Monocyte-derived dendritic cells from HLA-B27+ axial spondyloarthritis (SpA) patients display altered functional capacity and deregulated gene expression

International audienceINTRODUCTION:This study aimed to compare the functional capacity and gene expression profile of monocyte-derived dendritic cells (MD-DCs) in HLA-B27+ axial spondyloarthritis (SpA) patients and healthy controls.METHODS:MD-DCs were differentiated with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for seven days, starting from purified CD14+ monocytes and stimulated with lipopolysaccharide (LPS) for six and twenty four hours. Their capacity to stimulate allogeneic CD4+ T cells from unrelated healthy donor was tested. Transcriptomic study was performed with Affymetrix HuGene 1.0 ST microarrays. Gene expression levels were compared between patients and controls using a multivariate design under a linear model (LIMMA). Real-time quantitative PCR (qRT-PCR) was performed for validation of the most striking gene expression differences.RESULTS:The stimulatory capacity of allogeneic CD4+ T cells by MD-DCs from SpA patients was decreased. Transcriptomic analysis revealed 81 genes differentially expressed in MD-DCs between SpA patients and controls (P 1.5). Four selected genes were validated by qRT-PCR:ADAMTS15, CITED2, F13A1 and SELL. Expression levels of ADAMTS15 and CITED2, encoding a metallopeptidase and a transcription factor, respectively, were inversely correlated with each other (R = 0.75, P = 0.0003). Furthermore, in silico analysis identified several genes of the Wnt signaling pathway having expression co-regulated with CITED2.CONCLUSION:This study revealed altered function and gene expression pattern in MD-DCs from HLA-B27+ axial SpA. Co-expression study showed an inverse correlation between ADAMTS15 and CITED2. Moreover, the Wnt signaling pathway appeared as deregulated in SpA MD-DCs, a finding which may be connected to Th17-driven inflammatory responses

Systematic candidate gene investigations in the SPA2 locus (9q32) show an association between TNFSF8 and susceptibility to spondylarthritis

Objective. Our group previously identified a new susceptibility region linked to spondylarthritis (SpA) on chromosome 9q31-34. Fine mapping of this SPA2 locus allowed us to refine the peak of linkage to a 1.3-Mb interval. The objective of this study was to resequence most positional candidate genes lying in that region, to identify polymorphisms, and to examine their association with SpA. Methods. Variants screening was performed in 30 independent patients with SpA from families with a high linkage score to the SPA2 locus and 30 control subjects. The coding regions, intron-exon boundaries, and 5'- and 3'-flanking regions of ZNF618, A1L4R1_HUMAN (AF495724), AMBP, KIF12, ORM1, ORM2, C9ORF91, ENSESTG000000230601, and TNFSF8 were resequenced to identify polymorphisms. Selected variants were genotyped in an extended French cohort (442 patients and 268 control subjects overall). Replication was performed in a combined Belgian and Portuguese cohort (433 patients and 299 control subjects). Results. Variants screening allowed us to identify 98 polymorphisms, 5 of which were selected for further studies, based on statistical significance. The rare intronic single-nucleotide polymorphism (SNP) rs3181357, located in TNFSF8, was significantly associated with SpA in the French and the replication cohorts (odds ratio [OR] 2.03, P = 0.009 and OR 2.26, P = 0.0014, respectively) and in the pooled analysis (OR 2.14, P = 0.0001). Conclusion. Positional candidate gene screening in the SPA2 locus allowed us to identify and replicate an association between a rare SNP located in TNFSF8 and SpA. This new finding appears to be independent of an association with a haplotype near TNFSF15, which we recently reported

A Preclinical Validation of Iron Oxide Nanoparticles for Treatment of Perianal Fistulizing Crohn’s Disease

International audienceFistulizing anoperineal lesions are severe complications of Crohn’s disease (CD) that affect quality of life with a long-term risk of anal sphincter destruction, incontinence, permanent stoma, and anal cancer. Despite several surgical procedures, they relapse in about two-thirds of patients, mandating innovative treatments. Ultrasmall particles of iron oxide (USPIO) have been described to achieve in vivo rapid healing of deep wounds in the skin and liver of rats thanks to their nanobridging capability that could be adapted to fistula treatment. Our main purpose was to highlight preclinical data with USPIO for the treatment of perianal fistulizing CD. Twenty male Sprague Dawley rats with severe 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced proctitis were operated to generate two perianal fistulas per rat. At day 35, two inflammatory fistulas were obtained per rat and perineal magnetic resonance imaging (MRI) was performed. After a baseline MRI, a fistula tract was randomly drawn and topically treated either with saline or with USPIO for 1 min (n = 17 for each). The rats underwent a perineal MRI on postoperative days (POD) 1, 4, and 7 and were sacrificed for pathological examination. The primary outcome was the filling or closure of the fistula tract, including the external or internal openings. USPIO treatment allowed the closure and/or filling of all the treated fistulas from its application until POD 7 in comparison with the control fistulas (23%). The treatment with USPIO was safe, permanently closed the fistula along its entire length, including internal and external orifices, and paved new avenues for the treatment of perianal fistulizing Crohn’s disease

Comprehensive linkage and association analyses identify haplotype, near to the TNFSF15 gene, significantly associated with spondyloarthritis.

Spondyloarthritis (SpA) is a chronic inflammatory disorder with a strong genetic predisposition dominated by the role of HLA-B27. However, the contribution of other genes to the disease susceptibility has been clearly demonstrated. We previously reported significant evidence of linkage of SpA to chromosome 9q31-34. The current study aimed to characterize this locus, named SPA2. First, we performed a fine linkage mapping of SPA2 (24 cM) with 28 microsatellite markers in 149 multiplex families, which allowed us to reduce the area of investigation to an 18 cM (13 Mb) locus delimited by the markers D9S279 and D9S112. Second, we constructed a linkage disequilibrium (LD) map of this region with 1,536 tag single-nucleotide polymorphisms (SNPs) in 136 families (263 patients). The association was assessed using a transmission disequilibrium test. One tag SNP, rs4979459, yielded a significant P-value (4.9 x 10(-5)). Third, we performed an extension association study with rs4979459 and 30 surrounding SNPs in LD with it, in 287 families (668 patients), and in a sample of 139 cases and 163 controls. Strong association was observed in both familial and case/control datasets for several SNPs. In the replication study, carried with 8 SNPs in an independent sample of 232 cases and 149 controls, one SNP, rs6478105, yielded a nominal P-value<3 x 10(-2). Pooled case/control study (371 cases and 312 controls) as well as combined analysis of extension and replication data showed very significant association (P<5 x 10(-4)) for 6 of the 8 latter markers (rs7849556, rs10817669, rs10759734, rs6478105, rs10982396, and rs10733612). Finally, haplotype association investigations identified a strongly associated haplotype (P<8.8 x 10(-5)) consisting of these 6 SNPs and located in the direct vicinity of the TNFSF15 gene. In conclusion, we have identified within the SPA2 locus a haplotype strongly associated with predisposition to SpA which is located near to TNFSF15, one of the major candidate genes in this region

Interleukin 27 is a novel cytokine with anti-inflammatory effects against spondyloarthritis through the suppression of Th17 responses

International audienceIntroduction : Spondylarthritis (SpA) development in HLA-B27/human β2-microglobulin transgenic rat (B27-rat) is correlated with altered conventional dendritic cell (cDC) function that promotes an inflammatory pattern of CD4+T cells, including a biased expansion of pro-inflammatory Th 17 population and imbalance of regulatory T cells cytokine profile. Transcriptomic analysis revealed that cDCs from B27-rats under express IL-27, an anti-inflammatory cytokine which induces the differentiation of IL-10 + regulatory T cells and inhibits Th 17 cells. Methods : Here, we first investigated whether in vitro addition of exogenous IL-27 could reverse the inflammatory pattern observed in CD4 + T cells. Next, we performed preclinical assay using IL-27 to investigate whether in vivo treatment could prevent SpA development in B27-rats. Results : in vitro addition of IL-27 to cocultures of cDCs and CD4 + T cell subsets from B27-rats reduced IL-17 and enhanced IL-10 production by T cells. Likewise, IL-27 inhibited the production of IL-17 by CD4 + T cells from SpA patients. Interestingly, in vivo treatment with recombinant IL-27 starting before SpA onset, inhibited SpA development in B27-rats through the suppression of IL-17/TNF producing CD4 + T cells. Discussion : Overall, our results reveal a potent inhibitory effect of IL-27 and highlight this cytokine as a promising new therapeutic target in SpA, especially for SpA patients non responders to currently approved biotherapies

Monocyte-Derived Dendritic Cells (MDDCs) from Spondyloarthritis (SpA) Patients Exhbit a Coordinated Downregulation of the Cholesterol (chol) Biosynthesis Pathway That Relates to Lipid Overload

International audienceBackground/Purpose: Gene expression studies are useful to investigate the pathogenesis of complex diseases. The critical role of antigen-presenting cells such as dendritic cells (DCs) being suspected in SpA, we wished to study their particular biology, starting from unbiased transcriptomic experiment. Methods: MDDCs were obtained from circulating CD14+ monocytes after 7 days of culture with GMCSF + IL-4. Transcriptomes were profiled using Affymetrix microarrays (HuGene 1.0 ST) and further confirmed by RNA-seq (78 million paired-end reads (2×100 nt) per sample). Differentially expressed (DE) genes between SpA (ASAS criteria) and healthy controls (HC) were listed with LIMMA. Functional annotation was performed with DAVID and InnateDB. RT-qPCR was used to test the reproducibility of the signature over time. Intra-cellular lipid dropplets (LD) volume and number were quantified by Bodipy® labeling and confocal microscopy . Cellular content of chol pathway metabolites was quantified by mass spectrometry (MS). Results: For transcriptomic analysis, we generated 3 lists of DE genes (nominal p < 0.01) comparing (A) HLA-B27+ SpA (n=40) to B27-neg HC (n=30), (B) B27+ HC (n=44) to B27-neg HC and (C) B27+ SpA to B27+ HC. Subtraction A–B and intersection with C yielded a robust list of 68 genes affected by SpA controlling for unrelated HLA-B27 effect. Analysis of functional pathways revealed a significant overrepresentation of genes involved in chol biosynthesis and its regulation (p < 1×10-4). Five of the 6 genes in this pathway (SQLE, MSMO1, LDLR, INSIG1, SREBF2) were downregulated in SpA. These findings were confirmed by RNA-seq on the same samples and by qPCR on a new series of samples drawn from the same group of individuals (11 SpA vs. 10 HC). Using MS, we evidenced a significant increase of chol and 27-OH-chol content in MDDC from another panel of SpA (n = 14) compared to HC (n = 8) (p < 0.05), suggesting that downregulation of cholesterol synthesis might be secondary to chol overload. Consistently, there was a highly significant increase in the size (p = 5×10-4) and overall volume (p < 2×10-4) of LD in SpA (n=12) compared to HC (n=11) MDDCs (Figure). Importantly, there was no difference of total nor fractionated chol plasma levels between SpA and controls. Conclusion: Our study identified a downregulation of the chol synthesis pathway in MDDCs from SpA patients that seems to be secondary to lipid overload in those cells. Our findings are consistent with a state of pre-activation of those cells that could lead to a strong inflammatory response to endogenous or environmental stimuli