Induction of HLA-B27 heavy chain homodimer formation after activation in dendritic cells

Introduction Ankylosing spondylitis (AS) is a severe, chronic inflammatory arthritis, with a strong association to the human major histocompatibilty complex (MHC) class I allele human leucocyte antigen (HLA) B27. Disulfide-linked HLA-B27 heavy-chain homodimers have been implicated as novel structures involved in the aetiology of AS. We have studied the formation of HLA-B27 heavy-chain homodimers in human dendritic cells, which are key antigen-presenting cells and regulators of mammalian immune responses. Method Both an in vitro dendritic-like cell line and monocyte-derived dendritic cells from peripheral blood were studied. The KG-1 dendritic-like cell line was transfected with HLA-B27 cDNA constructs, and the cellular distribution, intracellular assembly and ability of HLA-B27 to form heavy-chain homodimers was compared with human monocyte-derived dendritic cells after stimulation with bacterial lipopolysaccharide (LPS). Results Immature KG-1 cells expressing HLA-B27 display an intracellular source of MHC class I heavy-chain homodimers partially overlapping with the Golgi bodies, but not the endoplasmic reticulum, which is lost at cell maturation with phorbyl-12-myristate-13-acetate (PMA) and ionomycin. Significantly, the formation of HLA-B27 homodimers in transfected KG-1 cells is induced by maturation, with a transient induction also seen in LPS-stimulated human monocyte-derived dendritic cells expressing HLA-B27. The weak association of wildtype HLA-B*2705 with the transporter associated with antigen processing could also be enhanced by mutation of residues at position 114 and 116 in the peptide-binding groove to those present in the HLA-B*2706 allele. Conclusion We have demonstrated that HLA-B27 heavy-chain homodimer formation can be induced by dendritic cell activation, implying that these novel structures may not be displayed to the immune system at all times. Our data suggests that the behaviour of HLA-B27 on dendritic cells may be important in the study of inflammatory arthritis.</p

High-content screening image dataset and quantitative image analysis of Salmonella infected human cells

This work was supported by the Medical Research Council Core funding the MRC LMCB (MC_U12266B) (JKV) and the EU FP7 Marie-Curie International Reintegration Grant PIRG08-GA-2010-276811 (JKV). ANA was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. ANA and SJP were also in part funded by ARUK (Grant 21261).Objectives Salmonella bacteria can induce the unfolded protein response, a cellular stress response to misfolding proteins within the endoplasmic reticulum. Salmonella can exploit the host unfolded protein response leading to enhanced bacterial replication which was in part mediated by the induction and/or enhanced endo-reticular membrane synthesis. We therefore wanted to establish a quantitative confocal imaging assay to measure endo-reticular membrane expansion following Salmonella infections of host cells. Data description High-content screening confocal fluorescence microscopic image set of Salmonella infected HeLa cells is presented. The images were collected with a PerkinElmer Opera LX high-content screening system in seven 96-well plates, 50 field-of-views and DAPI, endoplasmic reticulum tracker channels and Salmonella mCherry protein in each well. Totally 93,300 confocal fluorescence microscopic images were published in this dataset. An ImageJ high-content image analysis workflow was used to extract features. Cells were classified as infected and non-infected, the mean intensity of endoplasmic reticulum tracker under Salmonella bacteria was calculated. Statistical analysis was performed by an R script, quantifying infected and non-infected cells for wild-type and ΔsifA mutant cells. The dataset can be further used by researchers working with big data of endoplasmic reticulum fluorescence microscopic images, Salmonella bacterial infection images and human cancer cells.Publisher PDFPeer reviewe

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

AbstractThe oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101–164, and 203–259 in the peptide-binding and α3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication

A.N.A was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. I.L. was funded by an ARUK PhD studentship Ref No: 17868. A.N.A and S.J.P were also in part funded by ARUK (grant 21261)Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.Publisher PDFPeer reviewe