ROLE OF HLA-B35 AND ER STRESS IN SCLERODERMA PATIENTS WITH PULMONARY ARTERIAL HYPERTENSION

Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been implicated in various diseases. The presence of the HLA-B35 allele, Human antigen class I, has emerged as an important risk factor for the development of PAH in patients with lcSSc, however the mechanisms underlying this association have not been fully elucidated. We have recently reported that the presence of HLA-B35 contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by significantly increasing production of endothelin-1 (ET-1) and significantly decreasing endothelial NO synthase (eNOS). Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the ER stress and UPR in ECs and this mechanism can mediate the induction of ET-1 in patients with PAH. The goal of this study was to better understand the role of HLA-B35-induced ER stress/UPR in the development/progression of PAH disease in lcSSc patients. First we focused on the molecular mechanisms of ET-1 induction by HLA-B35. ER stress inducer, Thapsigargin (TG) and HLA-B35 induced ET-1 expression with similar potency in HDMECs. HLA-B35 or ER stress activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of X-box binding protein (XBP1), but did not affect the Activating Transcription Factor -6 (ATF6) pathways. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by HLA-B35/ER stress. Additional experiments have demonstrated that the JNK and NF-B pathways are also required for ET-1 upregulation by HLA-B35/ER stress. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-B complex was also increased. The functional role of c-JUN in responses to HLA-B35/ER stress was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. Then we focus on whether markers of ER stress/UPR were present in PBMCs from lcSSc-PAH patients and if the presence of HLA-B35 contributes to activation of the immune cells. Several ER stress/UPR genes, including Immunoglobulin-heavy-chain binding protein (BiP), ATF4 and ATF6 and a spliced form of XBP1 were upregulated in lcSSc PBMCs, with the highest levels in patients with PAH. Also selected HSP genes, particularly DNAJB1, and IFN-related genes were found at significantly elevated levels in PBMCs from lcSSc patients, while IRF4 was significantly decreased. There was a positive correlation between DNAJB1 and severity of PAH disease (PAP) (r = 0.56, p<0.05) and between ER stress markers and IL-6 levels (r = 0.53, p< 0.0001) in lcSSc PBMCs. When we stratified all PBMC samples based on the presence of the HLA-B35 allele, we could observe that HLA-B35 positive individuals showed higher levels of selected ER stress markers when compared to HLA-B35 negative individuals. Furthermore, patients carrying HLA-B35 antigen expressed higher levels of IL-6, a key inflammatory cytokine associated with development of PAH. This study demonstrates association between select ER stress/UPR markers and lcSSc-PAH suggesting that ER stress/UPR may contribute to the altered function of circulating immune cells in lcSSc. All these associations were enhanced by the presence of HLA-B35. In conclusion, we hypothesize that HLA-B35 may play a role in EC dysfunction inducing ET1 via ER stress/UPR. Also activation of ER stress/UPR, in combination with presence of HLA-B35, might drive the inflammatory process in lcSSc-PAH.