Ripk3-/-, or Gsdmd-/- Ripk3-/- SKG mice showed severe arthritis with expansion of arthritogenic Th17 cells in the draining LNs and inflamed joints, which was comparable to that in wild-type SKG mice. Despite the marked reduction of IL-1ß secretion from Gsdmd-/- or Ripk3-/- bone marrow-derived DCs by canonical stimuli, IL-1ß levels in the inflamed synovium were not affected in the absence of Gsdmd or Ripk3. Our results revealed that T cell-mediated autoimmune arthritis proceeds independently of the pyroptosis and necroptosis pathways.

Macrophage activation syndrome in adult dermatomyositis: a case-based review.

Macrophage activation syndrome (MAS) is a severe and life-threatening syndrome associated with autoimmune diseases, characterized by fever, hepatosplenomegaly, and pancytopenia. Dermatomyositis (DM) is one of the causes of MAS; however, its clinical characteristics in DM patients remain unclear. This study aimed to present a case of anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive DM complicated by MAS in a 29-year-old woman and to review the literatures including similar cases. Even though symptoms and cytopenia of our patient were refractory to combination therapy, including glucocorticoids, immunosuppressants, and plasma exchange, the administration of rituximab (RTX) resulted in rapid clinical improvement and glucocorticoid reduction. The literature review revealed 18 adult patients with DM associated MAS. Most patients developed MAS within 3 months from DM onset. A monotherapy of glucocorticoid was insufficient to control the disease, and the mortality of MAS in DM was higher than that of MAS in other rheumatic diseases, despite being treated by various means. RTX may be an effective treatment for patients with DM complicated by MAS who are refractory to conventional therapy. Anti-MDA5 antibody could influence the development of MAS; however, further investigations are needed to elucidate the association between myositis-specific antibody and MAS.

Strain-Specific Manifestation of Lupus-like Systemic Autoimmunity Caused by Zap70 Mutation.

A defect in TCR-proximal signaling is a major characteristic of CD4 T cells in systemic lupus erythematosus; however, it is not fully known how defects in TCR signaling lead to lupus-like systemic autoimmunity characterized by germinal center development and autoantibody production against nuclear Ags. In this study, we show that SKG mice, which develop autoimmune arthritis in a BALB/c background due to defective TCR signaling by a Zap70 mutation, develop lupus-like systemic autoimmune disease in the C57BL/6 (B6) background (B6SKG mice). B6SKG mice showed multiorgan inflammation with immune complex deposition and anti-dsDNA Ab production. Follicular helper T cells (Tfh), which help germinal center formation, were spontaneously expanded in B6SKG mice. Th cells secreting IFN-γ or IL-17 and regulatory T cells were also increased in B6SKG mice compared with wild-type B6 mice, with the regulatory T cell subpopulation losing the expression of CD25. Among the factors related to Tfh differentiation, the number of dendritic cells and the expression levels of the costimulatory molecules CD80, CD86, and ICOSL in dendritic cells but not in B cells were specifically increased in wild-type B6 mice compared with BALB/c mice. The inhibition of these costimulatory molecules suppressed Tfh development and lupus-like autoimmunity. Thus, a defect in TCR-proximal signaling leads to lupus-like systemic autoimmunity under the specific genetic background that facilitates Tfh development.

Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis.

Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention.

Identification of Novel and Noninvasive Biomarkers of Acute Cellular Rejection After Liver Transplantation by Protein Microarray.

BACKGROUND: Acute cellular rejection (ACR) is one of the main factors in transplanted organ failure in liver transplantation. A precise marker for diagnosing or predicting rejection is not currently available; therefore, invasive liver biopsy is standard procedure. To develop a noninvasive method for precise diagnosis of ACR, we evaluated autoantibodies from patient sera as potential biomarkers using protein microarrays (seromics). METHODS: Sera from hepatitis C virus-positive ACR patients were compared to three hepatitis C virus cirrhosis control groups and healthy volunteers. The control groups consisted of 2 no-ACR groups obtained on postoperative day 28 and 1 year after transplantation and a preoperative group obtained 1 day before transplantation. For validation, we evaluated whether the candidate antibodies can distinguish ACR from other types of liver dysfunction after liver transplantation using enzyme-linked immunosorbent assay. RESULTS: Seromic analysis by weighted average difference (WAD) ranking and Mann-Whitney U test revealed a significant increase of 57 autoantibodies in the sera of ACR patients with liver dysfunction. Among the 57 candidates, autoantibodies to charged multivesicular body protein 2B, potassium channel tetramerization domain containing 14, voltage gated subfamily A regulatory beta subunit 3, and triosephosphate isomerase 1 were regarded as potential biomarkers of ACR after liver transplantation. Using 20 ACR patients with variable backgrounds for validation, the autoantibodies to charged multivesicular body protein 2B and triosephosphate isomerase 1 were significantly increased in ACR patients compared to other control groups. CONCLUSIONS: A panel of autoantibodies identified by seromics as potential noninvasive biomarkers was clinically useful for diagnosing ACR after liver transplantation.

Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine.

OBJECTIVE: The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis. METHODS: We analyzed the fecal microbiota of patients with early RA and healthy controls, using 16S ribosomal RNA-based deep sequencing. We inoculated fecal samples from RA patients and healthy controls into germ-free arthritis-prone SKG mice and evaluated the immune responses. We also analyzed whether the lymphocytes of SKG mice harboring microbiota from RA patients react with the arthritis-related autoantigen 60S ribosomal protein L23a (RPL23A). RESULTS: A subpopulation of patients with early RA harbored intestinal microbiota dominated by Prevotella copri; SKG mice harboring microbiota from RA patients had an increased number of intestinal Th17 cells and developed severe arthritis when treated with zymosan. Lymphocytes in regional lymph nodes and the colon, but not the spleen, of these mice showed enhanced interleukin-17 (IL-17) responses to RPL23A. Naive SKG mouse T cells cocultured with P copri-stimulated dendritic cells produced IL-17 in response to RPL23A and rapidly induced arthritis. CONCLUSION: We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease.

T cells that mediate autoimmune diseases such as rheumatoid arthritis (RA) are difficult to characterize because they are likely to be deleted or inactivated in the thymus if the self antigens they recognize are ubiquitously expressed. One way to obtain and analyze these autoimmune T cells is to alter T cell receptor (TCR) signaling in developing T cells to change their sensitivity to thymic negative selection, thereby allowing their thymic production. From mice thus engineered to generate T cells mediating autoimmune arthritis, we isolated arthritogenic TCRs and characterized the self antigens they recognized. One of them was the ubiquitously expressed 60S ribosomal protein L23a (RPL23A), with which T cells and autoantibodies from RA patients reacted. This strategy may improve our understanding of the underlying drivers of autoimmunity.

Thymus, innate immunity and autoimmune arthritis: interplay of gene and environment.

A hypomorphic mutation of the gene encoding zeta-associated protein-70 (ZAP-70), a signaling molecule in T cells, produces autoimmune arthritis in mice under a microbially conventional condition but not in a clean environment. The genetic anomaly alters thymic selection of self-reactive T cells as well as natural regulatory T cells and their respective functions. Highly self-reactive polyclonal T cells, including arthritogenic ones, thus produced by the thymus strongly recognize self-antigens presented by antigen-presenting cells, stimulate them to up-regulate co-stimulatory molecules and secrete cytokines that drive naïve self-reactive T cells to differentiate into autoimmune effector Th17 cells. Administration of microbial products and activation of complement can facilitate the differentiation, evoking clinically overt arthritis in a microbially clean environment. Furthermore, mutation-dependent graded attenuation of T cell receptor signaling alters disease phenotypes and the dependency of disease occurrence on the environment. These findings provide a model of how genetic and environmental factors, in association, cause autoimmune diseases such as rheumatoid arthritis.

Graded attenuation of TCR signaling elicits distinct autoimmune diseases by altering thymic T cell selection and regulatory T cell function.

Mice with a mutation of the zeta-associated protein of 70 kDa gene (skg mutation) are genetically prone to develop autoimmune arthritis, depending on the environment. In a set of mice with the mutation, the amount of zeta-associated protein of 70 kDa protein as well as its tyrosine phosphorylation upon TCR stimulation decreased from +/+, skg/+, skg/skg, to skg/- mice in a stepwise manner. The reduction resulted in graded alterations of thymic positive and negative selection of self-reactive T cells and Foxp3(+) natural regulatory T cells (Tregs) and their respective functions. Consequently, skg/- mice spontaneously developed autoimmune arthritis even in a microbially clean environment, whereas skg/skg mice required stimulation through innate immunity for disease manifestation. After Treg depletion, organ-specific autoimmune diseases, especially autoimmune gastritis, predominantly developed in +/+, at a lesser incidence in skg/+, but not in skg/skg BALB/c mice, which suffered from other autoimmune diseases, especially autoimmune arthritis. In correlation with this change, gastritis-mediating TCR transgenic T cells were positively selected in +/+, less in skg/+, but not in skg/skg BALB/c mice. Similarly, on the genetic background of diabetes-prone NOD mice, diabetes spontaneously developed in +/+, at a lesser incidence in skg/+, but not in skg/skg mice, which instead succumbed to arthritis. Thus, the graded attenuation of TCR signaling alters the repertoire and the function of autoimmune T cells and natural Tregs in a progressive manner. It also changes the dependency of disease development on environmental stimuli. These findings collectively provide a model of how genetic anomaly of T cell signaling contributes to the development of autoimmune disease.

Complement drives Th17 cell differentiation and triggers autoimmune arthritis.

Activation of serum complement triggers Th17 cell-dependent spontaneous autoimmune disease in an animal model. In genetically autoimmune-prone SKG mice, administration of mannan or beta-glucan, both of which activate serum complement, evoked Th17 cell-mediated chronic autoimmune arthritis. C5a, a chief component of complement activation produced via all three complement pathways (i.e., lectin, classical, and alternative), stimulated tissue-resident macrophages, but not dendritic cells, to produce inflammatory cytokines including IL-6, in synergy with Toll-like receptor signaling or, notably, granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF secreted by activated T cells indeed enhanced in vitro IL-6 production by C5a-stimulated macrophages. In vivo, C5a receptor (C5aR) deficiency in SKG mice inhibited the differentiation/expansion of Th17 cells after mannan or beta-glucan treatment, and consequently suppressed the development of arthritis. Transfer of SKG T cells induced Th17 cell differentiation/expansion and produced arthritis in C5aR-sufficient recombination activating gene (RAG)-/- mice but not in C5aR-deficient RAG-/- recipients. In vivo macrophage depletion also inhibited disease development in SKG mice. Collectively, the data suggest that complement activation by exogenous or endogenous stimulation can initiate Th17 cell differentiation and expansion in certain autoimmune diseases and presumably in microbial infections. Blockade of C5aR may thus be beneficial for controlling Th17-mediated inflammation and autoimmune disease.

Reduction of opioid side effects by prophylactic measures of palliative care team may result in improved quality of life.

PURPOSE: In February 2002, the palliative care team was established in Ikeda Municipal Hospital to improve palliative care. We investigated changes in the incidences of side effects related to opioids, and evaluated palliative care team activities. METHODS: Regarding inpatients for whom narcotics were prescribed in our hospital in the years of 2002 (from October 1, 2002 until September 30, 2003), 2004 (from October 1, 2004 until September 30, 2005), and 2006 (from October 1, 2006 until September 30, 2007), we surveyed the rates at which laxatives or antiemetics were prescribed, frequency of defecation/its state before and after the start of narcotic therapy, frequency of nausea/vomiting, and dietary intake. RESULTS: The proportions of patients in whom laxatives were simultaneously prescribed during opioid therapy in 2002, 2004, and 2006 were 43.5%, 78.7%, and 75.6%, respectively. The proportions of those in whom antiemetics were combined with opioids were 45.7%, 78.7%, and 78.0%, respectively. The incidences of constipation were 50.0%, 39.3%, and 37.8%, respectively. Those of nausea/vomiting were 30.4%, 21.3%, and 9.8%, respectively. Those of anorexia were 65.3%, 39.4%, and 15.4%, respectively. CONCLUSIONS: These results suggest that palliative care team activities facilitated appropriate drug prescription during opioid therapy, reducing the appearance of side effects, with likelihood of improved quality of life.

Gamma/delta T cells are the predominant source of interleukin-17 in affected joints in collagen-induced arthritis, but not in rheumatoid arthritis.

OBJECTIVE: Although interleukin-17 (IL-17)-producing gamma/delta T cells were reported to play pathogenic roles in collagen-induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether gamma/delta T cells or CD4+ T cells are the predominant IL-17-producing cells, and to determine what stimulates gamma/delta T cells to secret IL-17 in mice with CIA. The involvement of IL-17-producing gamma/delta T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated. METHODS: IL-17-producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA. RESULTS: Gamma/delta T cells were the predominant population in IL-17-producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL-17-producing gamma/delta T cells expressed CC chemokine receptor 6, were maintained by IL-23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL-17 production by gamma/delta T cells was induced by IL-1beta plus IL-23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL-17-producing gamma/delta T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA. CONCLUSION: Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL-17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

Arthritis and pneumonitis produced by the same T cell clones from mice with spontaneous autoimmune arthritis.

SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.

Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model.

This report shows that interleukin (IL) 17-producing T helper type 17 (Th17) cells predominantly express CC chemokine receptor (CCR) 6 in an animal model of rheumatoid arthritis (RA). Th17 cells induced in vivo in normal mice via homeostatic proliferation similarly express CCR6, whereas those inducible in vitro by transforming growth factor beta and IL-6 additionally need IL-1 and neutralization of interferon (IFN) gamma and IL-4 for CCR6 expression. Forced expression of RORgamma t, a key transcription factor for Th17 cell differentiation, induces not only IL-17 but also CCR6 in naive T cells. Furthermore, Th17 cells produce CCL20, the known ligand for CCR6. Synoviocytes from arthritic joints of mice and humans also produce a large amount of CCL20, with a significant correlation (P = 0.014) between the amounts of IL-17 and CCL20 in RA joints. The CCL20 production by synoviocytes is augmented in vitro by IL-1beta, IL-17, or tumor necrosis factor alpha, and is suppressed by IFN-gamma or IL-4. Administration of blocking anti-CCR6 monoclonal antibody substantially inhibits mouse arthritis. Thus, the joint cytokine milieu formed by T cells and synovial cells controls the production of CCL20 and, consequently, the recruitment of CCR6+ arthritogenic Th17 cells to the inflamed joints. These results indicate that CCR6 expression contributes to Th17 cell function in autoimmune disease, especially in autoimmune arthritis such as RA.

T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis.

This report shows that highly self-reactive T cells produced in mice as a result of genetically altered thymic T cell selection spontaneously differentiate into interleukin (IL)-17-secreting CD4+ helper T (Th) cells (Th17 cells), which mediate an autoimmune arthritis that clinically and immunologically resembles rheumatoid arthritis (RA). The thymus-produced self-reactive T cells, which become activated in the periphery via recognition of major histocompatibility complex/self-peptide complexes, stimulate antigen-presenting cells (APCs) to secrete IL-6. APC-derived IL-6, together with T cell-derived IL-6, drives naive self-reactive T cells to differentiate into arthritogenic Th17 cells. Deficiency of either IL-17 or IL-6 completely inhibits arthritis development, whereas interferon (IFN)-gamma deficiency exacerbates it. The generation, differentiation, and persistence of arthritogenic Th17 cells per se are, however, insufficient for producing overt autoimmune arthritis. Yet overt disease is precipitated by further expansion and activation of autoimmune Th17 cells, for example, via IFN-gamma deficiency, homeostatic proliferation, or stimulation of innate immunity by microbial products. Thus, a genetically determined T cell self-reactivity forms a cytokine milieu that facilitates preferential differentiation of self-reactive T cells into Th17 cells. Extrinsic or intrinsic stimuli further expand these cells, thereby triggering autoimmune disease. Intervention in these events at cellular and molecular levels is useful to treat and prevent autoimmune disease, in particular RA.

Boc-Pro-Hyp-Gly-OBzl and Boc-Ala-Hyp-Gly-OBzl, two repeating triplets found in collagen.

The protected tripeptides benzyl N-{2-[N-(tert-butoxycarbonyl)prolyl]-4-hydroxyprolyl}glycinate or Boc-Pro-Hyp-Gly-OBzl, C24H33N3O7, and benzyl N-{2-[N-(tert-butoxycarbonyl)alanyl]-4-hydroxyprolyl}glycinate or Boc-Ala-Hyp-Gly-OBzl, C22H31N3O7, are the minimum repeating triplets found in collagen. Within the crystal structure of each are two independent peptide molecules with similar structures. The peptides are arranged antiparallel to one another and interact through hydrogen bonds involving the main chains and the 4-hydroxyprolyl groups. The structures exhibit characteristics of a triple helix, but the peptides tend to assume a sheet-like structure.

Spontaneous development of autoimmune arthritis due to genetic anomaly of T cell signal transduction: Part 1.

A point mutation of the gene encoding ZAP-70, a key signal transduction molecule in T cells, results in spontaneous development of T cell-mediated autoimmune arthritis in mice homozygous for the mutation. The genetic anomaly alters differentiation and selection of T cells in the thymus, leading to thymic production of arthritogenic autoimmune T cells. The arthritogenic T cells persist in the periphery and elicit arthritis when activated by microbial agents that stimulate innate immunity. This model is instrumental in understanding how genetic variations in T cell signal transduction, together with environmental influences, contribute to the development of autoimmune disease.