Дисертації з теми "Spondyloarthrites"

Les spondyloarthrites (SpA) sont des maladies inflammatoires chroniques articulaires qui se caractérisent par des atteintes de la colonne vertébrale et des articulations périphériques, en particulier des enthèses, souvent associées à des manifestations extra-articulaires telles que le psoriasis, l’uvéite, ou l’inflammation intestinale. Ces maladies complexes possèdent une forte composante génétique dominée par l'antigène HLA-B27 du complexe majeur d'histocompatibilité de classe I (CMH-I), présent chez plus de 80% des patients atteints de SpA. Découverte il y a 45 ans, l'association entre HLA-B27 et le développement des SpA reste inexpliquée. Plusieurs hypothèses ont été proposées pour expliquer cette association au niveau moléculaire. Cependant, la plupart se heurtent à des incohérences expérimentales qui semblent les invalider. Pour élucider les mécanismes moléculaires pathogènes liés au HLA-B27, nous avons utilisé une nouvelle approche. Drosophila melanogaster est un puissant modèle génétique qui a permis des avancées considérables dans la compréhension de nombreuses fonctions des cellules de métazoaires, ainsi que dans la description des processus cellulaires et moléculaires de nombreuses pathologies humaines. Nous avons établi plusieurs lignées de drosophiles transgéniques pour des formes d’HLA-B associées aux SpA ou pour une forme non associée à la maladie, ainsi que pour la chaîne invariante du CMH-I, la β2m humaine (hβ2m). L'expression des formes associées à la maladie, exclusivement en présence de la hβ2m, dans l'aile et dans l'œil de la drosophile conduit à l'apparition de deux phénotypes spécifiques. Mes résultats ont permis de mettre en évidence que le phénotype de perte des veines transversales de l’aile était associé à une perturbation de la signalisation par la voie des Bone Morphogenetic Protein (BMP). Cette perturbation est associée à une co-localisation de HLA-B27 avec le récepteur BMP de type I, Sax. Nos résultats préliminaires obtenus dans les cellules de patients atteints de SpA suggèrent l’existence d’une co-localisation analogue d’HLA-B27 avec le récepteur ALK2, orthologue de Sax. L'ensemble de nos résultats plaide en faveur d’un rôle pathogène de HLA-B27 passant par une dérégulation de la voie BMP à l’intersection des voies de l’ossification et de l’inflammation et pourrait donc s’appliquer à la physiopathologie des SpA
Spondyloarthritis (SpA) is a chronic inflammatory rheumatic disorder characterized by joint manifestations affecting the spine, peripheral joints and entheses, as well as extra-articular manifestations such as psoriasis, uveitis, or intestinal inflammation. This complex disorder has a strong genetic component dominated by the HLA-B27 antigen of the major histocompatibility complex class I (MHC-I), which is present in more than 80% of SpA patients. Discovered 45 years ago, the association between HLA-B27 and SpA development remains unexplained. Several hypotheses have been proposed to explain this association at the molecular level, but all face experimental inconsistencies that seem to invalidate them. Therefore, it appeared to us essential to elaborate new and yet unexplored approaches in order to better understand the molecular role of HLA-B27 in SpA development. Drosophila melanogaster is a powerful genetic model that has led to considerable advances in understanding numerous functions of metazoan cells, as well as in describing the cellular and molecular processes of many human pathologies. To elucidate the molecular pathogenic mechanisms associated with HLA-B27, we have established several transgenic Drosophila lines for SpA-associated and non-associated of HLA-B alleles, as well as for the MHC-I invariant chain, the human 2-microglobulin (hβ2m). Expression of the HLA-B27 alleles, in the presence of hβ2m, in the Drosophila wing and eye led to two specific phenotypes. The crossveinless wing phenotype is due to a disturbance in the Bone Morphogenetic Protein (BMP) signaling pathway. Interestingly, this misregulation is associated with a co-localization of HLA-B27 and the BMP type I receptor named Sax. Our preliminary results obtained in SpA patient cells suggest that HLA-B27 also colocalizes with ALK2 receptor, which is ortholog to Sax. Altogether, our results suggest that the pathogenic role of HLA-B27 in SpA may depend on a BMP signaling misregulation at the crosstalk between ossification and inflammation

La spondyloarthrite (SpA) est une maladie inflammatoire chronique fréquente. Chez l'Homme, l'association entre l'allèle HLA-B27 du complexe majeur d'histocompatibilité de classe-I (CMH-I) et le développement de la maladie a été démontrée il y a 50 ans, 70 à 90% des patients atteints de SpA étant porteurs de cet allèle. Cependant, le rôle exact du HLA-B27 dans la physiopathologie de la SpA reste inconnu. Le modèle du rat transgénique pour le HLA-B27 et la β2-microglobuline humaine (hβ2m) (rat B27), qui développe des manifestations similaires à la maladie humaine, a permis d'éclaircir certains aspects de la question. Notamment, l'implication des lymphocytes T (LT) CD4+ dans la SpA a pu être mise en évidence. Chez le rat B27, les LT CD4+ régulateurs (Treg) présentent un déséquilibre de la balance interleukine-10/interleukine-17 (IL-10/IL-17), respectivement anti- et pro- inflammatoires. D'autre part, une expansion de la sous-population des LT CD4+ helper 17 (Th17) pro-inflammatoires, producteurs d'IL-17 est observée aussi bien chez le rat B27 que chez les patients. Dans le but d'étudier les effets non-canoniques du HLA-B27, un modèle de Drosophila melanogaster transgénique pour le HLA-B27 et la hβ2m a été développé et a permis de mettre en évidence qu'une interaction entre le HLA-B27 et des récepteurs de type I de la voie BMP/TGFβ (BMPR1) altérait la formation des veines transversales de l'aile. Dans ce modèle, il a précédemment été montré une interaction entre le HLA-B27 et le récepteur Saxophone (Sax) conduisant à l'augmentation de la signalisation BMP. Notre étude a complété ces résultats en montrant qu'une signalisation aberrante passant par le BMPR1 Baboon (Babo) de la voie de l'activine/TGFβ participait aussi au phénotype anormal induit par l'expression du HLA-B27. Pour tenter d'extrapoler ces résultats à un mécanisme de pathogénicité du HLA-B27 au cours de la SpA, nous avons tout d'abord démontré qu'il existait une interaction spécifique entre HLA-B27 et l'activin receptor-like kinase 2 (ALK2), orthologue de Sax d'une part, mais aussi avec ALK5, le récepteur de type 1 au TGFβ orthologue de Babo dans les lymphocytes de rat B27. L'étude de SMAD2/3, le principal transducteur du signal TGFβ, dans les LT de rats B27 et non transgéniques révèle une phosphorylation basale plus faible et une amplitude de phosphorylation plus forte après stimulation par le TGFβ1. De façon concordante, nous avons observé que certains gènes induits par la signalisation TGFβ et impliqués dans la différenciation des Treg et Th17 (Foxp3, Rorc, Runx1) avaient une expression augmentée dans les LT CD4+ naïfs (Tn) de rat B27. Ensemble, ces résultats indiquent une possible activation précoce de la voie TGFβ dans les Tn des rats B27 suivie d'une boucle de rétrocontrôle négatif. De façon intéressante, le gène Tgfb1 lui-même était diminué. Compte tenu de l'importance du TGFβ1 autocrine produit par les LT pour prévenir l'inflammation chronique, ces observations ouvrent des perspectives pour mieux comprendre le rôle du HLA-B27 dans le développement de la SpA. Nous pourrons en particulier, étudier de façon plus approfondie la réponse des Tn de rats B27 au TGFβ1 par des méthodes multi-omiques (transciptomique, phosphoprotéomique, protéomique...). Enfin, compte tenu du rôle essentiel du TGFβ1 autocrine dans le maintien des profils Treg et Th17, une étude de la plasticité des Treg et Th17 des rats B27 serait pertinentes pour mieux comprendre la physiopathologie de la SpA
Spondyloarthritis (SpA) is a common chronic inflammatory disease. In human, the association between the HLA-B27 allele of the class-I major histocompatibility complex (MHC-I) and the development of this disease was demonstrated 50 years ago, with 70-90% of SpA patients carrying this allele. However, the exact role of HLA-B27 in the pathophysiology of SpA remains unknown. The model of rat transgenic for HLA-B27 and the human β2-microglobulin (hβ2m) (B27 rat), which develops manifestations similar to the human disease, has shed light on certain aspects of the question. In particular, the involvement of CD4+ T lymphocytes in SpA has been demonstrated. In the B27 rat, regulatory CD4+ T lymphocytes (Treg) exhibit an imbalance of the interleukin-10/interleukin-17 (IL-10/IL-17) ratio, which are anti-inflammatory and pro-inflammatory, respectively. On the other hand, an expansion of the sub-population of pro-inflammatory CD4+ T helper 17 (lymphocytes Th17), which produce IL-17, was observed in both B27 rats and SpA patients. To study the non-canonical effects of HLA-B27, a Drosophila melanogaster model transgenic for HLA-B27 and hβ2m was developed and demonstrated that an interaction between HLA-B27 and type I receptors of the BMP/TGFβ pathway (BMPR1s) altered the formation of the wing transverse veins. In this model, an interaction between HLA-B27 and the Saxophone (Sax) receptor has previously been shown to lead to increased BMP signalling. Our study complemented these results by showing that aberrant signaling via the BMPR1 Baboon (Babo) of the activin/TGFβ pathway also contributed to the abnormal phenotype induced by HLA-B27 expression. In an attempt to extrapolate these results to a mechanism of HLA-B27 pathogenicity in SpA, we first demonstrated that there was a specific interaction between HLA-B27 and activin receptor-like kinase 2 (ALK2), the orthologue of Sax, and also with ALK5, the type 1 receptor for TGFβ orthologue of Babo, in rat B27 lymphocytes. Study of SMAD2/3, the main transducer of the TGFβ signal, in T lymphocytes from B27 and nontransgenic rats revealed a lower basal phosphorylation and a higher amplitude of phosphorylation after stimulation by TGFβ1. Concordantly, we observed that several genes induced by TGFβ signaling and involved in Treg and Th17 differentiation (Foxp3, Rorc, Runx1) had increased expression in naive CD4+ T lymphocytes (Tn) from B27 rats. Taken together, these results indicate a possible early activation of the TGFβ pathway in B27 rat Tn followed by a negative feedback loop. Interestingly, the Tgfb1 gene itself was decreased. Given the importance of autocrine TGFβ1 produced by T lymphocytes in preventing chronic inflammation, these observations open up prospects for a better understanding of the role of HLA-B27 in the development of SpA. In particular, we propose to study in greater depth the response of Tn from B27 rats to TGFβ1 using multi-omics methods (transciptomic, phosphoproteomic, proteomic). Finally, given the essential role of autocrine TGFβ1 in the maintenance of Treg and Th17 profiles, a study of the plasticity of Treg and Th17 in B27 rat would be relevant to a better understanding of the pathophysiology of SpA

La spondyloarthrite (SpA) est une maladie multifactorielle avec une héritabilité estimée à plus de 90%, principalement en lien avec le HLA-B27. L'ensemble des facteurs de susceptibilité identifiés, incluant HLA-B27, expliquent moins du tiers de l'héritabilité. L'implication de variants rares pourrait expliquer une partie de cette héritabilité manquante. L'objectif de ce travail était d'identifier des variants rares associés à la SpA via une approche combinant analyses familiales et séquençage haut-débit. D'abord, nous avons séquencé une région de 1,4 Mb significativement liée à la SpA en 13q13 chez 71 patients et 21 témoins sains appartenant à des familles avec un score de liaison élevée dans cette région. Nous avons identifié un variant rare dans le gène FREM2 présent chez 9 malades d'une famille fortement liée à la région et non retrouvé dans d'autres familles ou cas isolés de SpA. Nous avons ensuite séquencé l'exome de 48 malades venant de 20 familles multiplex. Malheureusement, nous n'avons pas observé de variants récurrents entre les familles. Puis, nous nous sommes concentrés sur un deuxième pic de liaison génétique, déjà connu, sur le chromosome 9. L'étude de la famille la plus liée à cette région, qui comprend 12 patients, a conduit à l'identification de plusieurs variants rares codants ségrégeant avec la maladie. Cependant les études ultérieures ont montré des fréquences alléliques de ces variants équivalentes entres les cas et les témoins. Enfin, le séquençage du génome entier de 413 patients issus de 76 familles multiplex avec 4 malades ou plus a été réalisé. Nous avons identifié 1203 variants rares, codants et non synonymes et partagés par au moins tous les membres atteints d'une famille. Les analyses de validation génétique et fonctionnelle de ces variants sont en cours, tout comme l'analyse des variants non-codants. En conclusion, ces différentes approches suggèrent une importante hétérogénéité génétique de la SpA et soulignent également la difficulté de confirmer l'implication de variants rares dans les maladies complexes
Spondyloarthritis (SpA) is a multifactorial disease with an estimated heritability of over 90%, mainly related to HLA-B27. All identified susceptibility factors, including HLA-B27, explain less than one third of the heritability. The involvement of rare variants could explain part of this missing heritability. The aim of this work was to identify rare variants associated with SpA via a combined family analysis and high-throughput sequencing approach. First, we sequenced a 1.4 Mb region significantly linked to SpA at 13q13 in 71 patients and 21 healthy controls from families with a high linkage score in this region. We identified a rare variant in the FREM2 gene present in 9 patients from a family with high linkage to the region and not found in other families or isolated cases of SpA. We then sequenced the exome of 48 patients from 20 multiplex families. Unfortunately, we did not observe any recurrent variants between families. We then focused on a second, previously known genetic linkage peak on chromosome 9. The study of the family most linked to this region, which includes 12 patients, led to the identification of several rare coding variants segregating with the disease. However, subsequent studies have shown equivalent allelic frequencies of these variants between cases and controls. Finally, whole genome sequencing of 413 patients from 76 multiplex families with 4 or more patients was performed. We identified 1203 rare, coding, non-synonymous variants shared by at least all affected family members. Genetic and functional validation analyses of these variants are underway, as is the analysis of non-coding variants. In conclusion, these different approaches suggest significant genetic heterogeneity in SpA and also highlight the difficulty of confirming the involvement of rare variants in complex diseases

La Spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent, avec une prévalence de 0,43 % en France. Elle consiste en une atteinte prédominante du squelette axial, mais aussi des articulations périphériques, et peut conduire à une immobilité du rachis et des articulations sacro-iliaques. Des atteintes extra-articulaires sont fréquentes, telles qu'une uvéite, un psoriasis ou une maladie inflammatoire chronique de l'intestin. Les traitements actuels ne sont que symptomatiques, ciblant principalement les manifestations inflammatoires. L'étiologie de la SpA est multifactorielle avec une composante génétique dominée par l'association forte et bien connue avec l'allèle HLA-B27. Cependant, ce facteur génétique n'est clairement pas suffisant pour induire le développement de la maladie. L'objectif de ce projet de thèse était donc d'identifier d'autres facteurs génétiques à l'origine du développement de la SpA.Mon travail a porté sur l'analyse de deux jeux de données complémentaires, dans une perspective de biologie des systèmes. Dans une première partie, j'ai conduit une analyse de liaison dans 210 familles atteintes de la maladie représentant 1310 personnes génotypées avec des puces Affymetrix 250k. Une nouvelle région significativement liée à la SpA a été détectée en 13q13, avec un intervalle de 1,3 Mb défini par des haplotypes recombinants chez les patients.Ensuite, une analyse transcriptomique des cellules dendritiques dérivées des monocytes de 23 patients HLA-B27+, 23 témoins sains HLA-B27+ et 21 témoins sains HLA-B27-, et stimulées ou non par du LPS, a tenté de distinguer les gènes dont l'expression est modifiée par la maladie de ceux influencés par l'allèle HLA-B27 seul. L'annotation fonctionnelle et une analyse par réseau de gènes ont mis en évidence l'inhibition chez les patients des étapes précoces de la biosynthèse du cholestérol
Spondyloarthritis is a frequent chronic inflammatory rheumatism, with a prevalence of 0.43 % in France. This disease presents axial skeleton injuries, but also on peripheral joints, and can results in a total spinal and sacro-iliac motility loss. Extra-articular features including uveitis, psoriasis and inflammatory bowel disease are frequent. Current SpA treatments are only symptomatic, relieving inflammatory symptoms. SpA etiology is largely multifactorial with a genetic component dominated by the long-known strong association with the HLA-B27 allele. This allele, however, is not sufficient for the disease to occur. This thesis project objective was then to identify other genetic factors in the origin of SpA.My work was mainly divided in two complementary data analyses, in a way to get a systems biology approach. The first one consisted in proceed linking analyses on data from Affymetrix genotyping chips gathered from DNA of 1310 people grouped in 210 families. This study allowed notably to detect a new significantly linked region to SpA : 13q13, with an interval of 1.3 Mb. This part of genome is currently being sequenced to allow a better causal SNP identification.Secondly, an Affymetrix HumanGene 1.0 st transcriptomic chips analysis was performed on MD-DCs extracted from 68 people, stimulated or not by LPS during 6 or 24 hours. This cohort was grouped between 23 patients HLA-B27+, 23 healthy controls HLA-B27+ and 21 healthy controls HLA-B27-. I could notice that HLA-B27 allele is farly enough to considerably affect cell transcriptomic profiles, which encourages to include HLA-B27+ healthy controls. Otherwise, a gene network analysis allowed me to highlight on an inhibition of early steps of cholesterol biosyntthesis

Immune dysfunction in spondyloarthritis (SpA) causes significant morbidity to a large number of patients. An understanding of the underlying pathological processes involved in causing the disease is essential for the development and better targeting of therapies. I have shown for the first time in patients with SpA an overall expansion of peripheral blood and synovial fluid T cells producing GM-CSF and an expansion of GM-CSF positive Th17 cells. In addition, I have shown GM-CSF to be a major effector cytokine of joint-derived innate lymphoid cells. Surface phenotyping and transcriptional analysis of the IL-17A/GM-CSF double positive cells showed that whilst they are related to classic Th17 cells, they also have a uniquely activated transcriptional profile. Additionally, I have shown IL-7 to be a promoter of GM-CSF production by CD4 T lymphocytes in vitro. However, my data shows that polymorphisms of the IL7R, which have been shown to be associated with AS in genome-wide association studies, play a functional role through cell surface expression of the IL7R on CD14 monocytes. This genotypic expression of the IL7R on monocytes is only seen after activation with LPS or TNFa. I further show CD14 monocytes expressing IL7R to be present in SpA joints. My data shows that ROR?t, the master transcription factor of Th17 cells, can be therapeutically targeted using small molecule inhibitors in-vitro. These inhibitors lead to a specific suppression of both polyfunctional and IL-17A single positive Th17 cells. The work in this thesis has highlighted several areas of novel immune biology with potential therapeutic applications in SpA and across the spectrum of related inflammatory diseases.

La spondyloarthrite (SpA) est une pathologie rhumatologique caractérisée notamment par une inflammation et par des ossifications excessives se formant au niveau des enthèses. Il s’agit de zones de fortes contraintes mécaniques où les tendons et ligaments sont ancrés dans l’os via une zone fibrocartilagineuse. La sphingosine 1-phosphate (S1P) est un lipide bioactif qui joue un rôle important à la fois dans le remodelage osseux et la réponse inflammatoire. Notre objectif était donc d’explorer le rôle de la S1P dans l’ossification excessive de la SpA. Nous avons observé que les taux sériques de S1P des patients atteints de SpA sont significativement supérieurs à ceux de donneurs contrôles. Nous avons utilisé comme modèle des cultures primaires murines d’ostéoblastes, de chondrocytes et de ténocytes et des cultures organotypiques d’enthèse de souris. Nous avons observé que les enzymes de synthèse de la S1P, les sphingosine kinases 1 et 2, contribuent à la minéralisation des ostéoblastes et des chondrocytes. L’effet pro-minéralisant de la S1P est partiellement médié par deux de ses récepteurs (S1P1 et S1P3). De plus, la production de S1P est stimulée suite à un étirement cyclique dans les ostéoblastes et les chondrocytes, et après un traitement avec les cytokines TNF-α et IL-17 dans les chondrocytes. Finalement, l’inhibition générale du métabolisme de la S1P par le Fingolimod conduit à une diminution de la minéralisation dans les ostéoblastes et encore davantage dans les chondrocytes. Ces résultats suggèrent que le métabolisme de la S1P participe à l’ossification excessive de la SpA. Des études in vivo sont maintenant nécessaires pour valider cette possibilité
Spondyloarthritis (SpA) is a rheumatic disease characterized in particular by enthesis ectopic ossification and inflammation. Enthesis is a zone of concentration of mechanical stresses where ligaments and tendons attach to bone through fibrocartilaginous connections. Sphingosine 1-phosphate (S1P) is a bioactive lipid that plays an important role in both bone remodelling and in inflammatory response. Our aim was to explore the role of S1P in SpA excessive ossification. We observed that serum S1P concentrations in SpA patients are significantly higher compared to control donors. We used primary mouse osteoblasts, chondrocytes and tenocytes as cellular models and organotypic cultures of mice enthesis. We observed that S1P synthetizing enzymes, sphingosine kinases 1 and 2, stimulate osteoblasts’ and chondrocytes’ mineralizing process. S1P pro-mineralizing effect was partially mediated by two of the S1P receptors (S1P1 and S1P3). Moreover, S1P production was enhanced by cyclic strain in osteoblasts and chondrocytes and by pro-inflammatory cytokines (TNF-α and IL-17) in chondrocytes. Finally, the inhibition of S1P metabolic pathway by Fingolimod reduced the mineralization in cultured osteoblasts and even more in chondrocytes. These results suggest that S1P metabolism participates in SpA excessive ossification. In vivo studies are now needed to validate this possibility

There have been substantial advances in our understanding of the CD4+CD25+ regulatory T cell subset, but the possibility of an equivalent regulatory subset within the CD8+ T cell population has received less attention. In the course of studies investigating immunological abnormalities in patients with the inflammatory arthritis Ankylosing Spondylitis (AS), novel human CD8+ T cells that have a regulatory phenotype and function have been identified. CD8+/TCRαβ+ T cells were isolated from the peripheral blood of both AS patients and healthy donors and subsequently expanded as T cell lines using antilogous LPS activated dendritic cells. These lines contained IL-4 producing CD8+ T cells which were then cloned non-specifically by limiting dilution. Conventional CD8+ T cells are cytotoxic but these clones were not. They proliferated and produced cytokines in an autoreactive HLA class I restricted fashion; the cytokines produced included IL-4, IL-5 and TGFβ1, but not IFN-γ or IL-10. The clones expressed markers commonly associated with CD4+CD25+ regulatory T cells, including high levels of CTLA-4 and Foxp3. They suggested IFN-γ production and proliferation by CD4+ T cells in vitro in a cell contact dependent manner, which appeared to involve surface CTLA-4 since suppression could be blocked using an anti-CTLA-4 mAb. Increased numbers of these IL-4+ CD8+ T cells were found in AS patients’ PBMC stimulated directly ex vivo compared to healthy donors, and high numbers correlated with decreased numbers of pro-inflammatory IFN-γ producing T cells, suggesting they may actively regulate inflammation in vivo. Thus, human CD8+ regulatory T cells may be up-regulated in the peripheral blood under certain pathological conditions such as in response to chronic inflammation. AS is a remitting and relapsing inflammatory disease; therefore it is possible that these cells contribute to periods of remission by actively regulating inflammation in vivo.

Th17 cells are implicated in a variety of inflammatory disorders including Spondyloarthritis (SpA). Ankylosing spondylitis (AS), the most common SpA, is genetically associated with HLA-B27 and IL-23R polymorphisms, although the link remains unexplained. In addition to classically folded heterotrimers, HLA-B27 can form β2m-free homodimers (B272). B272, as well as free heavy chains, are able to interact with KIR3DL2. I have shown an expansion of KIR3DL2+ CD4+ T cells in peripheral blood of AS patients and HLA-B27+ healthy controls. Although KIR3DL2+ CD4+ T cells comprise a minority of CD4+ T cells in peripheral blood, KIR3DL2+ CD4+ T cells account for the majority of peripheral blood CD4+ T cell IL-23R expression. KIR3DL2+ CD4+ T cells from synovial fluid are also enriched for IL-23R expression compared to matched peripheral blood samples. Moreover, treatment exposure to rIL-1/23 significantly increases IL-17 production by KIR3DL2+ CD4+ T cells in AS patients but not RA patients and healthy controls. KIR3DL2+ CD4+ T cells from AS patients produce more IL-17 than KIR3DL2+ CD4+ T cells from HLA-B27- healthy controls. KIR3DL2+ CD4+ T cells from AS patients and healthy controls are also enriched for dual production of IL-17 and IFNγ, consistent with the theory that AS is a Th17 or a Th17/1 driven disease. I have shown that naïve CD4+ T cells express KIR3DL2 de novo after activation. Interaction with B272-expressing cells acts to maintain and/or further increase KIR3DL2 expression. Interaction with B272-expressing cells also increases expression of Bcl-2. I propose that interaction of KIR3DL2+ CD4+ T cells with B272 preferentially promotes the survival of these pro-inflammatory cytokine-producing KIR3DL2+ CD4+ T cells in SpA. HD6, a B272-specific monoclonal antibody, decreases IL-17 production by PBMCs from AS patients, making it an attractive candidate therapeutic reagent in the treatment of AS and other HLA-B27-associated SpA.

La recherche de nouvelle génération de marqueurs est cruciale pour une meilleure prise en charge des patients, que ce soit au niveau du diagnostic ou de la prédiction de la réponse au traitement. Contrairement aux approches standards visant une cible moléculaire précise, les techniques de spectroscopie vibrationnelle donnent accès à la constitution moléculaire globale d'un échantillon. Les travaux de l'unité BioSpecT ont montré que ces techniques permettaient d'apporter des informations sur l'organisation tissulaire complémentaires à l'histologie conventionnelle. Par exemple, l'analyse par imagerie spectrale de prélèvements tissulaires de lésions cancéreuses (côlon, peau, poumon) permet de mettre en évidence une hétérogénéité des zones tumorales et de caractériser finement le stroma. Une composante inflammatoire peut aussi être distinguer des autres structures. L'objectif de ce projet de thèse est d'exploiter les infor mations accessibles par spectroscopie vibrationnelle dans le cadre de pathologies spécifiques au côlon, afin d'extraire des marqueurs numériques caractérisant l'état physiopathologique des lésions étudiées. L'identification de tels marqueurs requiert de confronter les données spectroscopiques avec des informations de référence d'ordre clinique, histologique, biologique ou encore pharmacologique. Plus précisément, nous nous intéresserons principalement aux cancers du côlon métastatiques avec l'objectif d'identifier des marqueurs prédictifs de la réponse à la chimiothérapie. Pour les analyses, nous pourrons bénéficier non seulement d'échantillons de tissus humains (tumorothèque du CHU de Reims) mais également de xénogreffes réalisées chez la souris de façon à disposer de matériel obtenu selon des protocoles davantage standardisés. De manière complémentaire, une partie des travaux de recherche pourront être réalisés dans le cadre d'une étude débutante sur les maladies inflammatoires chr oniques de l'intestin, en focalisant les expérimentations sur la caractérisation de l'inflammation par spectroscopie vibrationnelle. Ces recherches seront menées en collaboration avec le service d'Oncologie Digestive (Pr O Bouché) du CHU de Reims, le laboratoire de Biopathologie (Pr A Marchal) du CHU de Reims et l'unité INSERM U1113 IRFAC (Dr D Guenot) de Strasbourg
The search for new generation markers is crucial for better patient management, both in terms of diagnosis and prediction of treatment response. Contrary to standard approaches targeting a specific molecular target, vibrational spectroscopy techniques give access to the global molecular constitution of a sample. The work of the BioSpecT unit has shown that these techniques can provide information on tissue organization that is complementary to conventional histology. For example, spectral imaging analysis of tissue samples from cancerous lesions (colon, skin, lung) can highlight the heterogeneity of tumor zones and finely characterize the stroma. An inflammatory component can also be distinguished from other structures. The objective of this thesis project is to exploit the information available by vibrational spectroscopy in the context of pathologies specific to the colon, in order to extract numerical markers charac terizing the physiopathological state of the lesions studied. The identification of such markers requires the comparison of spectroscopic data with clinical, histological, biological or pharmacological reference information. More precisely, we will focus on metastatic colon cancers with the objective of identifying predictive markers of response to chemotherapy. For the analyses, we will be able to benefit not only from human tissue samples (tumor library of the Reims University Hospital) but also from xenografts performed in mice in order to have material obtained according to more standardized protocols. In a complementary way, part of the research work could be carried out within the framework of a study on chronic inflammatory bowel diseases, by focusing the experiments on the characterization of inflammation by vibrational spectroscopy. This research will be carried out in collaboration with the Digestive Oncology Department (Pr O Bouché) of the Reims University Hospital, the Biopathology Laboratory (Pr A Marchal) of the Reims University Hospital and the INSERM U1113 IRFAC unit (Dr D Guenot) of Strasbourg

L’interface hôte/microbiote intestinal est un système d’interactions complexes dont le déséquilibre est associé au développement des maladies inflammatoires chroniques. Les traitements anti-TNFα sont très efficaces dans ces maladies, mais seulement chez certains patients. L’objectif de ce travail était de rechercher un lien entre composition du microbiote intestinal et réponse aux traitements anti-TNFα dans deux types de maladies inflammatoires chroniques, les spondyloarthrites et les maladies inflammatoires chroniques de l’intestin. Nous avons retrouvé des variations de la composition du microbiote intestinal après traitement par anti-TNFα chez des patients atteints de spondyloarthrite et avons identifié un nœud taxonomique prédictif de la réponse thérapeutique à trois mois. Ce nœud taxonomique, l’ordre des Burkholderiales, étant ainsi un biomarqueur potentiellement utilisable en pratique clinique, nous avons déposé une demande de brevet européen, qui est en cours d’instruction. Ce travail a été poursuivi par un nouveau protocole de recherche clinique incluant des patients atteints de spondyloarthrites mais aussi de maladies inflammatoires chroniques intestinales. Ce protocole est financé par le CHU de Bordeaux dans le cadre de l’Appel d’Offre Interne. Il permettra de valider les hypothèses de notre premier travail, en réalisant notamment des PCR quantitatives utilisant des amorces spécifiques de l’ordre des Burkholderiales. Nous avons de plus retrouvé in vitro pour la première fois à notre connaissance une modulation de la croissance bactérienne chez Bacteroides fragilis en réponse au TNFα humain
The host/gut microbiota interface is a system of complex interactions whose imbalance is associated with the development of chronic inflammatory diseases. Anti-TNFα treatments are very effective in these diseases, but only in some patients. The purpose of this work was to find a link between the composition of the intestinal microbiota and clinical response to anti-TNFα treatments in two types of chronic inflammatory diseases, spondyloarthritis and inflammatory bowel disease. We found variations in the composition of the intestinal microbiota after treatment with anti-TNFα in patients with spondyloarthritis and identified a taxonomic node predictive of the therapeutic response at 3 months. This taxonomic node, the Burkholderiales order, being a biomarker potentially usable in clinical practice, we have filed a European patent application, which is currently under investigation. This work was continued by a new clinical research protocol including patients with spondyloarthritis but also with inflammatory bowel diseases. This protocol is funded by the Bordeaux University Hospital as part of the internal call for tenders. It will validate the hypotheses of our first work, notably by performing quantitative PCRs using specific primers targeting the order of Burkholderiales. In vitro, we have also found for the first time, to our knowledge, a modulation of bacterial growth in Bacteroides fragilis in response to human TNFα

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompability Complex (MHC) class I antigen that is strongly associated with development of a group of closely related arthritic diseases, collectively known as the spondyloarthropathies (SpA). However, the mechanism by which HLA-B27 confers this susceptibility is unclear. Studies have shown that HLA-B27 heavy chains can form classical heterotrimers associated with peptide and β2-microglobulin (B27HT), and also non-classical heavy chain homodimers (B27₂). B27₂ assemble intracellularly during maturation and are also expressed at the cell surface following endosomal recycling of B27HT. A pathogenic role for B27₂ has been proposed in two of the current theories of pathogenesis: the B27 homodimer theory and the B27 misfolding and UPR theory. Yet, determinations of the extent, distribution, and triggers of B27₂ expression, as well as the functional consequences of its receptor interactions in AS pathogenesis, have been hampered by the lack of a specific detection reagent. Therefore, to investigate the role of B27₂ in AS, we generated a novel antibody to B27₂ – HD6 – using phage display technology, which binds to in vitro refolded B27₂ but not B27HT complexes by ELISA. This thesis provides evidence that HD6-reactive molecules, which include B27₂, are expressed at the cell surface in both cell lines and in the context of a disease setting. Recognition is B27-specific and strongly correlated with the magnitude of B27 expression, which could account for the lack of staining in some cell subsets. Moreover, staining was comparable in cell lines expressing the disease-associated B*27:05 and the less disease-associated subtype B*27:09. In addition, I have shown cells expressing physiologic levels of B27, including EBV-transformed BCLs and AS patient PBMCs, are capable of expressing the HD6 epitope upon low pH treatment. Interestingly, these ‘acid-inducible HD6’ molecules were absent from cells lacking a functional PLC. Finally, I have shown that HD6-reactive molecules can derive from pre-existing folding B27 molecules at the cell surface, which may be inhibited by the addition of exogenous B27-binding peptides. These findings are consistent with a mechanism of pathogenesis involving the surface expression and recognition of B27₂ and/or other aberrantly folded forms of B27, as proposed in the homodimer theory. HD6 will be a powerful tool to address the potential pathogenic role of B27₂ in SpA and may additionally have therapeutic potential.

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating naïve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.

La spondyloarthrite (SpA) est une maladie chronique inflammatoire affectant principalement les articulations axiales et périphériques et parfois des organes extra-articulaires comme les intestins. Le modèle du rat transgénique pour le HLA-B27 et la β2-microglobuline humaines (rat B27) développe des manifestations cliniques ressemblant à celles présentes chez l'Homme. Dans ce modèle, il a été montré de nombreux dysfonctionnements des cellules dendritiques conventionnelles (cDCs) CD103+, pouvant éventuellement concourir au développement de la SpA. La sous-population de cDCs CD4- exprimant XCR1, un récepteur chimiokinique impliqué dans leur migration, est décrite comme étant tolérogène. Aussi, dans ce travail de thèse, avons-nous souhaité examiner le devenir des cDCs XCR1+ dans ce modèle animal de SpA. Les sous populations de cDCs ont été isolée de la rate, des ganglions mésentériques (MLN) et de la lamina propria du colon de rats B27et de rats témoins non transgéniques (NTG) ou transgéniques pour le HLA-B7. Après digestion par la collagénase et isolation par gradient de densité, les DCs ont été caractérisées par cytométrie en flux ou par PCR en temps réel. La migration des cDCs de la muqueuse intestinale vers les MLN induite par la stimulation du TLR-7 par le Résiquimod a été étudiée. Nous avons observé que la fréquence de cDCs CD4- était réduite chez le rat B27 en comparaison avec celle des rats NTG. En outre, cette diminution n'était pas liée à une mortalité excessive des cDCs CD4- des rats B27. De manière intéressante, nous avons remarqué que la fréquence des cDCs XCR1+, sous-population particulière des cDCs CD4- était diminuée dans la rate, les MLN et la lamina propria colique. Enfin, nous avons observé qu'à la suite d'une stimulation du TLR7 par le Résiquimod, la migration des cDCs XCR1+ était significativement réduite chez les rats B27. Nos résultats démontrent pour la première fois une diminution de la proportion de la sous-population tolérogènes des cDCs XCR1+ dans les organes cibles de la SpA du rat B27, ce qui pourrait affecter le maintien de la tolérance au soi et le contrôle de l'inflammation
Spondyloarthritis (SpA) is a chronic inflammatory disease affecting primarily axial and peripheral joints and sometimes also extra-articular organs, such as the gut. Rats transgenic for HLA-B27 and human β2-microglobulin (B27-Tg rat) develop clinical manifestations resembling human disease. In this model, it has been shown that CD103+ conventional dendritic cells (cDCs) exhibited altered functions, likely promoting SpA development. CD4- cDCs sub-population expressing XCR1, a chemokine receptor involved in their migration have been described to be tolerogenic. Thus, in this study, we wished to examine the fate of XCR1+ cDCs in this animal model of SpA.cDCs populations were isolated from the spleen, mesenteric lymph nodes (MLN) and colonic lamina propria from B27-TG and control nontransgenic (NTG) and/or HLA-B7 transgenic rats after collagenase digestion and density gradient and characterized by flow cytometry or real time PCR. Migration of cDCs from intestinal mucosa to MLN was assessed, using stimulation of TLR-7 with Resiquimod. Interestingly, we observed a reduced frequency of CD4- DC in B27-Tg rats, as compared to NTG rats. Furthermore, this decrease was not due to excessive death of CD4- DCs in B27 rats. Interestingly, we observed a decreased frequency of the XCR1+ subpopulation among CD4- cDCs in the spleen, MLN and lamina propria from B27-Tg rats. Finally, we observed that after TLR-7 stimulation, the migration of XCR1+ cDCs to MLN was proportionally reduced in B27-Tg rats. Our results demonstrate for the first time a decreased proportion of tolerogenic XCR1+ cDCs subpopulation in SpA target organs in the B27-Tg rats, which may affect the maintenance of self-tolerance and control inflammation

L’objectiu d’aquesta recerca era avaluar el diagnòstic i el tractament de les formes primerenques d‘espondiloartritis a la practica diària. Per això, hem inclòs en aquesta tesi tres articles que reflecteixen els tres punts claus de la nostra recerca. Primer, hem validat els criteris ASAS per a les espondiloartritis axials en la pràctica clínica, avaluant el seu rendiment per a la classificació dels pacients però també per al diagnòstic, comparant-los amb altres criteris d'espondiloartritis, i en diferents subgrups d ‘espondiloartritis (com l’artritis psoriàsica, o l’artritis relacionada amb la malaltia inflamatòria intestinal). En segon lloc, donat que el braç “clínic” dels criteris ASAS per a les espondiloartritis axials no està completament reconegut per alguns col·legues i algunes instàncies sanitàries i de governs, vàrem investigar si les característiques demogràfiques fenotípiques i de la malaltia de pacients dels pacients que pertanyien al braç “imatge” i “clínic” dels criteris ASAS eren diferents, en un grup de espondiloartritis primerenques. Finalment, ens vam interessar a l’efecte terapèutic dels inhibidors del TNF alfa a la practica clínica i en les formes primerenques d ‘espondiloartritis, i en els potencials factors predictors de resposta (per exemple, el pertànyer al braç “imatge” dels criteris ASAS).
The objective of this research was to evaluate diagnosis and treatment of early axial spondyloarthritis in clinical practice. For this, we have included in this thesis three articles that reflect the three milestones of our research. First, we aimed to validate the ASAS criteria for axial spondyloarthritis in a clinical practice setting, by evaluating their performance both in terms of classification but also of diagnostic purposes, compared to other sets of criteria and in different spondyloarthritis presentations (e.g. psoriatic arthritis, IBD related-arthritis, etc..). Secondly, since the “clinical” arm of the ASAS criteria for axial spondyloarthritis is not well recognized by many in the medical community, we aimed to compare the demographics, phenotypic and disease characteristics of patients fulfilling the “imaging” and “clinical” arm of the ASAS. Finally, we aimed to evaluate the treatment effect of TNF-alpha blockers in clinical practices and the potential predictors of response (e.g. the fulfilment of the ASAS criteria for axial spondyloarthritis).

Background. The seronegative spondyloarthritis (SpA) are a group of chronic inflammatory diseases resulting from a complex interplay among genetic background (mainly represented by HLA-B27) and environmental factors, that leads to the activation of autoinflammation and the dysregulation of the immune-system. In many cases, an early diagnosis and an appropriate monitoring of disease activity can be difficult because of the overlap of clinical features. Furthermore, because of the indices of inflammation, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), are in the normal range in at least half of SpA patients with a clear expression of disease activity, a delay in diagnosis and consequently in treatment in these patients has been documented. This imparts a tremendous symptomatic burden and loss of function in these patients during the productive years of life. For all these reasons, much attention is currently devoted to the identification of biochemical and genetic biomarkers to be used in the diagnosis as well as prognostic factors in evaluating the treatment effectiveness. Among the genetic predisposing factors, a well-known role is that of HLA-B27, which contributes however to only 20–30% of the total heritability, whereas the whole major histocompatibility complex (MHC) accounts for about 40–50% of the genetic risk of developing SpA. This suggested that other genes are involved in pathogenetic mechanism. In fact, in addition to HLA-B27, a number of genetic factors in both, MHC and non-MHC locus, have been claimed to play a role in pathogenesis of SpA. In this context, because of TNF-α is primarily involved in the propagation and perpetuation of inflammation in SpA, the study of TNF-α genetic is of great interest. Several polymorphisms (SNPs) in genes involved in TNF-α signalling, as TNFA, TNFSF15, TNFR1 and TRADD genes, have been identified as associated with SpA, even if results are controversial. Of great interest are also variants in MEFV gene, involved in the pathogenesis of the autoinflammatory disorder Familial Mediterranean Fever (FMF). Recent studies have shown that the SpA, and in particular the ankylosing spondylitis (AS), are very common among patients affected by FMF and that these patients can present with AS as a sole manifestation. The present study, conducted in a cohort of 91 SpA patients and 223 controls, coming from a North-East Italian region, was aimed to identify biohumoral (biochemical and haematological) and genetic factors to support the diagnostic and prognostic (response to therapy) work-up of SpA diseases. In particular, in addition to biochemical and haematological indices, we investigated whether SNPs in the promoter region of TNFA, or SNPs in the autoinflammatory TNFRSF1A and MEFV genes, might concur with HLA-B27 in enhancing the risk of developing SpA disease and/or in predicting the response to anti-TNFα drugs. Methods. The study population comprised 91 patients with a diagnosis of SpA (mean age ± standard deviation: 52.1 ± 12.5 years; 57 males, 34 females) and 223 blood donors (mean age ± standard deviation: 46 ± 11 years; 146 males, 77 females) coming from Veneto Region, a North-East Italian region. Among patients, 36 had a diagnosis of AS and 55 patients of psoriatic arthritis (PsA), which were based on New York and CASPAR criteria respectively. The protocol of this study was approved by the Local Institutional Ethic Committee of University-Hospital of Padua, Italy (Prot.n. 3024P/13), and all participants gave written informed consent before entering the study. Demographic and physiological data, medical and familial history data were collected for each participant. Blood samples were collected and complete blood count, CRP, ESR, uric acid, prealbumin, alanina aminotransferase (ALT) and glucose were evaluated. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. HLA-B27 and TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) were assayed by Real Time-PCR. HLA-CW6 allele presence was analysed by molecular genetic testing using a commercially available CE-IVD microarray. Statistical analysis was performed using STATA software (version 13.1). Results. An higher number of circulating polymorphonuclear cells and higher CRP levels could be detected in SpA patients with respect to controls, and in PsA higher levels of ALT could be observed with respect not only to controls but also to AS. Anyway these indices were not highly elevated and often comprised within the reference intervals. As expected, HLA-B27 was associated with AS (χ2=120.1; p<0.0001). Although a slightly higher frequency of HLA-CW6 carriers was observed among patients with AS (about 6%) or PsA (about 13%) with respect to controls (about 4%), the difference was not statistically significant. Any single studied TNFA SNP was not associated with SpA diagnosis, nor with AS or PsA considered singly. The haplotypes deriving from the pairwise combinations of the five studied SNPs were also statistically inferred. The most frequent haplotypes in controls were selected as references, and only the haplotype -1031C/-308G was significantly associated with AS (p=0.015) exerting in this disease a protective role (Odds Ratio: 0.43; Confidence Interval 95%: 0.22-0.85). Three SNPs were identified in TNFRSF1A gene and among them, only the R92Q (Minor Allele Frequency- MAF=0.034) and the c.625+10A>G (MAF=0.479) were selected for their potential functional implications. Both SNPs were not associated with the presence of SpA (χ2=1.073 and p=0.300 for R92Q; χ2=4.721 and p=0.094 for c.625+10A>G), but c.625+10A>G was associated with the response to anti-TNF therapy, assessed by BASDAI score lower /equal or higher than 4 at 10 months (p=0.031). Twenty-one SNPs were identified in MEFV gene and among them, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF<0.025) except for R202Q (MAF=0.27). None was associated with SpA diagnosis (p>0.05). Conclusions. In conclusion the results of this study indicate the relevant role of TNF-TNFR pathway genetics in the complex network causing SpA and conditioning response to therapy. TNFA was shown to be a predisposing factor for SpA, but mainly for AS, among Italian patients, while genetics of the autoinflammatory gene MEFV appears of no impact in this setting. The haplotype resulting from TNFA-1031C/-308G, potentially associated with lower TNF-α production, exerts a protective role in AS, while the TNFRSF1A c.625+10A>G polymorphism emerged as a potential predictor of response to anti- TNFα therapy.
Introduzione. Le spondiloartriti sieronegative (SpA) sono un gruppo di malattie infiammatorie croniche risultanti da una complessa interazione tra fattori genetici (tra cui, HLA-B27 è il maggior predisponente) e ambientali. Ed è tale interazione ad indurre l'attivazione di processi autoinfiammatori e la disregolazione del sistema immunitario caratterizzanti la malattia. In molti casi, una diagnosi precoce ed un adeguato monitoraggio dell’ attività di malattia risultano difficili a causa della sovrapposizione delle caratteristiche cliniche tra le diverse forme. Il ritardo nella diagnosi e conseguentemente nel trattamento, è inoltre dovuto al fatto che, gli indici d’infiammazione comunemente utilizzati nella pratica clinica, la velocità di eritrosedimentazione (VES) ed la proteina C-reattiva (PCR), sono nella norma in almeno metà dei pazienti con chiara espressione dell’attività di malattia. Il ritardo nella diagnosi conferisce a questi pazienti un carico sintomatico importante ed una perdita di funzione durante gli anni di vita produttiva. Pertanto, forte attenzione è attualmente rivolta all’identificazione di marcatori biochimici e genetici utili alla diagnosi e di fattori prognostici necessari a valutare l'efficacia del trattamento. Tra i fattori genetici predisponenti, è noto il ruolo di HLA-B27, che contribuisce però solo per il 20-30% all'ereditarietà totale, mentre il complesso maggiore di istocompatibilità (MHC) rappresenta circa il 40-50% del rischio genetico di sviluppare la patologia. Questo dato ha suggerito il probabile coinvolgimento di altri geni nel meccanismo patogenetico. Studi di associazione genetica hanno permesso di identificare un certo numero di altri geni, associati alla patologia, sia nel locus MHC che in altri loci. In questo contesto, di grande interesse è lo studio della genetica di TNF-α, considerato il ruolo di tale citochina nel propagare e perdurare dell'infiammazione. Sebbene numerosi studi abbiano dimostrato l’associazione tra i polimorfismi di geni coinvolti nella via del segnale del TNF-α (es. TNFA, TNFSF15, TNFR1 e TRADD) e la patologia di SpA, i risultati sono discordanti. Di grande interesse sono anche le varianti del MEFV gene, coinvolto nella patogenesi della malattia autoinfiammatoria Febbre Mediterranea Familiare (FMF). Studi recenti hanno, infatti, dimostrato che le SpA, ed in particolare la spondilite anchilosante (AS), sono molto comuni tra i pazienti affetti da FMF e che questi pazienti possono presentarsi con AS come unica manifestazione. Questo studio, condotto su 91 pazienti e 223 controlli, provenienti da una regione italiana del Nord-Est, si propone di identificare fattori bioumorali (biochimici ed ematologici) e genetici al fine di supportare i processi diagnostici e prognostici (risposta alla terapia). In particolare, oltre ai parametri biochimici ed ematologici, è stato valutato se polimorfismi nella regione del promotore del gene TNFA, o dei geni TNFRSF1A e MEFV, possano concorrere con l’allele HLA-B27 all’aumento del rischio di sviluppare la malattia e/o nel predire la risposta agli inibitori del TNF-α. Metodi. La popolazione studiata comprendeva 91 pazienti con diagnosi di SpA (età media ± deviazione standard: 52.1 ± 12.5 anni; 57 maschi, 34 femmine) e 223 donatori di sangue (età media ± deviazione standard: 46 ± 11 anni; 146 maschi, 77 femmine) provenienti dalla Regione Veneto, una regione italiana del Nord-Est. Tra i pazienti, 36 presentavano AS e 55 artrite psoriasica (PsA), con diagnosi formulata sulla base dei criteri rispettivamente di New York e CASPAR. Il protocollo di questo studio è stato approvato dal Comitato Etico Istituzionale locale dell’Università-Azienda Ospedaliera di Padova, Italia (Prot.n. 3024P / 13), e tutti i soggetti arruolati hanno firmato un consenso informato prima di partecipare allo studio. Per ciascun soggetto arruolato, sono stati raccolti i dati demografici e fisiologici, la storia clinica e familiare. Sono stati raccolti poi, campioni di sangue, al fine di valutare l’emocromo e la VES, e di determinare i livelli di PCR, acido urico, prealbumina, alanina aminotransferasi (ALT) e glucosio. L’analisi molecolare dei geni MEFV (esoni 2,3,5 e 10) e TNFRSF1A (esoni 2,3,4 e 6) è avvenuta mediante sequenziamento diretto. La determinazione degli alleli HLA-B27 e dei polimorfismi del gene TNFA (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) è stata condotta mediante PCR in Real-Time. La determinazione degli alleli HLA-CW6 è avvenuta mediante un test genetico molecolare CE-IVD, disponibile in commercio, che adotta la tecnologia microarray. L’analisi statistica è stata effettuata utilizzando il software STATA (versione 13.1). Risultati. Un maggior numero di cellule polimorfonucleate circolanti e livelli di PCR più elevati sono stati rilevati nei pazienti affetti da SpA rispetto ai controlli. Inoltre, i pazienti affetti da PsA hanno mostrato livelli più elevati di ALT, non solo rispetto ai controlli, ma anche rispetto a pazienti affetti da AS. In ogni caso tali indici non erano molto elevati e spesso risultavano compresi entro gli intervalli di riferimento. Come atteso, gli alleli HLA-B27 sono risultati associati all’AS (χ2=120.1; p<0.0001). Sebbene una frequenza leggermente maggiore degli alleli HLA-CW6 sia stata osservata tra i pazienti con AS (circa il 6%) o PsA (circa il 13%) rispetto ai controlli (circa 4%), la differenza non è risultata essere statisticamente significativa. Nessuno dei polimorfismi del gene TNFA è risultato singolarmente associato alla diagnosi SpA, né a quella di AS o PsA, se valutate indipendentemente. Sono stati, poi, statisticamente dedotti gli aplotipi derivanti dalle coppie di combinazioni dei cinque polimorfismi studiati. Gli aplotipi più frequenti nei controlli sono stati selezionati come aplotipi di riferimento, e solo l’aplotipo -1031C/-308G è risultato significativamente associato con l’AS (p=0.015) esercitando in questa malattia un ruolo protettivo (odds ratio: 0.43; intervallo di confidenza al 95%: 0.22- 0.85). Tre polimorfismi sono stati identificati nel gene TNFRSF1A e tra questi, solo i polimorfismi R92Q (Frequenza dell’allele minore- MAF = 0.034) e c.625 + 10A> G (MAF = 0.479) sono stati selezionati a causa del potenziale ruolo funzionale. Entrambi i polimorfismi non sono risultati associati con la diagnosi di SpA (χ2 = 1.073 e p = 0.300 per R92Q; χ2 = 4.721 e p = 0.094 per c.625 + 10A> G). Il polimorfismo c.625 + 10A> G è però, risultato essere associato con la risposta alla terapia con anti-TNF, valutato sulla base di un punteggio BASDAI inferiore / uguale o superiore a 4, a 10 mesi dall’inizio della terapia (p = 0.031). Ventuno polimorfismi sono stati identificati nel gene MEFV e tra questi, 10 noti per il potenziale significato funzionale. Tali varianti alleliche sono risultate estremamente rare nella nostra popolazione (MAF <0.025) ad eccezione di R202Q (MAF = 0.27). Nessun polimorfismo è risultato essere associato con la diagnosi SpA (p> 0.05). Conclusioni. In conclusione, i risultati di questo studio suggeriscono il ruolo rilevante della genetica della via del segnale TNF-TNFR nel complesso sistema che induce la patogenesi di SpA e condiziona la risposta alla terapia. Il gene TNFA, nella popolazione oggetto di studio, si è dimostrato un fattore predisponente per lo sviluppo di SpA, ma soprattutto di AS. Al contrario, la genetica del gene MEFV non sembra mostrare alcun impatto in questo gruppo di malattie. L'aplotipo TNFA-1031C/-308G, potenzialmente associato alla produzione di livelli più bassi di TNF-α, sembra esercitare un ruolo protettivo nella patogenesi di AS, mentre è emerso che il polimorfismo c.625 TNFRSF1A + 10A> G costituisce un potenziale fattore predittivo di risposta alla terapia con anti-TNFα.

Background. The Spondyloarthritis (SpA) are a group of a multifactorial diseases characterised by a complex interplay between an inherited background and environmental factors that lead to immune response dysregulation and inflammation of the joints, mainly the sacro-ileal. Different from rheumatoid arthritis, there are no specific biomarkers for disease activity in the SpA that could be used in clinical practice. New biomarkers discovery could be helpful for early diagnosis, monitoring of disease activity, as well as for prognosis, outcome measures, and for assessing treatment efficacy. In SpA patients, macrophages infiltrating the inflamed joints, derive from circulating monocytes, express not only inflammatory cytokines, like TNF-α, IL-1β or TGF-β, but also enzymes causing tissue destruction and remodelling, like metalloproteinases. Metalloproteinases (MMPs), MMP3 in particular, have been reported to be highly expressed in synovial tissue and in peripheral blood of SpA patients. Recent studies have showed that MMP8 and MMP9, in particular, are produced by peripheral blood mononuclear cells (PBMCs) if they are stimulated by calprotectin (S100A8/S100A9 heterodimer). The SpA synovial tissue is characterized by an increased vascularization and an infiltrate composed of nucleated polymorphs, macrophages and lymphocytes. In these cells calcium signals are essential for various cellular functions, including proliferation, differentiation, apoptosis, and gene transcription. The aims of this work are to investigate whether the TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA expression levels and intracellular calcium ([Ca2+]i) fluxes variations in PBMCs might be associated with SpA. Methods. The study population comprised 64 patients with a diagnosis of SpA (39 males and 25 females; mean age±standard deviation: 39.5±13.2 years) and 100 healthy controls (58 males and 42 females; mean age±standard deviation: 46.68.5). Among patients, 26 (40.6%) had diagnosis of Ankylosing Spondylitis (AS), (modified New York criteria) and 38 (59.3%) had a diagnosis of Psoriatic Arthritis (PsA) (CASPAR criteria). Blood samples were collected and complete blood count, CRP, ESR, uric acid, ALT and glucose were evaluated. Relative quantification (Real Time PCR) of TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA were performed. Intracellular calcium ([Ca2+]i) fluxes were studied in patients and controls monocyte cells by a fluorescent microscope. Results. The mRNA expression levels in PBMCs of TNF-α, IL-1β, TGF-β were similar in AS and PsA patients when compared to controls. The variations of TNF-α, TGF-β and IL-1β were correlated each other. TNF-α mRNA expression levels also show a significant correlation if patient’s relatives with SpA where found (t=-2.5386, p=0.013). MMP8 and MMP9 mRNA expression levels did not vary between controls and patients, nor they were related to disease clinical activity indices. S100A9 mRNA expression did not vary, the expression of S100A8 (F=3.29, p=0.039) was reduced in PsA patients. S100A8 and S100A9 expression levels were significantly correlated with circulating inflammatory cells and S100A8 was correlated with CRP and ESR. Monocytes from healthy controls had evident and frequent ([Ca2+]i) oscillations, while SpA patients monocytes did not. The percentage of cells exhibiting ([Ca2+]i) oscillations profile was significantly lower in AS with respect to controls (F=6.15, p=0.003). The percentage of monocytes with intracellular calcium oscillations and the studied molecules were not correlated with the type of therapy or of drug used. Conclusions. SpA associates with a reduced expression of the inflammatory S100A8 calcium binding protein and with a decreased intracellular calcium fluxes in patients' cells compared to healthy subjects, suggesting that the presence of the disease affects the "on-off" mechanisms that regulate the concentration of intracellular calcium.
Introduzione. Le Spondiloartriti (SpA) sono un gruppo di malattie multifattoriali caratterizzate da una complessa interazione tra fattori genetici ed ambientali che determinano una disregolazione del sistema immunitario e l’attivazione di processi infiammatori a livello articolare, in particolar modo nelle articolazioni sacro-iliache. A differenza dell’artrite reumatoide, nelle SpA non esistono dei biomarcatori specifici di attività di malattia che vengono utilizzati nella pratica clinica. Pertanto, la ricerca di nuovi biomarcatori potrebbe essere d’ausilio per una diagnosi precoce e per un adeguato monitoraggio dell’attività di malattia, oltre che essere impiegati come fattori prognostici, misure di outcome e strumenti di valutazione dell’efficacia di trattamento.Nei pazienti con SpA, i macrofagi che infiltrano le articolazioni e che derivano principalmente dai monociti circolanti non esprimono solo citochine infiammatorie come TNF-α, IL-1β o TGF-β ma anche enzimi coinvolti nel rimodellamento tissutale come le metallo proteinasi di matrice (MMPs). La metalloproteinasi di matrice 3 (MMP-3), infatti, è riconosciuta come una molecola altamente espressa nel tessuto sinoviale e nel sangue periferico dei pazienti con SpA. Studi recenti hanno evidenziato che le metallo proteinasi di matrice 8 e 9 (MMP8 e MMP9) vengono prodotte dalle cellule mononucleate derivate da sangue periferico (PBMCs) quando vengono stimolate da calprotectina (eterodimero formato dalle proteine S100A8 e S100A9).Vi è poi una crescente evidenza del ruolo patogenetico nelle Spa svolto dalle cellule appartenenti all’immunità innata quali macrofagi, mastociti e neutrofili; il tessuto sinoviale dei pazienti con SpA infatti è caratterizzato da una elevata vascolarizzazione e quindi da una forte infiltrazione delle cellule immunitarie. In queste cellule i segnali di calcio intracellulare sono essenziali nella regolazione di numerose funzioni cellulari incluse proliferazione, differenziazione, apoptosi e trascrizione genica. Lo scopo di questo lavoro è stato quello di analizzare i livelli di espressione genica delle molecole TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9 e di valutare se le variazione dei flussi di calcio intracellulare ([Ca2+]i) nei PBMCs potrebbero essere associati alla presenza di SpA. Metodi.La popolazione studiata comprendeva 64 pazienti con diagnosi di SpA (età media ± deviazione standard: 39.5 ± 13.2 anni; 39 maschi, 25 femmine) e 100 controlli sani (età media ± deviazione standard: 46.6 ± 8.5 anni; 58 maschi, 42 femmine). Tra i pazienti, 26 (40.6%) presentavano spondilite anchilosante (AS) e 38 (59.3%) artrite psoriasica (PsA), con diagnosi formulata sulla base dei criteri rispettivamente di New York e CASPAR. Per ciascun soggetto arruolato, sono stati raccolti i dati demografici e fisiologici, la storia clinica e familiare. Sono stati raccolti poi, campioni di sangue, al fine di valutare l’emocromo e la VES, e di determinare i livelli di PCR, acido urico, prealbumina, alanina aminotransferasi (ALT) e glucosio. Per ciascun soggetto è stata effettuata un’analisi di espressione genica relativa (Real Time PCR) di TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9. La determinazione dei flussi di calico intracellulare ([Ca2+]i) nei pazienti e nei controlli è stata effettuate mediante microscopio ad epifluorescenza. Risultati. I livelli di espressione genica relativa nei PBMCs delle citochine infiammatorie of TNF-α, IL-1β, TGF-β erano simili nei pazienti con AS e PsA se comparati ai livelli di espressione nei controlli sani. Le variazioni dei livelli di espressione di TNF-α, TGF-β e IL-1β correlavano tra di loro. I livelli di espressione di TNF-α però risultavano correlati in maniera diretta, nei pazienti, con la presenza di una familiarità per la malattia (t=-2.5386, p=0.013). I livelli di espressione di MMP8 e MMP9 non risultavano essere associati con la diagnosi di SpA e non correlavano con gli indici clinici di attività di malattia.Anche i livelli di espressione di S100A9 non risultavano essere associati con la diagnosi di SpA mentre i livelli di espressione di S100A8 (F=3.29, p=0.039) erano ridotti nei pazienti con PsA.I livelli di espressione di S100A8 e S100A9 correlavano in maniera significativa con il numero di cellule infiammatorie circolanti e S100A8 correlava con i valori di PCR e VES. Dall’analisi dei dati ottenuti dai controlli sani e dai pazienti risultava evidente come la maggior parte dei monociti dei soggetti di controllo presentassero pulsazioni regolari di calcio intracellulare a differenza delle cellule ottenute da pazienti.I pazienti affetti da AS presentavano una ridotta percentuale di monociti con oscillazioni dei flussi di calcio intracellulare rispetto ai controlli sani (F=6.15, p=0.003). La percentuale di monociti con variazione di calcio intracellulare e l’ espressione delle molecole studiate non risultavano essere correlati ne con il tipo di terapia ne con il tipo di farmaco utilizzato. Conclusioni. In conclusione, I risultati di questo studio hanno evidenziato che nei pazienti con SpA vi è una ridotta espressione della proteina legante calcio S100A8 e vi è un decremento dei flussi di calcio intracellulare rispetto ai controlli sani, suggerendo che la presenza della malattia influenza i meccanismi "on-off" che regolano la concentrazione di calcio intracellulare.

The strong association of the human leukocyte antigen HLA-B27 (B27), with spondyloarthropathies (SpA), was discovered more than four decades ago, yet the role B27 plays in disease pathogenesis remains unclear. It has been demonstrated that HLA-B27 can form non-classical FHC forms, including B27 FHC homodimers (B272) and misfolded FHCs. Two leading theories of SpA pathogenesis proposed that both cell surface NC-B27 (the B27 homodimer theory) and intracellular (the B27 misfolding and UPR theory) can drive immune responses in SpA. Several studies demonstrated that cell surface NC-B27 binds to immune receptors and suggested that these interactions can activate Th17 immunity. My DPhil project focuses on the investigation of the cell surface expression of NC-B27 molecules in cells and tissues obtained from AS patients and B27 TG2 rats. I used both existing reagents and a novel HD6 antibody, generated against B272, to examine the expression of HLA-B27 by flow cytometry, confocal microscopy and immunoprecipitation. This thesis provides evidence that the expression of HD6- and HC10-reactive molecules in AS patients is rather restricted to specific cell types (e.g. osteoclasts) and possibly particular environmental factors and tissue types (e.g. synovial fluid and joint tissue). My data using B27 TG2 rats demonstrated that resident joint cells express HD6- and HC10-reactive molecules before the onset of clinical manifestations. Furthermore, I showed increased expression of NC-B27 on antigen presenting cells (APCs), and CD161+ and CD8+ cell populations in blood and secondary lymphoid organs. Based on these findings I hypothesized that cell surface expression of NC-B27 may not only be crucial for activation of Th17-driven immune responses, but may also shift the balance between immune tolerance and activation. Importantly, the expression of potentially pathogenic HD6-reactive molecules is restricted to specific cell and tissue types hence HD6 antibody may be a potential future therapeutic agent in SpA.

Spondyloarthritis (SpA) is an inflammatory disease comprising both ankylosing spondylitis and non-radiographic axial SpA. This thesis conducted four studies using data from DESIR, a longitudinal cohort of 708 French SpA patients, focusing on economic questions in the context of SpA treatment recommendations. The objectives of study 1 were to value health resource use and productivity loss among DESIR patients and identify factors associated with costs. Cost valuation was done using public data and factors associated with costs were identified in multivariate regression models. This study showed that anti-TNF agents were the greatest cost driver in DESIR and generated the cost outcomes necessary to perform economic evaluations using DESIR data. The objective of study 2 was to collaborate with rheumatologists to develop measurable definitions of adherence to SpA treatment recommendations. A Delphi survey was conducted with 15 rheumatologists, who developed measurable definitions for 6/11 recommendations. The study uncovered differences of opinion between rheumatologists and generated the classification system necessary to explore adherence to recommendations among DESIR patients. The objective of study 3 was to examine the effect of adherence to anti-TNF use recommendations on outcomes in DESIR. Employing alternate definitions of adherence, patients were classed as adherent anti-TNF users, non-adherent anti-TNF users, adherent non-users, and non-adherent non-users. Following one potential definition, non-adherent anti-TNF users had significantly increased non-biologic costs compared to adherent users, while non-adherent non-users had significantly worse health outcomes than adherent anti-TNF users. This study showed that the impact of adherence to anti-TNF recommendations is sensitive to the definition of adherence and highlighted the need to validate methods to measure adherence. The objective of study 4 was to explore whether the French anti-TNF access restrictions are the most cost-effective in that setting relative to other potential restrictions. Five separate datasets were created comprising DESIR patients satisfying different sets of anti-TNF access criteria. Incremental cost-effectiveness ratios (ICERs) associated with anti-TNF use were calculated in each subset in basecase and sensitivity analyses. A sensitivity analysis simulating a 24-week stopping rule among anti-TNF non-responders demonstrated the effect of non-response on anti-TNF cost-effectiveness. The study underscored the need for evidence-informed anti-TNF access restrictions.
Medicine, Faculty of
Population and Public Health (SPPH), School of
Graduate

Die axiale Spondyloarthritis (axSpA) ist eine chronische entzündlich-rheumatische Erkrankung unbekannter Ursache, die durch Entzündungen in den Sakroiliakalgelenken (SIG) und an den Gelenken der Wirbelsäule gekennzeichnet ist. Darüber hinaus kann es im Krankheitsverlauf zu einer Ankylose in den SIG und zur Entwicklung von Syndesmophyten an den Wirbelkörpern kommen. Da ein Großteil der axSpA-Patienten auch sub-klinische mukosale Entzündungen aufweist, werden mukosale Antigene als Trigger der Entzündung diskutiert. Für die Diagnose und Prognose der axSpA existieren bisher wenige serologische Marker mit hoher Sensitivität. In dieser Arbeit konnte gezeigt werden, dass die Serumkonzentrationen von CTX-II, BMP-2 und LBP ein hohes diagnostisches Potenzial für die axSpA aufweisen, während die Serumkonzentrationen von BMP-2, PINP und VEGF als Marker für die Vorhersage röntgenolgischer Progression geeignet sein könnten. Weiterhin konnten erhöhte LPS-, LBP- und IL-6-Serum-Konzentrationen bei axSpA-Patienten nachgewiesen werden, die auf eine Translokation bakterieller Antigene hinweisen. Die Charakterisierung der Monoyzten zeigte erhöhte Frequenzen der CD14++CD16- und einen verminderten Anteil an CD14++CD16+ Monozyten in Patienten mit axSpA. Funktionell wiesen die Monozyten von axSpA-Patienten eine in vivo Präaktivierung mit erhöhter spontaner und durch suboptimale bakterielle Stimuli induzierter Freisetzung proinflammatorischer Zytokine bei gleichzeitig verminderter Reaktivität auf LPS in vitro auf. Diese Präaktivierung war bei Patienten unter Standardtherapie, nicht aber unter TNF-Blocker-Therapie, nachweisbar. Interessanterweise bestand bei Patienten unter Standardtherapie ein Zusammenhang zwischen der Krankheitsaktivität und der Frequenz zytokinproduzierender Monozyten. Somit konnten mit dieser Arbeit Biomarker mit diagnostischer und prognostischer Bedeutung für die axSpA identifiziert werden, deren Bedeutung in unabhängigen Kohorten weiter untersucht werden muss.
Axial Spondyloarthitis (axSpA) is a chronic inflammatory-rheumatic disease of unknown cause. It is characterized by inflammations of the sacroiliac joints (SIG) and the spinal joints. In addition an ankylosis in the SIG can develop in the progression of the disease as well as syndesmophytes at the vertebral body. Since the majority of axSpA-patients also have sub-clinical mucosal inflammations, mucosal anti-gens are discussed as triggers of the inflammation. For the diagnosis and prognosis of axSpA there are only a few serological markers with high sensitivity and specificity until now. In this thesis it could be shown that the serum concentration of CTX-II, BMP-2 and LBP exhibit a high diagnostic potential for axSpA, while the serum concentration of BMP-2, PINP and VEGF could be suitable markers for the forecast of radiographic progression. Furthermore increased LPS-, LBP- and IL-6-serum concentrations could be verified, which can be an indicator for the translocation of bacterial antigenes. The monocyte characterization showed increased frequencies of the pro-inflammatory CD14++CD16- sub population and a reduced portion of CD14++CD16+ monocytes in patients with axSpA. Functionally the monocytes of axSpA-patients showed an in vivo pre-activation with increased spontaneous and by suboptimal bacterial stimuli induced release of pro-inflammatory cytokines with simultaneously reduced reactivity towards LPS in vitro. This pre-activation could be detected for patients undergoing standard therapy, but not for those under TNF-blocker-therapy. Interestingly for the standard therapy patients there was a connection between the activity of the disease, meaning the BASDAI, and the frequency of the cytokine-producing monocytes. Therefore biomarkers with diagnostic and prognostic relevance could be identified in line with this thesis. The significance of these biomarkers has to be researched further in independent cohorts.

En la tesi que presento s'avaluen les implicacions de la presencia de l'HLA-B27 en el fenotip i les comorbilitats de pacients amb Espondiloartritis Axial
En la tesis que presento se evalúan las implicaciones de la presencia del HLA-B27 en el fenotipo y las comorbilidades de pacientes con Espondiloartritis Axial
The present thesis assesses the implications of the presence of HLA-B27 in phenotype and comorbidities in Axial Spondyloarthritis patients

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF
The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers

La spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent avec une prévalence de 0,43% en France, fortement associée à HLA-B27. À l’heure actuelle, il n’existe aucun traitement curatif et les mécanismes physiopathologiques impliqués restent méconnus. Afin de mieux comprendre les mécanismes immunologiques impliqués dans le développement de la SpA, nous avons étudié deux populations cellulaires clé, les cellules dendritiques (DC) et les lymphocytes T (LT) CD4+, chez le rat transgénique pour le HLA-B27 et la β2 microglobuline humaine (rat B27) qui développe spontanément tous les symptômes de la SpA. Il a été démontré que l’accumulation de lymphocytes T helper producteurs d’interleukine 17 (IL-17) pathogénique (lymphocyte Th17), et plusieurs défauts fonctionnels des cellules dendritiques (DCs) sont corrélés avec le développement de la SpA chez les rats B27.Nous nous sommes tout d’abord intéressés aux lymphocytes T régulateurs (Treg), dont le rôle est d’empêcher l’établissement d’une réponse immune pathogène pour l’hôte, chez le rat B27. Nous avons découvert que les Treg de rats B27 présentent un phénotype pro-inflammatoire (surexpression d’IL-17 et sous-expression d’IL-10 anti-inflammatoire), lié à la surexpression de la molécule ICOS. De plus, la sévérité des signes cliniques chez les rats B27 n’exprimant pas ICOS (rats B27 ICOS KO) est diminuée comparé aux animaux HLA-B27 sauvages. Cette protection partielle est corrélée à une réduction de la proportion de lymphocytes Th17. Ces résultats mettent en lumière le rôle majeur d’ICOS dans la physiopathologie de la SpA du rat.La deuxième partie de ce travail s’est concentrée sur les conséquences de la sous-expression d’IL-27 par les DC de rats B27, cytokine connue pour inhiber le développement des Th17. Nous avons observé que l’addition d’IL-27 exogène permet de diminuer la production d’IL-17 et d’augmenter la synthèse d’IL-10 anti-inflammatoire par les LT différenciés (T effecteurs et Treg) et les LT naïfs de rats B27 différenciés in vitro. De façon intéressante, l’IL-27 réduit également la synthèse d’IL-17 par les LT CD4+ circulants de patients atteints de SpA.Ces travaux démontrent pour la première fois le rôle clé de l’IL-27 et d’ICOS dans le contrôle de l’inflammation chez le rat B27 et suggèrent fortement que ces deux molécules sont de nouvelles cibles thérapeutiques prometteuses dans la SpA
Spondyloarthritis (SpA) is a frequent chronic rheumatic inflammatory disorder with a prevalence of 0.43% in France and closely associated to HLA-B27. To date, there is no curative treatment and pathophysiological mechanisms involved in this pathology remain elusive. To better understand these mechanisms, we studied two crucial cell populations, dendritic cells (DC) and CD4+ T cells in rats transgenic for HLA-B27 and human β2 microglobulin (B27 rats) which spontaneously develop a phenotype closely resembling human spndyloarthritis. Previous studies demonstrated that accumulation of pathogenic IL-17 producing T cells (Th17 cells) and several function defects of DCs are correlated with SpA development in B27 rats.First, we focused on regulatory T cells, whose role is to prevent the establishment of pathogenic immune responses. We discovered that Treg from B27 rats have a pro-inflammatory phenotype. They overexpress IL-17 and underexpress anti-inflammatory IL-10, linked to ICOS overexpression. Furthermore, B27 rats knock-out for ICOS (B27 ICOS KO rats) have reduced severity of clinical symptoms compared to B27 ICOS WT rats. This protective effect is correlated with a reduced proportion of Th17 cells. These results highlight the crucial role of ICOS in rat SpA physiopathology.In the second part of this work we studied the consequences of IL-27 underexpression by B27 DC, a cytokine known to inhibit Th17 development. Addition of exogenous IL-27 reduces IL-17 and increases IL-10 productions by differentiated T cells (Teff and Treg) and by naive T cells polarized in vitro. Interestingly, IL-27 also reduces IL-17 production by circulating CD4+ T cells isolated from blood of SpA patients.This work demonstrate for the first time the key role of IL-27 and ICOS in the control of inflammation in B27 rats and highly suggest that these molecules may be new promising therapeutic targets in SpA

La spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent et invalidant. Plus d’une vingtaine de locus de susceptibilité à la maladie ont été identifiés à ce jour, dont HLA-B27 situé dans le complexe majeur d’histocompatibilité (CMH). L’objectif de ce travail était d’identifier de nouveaux facteurs génétiques de susceptibilité à la SpA grâce à une double approche d’études familiales et de génomique fonctionnelle. Dans la première partie, nous avons génotypé des familles multiplex de SpA. L’analyse de liaison non paramétrique a révélé la présence, en plus du CMH, d’un nouveau locus significativement lié à la SpA en 13q13. L’étude de ce locus nous a permis de restreindre la région d’intérêt à un intervalle de 1,3 Mb, dont le séquençage est en cours. Par ailleurs, l’étude d’association intra-familiale a identifié un SNP intronique de MAPK14 significativement associé à la SpA. Enfin, nous avons montré que l’un des SNPs du gène IL23R connu pour être associé à la spondylarthrite ankylosante était en fait associé à la présence d’une sacro-iliite radiologique dans la SpA. Parallèlement aux études familiales, nous avons comparé le transcriptome de cellules dendritiques de neuf patients atteints de SpA à celui de dix témoins sains. Nous avons ainsi identifié 81 gènes différentiellement exprimés. Nous avons aussi montré que l’expression génique d’ERAP1 (et à un moindre degré son expression protéique et son niveau d’activité enzymatique) étaient sous le contrôle de polymorphismes de ce gène associés à la SpA
Spondyloarthritis (SpA) is a frequent and disabling chronic rheumatic disease. To date, more than 20 susceptibility loci have been identified, including HLA-B27 in the major histocompatibility complex (MHC). Most of the disease heritability remains to be elucidated. The aim of the study was to identify new genetic factors of susceptibility to SpA using an approach combining genetics and functional genomics. In the first part of this work, we genotyped SpA multiplex families with microarrays of 250,000 SNPs. Non parametric linkage analysis revealed a new locus significantly linked to SpA outside the MHC, in 13q13. Further studies on this locus allowed us to map the disease interval to a 1.3 Mb region, which will be soon sequenced. Moreover, family-based association study identified a significant association between one intronic SNP in MAPK14 and SpA. We also showed that one of the known ankylosing spondylitis-associated SNP in IL23R was indeed associated with sacroiliitis in SpA. We have also compared dendritic cells gene expression between nine SpA patients and ten controls and identified 81 genes differentially expressed. Moreover, we showed that ERAP1 gene expression (and at a less extent protein expression and enzymatic activity) is under the control of several polymorphisms in the gene which has previously been associated with SpA

Ankylosing spondylitis (AS) is an inherited chronic immune-mediated disease, with a complex genetic and immunological basis. This thesis examined the effect of ankylosing spondylitis-associated genetic changes on DNA methylation and gene expression in isolated immune cell subsets. This study confirmed several pathways associated with disease, such as CD8+T-cell activation, and posed new evidence for the mechanisms of disease. The final chapter developed a signature for distinguishing individuals who have AS from healthy individuals by leveraging genetic profiling, gene expression and DNA methylation. This is the largest study of gene expression or DNA methylation in ankylosing spondylitis to date.

Introdução: A Espondiloartrite axial (EpA-ax) é uma doença inflamatória e crônica do grupo das Espondiloartrites (EpA). Ela acomete o sistema músculo esquelético ocasionando inflamação das articulações axiais (especialmente da sacroilíaca). Quando essas manifestações são evidenciadas por exames de imagem, temos a caracterização da Espondilite Anquilosante (EA). Do contrário, chamamos de EpA-axial não radiográfica (EpA-ax-nr). A Espondilite Anquilosante (EA) se caracteriza pelo envolvimento inflamatório de articulações do esqueleto axial e apendicular. Seu diagnóstico é baseado em achados clínicos e radiológicos, além da elevação de marcadores inflamatórios e presença do HLA-B27. Várias são as metodologias que se propõem a identificar o HLA-B27 e a Polymerase Chain Reaction (PCR) é tida como referência. Sua ampla utilização esbarra, no entanto, em questões que levam em conta o custo, oferta do exame e o tempo até a obtenção do resultado. Neste contexto, a Citometria de Fluxo (CF) surge como uma alternativa capaz de ajudar o médico na identificação deste marcador genético que pode ser importante para o diagnóstico e prognóstico dessa doença. Objetivo: Avaliar a correlação entre as técnicas laboratoriais mais utilizadas na prática clínica (CF e PCR), comparando sensibilidade e especificidade para detecção do HLA-B27 em pacientes com diagnóstico estabelecido de EpA-ax. Métodos: Estudo transversal, comparativo, com pacientes maiores de 18 anos de idade selecionados por conveniência, coletados ao longo de 2015, com diagnóstico estabelecido de EpA-ax, segundo os do grupo internacional ASAS (working group - Assessment of SpondyloArthritis Intenational Society). Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. O teste Exato de Fisher foi utilizado para comparar as variáveis categóricas, o teste t de Student, para variáveis quantitativas com distribuição simétrica de amostras independentes. E as variáveis com distribuição assimétrica foram comparadas pelo teste de Mann-Whitney. Resultados: O coeficiente Kappa obtido para a determinação da concordância entre os testes foi de 0,454. Foram incluídos no estudo 62 pacientes, desses, sessenta preencheram os critérios para diagnóstico de EA e 2 para EpA-ax não radiográfica (EpA-ax-nr), 64,5% dos pacientes eram do sexo masculino, 88,7% se autodeclararam brancos, a idade média (± desvio padrão) foi de 54,5±12 anos e o tempo mediano (percentis 25 e 75) de diagnóstico de 14 (9 e 24) anos. Dentre as características clínicas apresentadas pela população estudada houve diferença estatisticamente significativa para a artrite periférica, sendo mais frequente no grupo HLA-B27 negativo que no grupo HLA-B27 positivo (P=0,032). Na análise de correlação, 90,3% apresentaram tipagem HLA-B27 positiva por CF e 79,0% pela técnica de PCR. Tendo o PCR como padrão ouro, a CF apresentou uma sensibilidade de 98,0%, especificidade de 38,5% e uma acurácia de 85,5%. Conclusão: Apesar da baixa especificidade apresentada pela CF, nosso estudo demonstrou que a CF tem alta sensibilidade e boa acurácia o que a torna uma boa alternativa para ser utilizada como um teste de triagem na busca da caracterização da doença. Apesar da moderada concordância com a técnica de referência, a CF poderia ajudar o médico a excluir resultados falsamente negativos, racionalizando assim a investigação laboratorial para o diagnóstico da EA.
Introduction: Axial spondyloarthritis (SpA-ax) is an inflammatory and chronic disease of the Spondyloarthritis (SpA) group. It affects the skeletal muscle system causing inflammation of the axial joints (especially of the sacroiliac). When these manifestations are evidenced by imaging tests, a characterization of Ankylosing Spondylitis (AS). Otherwise, we call the non-radiographic SpA-axial (SpA-ax-nr). AS marked by the inflammatory involvement of the axial and appendicular skeletal joints. The diagnosis is based on clinical and radiological findings, as well as the on the elevation of inflammatory markers and presence of HLA-B27. There are several methodologies to identify the HLA-B27 gene and a Polymerase Chain Reaction (PCR) is considered the reference method. However, its use on a large scale does not progress because it takes into account the cost, offer of the exam and the running time to obtain the result. In this context, Flow Cytometry (FC) emerges as an alternative method, helping with the physician in the determination of this genetic marker, important for the diagnosis and prognosis of disease. Objective: To evaluate the correlation between FC and PCR, comparing sensitivity and specificity for detection of HLA-B27 in patients with established diagnosis of SpA-ax. Methods: A cross sectional study including 62 patients recruited during 2015 month was conducted in Hospital de Clínicas de Porto Alegre, an university public hospital. The sample, recruited by convenience in the SpA clinic, was composed of patients ≥ 18 years old, fulfilling the Assessment of Spondyloarthritis (ASAS) criteria SpA-ax. All participants underwent HLA-B27 typing through FC and PCR. The kappa statistic was used to calculate the concordance between the FC and PCR. Taking PCR as the gold standard, sensitivity and specificity of FC to detect HLA-B27 were calculated. Results: The Kappa coefficient obtained to determine the agreement between the tests was 0.454. Sixty two patients were included in the study, sixty met the criteria for diagnosis of AS and two for SpA-ax-nr, 64.5% of the patients were male, 88.7% were self-declared mean age (± standard deviation) was 54.5 ± 12 years and median time (25th and 75th percentiles) for diagnosis was 14 (9 and 24) years. Among the clinical characteristics presented by the population studied, there was a statistically significant difference for peripheral arthritis, wich was more frequent in the HLA-B27 negative group than in the HLA-B27 positive group (P = 0.032). In the correlation analysis, 90.3% presented HLA-B27 positive typing by FC and 79.0% by PCR technique. When PCR was considered the gold standard, CF had a sensitivity of 98.0%, specificity of 38.5% and an accuracy of 85.5%. Conclusion: Despite the low specificity presented by FC, our study demonstrated that FC has high sensitivity and good accuracy, which makes it a good alternative to be used as a screening test in the search for the characterization of the disease. Despite the moderate agreement with the reference technique, FC could help the physician to exclude falsely negative results, thus rationalizing laboratory investigation for the diagnosis of AS.

Recently several studies have focused on the use of magnetic resonance imaging (MRI) and new biomarkers that might facilitate early diagnosis of axial spondyloarthritis (axSpA) and identify individuals at higher risk of developing the disease. Objectives:The study aimed to identify:1)potentially useful biomarkers for early diagnosis of axSpA and their correlations with disease activity and imaging indices;2)the prevalence of spine and pelvis MRI lesions in patients (pts) with low back pain (LBP);3)the correlation between the site of axial pain and of MRI-lesions.Material and Methods:Seventy-two pts with LBP (≥3 months, ≤2 years, onset ≤45 years) underwent a physical examination, questionnaires, laboratory tests, X-rays and MRI of the spine and sacroiliac joints (SIJ) at baseline and during a follow-up period of 24 months.Two expert rheumatologists formulated axSpA diagnosis and assessed fulfilment of Assessment of SpondyloArthritis International Society ASAS criteria.Disease activity and physical functioning were assessed using imaging,clinical and serological indices: Bath Ankylosing Spondylitis Metrology Index (BASMI);Maastricht Ankylosing enthesitis Spondilities Score (MASES);Bath Ankylosing Spondylitis Disease Activity Index (BASDAI);Bath Ankylosing Spondylitis Functional Index (BASFI);Ankylosing Spondylitis disease activity score (ASDAS);Visual Analogue Scale (VAS pain);VAS night pain;VAS disease activity;Bath Ankylosing Spondylitis Patient Global Score (BASG1);BASG2;Health Assessment Questionnaire (HAQ);ESR;serum ultrasensitive CRP (hs-CRP);matrix metalloproteinase (MMP3);interleukin (IL) IL-22, IL-17, IL-23.Spine and SIJ MRI and X-rays were scored independently by 2 readers following the SPARCC, mSASSS and NY-criteria.The axial pain and localization of MRI-lesions were classified into 4 sites:cervical/thoracic/lumbar spine and buttocks.The association between the site of pain and MRI-lesions was evaluated through Odds Ratio (OR).The Spearman test and Kruskal Wallis test were used to compare all indices in these cohorts. Results: Patients were diagnosed with axSpA and classified in accordance ASAS criteria as: 1)21 pts classified according to axSpA imaging arm+;2)29 pts classified according to axSpA clinical arm+;3)25 pts not fulfilling ASAS criteria. The median LBP onset was 28.51±8.05 years, 45.3% were male, HLA-B27 was positive in 38.7% pts;56% pts showed bone marrow edema (BME) in spine-MRI and 61.3% pts in SIJ-MRI. Eigtheen (24%) pts presented a negative BME SIJ-MRI with a positive BME spine-MRI. OR between site of pain and site of BME lesions was 20.78 (p=NS),163.93 (p=0.0006);0.34 (p=NS) for cervical/thoracic/lumbar spine and 304.88 (p=0.0203) for buttocks.A significant correlation between thoracic pain/enthesitis on thoracic district (OR=32.69;p= 0.0336) was also found. There was a significant difference between the three cohorts with regard to the prevalence of radiographic sacroiliitis,active sacroiliitis on MRI and the SPARCC SIJ score.There were no differences in these groups regarding ILs, MMP-3 and hsCRP.There was found a correlation between IL-22 and some clinical indices.The correlation between mSASSS, MMP3 and hsCRP was an interesting finding. Conclusions:The site of pain correlated in a statistically significant manner with BME lesions in thoracic and buttock districts.Since positive spine-MRI images were observed in absence of sacroiliitis, we can be therefore hyphothezise that this finding could have a diagnostic significance in suspected axSpA.Although not significantly higher in any of the three groups, IL-22, MMP3 and hsCRP values correlated with some disease activity indices and with mSASSS.Larger observational studies are warranted to confirm these preliminary findings.
Recenti studi si sono concentrati sull’impiego della risonanza magnetica (RMN) e di nuovi biomarcatori che potrebbe facilitare la diagnosi precoce della spondiloartrite assiale (axSpA) e identificare gli individui a più alto rischio di sviluppare la malattia. Scopo del lavoro:Valutare:1)biomarcatori potenzialmente utili per la diagnosi precoce di axSpA e le loro correlazioni con gli indici di attività di malattia e di imaging;2)la prevalenza delle lesioni alla RMN del rachide e del bacino in pazienti (pts) con lombalgia (LBP);3)la correlazione tra la sede del dolore assiale e la localizzazione delle lesioni alla RMN.Materiali e Metodi:Settantadue pts con LBP (≥3 mesi,≤2 anni, insorgenza≤45 anni) sono stati valutati con:esame obiettivo, questionari, indagini sierologiche, radiografie e risonanza magnetica del rachide e delle articolazioni sacro-iliache (SI) al basale e durante un periodo di follow-up di 24 mesi.Due reumatologi esperti hanno formulato la diagnosi axSpA secondo i criteri ASAS “SpondyloArthritis International Society” per axSpA.Sono stati valutati i seguenti indici:Bath Ankylosing Spondylitis Metrology Index (BASMI);Maastricht Ankylosing enthesitis Spondilities Score (MASES);Bath Ankylosing Spondylitis Disease Activity Index (BASDAI);Bath Ankylosing Spondylitis Functional Index (BASFI);Ankylosing Spondylitis disease activity score (ASDAS);Visual Analogue Scale (VAS pain);VAS night pain;VAS disease activity;Bath Ankylosing Spondylitis Patient Global Score (BASG1);BASG2;Health Assessment Questionnaire (HAQ);VES;PCR ultrasensibile (hs-PCR);la metallo-proteinasi 3 (MMP3);interleuchine (IL) IL-22, IL-17, IL-23.Le immagini alla radiografia e alla RMN sono state valutate in modo indipendente da 2 radiologi secondo i criteri SPARCC, mSASSS e mNY.La sede del dolore assiale e delle lesioni alla RMN sono state classificate in 4 siti: colonna cervicale/toracica/lombare e regione glutea.L'associazione tra la sede del dolore e delle lesioni alla RMN è stata valutata tramite Odds Ratio (OR).I test Spearman e Kruskal Wallis sono stati usati per confrontare tutti gli indici di queste coorti.Risultati: I pts sono stati diagnosticati come axSpA e classificati secondo i criteri ASAS come: 1)21 pts classificati axSpA secondo il braccio dell’imaging; 2)29 pts classificati axSpA secondo il braccio clinico; 3)25 pts che non soddisfacevano i criteri ASAS.L'età media di insorgenza della LBP era 28,51 anni, il 45,3% era di sesso maschile,l'HLA-B27 era positivo nel 38,7%.Il 56% dei pts mostrava segni di edema midollare osseo (BME) a carico della RMN del rachide e il 61,3% a carico della RMN delle SI.Diciotto pts (24%) presentavano una RMN delle SI negativa per BME con una RMN del rachide positiva.L’OR tra la sede del dolore e delle lesioni BME era rispettivamente:20.78(p=NS); 163.93(p=0.0006); 0,34(p=NS)per la colonna cervicale/toracica/lombare e 304,88 (p=0,0203) per la regione glutea.È stata anche trovata una correlazione significativa tra dolore toracico/entesite del distretto toracico (OR=32.69; p=0.0336). C'era una differenza significativa tra le tre coorti per quanto riguarda la prevalenza di sacroileite radiografica, sacroileite attiva alla RMN e punteggio SPARCC-SI. È stata trovata una correlazione tra IL-22 e alcuni indici clinici.La correlazione tra mSASSS, MMP3 e hsPCR è stata una scoperta interessante.Conclusioni:Il sito del dolore si correla significativamente con le lesioni BME nei distretti toracico e gluteo. Dato che sono state osservate immagini positive alla RMN del rachide in assenza di sacroileite, riteniamo che lo studio del rachide possa avere un valore aggiunto nella diagnosi di early axSpA.I valori di IL-22, MMP3 e hsPCR correlano con alcuni indici di attività della malattia e con mSASSS.Sono necessari studi osservazionali più ampi per confermare questi risultati preliminari.

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Introduction: Spondyloarthritis is a group of diseases with clinical, laboratory and image similar. Analysis of clinical manifestations of spondyloarthritis in patients with and without the HLA-B*27 were performed, but the results revealed great heterogeneity. Objectives: The aim of this study was to evaluate the clinical manifestations of spondyloarthritis according to the classification criteria of the European Study Group Spondyloarthropathy (ESSG) in the presence and absence of the HLA-B*27. Patients and methods: From a total of 156 patients with clinical suspicion referred for the investigation of gene HLA-B*27, 73 were diagnosed with spondyloarthritis according to the criteria of ESSG. The HLA-B*27 were identified using commercial kits (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Clinical data were collected from medical records of patients. The results were compared using the chi-square or Fisher exact test. The values of Odds Ratio (OR) and confidence interval of 95% were also calculated. The p-value equal to or less than 0.05 was considered significant. Results: Of the 73 selected patients, 53 (72.6%) were male and 20 (27.4%) were female. The mean age was 49.4 and did not differ between genders (p = 0337). The spondyloarthritis found among the 73 patients were: ankylosing spondylitis (n = 47; 64.4%), psoriatic spondyloarthritis (n = 9; 12.3%), undifferentiated spondyloarthritis (n = 9; 12.3%), spondyloarthritis enteropathica (n = 6; 8.2%) and reactive arthritis (n = 2; 2.7%). The average age of onset was equal to 39.1 (± 11.7) and did not differ between genders (p = 0.9057). Of the total, 35 (47.9%) patients were HLA-B*27 positive and 38 (52.1%) were negative. This gene was positively associated with ankylosing spondylitis (OR: 5.37, 95% CI: 1813-15905, p = 0.003) and negatively with spondyloarthritis enteropathica (OR: 0.07, 95% CI: 0003-1301, p = 0.025). The sacroiliitis was associated with the presence of the gene (OR: 10 552, 95% CI: 1260-88256, p = 0.014) and intestinal injury absence (OR: 0.195, 95% CI: 0038-0978, p = 0.048). Conclusions: The HLA-B * 27 was associated with ankylosing spondylitis, enteropathic but not to spondyloarthritis. The radiological signs of sacroiliitis prevailed in patients positive for HLA-B*27, while intestinal involvement was associated with the absence of this gene. especially in cases of dystrophic scoliosis. In both cases, studies with larger samples are needed to assess whether these trends are evident.
Introdução: Espondiloartrite é um grupo de doenças com características clínicas, laboratoriais e imagenológicas semelhantes. Análises das manifestações clínicas das espondiloartrites em pacientes com e sem o gene HLA-B*27 foram realizadas, mas os resultados revelaram grande heterogeneidade. Objetivos: O objetivo deste trabalho foi avaliar as manifestações clínicas das espondiloartrites utilizadas nos critérios de classificação do European Spondyloarthropathy Study Group (ESSG) na presença e na ausência do gene HLA-B*27. Casuística e método: De um total de 156 pacientes com suspeita clínica, encaminhados para investigação do gene HLA-B*27, 73 tiveram diagnóstico de espondiloartrites confirmado, de acordo com os critérios do ESSG. O gene HLA-B*27 foi identificado com o uso de kits comerciais (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Os dados clínicos foram colhidos dos prontuários médicos dos pacientes. Os resultados foram comparados com o uso dos testes Qui-quadrado ou o teste exato de Fisher. Os valores de Odds Ratio (OR) e intervalo de confiança a 95% também foram calculados. O valor p igual ou menor que 0,05 foi considerado significante. Resultados: Dos 73 pacientes selecionados, 53 (72,6%) eram do sexo masculino e 20 (27,4%), femininos. A média de idade foi igual a 49,4 e não diferiu entre os sexos (p=0.337). As espondiloartrites encontradas entre os 73 pacientes foram: espondilite anquilosante (n=47; 64,4%), espondiloartrite psoriásica (n=9; 12,3%), espondiloartrite indiferenciada (n=9; 12,3%), espondiloartrite enteropática (n=6; 8,2%) e artrite reativa (n=2; 2,7%). A média de idade de início dos sintomas foi igual a 39,1 (±11.7) e não diferiu entre os sexos (p=0,9057). Do total, 35 (47.9%) pacientes eram HLA-B*27 positivo e 38 (52.1%), negativos. Este gene foi associado positivamente à espondilite anquilosante (OR: 5.37; IC 95%: 1.813-15.905; p=0.003) e negativamente à espondiloartrite enteropática (OR: 0.07; IC 95%: 0.003-1.301; p=0.025). A sacroiliíte se associou à presença do gene (OR: 10.552; IC 95%: 1.260-88.256; p=0.014) e o comprometimento intestinal à ausência (OR: 0.195; IC 95%: 0.038-0.978; p=0.048). Conclusões: O gene HLA-B*27 foi associado à espondilite anquilosante, mas não à espondiloartrite enteropática. Os sinais radiológicos de sacroiliíte prevaleceram nos pacientes positivos para o gene HLA-B*27, enquanto o comprometimento intestinal foi associado à ausência deste gene.

La spondylarthrite ankylosante (SA), la forme la plus commune des spondylarthrites (SpA), est fortement associée à la molécule du CMH de classe I HLA-B27 mais le rôle de cet antigène d'histocompatibilité dans le développement de ces pathologies reste encore inexpliqué. L’étude des rats transgéniques HLA-B27, développant une pathologie inflammatoire spontanée ressemblant aux SpA, a permis de confirmer l’implication directe du HLA-B27 et de corréler l’apparition des symptômes avec une forte expression de cette molécule. De plus, il a été montré que l’HLA-B27 présentait une propension particulière au mauvais repliement et à la formation d’oligomères de chaînes lourdes. L’objectif de mon travail de thèse était de déterminer si le trafic et/ou la formation d’oligomères du HLA-B27 étaient corrélés à sa surexpression. Pour cela, notre équipe a développé des protéines de fusion (HLA-BYFP et HLA-BRLuc) ainsi que la technique BRET afin d’étudier les interactions HLA-B/HLA-B. Au moyen de ce système expérimental, nous avons montré la formation de vésicules intracellulaires riches en protéines HLA-B mal repliées lorsqu’elles étaient fortement exprimées, qu'il s'agisse d'allèles associés à la SA (HLA-B*2702, -05, et -07) ou non (HLA-B*2706, et -09, HLA-B*0702). Cependant, ce phénomène était significativement plus prononcé pour les sous-types associés à la SA. Dans les conditions de forte expression, nous avons également observé que les sous-types associés à la SA formaient des oligomères qui se comportent différemment de ceux formés par la protéine HLA-B7. Ce phénomène ne semble pas être dû au déclenchement de la réponse cellulaire « Unfolded Protein Response » (UPR) et n’est pas abrogé par l’inhibition du protéasome
Ankylosing spondylitis (AS), the most common form of spondyloarthritis (SpA), is strongly associated with the MHC class I HLA-B27 molecule. Although this association has been largely studied, mechanisms of pathology remain unclear. Development of a spontaneous inflammatory disease resembling human SpA in HLA-B27 transgenic rats confirmed the direct involvement of HLA-B27 and allowed to associate disease development with high expression levels of this molecule. Moreover, the HLA-B27 protein has an enhanced propensity to misfold and form aberrant disulfide linked heavy chain oligomers in the endoplasmic reticulum and at the cell surface. The goal of my thesis work was to determine if the HLA-B27 traffic and/or its ability to form oligomers are involved in this requirement of overexpression. For that, our team has developed fusion proteins ((HLA-BYFP and HLA-BRLuc) and the BRET technique to study, in vitro, the HLA-B/HLA-B interactions. Using this experimental system, we have shown the formation of intracellular vesicles, in which misfolded/unfolded HLA-B proteins accumulated when they were highly expressed, for both AS-associated alleles (HLA-B*2702, -05, et -07) or not (HLA-B*2706, et -09, HLA-B*0702). This phenomenon is strongly pronounced for AS-associated subtypes. For high-level expression, we also observed that the AS-associated subtypes form oligomers that behave differently from those formed by the HLA-B7 control protein. This phenomenon doesn’t appear to be due to unfolded protein response (UPR) triggering and is not abrogated by proteasome inhibition