Pauline Soulas-Sprauel

Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: Decreased memory B cells and membrane CD19 expression

B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce autoantibodies. Several B cell phenotype characteristics such as the expansion of activated populations, and of a newly identified memory compartment have already been reported. These results are not easy to interpret because of the clinical heterogeneity of SLE, as well as the difficulties to establish homogeneous and well defined groups taking in consideration the activity of the disease and the various therapies. However, although many mediators and mechanisms can contribute to the clinical presentation and subsequent progression of individuals with SLE, several data suggest that some intrinsic B cells abnormalities may be central to the disease process. In this view, we have analysed the phenotype of B cells from 18 patients with quiescent diseases (mean SLEDAI score below 2) and from 11 healthy controls. B cell surface marker expression was determined by flow cytometry. We analysed the main B cell sub-populations. We demonstrate the persistence of plasmocyte-differentiated and -activated B cells even in quiescent patients. However, quiescent patients display a decrease in memory B cells that could reflect the control of their disease. Above all, we describe a lower membrane expression of the CD19 protein on all B cells in every patient compared to controls. This lower CD19 expression is associated with reduced CD45 levels. It is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. These findings suggest certain mechanisms of lupus development.

B Cell Signature during Inactive Systemic Lupus Is Heterogeneous: Toward a Biological Dissection of Lupus

Systemic lupus erythematosous (SLE) is an autoimmune disease with an important clinical and biological heterogeneity. B lymphocytes appear central to the development of SLE which is characterized by the production of a large variety of autoantibodies and hypergammaglobulinemia. In mice, immature B cells from spontaneous lupus prone animals are able to produce autoantibodies when transferred into immunodeficient mice, strongly suggesting the existence of intrinsic B cell defects during lupus. In order to approach these defects in humans, we compared the peripheral B cell transcriptomas of quiescent lupus patients to normal B cell transcriptomas. When the statistical analysis is performed on the entire group of patients, the differences between patients and controls appear quite weak with only 14 mRNA genes having a false discovery rate ranging between 11 and 17%, with 6 underexpressed genes (PMEPA1, TLR10, TRAF3IP2, LDOC1L, CD1C and EGR1). However, unforced hierarchical clustering of the microarrays reveals a subgroup of lupus patients distinct from both the controls and the other lupus patients. This subgroup has no detectable clinical or immunological phenotypic peculiarity compared to the other patients, but is characterized by 1/an IL-4 signature and 2/the abnormal expression of a large set of genes with an extremely low false discovery rate, mainly pointing to the biological function of the endoplasmic reticulum, and more precisely to genes implicated in the Unfolded Protein Response, suggesting that B cells entered an incomplete BLIMP1 dependent plasmacytic differentiation which was undetectable by immunophenotyping. Thus, this microarray analysis of B cells during quiescent lupus suggests that, despite a similar lupus phenotype, different biological roads can lead to human lupus.

Synovial fibroblasts promote immunoglobulin class switching by a mechanism involving BAFF

Fibroblast‐like synoviocytes (FLSs) are important actors in rheumatoid arthritis (RA) pathogenesis. The autoimmune nature of RA is attributed to autoantibody production, which confers to B cells a predominant role in RA. Several arguments support an induction of class switch recombination (CSR) in RA synovium, causing – in conjunction with somatic hypermutation – the production of potentially pathogenic IgG. To determine whether RA FLSs can directly promote CSR and to analyze the role of external factors like TLR signals and BAFF (B cell activating factor) family cytokines in this FLS‐B cell crosstalk, we performed cocultures of blood B cells (from normal individuals or RA patients) with RA FLSs and analyzed CSR induction by quantification of AICDA (encoding activation‐induced cytidine deaminase, AID) and switch circular transcripts expression, and IgG secretion. RA FLSs – and to a lesser extent osteoarthritis or control FLSs – promoted CSR, and TLR3 stimulation potentialized it. In addition, induction of CSR by RA FLSs was totally dependent on cell–cell contact in basal conditions, and partially dependent in the case of TLR3 stimulation. Finally, we showed that the mechanism by which RA FLSs induce CSR is mostly BAFF‐dependent. Our results support the hypothesis that CSR can be induced outside the ectopic lymphoid structures in RA.

MyD88 Negatively Controls Hypergammaglobulinemia with Autoantibody Production during Bacterial Infection

ABSTRACT A large body of evidence has convincingly shown that Toll-like receptors are necessary sensors for infections with pathogens, but their activation was also suggested to generate autoimmunity. During experimental infections, the lack of these sensors or of their signaling molecules should lead to a deficient immune response. We found out that MyD88, the major adaptor of the Toll/interleukin-1 (Toll/IL-1) receptor signaling pathway, can actually act as a negative regulator of B-cell function in some settings. MyD88-deficient mice infected by Borrelia burgdorferi developed extreme hypergammaglobulinemia compared to wild-type animals, with high levels of immunoglobulin M (IgM) autoantibodies. In vivo, cell transfer experiments and cell blocking assays showed that this phenotype was not linked to the absence of MyD88 in B cells but rather to CD4 T-cell and likely dendritic cell dysfunctions leading to a Th1-to-Th2 cytokine switch. In addition, our results suggest a relative defect in the Ig class switch recombination process, since MyD88 knockout mice developed mostly IgM antibodies. Collectively, these data emphasize the complex role of the Toll/IL-1 receptor pathway in tuning the immune response against infection and avoiding autoimmunity.

PTPN22 inhibition resets defective human central B cell tolerance

Inhibiting the human autoimmunity risk factor PTPN22 restores B cell tolerance in a humanized mouse. Restoring tolerance Therapies that target and eliminate B cells have had some success in patients with autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. However, restoring tolerance in self-reactive B cells that lead to disease remains an unmet goal. The 1858T allele of protein tyrosine phosphatase nonreceptor type 22 (PTPN22 T) has been associated with autoreactive B cells in autoimmune disease in humans. Now, Schickel et al. report that this allele interferes with the development of central B cell tolerance in a humanized mouse model of autoimmunity and that blocking PTPN22 can restore this defect. If these observations hold true in humans, targeting PTPN22 may be able to restore B cell tolerance in patients with autoimmune disease. The 1858T protein tyrosine phosphatase nonreceptor type 22 (PTPN22 T) allele is one of the main risk factors associated with many autoimmune diseases and correlates with a defective removal of developing autoreactive B cells in humans. To determine whether inhibiting PTPN22 favors the elimination of autoreactive B cells, we first demonstrated that the PTPN22 T allele interfered with the establishment of central B cell tolerance using NOD-scid-common γ chain knockout (NSG) mice engrafted with human hematopoietic stem cells expressing this allele. In contrast, the inhibition of either PTPN22 enzymatic activity or its expression by RNA interference restored defective central B cell tolerance in this model. Thus, PTPN22 blockade may represent a therapeutic strategy for the prevention or treatment of autoimmunity.

Carabin deficiency in B cells increases BCR-TLR9 costimulation-induced autoimmunity

The mechanisms behind flares of human autoimmune diseases in general, and of systemic lupus in particular, are poorly understood. The present scenario proposes that predisposing gene defects favour clinical flares under the influence of external stimuli. Here, we show that Carabin is low in B cells of (NZB × NZW) F1 mice (murine SLE model) long before the disease onset, and is low in B cells of lupus patients during the inactive phases of the disease. Using knock‐out and B‐cell‐conditional knock‐out murine models, we identify Carabin as a new negative regulator of B‐cell function, whose deficiency in B cells speeds up early B‐cell responses and makes the mice more susceptible to anti‐dsDNA production and renal lupus flare after stimulation with a Toll‐like Receptor 9 agonist, CpG‐DNA. Finally, in vitro analysis of NFκB activation and Erk phosphorylation in TLR9‐ and B‐cell receptor (BCR)‐stimulated Carabin‐deficient B cells strongly suggests how the internal defect synergizes with the external stimulus and proposes Carabin as a natural inhibitor of the potentially dangerous crosstalk between BCR and TLR9 pathways in self‐reactive B cells.

Carabin Protects Against Cardiac Hypertrophy by Blocking Calcineurin, Ras, and Ca2+/Calmodulin-Dependent Protein Kinase II Signaling

Background— Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood. Methods and Results— Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin−/−) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P<0.0001). Conversely, compensation of Carabin loss through a cardiotropic adeno-associated viral vector encoding Carabin prevented transverse aortic constriction–induced cardiac hypertrophy with preserved fractional shortening (39.9±1.2% versus 25.9±2.6% in control plus transverse aortic constriction mice; P<0.0001). Carabin also conferred protection against adrenergic receptor–induced hypertrophy in isolated cardiomyocytes. Mechanistically, Carabin carries out a tripartite suppressive function. Indeed, Carabin, through its calcineurin-interacting site and Ras/Rab GTPase–activating protein domain, functions as an endogenous inhibitor of calcineurin and Ras/extracellular signal-regulated kinase prohypertrophic signaling. Moreover, Carabin reduced Ca2+/calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras–GTPase–activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin. Conclusions— Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca2+/calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure.

Carabin Protects Against Cardiac Hypertrophy by Blocking Calcineurin, Ras and CaMKII Signaling

Background— Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood. Methods and Results— Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin−/−) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P<0.0001). Conversely, compensation of Carabin loss through a cardiotropic adeno-associated viral vector encoding Carabin prevented transverse aortic constriction–induced cardiac hypertrophy with preserved fractional shortening (39.9±1.2% versus 25.9±2.6% in control plus transverse aortic constriction mice; P<0.0001). Carabin also conferred protection against adrenergic receptor–induced hypertrophy in isolated cardiomyocytes. Mechanistically, Carabin carries out a tripartite suppressive function. Indeed, Carabin, through its calcineurin-interacting site and Ras/Rab GTPase–activating protein domain, functions as an endogenous inhibitor of calcineurin and Ras/extracellular signal-regulated kinase prohypertrophic signaling. Moreover, Carabin reduced Ca2+/calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras–GTPase–activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin. Conclusions— Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca2+/calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure.

Immunological phenotype of the murine Lrba knockout

Biallelic mutations in the human lipopolysaccharide responsive beige‐like anchor (LRBA) gene lead to a primary immunodeficiency known as LRBA deficiency, characterized by a broad range of clinical manifestations including autoimmunity, organomegaly, hypogammaglobulinemia and recurrent infections. Considering the phenotypic heterogeneity in patients and the severity of the disease, our aim was to assess the role of LRBA in immune cells and to understand the underlying pathomechanisms through the study of a Lrba knockout (Lrba−/−) mouse model. LRBA‐deficient mice did not show severe clinical or immunological signs of disease, either at steady state under specific‐pathogen‐free conditions, after vaccination with T‐dependent and T‐independent antigens, or in the context of acute infections with lymphocytic choriomeningitis virus (LCMV) or Salmonella Typhimurium. Although Lrba−/− mice were able to produce normal serum immunoglobulin M (IgM) and IgG and to mount a specific immune response after immunization, they showed elevated serum and secretory basal IgA levels. LRBA was dispensable for B‐ and T‐cell development, as well as for in vitro B‐cell proliferation, survival, isotype switching and plasmablast differentiation. Interestingly, Lrba−/− mice displayed decreased cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA‐4) expression by regulatory T cells and activated conventional CD4+ and CD8+ T lymphocytes, reduced frequency of peritoneal B‐1a cells along with diminished interleukin‐10 production and increased percentages of T follicular helper cells in Peyers patches, but without developing overt signs of autoimmunity. Our findings expand the role of LRBA in immune regulatory mechanisms previously reported in patients, and suggest a novel role in IgA production that is crucial for the protection of mucosal surfaces and gut‐associated immune tolerance.

Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation

Systemic Lupus Erythematosus (SLE) is a severe systemic autoimmune disease, characterized by multi‐organ damages, triggered by an autoantibody‐mediated inflammation, and with a complex genetic influence. It is today accepted that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility and contributing to a phenotypic trait to the disease. One of the ways to find these gene variations consists in comprehensive analysis of gene expression variation in a precise cell type, which can constitute a good complementary strategy to genome wide association studies. Using this strategy, and considering the central role of B cells in SLE, we analyzed the B cell transcriptome of quiescent SLE patients, and identified an overexpression of FKBP11, coding for a cytoplasmic putative peptidyl‐prolyl cis/trans isomerase and chaperone enzyme. To understand the consequences of FKBP11 overexpression on B cell function and on autoimmunitys development, we created lentiviral transgenic mice reproducing this gene expression variation. We showed that high expression of Fkbp11 reproduces by itself two phenotypic traits of SLE in mice: breakdown of B cell tolerance against DNA and initiation of plasma cell differentiation by acting upstream of Pax5 master regulator gene.