Misregulations induced by the major Spondyloarthritis susceptibility factor HLA-B27: from Drosophila to patients' cells – Fly2HumanSPA

Drosophila transgenic lines expressing SpA-associated-or non-associated HLA-B alleles associated to hß2m are used to study non-canonical effects of HLA-B27. Genetic and cell biology approaches are conducted in Drosophila model, in immune cells extracted from SpA-rat model and patient, to characterize BMP/TGFß signaling pathway misregulation. Mass spectrometry analyses are performed to identify shared peptides specifically presented by HLA-B27 in both Drosophila and human cells. HLA-B27 impact on gut homeostasis is studied by confocal microscopy in Drosophila expressing HLA B alleles specifically in immune or intestinal cells.

Our previous results showed that HLA-B27 impaired the negative control exerted by Sax on BMP signaling in Drosophila, resulting in heightened activity of the pathway. HLA-B27 interacted physically both with Sax in Drosophila and with ALK2, the human ortholog of Sax found at the surface of SpA patient’s cells. Therefore, HLA-B27 may also antagonize ALK2 and in particular, its inhibitory function on the Activin pathway, resulting in heightened response to Activin in situation of inflammation. In mammals, ALK2 and ALK1 Sax orthologs are also implicated in the negative control of TGFß/BMP signaling. It has notably been shown in mouse that ALK2 bound Activin A without inducing subsequent phosphorylation of R-Smad, thereby limiting Activin signaling. Interestingly, in fibrodysplasia ossificans progressiva, the loss of such inhibitory function of ALK2 in the context of inflammation leads to aberrant ossification mediated by Activin that mimics a major characteristic of SpA pathogenesis.
Our prediction is that expression of HLA-B27/hß2m conformers would result in a dominant negative effect on interacting with BMPR1s in mammal cells. Therefore, we are exploring if the interaction between HLA-B27 and BMPR1 can enhance the ability of cells to respond to TGFß/BMP ligands and if such signaling can participate to the pathogenic effect of HLA-B27.

The interaction between HLA-B27 and various receptors of BMP/TGFß is tested in immune mammals and Drosophila cells. Our preliminary results confirm that HLA-B27 expression in hemocytes specifically decrease Drosophila lifespan. The characterization of the effect of HLA-B27 expression in intestinal cells on gut homeostasis is in progress.

The different SpA animal models we established led to converging results that are coherent with clinical data. The use of Drosophila is particularly original in the study of a pathology involving innate immunity leading to results that appear conserved from insects to patients. The “Drosophila peptidome” analysis and its comparison with the human peptidome presented by HLA-B27 could bring therapeutic and prophylactic tools. Our study of the role of other tissues may raise new leads to describe the impact of HLA-B27 on SpA. Additionally, this project could bring some insights into the link between SpA and IBD using the Drosophila model. Studying the role of HLA-B27 on the gut physiology could help understand the gut microbiota dysbiosis that has been observed by Partner 2 in SpA patients.

Spondyloarthritis (SpA) are chronic inflammatory rheumatic diseases affecting both axial and peripheral skeleton joints and leading to bone growth and fusion. The major histocompatibility complex class I antigen, HLA-B27 is the strongest genetic factor associated with disease predisposition. HLA B27 associates with human ß2-microglobulin (hß2m), to present antigenic peptides to T CD8+ cells.

Despite 46 years of research, the mechanism by which HLA-B27 predisposes to SpA remains unsolved, although hypotheses have speculated either on the presentation of particular peptides to CD8+ T cells or on non-canonical functions. Partners 1 and 2 collaborated to produce Drosophila transgenic for HLA-B27 in combination with hß2m, speculating that this simplified animal model could facilitate deciphering of the non-canonical effects of the molecule in the absence of its role on adaptive immunity that may mask some of its pathogenic effects.

Interestingly transgenic flies, carrying two SpA-associated HLA-B27 subtypes -but not SpA-non-associated HLA-B7 allele- developed abnormal phenotypes, only in the presence of hß2m that allows localization of well-folded HLA-B27 molecules at the cell surface. Partner 1 showed that this phenotype was due to a disturbance of the bone morphogenetic proteins (BMP) signaling pathway. BMPs are members of the TGF-ß superfamily. In Drosophila, HLAB27/hß2m repressed Sax BMPR1 function and increased BMP signaling. Consistently, Partner 1&2 showed that HLA-B27/hß2m well-folded conformers co-localized with Sax, and with the Sax mammalian ortholog ALK2 in immune cells from SpA patients.

The hypothesis of an altered BMP pathway in SpA patients is also supported by Partner 2 and others showing dysregulation of BMP effectors or ligands in dendritic cells from HLA-B27 transgenic rat or in patients’ serum respectively. These results indicate that the pathogenic role of HLA-B27 in SpA may result from a TGFß/BMP signaling misregulation at the crossroad between inflammation and ossification.

The TGFß superfamily also contains key regulators of intestinal homeostasis in both Drosophila and human. Intestinal in?ammatory diseases are frequently associated with SpA. The effect of HLA-B27 on these diseases remains unclear and could result from HLA-B27-mediated misregulation in either immune or intestinal cells. Upon infection, BMP signaling regulates hemocyte (Drosophila macrophage-like cells) proliferation and adhesion. BMPs are also an essential signal secreted by hemocytes to trigger intestinal regeneration. Additionally, Partner 1 showed that HLA-B*2705 in the hemocytes -unlike SpA non-associated alleles- decreases Drosophila lifespan, and we hypothesize that this reduced lifespan may result from intestinal dyshomeostasis through HLA-B27 action on BMP signaling in hemocytes.

This project will be realized by a Consortium already engaged in collaboration. Our objective is to decipher misregulation of the TGFß/BMP pathway induced by HLA B27.
Task 1 will be to further identify receptors of the TGFß superfamily that interact with and are misregulated by HLA-B27 and to characterize their effects on BMP signaling both in Drosophila and patients’ cells.
Task 2 will characterize HLA-B27 peptidome in Drosophila and compare it to rat and human peptidomes to identify specific peptides that could modulate HLA-B27 processing in mammalian cells.
Task 3 will address the role of HLA-B27 on hemocyte and intestinal cell homeostasis. The expression of HLA-B27 will be driven either in Drosophila hemocytes or in intestinal cells and its impact on intestine regeneration after infection will be studied.

Drosophila is a novel model to study non-canonical effects of HLA-B27. It is complementary to the HLA-B27 transgenic rat model and patients’ cells also used by the consortium in this project. The results may have important translational impacts to patients with SpA in terms of novel therapeutic interventions.