Toll-like receptor 3 increases antigen-presenting cell responses to a pro-apoptotic stimulus, yet does not contribute to systemic lupus erythematosus genetic susceptibility.

OBJECTIVES: TLR3 mediates skin solar injury by binding nuclear material released from apoptotic keratinocytes, resulting in the production of pro-inflammatory cytokines. Because the TLR3 gene is located in 4q35, a known systemic lupus erythematosus (SLE) susceptibility locus, we wondered whether TLR3 single nucleotide polymorphisms (SNPs) were associated with inflammatory mechanisms relevant to the development of SLE, and disease susceptibility. METHODS: Functional assays were carried out in TLR3-transfected HEK293 cells and in monocyte-derived dendritic cells (moDCs). TLR3 and IFNß immunofluorescence studies were performed in skin samples from 7 SLE patients and 3 controls. We performed a SNP association study in a discovery cohort of 153 patients and 105 controls, followed by a confirmation study in an independent cohort of 1,380 patients and 2,104 controls. RESULTS: TLR3 and IFNß are overexpressed in SLE skin lesions. TLR3 overexpression in HEK293 cells amplifies their sensitivity to a pro-apoptotic stimulus. Taking advantage of a naturally occurring polymorphic TLR3 variant (rs3775291) that weakly versus strongly responds to poly I:C stimulation, we found that TLR3 is associated with amplified apoptotic responses, production of the Ro/SSA autoantigen and increased maturation of myeloid-derived dendritic cells (moDC) after exposure to UV irradiation. However, TLR3 SNPs are not associated with susceptibility to SLE in a large population of patients and controls. CONCLUSIONS: TLR3 is overexpressed in SLE skin lesions and amplifies apoptotic and inflammatory responses to UV-irradiation in antigen-presenting cells in vitro. However, TLR3 SNPs do not impact susceptibility to the development of the disease.

STAT3 phosphorylation mediates the stimulatory effects of interferon alpha on B cell differentiation and activation in SLE.

OBJECTIVE: Type I IFNs play a well-known role in the pathogenesis of SLE, through activation of CD4 T and antigen-presenting cells. Here, we investigated the effects of IFN alpha (IFNα) on SLE B cell activation and differentiation. METHODS: Peripheral blood mononuclear cells (PBMCs) and purified total or naïve B cells were obtained from healthy controls and SLE patients. The effects of IFNα on B cell differentiation were studied by flow cytometry. The role of STAT3 in B cell responses to IFNα was studied using pharmacological inhibitors and PBMCs from STAT3-deficient individuals. RESULTS: Incubation of normal PBMCs with IFNα induces a B cell differentiation pattern as observed spontaneously in SLE PBMCs. IFNα displays direct stimulatory effects on purified naïve B cells from healthy individuals, as evidenced by a significant induction of cell surface CD38 and CD95 in the presence of the cytokine. In purified naïve B cells, IFNα also induces STAT3 phosphorylation. IFNα-induced naïve B cell differentiation in total PBMCs is significantly inhibited in the presence of STAT3 inhibitors, or in PBMCs from individuals with STAT3 loss of function mutations. Spontaneous levels of STAT3, but not STAT1, phosphorylation are significantly higher in total B cells from SLE patients compared with controls. Pharmacological STAT3 inhibition in SLE PBMCs inhibits naïve B cell activation and differentiation. CONCLUSION: IFNα displays direct stimulatory effects on B cell differentiation and activation in SLE. STAT3 phosphorylation mediates the effects of IFNα stimulation in naïve B cells, an observation that opens new therapeutic perspectives in SLE.

Overexpression of ubiquitin-specific peptidase 15 in systemic sclerosis fibroblasts increases response to transforming growth factor ß.

OBJECTIVE: Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-ß signalling in primary fibroblast cell lines. METHODS: High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-ß stimulation. RESULTS: Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-ß signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-ß induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-ß-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action. CONCLUSION: Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-ß, and is a potential therapeutic target in SSc.

Multi-center harmonization of flow cytometers in the context of the European "PRECISESADS" project.

The innovative medicine initiative project called PRECISESADS will study 2.500 individuals affected by systemic autoimmune diseases (SADs) and controls. Among extensive OMICS approaches, multi-parameter flow cytometry analyses will be performed in eleven different centers. Therefore, the integration of all data in common bioinformatical and biostatistical investigations requires a fine mirroring of all instruments. We describe here the procedure elaborated to achieve this prerequisite. One flow cytometer chosen as reference instrument fixed the mean fluorescence intensities (MFIs) of 8 different fluorochrome-conjugated antibodies (Abs) using VersaComp Ab capture beads. The ten other centers adjusted their own PMT voltages to reach the same MFIs. Subsequently, all centers acquired Rainbow 8-peak beads data on a daily basis to follow the stability of their instrument overtime. One blood sample has been dispatched and concomitantly stained in all centers. Comparison of leukocytes frequencies and cell surface marker MFIs demonstrated the close sensitivity of all flow cytometers, allowing a multicenter analysis. The effective multi-center harmonization enables the constitution of a workable wide flow cytometry database for the identification of specific molecular signatures in individuals with SADs.

Interferon α kinoid induces neutralizing anti-interferon α antibodies that decrease the expression of interferon-induced and B cell activation associated transcripts: analysis of extended follow-up data from the interferon α kinoid phase I/II study.

OBJECTIVE: IFN α Kinoid (IFN-K) is a therapeutic vaccine composed of IFNα2b coupled to a carrier protein. In a phase I/II placebo-controlled trial, we observed that IFN-K significantly decreases the IFN gene signature in whole blood RNA samples from SLE patients. Here, we analysed extended follow-up data from IFN-K-treated patients, in order to evaluate persistence of neutralizing anti-IFNα Abs antibodies (Abs), and gene expression profiling. METHODS: Serum and whole blood RNA samples were obtained in IFN-K-treated patients included in the follow-up study, in order to determine binding and neutralizing anti-IFNα Ab titres, and perform high-throughput transcriptomic studies. RESULTS: Neutralization studies of 13 IFNα subtypes demonstrated the polyclonal nature of the Ab response induced by IFN-K. Follow-up analyses in six patients confirmed a significant correlation between neutralizing anti-IFNα Ab titres and decrease in IFN scores compared to baseline. These analyses also revealed an inhibitory effect of IFNα blockade on the expression of B cell associated transcripts. CONCLUSIONS: IFN-K induces a polyclonal anti-IFNα response that decreases IFN- and B cell-associated transcripts. TRIAL REGISTRATION: ClinicalTrials.gov, clinicaltrials.gov, NCT01058343.

Higher expression of TNFα-induced genes in the synovium of patients with early rheumatoid arthritis correlates with disease activity, and predicts absence of response to first line therapy.

BACKGROUND: IL6-related T cell activation and TNFα-dependent cell proliferation are major targets of therapy in the RA synovium. We investigated whether expression of these pathways in RA synovial biopsies is associated with disease activity and response to therapy. METHOD: Correlation and gene set enrichment studies were performed using gene expression profiles from RA synovial biopsies. Immunostaining experiments of GADD45B and PDE4D were performed on independent additional sets of early untreated RA samples, obtained in two different centers by needle-arthroscopy or US-guided biopsies. RESULTS: In 65 RA synovial biopsies, transcripts correlating with disease activity were strongly enriched in TNFα-induced genes. Out of the individual variables used in disease-activity scores, tender joint count, swollen joint count and physician's global assessment, but not CRP or patient's global assessment displayed a similar correlation with the expression of TNFα-dependent genes. In addition, TNFα-induced genes were also significantly enriched in transcripts over-expressed in synovial biopsy samples obtained from poor-responders to methotrexate or tocilizumab, prior to initiation of therapy. GADD45B (induced by TNFα in monocytes) and PDE4D (induced by TNFα in FLS) immunostaining was significantly higher in overall poor-responders to therapy in 46 independent baseline samples obtained from early untreated RA patients prior to initiation of therapy. GADD45B (but not PDE4D) immunostaining was significantly higher in the sub-group of patients with poor-response to methotrexate therapy, and this was confirmed in another population of methotrexate-treated patients. CONCLUSION: Higher expression of TNFα-induced transcripts in early RA synovitis is associated with higher disease activity, and predicts poor response to first-line therapy. That over-expression of TNFα-induced genes predicts poor-response to therapy regardless of the drug administered, indicates that this molecular signature is associated with disease severity, rather than with specific pathways of escape to therapy.

Heterogeneity of synovial molecular patterns in patients with arthritis.

OBJECTIVES: Early diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. Previously, we performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Here, we wanted to further explore the consistency of the gene expression signals in synovial biopsies of patients with arthritis, using low-density platforms. METHODS: Low-density assays (cDNA microarray and microfluidics qPCR) were designed, based on the results of the high-density microarray data. Knee synovial biopsies were obtained from patients with RA, spondyloarthropathies (SA) or osteoarthritis (OA) (n = 39), and also from patients with initial undifferentiated arthritis (UA) (n = 49). RESULTS: According to high-density microarray data, several molecular pathways are differentially expressed in patients with RA, SA and OA: T and B cell activation, chromatin remodelling, RAS GTPase activation and extracellular matrix regulation. Strikingly, disease activity (DAS28-CRP) has a significant influence on gene expression patterns in RA samples. Using the low-density assays, samples from patients with OA are easily discriminated from RA and SA samples. However, overlapping molecular patterns are found, in particular between RA and SA biopsies. Therefore, prediction of the clinical diagnosis based on gene expression data results in a diagnostic accuracy of 56.8%, which is increased up to 98.6% by the addition of specific clinical symptoms in the prediction algorithm. Similar observations are made in initial UA samples, in which overlapping molecular patterns also impact the accuracy of the diagnostic algorithm. When clinical symptoms are added, the diagnostic accuracy is strongly improved. CONCLUSIONS: Gene expression signatures are overall different in patients with OA, RA and SA, but overlapping molecular signatures are found in patients with these conditions. Therefore, an accurate diagnosis in patients with UA requires a combination of gene expression and clinical data.

Global molecular effects of tocilizumab therapy in rheumatoid arthritis synovium.

OBJECTIVE: To investigate the global molecular effects of tocilizumab (TCZ) in comparison with methotrexate (MTX) treatment in synovial biopsy tissue obtained from patients with previously untreated rheumatoid arthritis (RA) before therapy (T0) and 12 weeks after the initiation of therapy (T12), and to compare the results with previous gene expression data obtained in synovial biopsy tissue from adalimumab (ADA)- and rituximab (RTX)-treated patients with RA. METHODS: Paired synovial biopsy samples were obtained at T0 and T12 from the affected knee of TCZ-treated RA patients and MTX-treated RA patients. Gene expression studies were performed using GeneChip Human Genome U133 Plus 2.0 microarrays, and confirmatory quantitative real-time reverse transcription-polymerase chain reaction experiments were performed on selected transcripts. The effects of TCZ and MTX on synovial cell populations and histologic characteristics were assessed by immunohistochemistry. RESULTS: Gene expression studies showed that blockade of the interleukin-6 receptor (IL-6R) gene (IL6R) using TCZ induced a significant decrease in the expression of numerous chemokine and T cell activation genes in the RA synovium. These effects strongly correlated with the molecular effects of MTX and RTX therapy on RA synovial tissue, but differed from the molecular changes induced by ADA (decreased expression of genes involved in cell proliferation). CONCLUSION: The molecular similarities between the effects of TCZ, RTX, and MTX therapies in the RA synovium indicate that B cell- and IL-6-dependent pathways play synergistic roles in the pathogenesis of the disease, in particular through activation of T cell responses. Moreover, these results open perspectives for the individualization of therapeutic decisions, based on a better knowledge of the synovial molecular effects of each type of RA therapy.

Type I interferon blockade in systemic lupus erythematosus: where do we stand?

SLE is an autoimmune condition characterized by loss of tolerance to chromatin constituents and the production of ANAs. The majority of SLE patients display spontaneous expression of type I IFN-induced genes in circulating mononuclear cells and peripheral tissues, and type I IFNs play a role in the pathogenesis of the disease via the sustained activation of autoreactive T and B cells necessary for the production of pathogenic autoantibodies. Several IFN-blocking strategies are currently being evaluated in clinical trials: monoclonal antibodies directed against IFN-α and type I IFN-α receptor (IFNAR), as well as active immunization against IFN-α. This review describes the rationale behind these trials and the results obtained, and discusses the perspectives for further development of these drugs.

Analysis of sialoadhesin expression on mouse alveolar macrophages.

Sialoadhesin (Sn) is a macrophage-restricted receptor that was first characterised on mouse resident bone marrow macrophages as a receptor that mediates the binding, without ingestion, of sheep erythrocytes. Sn is highly conserved in mammals but its expression on tissue macrophages is heterogeneous. In the mouse, high levels of erythrocytes binding are shown on macrophages from lymphoid tissues but a low erythrocytes binding activity is detectable on macrophages isolated from the broncho-alveolar space. Yet, Sn expression has been demonstrated on human, rat and pig alveolar macrophages (AM) using methods of molecular biology. Therefore, the present study aimed to investigate the expression of Sn on mouse AM in order to confirm the presence of the protein on this population of murine macrophages. Using cytometrical analyses, we showed that Sn was expressed on mouse AM surface. Following desialylation, AM largely bound erythrocytes and this binding was inhibited by 3D6, an anti-mouse Sn monoclonal antibody, in a dose-dependent manner. This indicates that Sn is expressed on mouse AM but that the sialic acid binding activity mediated by this molecule is naturally masked by endogenous sialic acid within the glycocalyx on the cell surface.

PEGylation of anti-sialoadhesin monoclonal antibodies enhances their inhibitory potencies without impairing endocytosis in mouse peritoneal macrophages.

Poly(ethylene glycol) (PEG) 5 kDa and 20 kDa have been previously conjugated to two anti-sialoadhesin (Sn) monoclonal antibodies (mAbs), SER-4 and 3D6, and shown to dramatically increase their inhibitory potency in solid-phase red blood cell binding assays. In the present study, we evaluated the effect of anti-Sn SER-4 and 3D6 mAbs PEGylation on their inhibition of cell adhesion in mouse peritoneal macrophages. We also examined whether Sn-mediated PEGylation could affect plasma membrane functions of macrophages as to prevent accessibility, binding, and endocytosis of macromolecules and particles. Conjugation of PEG to plasma membrane is known to cause immune tolerance by impairing protein-protein and cell-cell interactions. PEGylation of SER-4 and 3D6 mAbs increased by 4-fold their inhibition of Sn-mediated erythrocyte binding to macrophages. PEGylated SER-4 and 3D6 mAbs did not impair macrophage membrane integrity, cell metabolism, nor pinocytosis of macromolecules and phagocytosis of latex particles. Thus, PEGylation of antibodies directed to cell surface receptors could be potentially exploited in a therapeutic setting to increase inhibitory potency of antibodies without impairing vital functions of cells.

The inhibitory potencies of monoclonal antibodies to the macrophage adhesion molecule sialoadhesin are greatly increased following PEGylation.

PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.