Deficiency and altered phenotype of mucosal-associated invariant T cells in systemic sclerosis.

Objective: Systemic sclerosis is an autoimmune disease characterized by fibrosis of the skin and internal organs including the lung. Mucosal-associated invariant T cells are innate-like T lymphocytes able to produce various cytokines and cytotoxic mediators such as granzyme B. A large body of evidence supports a role of mucosal-associated invariant T cells in autoimmune disease but more recent reports suggest also a potential role in fibrotic conditions. Therefore, we herein addressed the question as whether mucosal-associated invariant T cells may have an altered profile in systemic sclerosis. Methods: Mucosal-associated invariant T cell frequency was analyzed by flow cytometry, using fresh peripheral blood from 74 consecutive systemic sclerosis patients who were compared to 44 healthy donors. In addition, in-depth mucosal-associated invariant T cell phenotype and function were analyzed in unselected 29 women with systemic sclerosis who were compared to 23 healthy women donors. Results: Proportion of circulating mucosal-associated invariant T cells was significantly reduced by 68% in systemic sclerosis compared to healthy donors (0.78% in systemic sclerosis vs 2.5%, p < 0.0001). Within systemic sclerosis subsets, mucosal-associated invariant T cells were reduced in patients with interstitial lung disease (systemic sclerosis-interstitial lung disease) (0.56% vs 0.96% in patients without interstitial lung disease, p = 0.04). Moreover, in systemic sclerosis patients, mucosal-associated invariant T cells displayed an activated phenotype indicated by markedly increased CD69+ mucosal-associated invariant T cell frequency (20% mucosal-associated invariant T cell CD69+ compared to 9.4% in healthy donors, p = 0.0014). Interestingly, mucosal-associated invariant T cells from systemic sclerosis-interstitial lung disease patients had a more pronounced altered phenotype compared to systemic sclerosis without interstitial lung disease with a correlation between mucosal-associated invariant T cells expressing CCR6+ and mucosal-associated invariant T cell frequency (r = 0.8, p = 0.006). Conclusion: Circulating mucosal-associated invariant T cells were reduced and exhibited an activated phenotype in systemic sclerosis patients. This peripheral mucosal-associated invariant T cell deficiency may be related to enhanced apoptosis and/or homing in inflamed tissue, particularly in systemic sclerosis-interstitial lung disease patients.

Relevance of circulating Semaphorin 4A for rheumatoid arthritis response to treatment.

The lack of validated tools to predict rheumatoid arthritis (RA) disease course warrants the development of new reliable biomarkers. Our aim was to evaluate the merit of circulating SEMA4A for the prediction of outcomes in patients with RA. In a first cohort of 101 consecutive RA patients followed up for 41 ± 15 months, increased baseline SEMA4A levels were identified as an independent predictor of treatment failure (hazard ratio, HR 2.71, 95% CI 1.14-6.43), defined by the occurrence of patient-reported flares and initiation or change of targeted therapy. The highest predictive value of treatment failure was obtained with the combination of increased circulating SEMA4A and/or Disease Activity Score (DAS) 28-CRP > 3.2 and/or active synovitis on doppler ultrasound (HR 10.42, 95% CI 1.41-76.94). In a second independent cohort of 40 consecutive RA patients who initiated new therapy because of insufficient disease control, baseline SEMA4A levels were significantly higher in patients who further experienced none or moderate response, and SEMA4A concentrations were markedly decreased in the group of patients with good clinical response as compared to non-responders. Circulating SEMA4A appears as an appealing biomarker in RA with ability to predict treatment failure, and with association with response to therapy.

Driving Role of Interleukin-2-Related Regulatory CD4+ T Cell Deficiency in the Development of Lung Fibrosis and Vascular Remodeling in a Mouse Model of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by severe lung outcomes resulting in reduced life expectancy. Fra-2-transgenic mice offer the opportunity to decipher the relationships between the immune system and lung fibrosis. This study was undertaken to investigate whether the Fra-2-transgenic mouse lung phenotype may result from an imbalance between the effector and regulatory arms in the CD4+ T cell compartment. METHODS: We first used multicolor flow cytometry to extensively characterize homeostasis and the phenotype of peripheral CD4+ T cells from Fra-2-transgenic mice and control mice. We then tested different treatments for their effectiveness in restoring CD4+ Treg cell homeostasis, including adoptive transfer of Treg cells and treatment with low-dose interleukin-2 (IL-2). RESULTS: Fra-2-transgenic mice demonstrated a marked decrease in the proportion and absolute number of peripheral Treg cells that preceded accumulation of activated, T helper cell type 2-polarized, CD4+ T cells. This defect in Treg cell homeostasis was derived from a combination of mechanisms including impaired generation of these cells in both the thymus and the periphery. The impaired ability of peripheral conventional CD4+ T cells to produce IL-2 may greatly contribute to Treg cell deficiency in Fra-2-transgenic mice. Notably, adoptive transfer of Treg cells, low-dose IL-2 therapy, or combination therapy changed the phenotype of Fra-2-transgenic mice, resulting in a significant reduction in pulmonary parenchymal fibrosis and vascular remodeling in the lungs. CONCLUSION: Immunotherapies for restoring Treg cell homeostasis could be relevant in SSc. An intervention based on low-dose IL-2 injections, as is already proposed in other autoimmune diseases, could be the most suitable treatment modality for restoring Treg cell homeostasis for future research.

Aire-dependent transcripts escape Raver2-induced splice-event inclusion in the thymic epithelium.

Aire allows medullary thymic epithelial cells (mTECs) to express and present a large number of self-antigens for central tolerance. Although mTECs express a high diversity of self-antigen splice isoforms, the extent and regulation of alternative splicing events (ASEs) in their transcripts, notably in those induced by Aire, is unknown. In contrast to Aire-neutral genes, we find that transcripts of Aire-sensitive genes show only a low number of ASEs in mTECs, with about a quarter present in peripheral tissues excluded from the thymus. We identify Raver2, as a splicing-related factor overexpressed in mTECs and dependent on H3K36me3 marks, that promotes ASEs in transcripts of Aire-neutral genes, leaving Aire-sensitive ones unaffected. H3K36me3 profiling reveals its depletion at Aire-sensitive genes and supports a mechanism that is preceding Aire expression leading to transcripts of Aire-sensitive genes with low ASEs that escape Raver2-induced alternative splicing. The lack of ASEs in Aire-induced transcripts would result in an incomplete Aire-dependent negative selection of autoreactive T cells, thus highlighting the need of complementary tolerance mechanisms to prevent activation of these cells in the periphery.

Semaphorins: From Angiogenesis to Inflammation in Rheumatoid Arthritis.

OBJECTIVE: To study the potential role of semaphorins in the pathogenesis of rheumatoid arthritis (RA). METHODS: Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in RA endothelial cells (ECs) and control ECs derived from circulating progenitors. Expression of class 3 and class 4 semaphorins and their receptors in the serum of RA patients and healthy controls was assessed by immunohistochemical analysis in synovial tissue and by enzyme-linked immunosorbent assay. RESULTS: Microarray analysis revealed differential expression of class 3 and class 4 semaphorins and their receptors in RA ECs. Semaphorin 4A (SEMA4A), plexin D1, and neuropilin 1 messenger RNA (mRNA) levels were markedly increased in RA ECs by 1.75-, 2.21-, and 1.68-fold, respectively. Stimulation with tumor necrosis factor (TNF) led to a 2-fold increase in SEMA4A mRNA levels in RA ECs, and deficient SEMA4A expression modified RA EC angiogenic properties. Class 3 and class 4 semaphorins as well as their receptors were overexpressed in RA synovial tissue. A respective 1.30-fold increase and 1.54-fold increase in SEMA4A and SEMA3E, as well as a 24% decrease in SEMA3A, was observed in the serum of RA patients. Serum levels of SEMA4A, SEMA4D, and SEMA3A correlated with levels of inflammation and proangiogenic markers. In 2 independent cohorts of patients with low disease activity or with RA in remission, the presence of SEMA4A identified patients with residual disease activity. CONCLUSION: Gene expression profiling of ECs identified class 3 and class 4 semaphorins as potential biomarkers and therapeutic candidates in RA, with confirmed overexpression in ECs, synovial vessels, and serum, and correlation with validated markers of inflammation and angiogenesis. Thus, semaphorins might be novel and appealing EC-derived inflammatory and proangiogenic targets in RA.

Estrogens Counteract the Profibrotic Effects of TGF-ß and their Inhibition Exacerbates Experimental Dermal Fibrosis.

Systemic sclerosis primarily affects women. This sex bias raises the question on the role female hormones could play in the development of fibrosis, which is largely unknown. Our aim was to evaluate the effects of estrogens in the development of experimental dermal fibrosis, in the mouse models of bleomycin-induced dermal fibrosis and tight skin (Tsk-1) mice, and on the activation of dermal fibroblasts by transforming growth factor-ß (TGF-ß). Estrogen inhibition, obtained through gene inactivation for the estrogen receptor-αknockout or treatment with tamoxifen, exacerbated skin fibrosis in the bleomycin model and in the Tsk-1 mice. In the dermal fibroblasts, treatment with 17-ß-estradiol significantly decreased the stimulatory effects of TGF-ß on collagen synthesis and myofibroblast differentiation, decreased the activation of canonical TGF-ß signaling, and markedly reduced the expression of the TGF-ß target genes. Tamoxifen reversed the inhibitory effects of estrogens by restoring Smad2/3 phosphorylation and TGF-ß-induced collagen synthesis. Our results demonstrate a beneficial effect of estrogens in dermal fibrosis. Estrogens reduce the TGF-ß-dependent activation of dermal fibroblasts, and estrogen inhibition leads to a more severe experimental dermal fibrosis. These findings are consistent with the prominent development of systemic sclerosis in postmenopausal women and the greater severity of the disease in men.

The classical NLRP3 inflammasome controls FADD unconventional secretion through microvesicle shedding.

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1ß (IL-1ß) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1ß release and pyroptosis and, reciprocally, that IL-1ß release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1ß, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K+ efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.

Linking systemic angiogenic markers to synovial vascularization in rheumatoid arthritis.

BACKGROUND: Neoangiogenesis is a crucial event to promote the development of the hyperplasic proliferative pathologic synovium in Rheumatoid arthritis (RA). Ultrasound (US) is sensitive for detection of power Doppler (PD) vascularization. OBJECTIVE: To explore the associations between a set of complementary circulating angiogenic markers and a comprehensive US assessment in patients with RA. PATIENTS AND METHODS: Serum levels of eight angiogenic markers were measured by quantitative ELISAs in a total of 125 patients with RA, who were all systematically assessed in parallel by PDUS, performed on 32 joints. RESULTS: Serum levels of soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) and Tie-2 were more likely to be increased in patients with synovial hyperemia detected on at least one joint (Power Doppler grade ≥1). sVCAM-1, Tie-2 and Angiostatin concentrations gradually increased together with the grade of the semiquantitative PDUS scale and concentrations of these three markers were markedly increased in patients with moderate to marked hyperemia (Power Doppler grade 2 and 3). Levels of sVCAM-1, Tie-2, and Angiostatin correlated with a global arthritis sum score, defined by the sum of the semiquantitative PDUS scores for all joints examined. Levels of Tie-2 and Placenta Growth Factor (PlGF) were associated with PDUS features indicating residual disease activity. CONCLUSION: Our results support the relevance of measuring serum levels of vascular markers to evaluate the intensity and extent of synovial vascularization. Angiogenic markers, and particularly Tie-2, could be a valuable surrogate of active synovitis and their place in relation to PDUS in clinical practice deserve further investigation.

Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces.

BACKGROUND: The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites. RESULTS: By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite. CONCLUSIONS: This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.

AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity.

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1(KO)) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1(KO) tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1(KO) population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo.

Spire-1 contributes to the invadosome and its associated invasive properties.

Cancer cells have an increased ability to squeeze through extracellular matrix gaps that they create by promoting proteolysis of its components. Major sites of degradation are specialized micro-domains in the plasma membrane collectively named invadosomes where the Arp2/3 complex and formin proteins cooperate to spatio-temporally control actin nucleation and the folding of a dynamic F-actin core. At invadosomes, proper coupling of exo-endocytosis allows polarized delivery of proteases that facilitate degradation of ECM and disruption of the cellular barrier. We investigated the contribution of the actin nucleator Spire-1 to invadosome structure and function, using Src-activated cells and cancer cells. We found that Spire-1 is specifically recruited at invadosomes and is part of a multi-molecular complex containing Src kinase, the formin mDia1 and actin. Spire-1 interacts with the Rab3A GTPase, a key player in the regulation of exocytosis that is present at invadosomes. Finally, over- and under-expression of Spire-1 resulted in cells with an increased or decreased potential for matrix degradation, respectively, therefore suggesting a functional interplay of Spire-1 with both actin nucleation and vesicular trafficking that might impact on cell invasive and metastatic behavior.

Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion.

Toxoplasma gondii, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, and progressively propels itself through the junction, inside a newly formed vacuole that encloses the entering parasite. Little is known about how a parasite that is a few microns in diameter overcomes the host cell cortical actin barrier to achieve the remarkably rapid process of internalization (less than a few seconds). Using correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis we identified that toxofilin, an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy of cells expressing toxofilin showed that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, were found to be impaired in cortical actin disassembly and exhibited delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell.

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.

Host cell entry by apicomplexa parasites requires actin polymerization in the host cell.

Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.