Julie Ducreux

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.

Global molecular effects of tocilizumab therapy in rheumatoid arthritis synovium

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.

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.

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

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.

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.

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

BackgroundIL6-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.MethodCorrelation 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.ResultsIn 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.ConclusionHigher 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.

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.

Tocilizumab and rituximab, but not adalimumab, display highly concordant molecular effects in the rheumatoid arthritis synovium

Objectives The clinical effects of biologics in patients with rheumatoid arthritis (RA) are well known. By contrast, little is known about their molecular effects in the RA synovium, and how administration of these agents can be tailored according to individual disease status. Here, we compared the effects of three biologics on global synovial gene expression profiles in RA patients. Methods Previously reported high-density transcriptomic data (GeneChip HGU133 Plus 2.0 slides) were recovered, obtained in our laboratory using using synovial biopsies from patients with osteoarthritis (OA, n=5), early untreated RA (n=7), Methotrexate-resistant RA patients before and 3 months after Adalimumab therapy (n= 2 x 8), anti-TNF-resistant RA patients before and 3 months after Rituximab therapy (n= 2 x 12). In addition, new hybridizations were performed with synovial biopsies from untreated early RA patients before and 3 months following Tocilizumab therapy (n= 2 x 12). Results Comparison of RA and OA synovial biopsies led to the identification of 885 RA specific probe sets (535 are up-regulated and 350 are down-regulated in RA synovial tissue). Most (88%) of the probe sets up-regulated in the early RA synovium are down-regulated by Tocilizumab and Rituximab in their respective patients’ populations; conversely 60% of the transcripts down-regulated in RA synovial tissue are up-regulated by both therapies. Changes in gene expression induced by Tocilizumab and Rituximab are highly correlated (r = 0.5797, p < 0.0001); by contrast, much less similarities are found when Tocilizumab or Rituximab is compared to Adalimumab. When the same analyses are applied on all probe sets expressed in the synovium, the same similarities between Tocilizumab and Rituximab are found (r = 0.4116, p < 0.0001), while the effects of Adalimumab appear to be different. 7 out of 12 early RA patients treated with Tocilizumab were in SDAI remission 6 months after initiation of therapy. Patients who did not reach remission had higher baseline expression of IL-6 induced transcripts, including immunoglobulin genes. By contrast, expression of immunoglobulin genes is higher in RA patients who respond to Rituximab therapy. Conclusions Genes differentially expressed in early RA versus OA synovial biopsies are IL-6 and B-cell dependent. The concordance between the molecular effects of Rituximab and Tocilizumab suggests that IL-6 producing B cells are major players in the physiopathology of the disease. Tocilizumab therapy could be less efficient in patients displaying a very strong synovial IL-6 signature. Our results suggest that administration of Rituximab could bypass this obstacle in such patients. Further studies are needed to confirm whether therapy can be tailored according to the molecular patterns found in individual patients. Disclosure of Interest None Declared

THU0108 Global molecular effects of tocilizumab therapy in synovial biopsies of early RA patients

Background Tocilizumab is an approved humanized anti-IL-6 Receptor antibody with proven therapeutic benefits in the treatment of patients with RA. Objectives This study aimed to investigate the global molecular effects of Tocilizumab versus Methotrexate therapy in synovial biopsy samples obtained from early RA patients harvested prospectively before and 12 weeks after administration of the drug. The results were compared with our previous data, generated in prospective cohorts of Adalimumab- and Rituximab-treated (Methotrexate- and anti-TNF-resistant, respectively) RA patients. Methods Paired synovial biopsy samples were obtained from the affected knee of early RA patients before and 12 weeks after initiation of Tocilizumab (n=12) or Methotrexate (n=8) therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR) experiments were performed to confirm the differential expression of selected transcripts. Results We found that Tocilizumab induces a significant down-regulation of genes included in specific pathways: cytokines (IL-6, IL-7, IL-22, ...) & chemokines (CCL8, CCL11, CCL13, CCL19, CCL20, CXCL5, CXCL13, ...), and T cell activation. By contrast, Tocilizumab induces a significant up-regulation of genes associated with healing processes. These effects are significantly more pronounced as compared with the effects of Methotrexate, Rituximab, and Adalimumab therapies. Real-time qPCR experiments, performed until now, confirm the down-regulation of CXCL13 and up-regulation of BMPR1A expression following Tocilizumab treatment. Conclusions Tocilizumab displays distinct molecular effects on synovial biopsies of RA patients. These results open perspectives for the individualization of therapeutic decisions, based on the molecular profiles of the patients. Disclosure of Interest None Declared