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

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.

HLA-B27 homodimers and free H chains are stronger ligands for leukocyte Ig-like receptor B2 than classical HLA class I.

Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with ß(2)-microglobulin (ß2m) and peptide and (ß2m free) free H chain (FHC) forms including B27 dimers (termed B27(2)) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K(D) of 5.3 ± 1.5 µM but did not bind B27 FHC. LILRB2 bound to B27(2) and B27 FHC and B27 heterotrimers with K(D)s of 2.5, 2.6, and 22 ± 6 µM, respectively. Domain exchange experiments showed that B27(2) bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K(D)s of 15.0 ± 0.8 and 16.0 ± 2.0 µM, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.

Tapasin dependence of major histocompatibility complex class I molecules correlates with their conformational flexibility.

Major histocompatibility complex (MHC) class I molecules present cell internally derived peptides at the plasma membrane for surveillance by cytotoxic T lymphocytes. The surface expression of most class I molecules at least partially depends on the endoplasmic reticulum protein, tapasin, which helps them to bind peptides of the right length and sequence. To determine what makes a class I molecule dependent on support by tapasin, we have conducted in silico molecular dynamics (MD) studies and laboratory experiments to assess the conformational state of tapasin-dependent and -independent class I molecules. We find that in the absence of peptide, the region around the F pocket of the peptide binding groove of the tapasin-dependent molecule HLA-B*44:02 is in a disordered conformational state and that it is converted to a conformationally stable state by tapasin. This novel chaperone function of tapasin has not been described previously. We demonstrate that the disordered state of class I is caused by the presence of two adjacent acidic residues in the bottom of the F pocket of class I, and we suggest that conformational disorder is a common feature of tapasin-dependent class I molecules, making them essentially unable to bind peptides on their own. MD simulations are a useful tool to predict such conformational disorder of class I molecules.

Salmonella Exhibit Altered Cellular Localization in the Presence of HLA-B27 and Codistribute with Endo-Reticular Membrane.

Salmonella enteritica (S. enteritica) induce and require unfolded protein response (UPR) pathways for intracellular replication. Salmonella infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B∗27 : 05. S. enteritica normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the Salmonella-containing vacuole (SCV). Changes in cellular localization of infecting Salmonella can alter their ability to replicate. We therefore used isogenic epithelial cell lines expressing physiological levels of HLA-B∗27 : 05 heavy chain (HC) and a control HLA-B allele, HLA-B∗35 : 01.HC to determine any changes in Salmonella localization within epithelial cells. Expression of HLA-B∗27 : 05 but not HLA-B∗35 : 01 was associated with a quantifiable change in S. enteritica cellular distribution away from the Golgi apparatus. Furthermore, the Salmonella requirements for UPR induction and the consequences of the concomitant endoplasmic reticulum (ER) membrane expansion were determined. Using confocal imaging, S. enteritica bacteria exhibited a significant and quantifiable codistribution with endo-reticular membrane as determined by ER tracker staining. Isogenic S. enterica Typhimurium mutant strains, which can infect but exhibit impaired intracellular growth, demonstrated that the activation of the UPR was dependent on an integral intracellular niche. Therefore, these data identify cellular changes accompanying Salmonella induction of the UPR and in the presence of HLA-B27.

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (ß2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

Rapid acidification and alkylation: redox analysis of the MHC class I pathway.

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.

Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

OBJECTIVE: HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. METHODS: HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. RESULTS: We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. CONCLUSION: The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease.

The MHC Class I heavy chain structurally conserved cysteines 101 and 164 participate in HLA-B27 dimer formation.

AIMS: The human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies (SpAs). The unusual biochemistry of HLA-B27 has been proposed to participate in disease development, especially the enhanced ability of HLA-B27 to form several heavy chain-dimer populations. HLA-B27 possesses three unpaired cysteine (C) residues at position 67, 308, and 325, in addition to the four conserved cysteine residues at p101, 164, 203, and 259. C67 was proposed to participate in dimer formation of recombinant HLA-B27 protein and in vivo heavy chain-dimers. However, the structurally conserved C164 was demonstrated to participate in endoplasmic reticulum (ER) resident heavy chain-dimer formation. We therefore wanted to determine whether these aggregates involve cysteines other than C164 and the basis for the difference between the observed heavy chain-dimer species. RESULTS: We determined that C164 and C101 can form distinct dimer structures and that the heterogenous nature of heavy chain-dimer species is due to differences in both redox status and conformation. Different HLA-B27 dimer populations can be found in physiologically relevant cell types derived from HLA-B27-positive patients with inflammatory arthritis. In addition, HLA-B27 dimer formation can be correlated with cellular stress induction. INNOVATION: The use of both mutagenesis and manipulating cellular redox environments demonstrates that HLA-B27 dimerization requires both specific cysteine?cysteine interactions and conformations with differing redox states. CONCLUSION: HLA-B27 heavy chain-dimerization is a complex process and these findings provide an insight into HLA-B27 misfolding and a potential contribution to inflammatory disease development.

Pathogenicity of Misfolded and Dimeric HLA-B27 Molecules.

The association between HLA-B27 and the group of autoimmune inflammatory arthritic diseases, the spondyloarthropathies (SpAs) which include ankylosing spondylitis (AS) and Reactive Arthritis (ReA), has been well established and remains the strongest association between any HLA molecule and autoimmune disease. The mechanism behind this striking association remains elusive; however animal model and biochemical data suggest that HLA-B27 misfolding may be key to understanding its association with the SpAs. Recent investigations have focused on the unusual biochemical structures of HLA-B27 and their potential role in SpA pathogenesis. Here we discuss how these unusual biochemical structures may participate in cellular events leading to chronic inflammation and thus disease progression.

The oxidative folding and misfolding of human leukocyte antigen-b27.

The major histocompatibility complex class I molecule human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies. Many autoimmune diseases exhibit associations with major histocompatibility complex molecules encoded within the class II locus with defined immune responses either mediated by T or B-lymphocytes. Despite the association being known for over 30 years, no defined immune response and target autoantigens have been characterized for the spondyloarthropathies. Thus, the mechanism and role of HLA-B27 in disease pathogenesis remains undetermined. One hypothesis that has recently received much attention has focused around the enhanced propensity for HLA-B27 to misfold and the increased tendency of the heavy chain to dimerize. The misfolding of HLA-B27 has been associated with its redox status and this is postulated to be involved in disease development. Here we discuss the impact of the redox status on HLA-B27 biosynthesis and function.

Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity.

Stable presentation of peptide epitope by major histocompatibility complex (MHC) class I molecules is a prerequisite for the efficient expansion of CD8(+) T cells. The construction of single-chain MHC class I molecules in which the peptide, ß(2)-microglobulin, and MHC heavy chain are all joined together via flexible linkers increases peptide-MHC stability. We have expressed two T cell epitopes that may be useful in leukemia treatment as single-chain MHC class I molecules, aiming to develop a system for the expansion of antigen-specific CD8(+) T cells in vitro. Disulfide trap versions of these single-chain MHC molecules were also created to improve anchoring of the peptides in the MHC molecule. Unexpectedly, we observed that soluble disulfide trap single-chain molecules expressed in eukaryotic cells were prone to homodimerization, depending on the binding affinity of the peptide epitope. The dimers were remarkably stable and efficiently recognized by conformation-specific antibodies, suggesting that they consisted of largely correctly folded molecules. However, dimerization was not observed when the disulfide trap molecules were expressed as full-length, transmembrane-anchored molecules. Our results further emphasize the importance of peptide binding affinity for the efficient folding of MHC class I molecules.

Novel detection of in vivo HLA-B27 conformations correlates with ankylosing spondylitis association.

OBJECTIVE: The class I major histocompatibility complex (MHC) molecule HLA-B27 exhibits a strong association with the autoimmune inflammatory arthritis disorder ankylosing spondylitis (AS) and with other related spondylarthropathies. In the absence of both a defined autoimmune response and a target autoantigen(s), the propensity of HLA-B27 to misfold has been hypothesized to be a major parameter in disease pathogenesis. We undertook this study to test the hypothesis that HLA-B27 misfolding is due to exposure of cysteine residues within the heavy chain to the oxidizing environment of the endoplasmic reticulum. METHODS: A rapid acidification and alkylation modification method was used to examine cysteine residue exposure and accessibility within AS-associated and non-AS-associated HLA-B27 subtypes. RESULTS: This novel approach to probing in vivo class I MHC structure revealed that the HLA-B27 heavy chain adopts conformations not previously described. Furthermore, amino acid residues specific to subtypes HLA-B*2706, B*2709, and B*2704 can have an impact on these novel conformations and on cysteine residue exposure. CONCLUSION: HLA-B27 can adopt novel conformations, resulting in differential accessibility of cysteine residues, which can explain the propensity to misfold. Cysteine exposure in the HLA-B27 heavy chain is also affected by residues within the 114 and 116 regions, thereby providing a potential biochemical basis for the association of HLA-B27 subtypes with AS.