HLA-B27 Subtypes Predisposing to Ankylosing Spondylitis Accumulate in an Endoplasmic Reticulum-Derived Compartment Apart From the Peptide-Loading Complex.

OBJECTIVE: It was previously shown that HLA-B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non-SpA-associated HLA-B*07:02 and HLA-B*27:06. This study was undertaken to characterize the nature and content of HLA-B-containing vesicles and to further examine their relevance to SpA predisposition. METHODS: Vesicles containing HLA-B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, by microscopy. The nature and content of HLA-B-containing vesicles were characterized in colocalization experiments with appropriate markers. RESULTS: The SpA-associated HLA-B*27:04 subtype accumulated at higher levels (P < 10-5 ) in cytoplasmic vesicles compared to HLA-B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA-B heavy chain along with ß2 m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose-regulated protein 94-kd) and belonged to the ER but were distinct from the peptide-loading complex (PLC). Similar vesicles were observed in immune cells from HLA-B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA-B27/hß2 m transgenic rats, correlating with SpA susceptibility. CONCLUSION: Accumulation of misfolded HLA-B heavy chain along with ß2 m and ER chaperones into ER-derived vesicles distinct from the PLC is a characteristic feature of HLA-B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA-B27 pathogenicity, via a noncanonical mechanism.

HLA-B27 alters BMP/TGFß signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis.

OBJECTIVES: The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. METHODS: We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human ß2-microglobulin (hß2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA. RESULTS: Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hß2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor ß (TGFß). CONCLUSIONS: Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFß/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.

Tolerogenic XCR1+ dendritic cell population is dysregulated in HLA-B27 transgenic rat model of spondyloarthritis.

BACKGROUND: 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 ß2-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- cDC subpopulation expressing XCR1, a chemokine receptor involved in their migration, have been described to be tolerogenic in steady state. Thus, in this study, we wished to examine the fate of XCR1+ cDCs in this animal model of SpA. METHODS: cDC 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 with flow cytometry or real-time PCR. Migration of cDCs from intestinal mucosa to MLN was assessed, using TLR-7 stimulation with Resiquimod. RESULTS: We observed a reduced frequency of cCD4- DCs in B27-Tg rats, as compared to control rats. Furthermore, such decrease was not due to excessive death of CD4- cDCs in B27-Tg rats. Interestingly, we observed a decrease frequency of the XCR1+ subpopulation among CD4- cDCs in the spleen, MLN, and lamina propria from B27-Tg rats. Finally, after TLR-7 stimulation, the migration of XCR1+ cDCs to MLN was proportionally reduced in B27-Tg rats. CONCLUSION: Our results demonstrate for the first time a decreased proportion of the tolerogenic XCR1+ cDC subpopulation in SpA target organs in B27-Tg rat, which may affect the maintenance of self-tolerance and control of inflammation.

FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation.

Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance.

HLA-B27 subtype oligomerization and intracellular accumulation patterns correlate with predisposition to spondyloarthritis.

OBJECTIVE: Mechanisms underlying the striking association of spondyloarthritis (SpA) with the class I major histocompatibility complex molecule HLA-B27 remain poorly understood. SpA-like disease develops spontaneously in B*2705-transgenic rats, in conjunction with high HLA-B27 expression levels. This study was undertaken to examine the effects of increased expression of HLA-B27 alleles that are differentially associated with SpA on oligomerization and intracellular redistribution. METHODS: HeLa cells were transfected with complementary DNA encoding for HLA-B proteins fused to yellow fluorescent protein and/or Renilla luciferase and harvested at an early phase and a later phase of expression. We monitored HLA-B intracellular trafficking and localization by means of microscopy and live-cell imaging. Bioluminescence resonance energy transfer (BRET) and Western blotting were used to monitor HLA-B oligomerization. RESULTS: At low expression levels, BRET signals were similarly elevated for all SpA-associated HLA-B27 alleles tested, but were lower for the nonassociated B*2706. Of note, at higher expression levels, HLA-B27 signals remained steady while signal for HLA-B7 decreased sharply, reaching the level observed for B*2706. This was due at least in part to a decreased oligomer proportion without unfolded protein response outbreak. Such differential behavior was not abrogated by proteasome inhibition. With increased expression, all HLA-B proteins accumulated to a high density in cytoplasmic vesicles with labile form and size. The extent of this phenomenon was closely correlated with the level of association with predisposition to SpA. CONCLUSION: To our knowledge, this is the first report of a correlation between the level of predisposition to SpA conferred by HLA-B27 alleles and their biochemical behavior. These findings open new perspectives for understanding the pathogenicity of HLA-B27.