Alessandra B. Pernis

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5−CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5− ‘peripheral helper’ T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Enhanced rho-associated protein kinase activation in patients with systemic lupus erythematosus.

OBJECTIVE Rho-associated protein kinases (ROCKs) have been implicated in the pathogenesis of cardiovascular and renal disorders. We recently showed that ROCKs could regulate the differentiation of murine Th17 cells and the production of interleukin-17 (IL-17) and IL-21, two cytokines associated with systemic lupus erythematosus (SLE). The goal of this study was to assess ROCK activation in human Th17 cells and to evaluate ROCK activity in SLE patients. METHODS An enzyme-linked immunosorbent assay (ELISA)-based ROCK activity assay was used to evaluate ROCK activity in human cord blood CD4+ T cells differentiated under Th0 or Th17 conditions. We then performed a cross-sectional analysis of 28 SLE patients and 25 healthy matched controls. ROCK activity in peripheral blood mononuclear cell (PBMC) lysates was determined by ELISA. Cytokine and chemokine profiles were analyzed by ELISA. RESULTS Human cord blood CD4+ T cells differentiated under Th17 conditions expressed higher levels of ROCK activity than did CD4+ T cells stimulated under Th0 conditions. Production of IL-17 and IL-21 was inhibited by the addition of a ROCK inhibitor. SLE PBMCs expressed significantly higher levels of ROCK activity than did healthy control PBMCs (1.25 versus 0.56; P = 0.0015). Sixteen SLE patients (57%) expressed high levels of ROCK (optical density at 450 nm >1). Disease duration, lymphocyte count, and azathioprine use were shown to be significant independent predictors of ROCK activity in multivariable analyses. CONCLUSION Consistent with previous results in the murine system, increased ROCK activation was associated with Th17 cell differentiation. Moreover, enhanced ROCK activity was observed in a subgroup of SLE patients. These data support the concept that the ROCK pathway could represent an important therapeutic target for SLE.

Epigenetics and the IRFs: A complex interplay in the control of immunity and autoimmunity

Abstract Effective immune responses require the coordinated activation and differentiation of several cell types, including T-cells, B-cells and myeloid cells. Abnormalities in the appropriate regulation of these processes underlie the pathogenesis of many autoimmune disorders, including systemic lupus erythematosus (SLE). Recent studies have revealed that, in addition to sequence-specific DNA-binding factors, the chromatin landscape of a cell can play a pivotal role in controlling these processes and in regulating the onset of autoimmunity. Interferon regulatory factors (IRFs) are emerging as critical regulators of the activation and differentiation of immune cells and deregulation in the expression and/or function of members of the IRF family has increasingly been linked to the pathogenesis of lupus. In this review, we will provide a brief overview of the role of different IRFs in immune responses and SLE development and discuss studies, which highlight the intricate relationship of this family of transcription factors with the epigenetic machinery.

A Murine Autoimmune Model of Rheumatoid Arthritis and Systemic Lupus Erythematosus Associated with Deregulated Production of IL-17 and IL-21

T-helper cell 17 (Th17) cells play an important role in the pathogenesis of many autoimmune disorders including Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). In this chapter we describe a murine model where deregulated production of IL-17 and IL-21 can lead to either lupus-like disease or RA-like symptoms depending on the genetic background. We delineate the key techniques that can be used to dissect the mechanisms responsible for the pathogenesis of these diseases at both a cellular and molecular level including in vitro Th17 cell differentiation, chromatin immunoprecipitation assays, and retroviral transduction experiments. We also describe the methodologies that can be utilized to monitor the classic clinical findings of RA and SLE in murine models. Given the broad involvement of deregulated production of IL-17 and IL-21 in autoimmunity, many of these techniques could also be valuable for the investigation of these pathways in murine models of other autoimmune diseases.

The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity

Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1–4E-BP–eIF4E axis, secondary to aberrant assembly of a raptor–p62–TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (TFH) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of TFH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

Increased rho kinase activity in temporal artery biopsies from patients with giant cell arteritis

OBJECTIVE Aberrant rho kinase (ROCK) activity is implicated in the pathogenesis of several vascular diseases and is associated with Th17 differentiation. Th17 immune response is recognized in the pathogenesis of GCA. The aim of this study was to assess ROCK activity in GCA. METHODS All patients who underwent temporal artery biopsy (TAB) at a tertiary care centre over 5 years were identified and charts reviewed. Subjects were categorized into three groups: TAB-positive GCA, TAB-negative GCA and age- and sex-matched controls. TABs were stained for phosphorylated ezrin/radixin/moesin (pERM), a surrogate of ROCK activity, and reviewed by a pathologist blinded to clinical status. Three areas were scored for staining intensity on a scale of 0-2, with a maximum possible score of 6. RESULTS Nineteen subjects with TAB-positive GCA, 17 with TAB-negative GCA and 18 controls were analysed. Compared with controls, GCA subjects with either positive or negative TABs had significantly higher pERM intensity scores (P = 0.0109). Adjusting for diabetes, hypertension, prednisone and statin use, GCA subjects still had higher pERM scores [odds ratio 7.3 (95% CI 1.9, 25.9), P = 0.0046]. The high pERM score had a sensitivity of 90% and a negative predictive value of 91% for the diagnosis of GCA in those with a negative TAB, compared with 51% sensitivity for histopathology alone. CONCLUSION Subjects with GCA had more intense pERM staining in TAB specimens compared with age- and sex-matched controls, regardless of whether TAB was positive or negative by routine histopathology, suggesting increased ROCK activity in GCA. The ROCK pathway warrants further investigation in GCA, as it may have diagnostic significance in enhancing the sensitivity of TAB.

Regulation of Effector Treg Cells in Murine Lupus

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF‐4) is an essential controller of effector Treg cells and regulates their ability to express interleukin‐10 (IL‐10). In non–Treg cells, IRF‐4 activity is modulated by interactions with DEF‐6 and its homolog switch‐associated protein 70 (SWAP‐70). Although mice lacking both DEF‐6 and SWAP‐70 (double‐knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF‐6 and SWAP‐70 to restrain IRF‐4 activity.

Regulation of effector Tregs in murine lupus

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF‐4) is an essential controller of effector Treg cells and regulates their ability to express interleukin‐10 (IL‐10). In non–Treg cells, IRF‐4 activity is modulated by interactions with DEF‐6 and its homolog switch‐associated protein 70 (SWAP‐70). Although mice lacking both DEF‐6 and SWAP‐70 (double‐knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF‐6 and SWAP‐70 to restrain IRF‐4 activity.

IRF4-Dependent and IRF4-Independent Pathways Contribute to DC Dysfunction in Lupus

Interferon Regulatory Factors (IRFs) play fundamental roles in dendritic cell (DC) differentiation and function. In particular, IRFs are critical transducers of TLR signaling and dysregulation in this family of factors is associated with the development of autoimmune disorders such as Systemic Lupus Erythematosus (SLE). While several IRFs are expressed in DCs their relative contribution to the aberrant phenotypic and functional characteristics that DCs acquire in autoimmune disease has not been fully delineated. Mice deficient in both DEF6 and SWAP-70 (= Double-knock-out or DKO mice), two members of a unique family of molecules that restrain IRF4 function, spontaneously develop a lupus-like disease. Although autoimmunity in DKO mice is accompanied by dysregulated IRF4 activity in both T and B cells, SWAP-70 is also known to regulate multiple aspects of DC biology leading us to directly evaluate DC development and function in these mice. By monitoring Blimp1 expression and IL-10 competency in DKO mice we demonstrate that DCs in these mice exhibit dysregulated IL-10 production, which is accompanied by aberrant Blimp1 expression in the spleen but not in the peripheral lymph nodes. We furthermore show that DCs from these mice are hyper-responsive to multiple TLR ligands and that IRF4 plays a differential role in in these responses by being required for the TLR4-mediated but not the TLR9-mediated upregulation of IL-10 expression. Thus, DC dysfunction in lupus-prone mice relies on both IRF4-dependent and IRF4-independent pathways.

Rho Kinases in Autoimmune Diseases

The Rho kinases, or ROCKs, are a family of serine-threonine kinases that serve as key downstream effectors for Rho GTPases. The ROCKs are increasingly recognized as critical coordinators of a tissue response to injury due to their ability to modulate a wide range of biological processes. Dysregulated ROCK activity has been implicated in several human pathophysiological conditions ranging from cardiovascular and renal disorders to fibrotic diseases. In recent years, an important role for the ROCKs in the regulation of immune responses is also being uncovered. We provide an overview of the role of the ROCKs in immune cells and discuss studies that highlight the emerging involvement of this family of kinases in the pathogenesis of autoimmune diseases. Given the potential promise of the ROCKs as therapeutic targets, we also outline the approaches that could be employed to inhibit the ROCKs in autoimmune disorders.