WHY IS HLA-B27 LINKED TO ARTRITIC DISEASES?
The MHC class I allele HLA-B27 is very strongly correlated with development of autoimmune spondyloarthritis, psoriasic artritic, IBD and uveitis, although the mechanism underlying this disease remains unknown.
HLA in health and disease
The prevalence of HLA-B27 varies markedly in the general population. For example, about 8% of Caucasians, 4% of North Africans, 2-9% of Chinese, and 0.1-0.5% of persons of Japanese descent possess this gene. In northern Scandinavia (Lapland), 24% of people are HLA-B27 positive, while 1.8% have associated ankylosing spondylitis. Even though this gene is associated with a wide range of pathology, it does not appear to be the sole mediator in the development of the disease. For example, while 90% of people affected by ankylosing spondylitis (AS) are HLA-B27 positive, only a fraction of people with HLA-B27 does ever develop AS .
Class I molecules classically associate in the endoplasmic reticulum (ER) with beta2-microglobulin (beta(2)m) and antigenic peptides for cell surface expression and presentation to T cells.
There are many hypothesis that can show this relation and the main are listed in the following table:
|• HLA B27 is in genetic linkage with a disease‐associated gene|
• HLA B27 binds and presents ‘arthritogenic’ peptides to T cells
• HLA B27 is involved in thymic selection of a T‐cell repertoire that is susceptible to spondyloarthritis
• HLA B27 has an unusual cell biology compared with other HLA class 1 molecules
• The HLA B27 free cysteine at position 67 can be chemically modified, leading to an ‘altered self’
• There is cross reactivity between antibodies directed at bacterial protein(s) and HLA B27
• HLA B27 is a receptor for a bacterial ligand
• Interaction of HLA B27 with a bacterial superantigen causes non‐specific T‐cell stimulation
• HLA B27‐derived peptides are presented by HLA class 2 molecules to CD4+ T cells
All of these diseases can be grouped as in the table:
The receptor theory states that a class I major histocompatibility complex (MHC) molecule acts as a receptor cavity that binds some as yet undefined, external, environmental polypeptides, which then somehow increases the susceptibility of the subject to develop the disease.
Ankylosing spondylitis is caused by Klebsiella. Evidence from immunogenetic, microbiologic, and serologic studies
At now, however, the nature of the "arthritogenic" polypeptides present in the HLA-B27 pocket have not been identified. In 2009 was demostrated by Taurog JD et coll. that there was no significant difference in disease prevalence or severity between CD8a–/– rats and CD8a/– rats: all of the previously described disease manifestations in HLA–B27/Hu2m-transgenic rats arise in the absence of any functional CD8 T cells. So it seems unlikely that classic T cell recognition of HLA–B27 is of primary importance in this animal model. Anyway, the possibility of a secondary role of a CD8- dependent mechanism cannot, of course, be entirely excluded.
Spondylarthritis in HLA-B27/human beta2-microglobulin-transgenic rats is not prevented by lack of CD8
The molecular mimicry hypotesis is based on a cross-reactivity between some bacterial antigen, such as Chlamydia, Salmonella, Yersinia, E. Coli and Klebsiella, on one side, and some human peptides, on the other side. If there is cross-reactivity between HLA antigens and bacteria, then infection by such microorganisms would lead to the production of antibodies, which would have both antimicrobial and autoimmune activity.
For example some homologies are described between HLA-B27 itself (area from aminoacid 70 to 78, in the variable of a1-helix region) and YadA (Yersinia adhesion, responsable for serum resistance, cell adherence, resistance to phagocytosis, and binding to soluble collagen), OmpH (a cationic outer membrane protein of Salmonella Typhimurium, that probably interacts with LPS and is involved in the membrane integrity) and the Klebsiella pneumoniae nitrogenase (which shares four aminoacids with YadA). These evidences are summerized in the following table:
|Protein||Residues||Amino acid sequence||Reference|
|HLA B*2705||63-84||ETQICKAKA QTDRE DLRTLLRY||Lopez de Castro (1989)|
|HLA B*2701||63-84||ETQICKAKA QTYRE NLRTALRY|
|HLA B*2702||63-84||ETQICKAKA QTDRE NLRIALRY|
|HLA B*2703||63-84||ETQICKAKA QTDRE DLRTLLRY|
|HLA B*2704||63-84||ETQICKAKA QTDRE SLRTLLRY|
|HLA B*2706||63-84||ETQICKAKA QTDRE SLRTLLRY|
|YadA protein of Y.ent||171-188||____AIGDRSK TDRE NSVSIGH||Skurnik & Wolf-Watz (1989)|
|Klebsiella pneumoniae nitrogenase||185-194||______CNSR QTDRE DELI___||Schwimmbeck et al. (1987)|
Molecular mimicry between HLA B27 and Yersinia, Salmonella, Shigella and Klebsiella within the same region of HLA al-helix
Ankylosing spondylitis is caused by Klebsiella. Evidence from immunogenetic, microbiologic, and serologic studies
The role of Klebsiella pneumoniae nitrogenase is described in Ebringer's work: he proposed that if the bacteria were to be present in the gut or the bowel mucosa, it could be expected that the related lymph nodes, which are present in the mesentery of the gut and the pelvis, would be closely related to both the lumbar spine and sacroiliac joints. Therefore, high titers of antibodies would be expected in these areas, which are the main pathologic sites of disease expression in AS. Continued production of high titers of antibodies would lead to inflammation around the lumbar spine and sacroiliac joints, thus presence of clinical signs and symptoms related to those areas would be expected in such patients. In situations of persistent infections, the antibodies would enter from the mesenteric and pelvic lymph nodes into the general circulation, therefore such antibodies should not only be detectable in the blood, but would also affect more distal sites, such as the neck, uvea, and peripheral large joints.
In agreement of this work elevated levels of antibody to Klebsiella pneumoniae are found in both active Crohn’s disease (CD) and active AS patients compared with healthy controls.
Levels of class-specific IgM, IgG and IgA antibodies to K.pneumoniae were also significantly high in early and late CD and AS compared with healty controls (P<0.001). In the same article it's reported that these antibodies can recognize collagens type I, III, IV and V.
Correletion between the immune responses to collagens type I, III, IV and V and Klebsiella pneumoniae in patients with Crohn’s disease and ankylosing spondylitis
Give the above it is suggested that the role of Klebsiella is one of the most important in the development of this autoimmune disease. We have then decided to look for a possible human peptide that is similar to Klebsiella nitrogenase and that is expressed just in the tissues affected in this kind of pathology. Infact all the autoantibodies found in the studies reported recognize ubiquitary proteins.
From the evidence that osteopontin expression is decreased in uveitis and that this protein is present in high levels in skeletal-muscle tissue, we try to make a blast between osteopontin and Klebsiella nitrogenase.
Osteopontin and Fibronectin Levels Are Decreased in Vitreous of Autoimmune Uveitis and Retinal Expression of Both Proteins Indicates ECM Re-Modeling
|Score = 16.5 bits (31), Expect = 0.68, Method: Compositional matrix adjust.|
Identities = 15/61 (25%), Positives = 25/61 (41%), Gaps = 7/61 (11%)
Osteop. SH KQ SRL Y KRK A NDES N EHSD V IDSQ---- EL SKVSR EF HSH E FHSH ED MLVVDPKSK EE D
BLAST--+§ KQ +§§ Y +§§ A §§§§ N §§§§ V +§+§§§§§ EL §§+§§ EF §§§ E §§§§ ED §§++§§§+§ EE +
Kl.nitr.--AC KQ ANE Y RTL A QKIV N NTMK V VPTPCTMD EL ESLLM EF GIM E ---E ED TSI IGKTAA EE N
Our results show a minimal sequence analogy, but we can't exclude that exists a tertiary-structure analogy or that some other proteins are involved. To prove this link, it might be important to test the immunoflorescence pattern in a patologic tissue: to confirm our hypothesis the pattern could be cell surface or cytosolic.
II) INDEPENDENT OF ANTIGEN-SPECIFICITY
HLA-B27 misfolding and spondyloarthropathies.
The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong to a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with beta2m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface; but might happen on the cell surface too. The evidences indicate that misfolding occurs in the ER prior to b2m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation ofmisfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR).
The UPR maintains ER homeostasis, initially by dampening the flux of protein into this organelle, and then by expanding its capacity to fold, secrete, and/or degrade protein. However, depending on the magnitude and duration of ER stress, and the type of cell that is affected, the UPR can result in apoptosis.
The UPR has been implicated in the pathogenesis of a number of protein misfolding diseases, in part through cell death.
It is recently found that XBP1, a transcription factor induced by UPR activation, mediates synergistic type I IFN induction in cells exposed to certain pattern recognition receptor (PRR) agonists (‘UPR-PRR synergy’). Moreover there is increasing recognition that the UPR plays a role in immune modulation with potential links to inflammatory disease pathogenesis.
So, the misfolding of HLA-B27 has thus been associated with its redox status. Newly synthesized MHC class I heavy chains associate with calnexin, which, upon β2-microglobulin association, is displaced by calreticulin. Both calnexin and calreticulin can recruit the oxidoreductase ERp57 into these early folding intermediates. Upon calreticulin association, the partially folded MHC class I molecule associates with the transporter associated with antigen processing (TAP) via the MHC class I-specific accessory molecule tapasin, thus forming the peptide loading complex. As part of the peptide loading complex, tapasin is disulfide-bonded to ERp57, and its function promotes the binding of optimal peptides by MHC class I molecules.
The oxidative folding and misfolding of human leukocyte antigen-b27
HLA-B27 misfolding and UPR (unfolded protein response) activation in macrophages can result in enhanced induction of the pro-Th17 cytokine, IL-23.
IL-23 cytokine is comprised of two subunits and plays a key role in driving memory CD4+ T cells (‘Th17′) to produce pro-inflammatory cytokines including IL-17.
Infact, IL-23 was also increased in the colon tissue from B27/Hubeta(2)m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17-expressing T cells.
p<>. These results suggest a possible link between HLA-B27 misfolding and immune dysregulation in this animal model with implications for human disease.
HLA-B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic rats
HLA-B27 upregulation may occur initially as a result of antigen presenting cell stimulation with TLR agonists from commensal microorganisms and/or low level IFN-γ production from innate immune cells such as NK cells. UPR activation is superimposed on macrophage activation because of HLA-B27 misfolding, resulting in greater IL-23 production in response to TLR agonists. This, in turn, stimulates Th17 cells to produce IL-17. Th1 activation, and/or double positive IL-17/IFN-γ producing cells, may help to sustainHLA-B27 expression thus perpetuating this cycle.
HLA-B27 Heavy chain homodimer formation
We know that the main natural function of HLA B27 is an immunological one, namely to bind and present short antigenic peptides to cytotoxic T lymphocytes in both healthy and spondyloarthropathy affected individuals. Normally MHC class I heavy chains associate with β2-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface, but in spondyloarthropathy patients there is an abnormal presence of HLA B27 homodimers, specially expressed in a variety of lymphoid cell lines.
Formation of HLA-B27 homodimers and their relationship to assembly kinetics.
The MHC Class I heavy chain structurally conserved cysteines 101 and 164 participate in HLA-B27 dimer formation.
p<>.Indeed the main aspect to be investigated is the unusual possibility for the HLA B27 to form heavy chain homodimers not in vitro only. HLA-B27 forms, in fact, disulfide-bonded homodimers in vivo by two different pathways.
In vitro folding studies revealed the capacity of HLA-B27 heavy chains to form dimeric structures via a disulfide bridge at the relatively rare unpaired cysteine at position 67 (Cys67) in the α1 helical region of the peptide binding groove. However, the same thing could happen if the cysteine at position 164 is involved. In both cases, HLA-B27 dimer formation can be correlated with cellular stress induction.
Furthermore, two species of HC-dimers (the ER ones and surface ones) have been detected: one within the ER and another at the cell surface. These populations of homodimers appear to be distinct from each other, since cell surface homodimers arise from recycling of HLA-B27-β2-microglobulin complexes through an endocytic compartment, whereas ER-resident homodimers do not appear to transit the secretory pathway.
Experiments with inhibitors indicate that HLA-B27 homodimers can arise from cell-surface heterodimers via an endosome-dependent recycling pathway. HLA-B27 homodimer expression on the cell surface of 721.220 is dependent on the unpaired cysteine 67 and is inhibited by restoration of tapasin function or by incubation with peptides that bind strongly to HLA-B27 heterodimers.
Usually, HLA-B27 homodimers formed in the ER appear unable to egress to the cell surface in human cells, but they are abundantly expressed on surface of a variety of lymphoid cell lines.
Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling.
In fact, preliminary data suggest that significant numbers of T cells from patients with spondyloarthropathy express a ligand for HLA B27 homodimers, proposing the testable new hypothesis that HLA B27 heavy chain dimerization may be involved in the pathogenesis of spondyloarthritis.
Despite the absence of β2m, previously considered essential to the structural integrity of all mature HLA complexes, the homodimers maintains the conformation of a significant part of its peptide‐binding groove, as evidenced by the binding of monoclonal antibodies such as W6/32, and is capable of binding peptide.
One possible mechanism by which expression of homodimers could lead to joint inflammation would be by the presentation of peptides to either CD4+ or CD8+ T cells: peptide presentation by MHC class 1 molecules to CD4+ T cells is rare, but might be favoured for homodimers by partial unwinding of the α1 helix, making this complex more similar to a class 2 molecule.
Another possible mechanism involved is that the HLA B27 homodimers might be a ligand for natural killer (NK) receptors or other receptors. Alternatively, HC-dimers within the ER may induce ER stress responses.
The homodimer formation might only occur under particular conditions: a relative lack of β2m (as in the β2m‐deficient mice) or of suitable peptides (as in cell lines with defects in their antigen‐presenting apparatus); similar conditions could occur during infection with intracellular bacteria, some of which are known to trigger rheumatoid arthritis.
This hypothetical model for the role of HLA-B27 homodimers in the pathogenesis of spondyloarthritis could be rappresented as:
Moreover the cell‐surface expression of HLA B27 homodimers results in a local inflammatory response, through either cellular or humoral mechanisms. If HC‐B27 is indeed a pro‐inflammatory ligand, its production in certain tissues at certain times might explain the pathogenic role of HLA B27 (in this way HLA B27 homodimers inhibitors or monoclonal antibodies could used as therapeutic strategies).