Chronic inflammation causes target organ damage in patients with systemic autoimmune diseases. The factors that allow this protracted response are poorly understood. We analyzed the transcriptional regulation of PPP2R2B (B55ß), a molecule necessary for the termination of the immune response, in patients with autoimmune diseases. Altered expression of B55ß conditioned resistance to cytokine withdrawal-induced death (CWID) in patients with autoimmune diseases. The impaired upregulation of B55ß was caused by inflammation-driven hypermethylation of specific cytosines located within a regulatory element of PPP2R2B preventing CTCF binding. This phenotype could be induced in healthy T cells by exposure to TNF-α. Our results reveal a gene whose expression is affected by an acquired defect, through an epigenetic mechanism, in the setting of systemic autoimmunity. Because failure to remove activated T cells through CWID could contribute to autoimmune pathology, this mechanism illustrates a vicious cycle through which autoimmune inflammation contributes to its own perpetuation.
Iris K. Madera-Salcedo, Beatriz E. Sánchez-Hernández, Yevgeniya Svyryd, Marcela Esquivel-Velázquez, Noé Rodríguez-Rodríguez, María Isabel Trejo-Zambrano, H. Benjamín García-González, Gabriela Hernández-Molina, Osvaldo M. Mutchinick, Jorge Alcocer-Varela, Florencia Rosetti, José C. Crispín
Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin–dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell–dependent autoimmune diseases.
Michihito Kono, Kayaho Maeda, Irina Stocton-Gavanescu, Wenliang Pan, Masataka Umeda, Eri Katsuyama, Catalina Burbano, Seo Yeon K. Orite, Milena Vukelic, Maria G. Tsokos, Nobuya Yoshida, George C. Tsokos
Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.
Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor
The central nervous system manifestations of systemic lupus erythematosus (SLE) remain poorly understood. Given the well-defined role of autoantibodies in other lupus manifestations, extensive work has gone into the identification of neuropathic autoantibodies. However, attempts to translate these findings to patients with SLE have yielded mixed results. We used the MRL/MpJ-Faslpr/lpr mouse, a well-established, spontaneous model of SLE, to establish the immune effectors responsible for brain disease. Transcriptomic analysis of the MRL/MpJ-Faslpr/lpr choroid plexus revealed an expression signature driving tertiary lymphoid structure formation, including chemokines related to stromal reorganization and lymphocyte compartmentalization. Additionally, transcriptional profiles indicated various stages of lymphocyte activation and germinal center formation. The extensive choroid plexus infiltrate present in MRL/MpJ-Faslpr/lpr mice with overt neurobehavioral deficits included locally proliferating B and T cells, intercellular interactions between lymphocytes and antigen-presenting cells, as well as evidence for in situ somatic hypermutation and class switch recombination. Furthermore, the choroid plexus was a site for trafficking lymphocytes into the brain. Finally, histological evaluation in human lupus patients with neuropsychiatric manifestations revealed increased leukocyte migration through the choroid plexus. These studies identify a potential new pathway underlying neuropsychiatric lupus and support tertiary lymphoid structure formation in the choroid plexus as a novel mechanism of brain-immune interfacing.
Ariel D. Stock, Evan Der, Sivan Gelb, Michelle Huang, Karen Weidenheim, Ayal Ben-Zvi, Chaim Putterman
Alteration of innate immune cells in the lungs can promote loss of peripheral tolerance that leads to autoimmune responses in cigarette smokers. Development of autoimmunity in smokers with emphysema is also strongly linked to the expansion of autoreactive T helper (Th) cells expressing interferon gamma (Th1), and interleukin 17A (Th17). However, the mechanisms responsible for enhanced self-recognition and reduced immune tolerance in smoker with emphysema remain less clear. Here we show that C1q, a component of the complement protein 1 complex (C1), is downregulated in lung CD1a+ antigen presenting cells (APCs) isolated from emphysematous human, and mouse lung APCs after chronic cigarette smoke exposure. C1q potentiated the function of APCs to differentiate CD4+ T cells to Tregs, while it inhibited Th17 cell development and proliferation. Mice deficient in C1q that were exposed to chronic smoke exhibited exaggerated lung inflammation marked by increased Th17 cells, while reconstitution of C1q in the lungs enhanced Tregs abundance, dampened smoke-induced lung inflammation, and reversed established emphysema. Our findings demonstrate that cigarette smoke-mediated loss of C1q could play a key role in reduced peripheral tolerance, which could be explored to treat emphysema.
Xiaoyi Yuan, Cheng-Yen Chang, Ran You, Ming Shan, Bon Hee Gu, Matthew Madison, Gretchen Diehl, Sarah Perusich, Li-Zhen Song, Lorraine Cornwell, Roger D. Rossen, Rick Wetsel, Rajapakshe Kimal, Cristian Coarfa, Holger Eltzschig, David B. Corry, Farrah Kheradmand
IgG antinuclear antibodies (ANAs) are a dominant feature of several autoimmune diseases. We previously showed that systemic lupus erythematosus (SLE) is characterized by increased ANA+ IgG plasmablasts/plasma cells (PCs) through aberrant IgG PC differentiation rather than an antigen-specific tolerance defect. Here, we aimed to understand the differentiation pathways resulting in ANA+ IgG PCs in SLE patients. We demonstrate distinct profiles of ANA+ antigen-experienced B cells in SLE patients, characterized by either a high frequency of PCs or a high frequency of IgG+ memory B cells. This classification of SLE patients was unrelated to disease activity and remained stable over time in almost all patients, suggesting minimal influence of disease activity. A similar classification applies to antigen-specific B cell subsets in mice following primary immunization with T-independent and T-dependent antigens as well as in lupus-prone mouse models (MRL/lpr and NZB/W). We further show that, in both lupus-prone mice and SLE patients, the classification correlates with the serum autoantibody profile. In this study, we identified B cell phenotypes that we propose reflect an extrafollicular pathway for PC differentiation or a germinal center pathway, respectively. The classification we propose can be used to stratify patients for longitudinal studies and clinical trials.
Jolien Suurmond, Yemil Atisha-Fregoso, Ashley N. Barlev, Silvia A. Calderon, Meggan C. Mackay, Cynthia Aranow, Betty Diamond
Autoimmune disease is 4 times more common in women than men. This bias is largely unexplained. Female skin is “autoimmunity prone,” showing upregulation of many proinflammatory genes, even in healthy women. We previously identified VGLL3 as a putative transcription cofactor enriched in female skin. Here, we demonstrate that skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash and systemic autoimmune disease that involves B cell expansion, autoantibody production, immune complex deposition, and end-organ damage. Excess epidermal VGLL3 drives a proinflammatory gene expression program that overlaps with both female skin and cutaneous lupus. This includes increased B cell–activating factor (BAFF), the only current biologic target in systemic lupus erythematosus (SLE); IFN-κ, a key inflammatory mediator in cutaneous lupus; and CXCL13, a biomarker of early-onset SLE and renal involvement. Our results demonstrate that skin-targeted overexpression of the female-biased factor VGLL3 is sufficient to drive cutaneous and systemic autoimmune disease that is strikingly similar to SLE. This work strongly implicates VGLL3 as a pivotal orchestrator of sex-biased autoimmunity.
Allison C. Billi, Mehrnaz Gharaee-Kermani, Joseph Fullmer, Lam C. Tsoi, Brett D. Hill, Dennis Gruszka, Jessica Ludwig, Xianying Xing, Shannon Estadt, Sonya J. Wolf, Syed Monem Rizvi, Celine C. Berthier, Jeffrey B. Hodgin, Maria A. Beamer, Mrinal K. Sarkar, Yun Liang, Ranjitha Uppala, Shuai Shao, Chang Zeng, Paul W. Harms, Monique E. Verhaegen, John J. Voorhees, Fei Wen, Nicole L. Ward, Andrzej A. Dlugosz, J. Michelle Kahlenberg, Johann E. Gudjonsson
Biased agonism is a paradigm that may explain the selective activation of a signaling pathway via a GPCR that activates multiple signals. The autoantibody-induced inactivation of the calcium-sensing receptor (CaSR) causes acquired hypocalciuric hypercalcemia (AHH). Here, we describe an instructive case of AHH in which severe hypercalcemia was accompanied by an increased CaSR antibody titer. These autoantibodies operated as biased allosteric modulators of CaSR by targeting its Venus flytrap domain near the Ca2+-binding site. A positive allosteric modulator of CaSR, cinacalcet, which targets its transmembrane domain, overcame this autoantibody effect and successfully corrected the hypercalcemia in this patient. Hence, this is the first study to our knowledge that identifies the interaction site of a disease-causing GPCR autoantibody working as its biased allosteric modulator and demonstrates that cinacalcet can correct the AHH autoantibody effects both in vitro and in our AHH patient. Our observations provide potentially new insights into how biased agonism works and how to design a biased allosteric modulator of a GPCR. Our observations also indicate that the diagnosis of AHH is important because the severity of hypercalcemia may become fatal if the autoantibody titer increases. Calcimimetics may serve as good treatment options for some patients with severe AHH.
Noriko Makita, Takao Ando, Junichiro Sato, Katsunori Manaka, Koji Mitani, Yasuko Kikuchi, Takayoshi Niwa, Masanori Ootaki, Yuko Takeba, Naoki Matsumoto, Atsushi Kawakami, Toshihisa Ogawa, Masaomi Nangaku, Taroh Iiri
Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet β cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet β cell mass or proliferation. However, improved β cell function was reflected by a partial recovery of MafA transcription factor expression, a sensitive marker of islet β cell stress that is important to adult β cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving reactive oxygen species (ROS) handling in NOD islets. This study reveals a novel mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D β cells.
Christopher S. Wilson, Jason M. Spaeth, Jay Karp, Blair T. Stocks, Emilee M. Hoopes, Roland W. Stein, Daniel J. Moore
Paraneoplastic neurological disorders result from an autoimmune response against neural self-antigens that are ectopically expressed in neoplastic cells. In paraneoplastic disorders associated to autoantibodies against intracellular proteins, such as paraneoplastic cerebellar degeneration (PCD), current data point to a major role of cell-mediated immunity. In an animal model, in which a neo–self-antigen was expressed in both Purkinje neurons and implanted breast tumor cells, immune checkpoint blockade led to complete tumor control at the expense of cerebellum infiltration by T cells and Purkinje neuron loss, thereby mimicking PCD. Here, we identify 2 potential therapeutic targets expressed by cerebellum-infiltrating T cells in this model, namely α4 integrin and IFN-γ. Mice with PCD were treated with anti-α4 integrin antibodies or neutralizing anti–IFN-γ antibodies at the onset of neurological signs. Although blocking α4 integrin had little or no impact on disease development, treatment using the anti–IFN-γ antibody led to almost complete protection from PCD. These findings strongly suggest that the production of IFN-γ by cerebellum-invading T cells plays a major role in Purkinje neuron death. Our successful preclinical use of neutralizing anti–IFN-γ antibody for the treatment of PCD offers a potentially new therapeutic opportunity for cancer patients at the onset of paraneoplastic neurological disorders.
Lidia Yshii, Béatrice Pignolet, Emilie Mauré, Mandy Pierau, Monika Brunner-Weinzierl, Oliver Hartley, Jan Bauer, Roland Liblau
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