Evidence for Altered Cytoskeleton Mobilization Pathway in Splenic Dendritic Cells (DC) from HLA-B27/human b2 microglobulin Transgenic Rats (B27-rats)

Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27-transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLA-B7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. First, all proteins involved in protein processing and MHC I assembly were upregulated in B27 DCs, illustrating the higher pressure on the ER due to misfolding of the HLA-B27 heavy chain. Second, all proteins directly influencing actin-dynamics were downregulated. We showed earlier that this not only influences motility, but also plays an important role in deficient immunological synapse formation. Third, the key thiol protease Cathepsin S involved in MHC II synthesis was downregulated, which led us to quantify RT1-B and RT1-D surface expression. Downregulation concerned both CD4+ and CD4- OX62+ HLA-B27 DC subpopulations and maturation enlarged differences in both population bias and expression intensity. Deficient actin dynamics could also contribute to this lower MHC II surface expression. Study of sensitivity to MHC class II-mediated apoptosis by antibody stimulation showed that compared to NTG, both B7 and B27 CD4+ DC were more prone to apoptosis but did not mutually differ. In contrast, overnight culturing resulted in a higher cell death in B27 than in control CD4- DC, even without antibody stimulation. Interestingly, decreased actin dynamics could also be involved in DC apoptosis. Conclusions: We have demonstrated that DCs are a very vulnerable cell type in HLA-B27 rats. Deficient cytoskeletal dynamics could immobilize matured DC in the tissue or induce aberrant migration patterns upon activation. On top of that abnormal intracellular trafficking and membrane organization together with a reduced expression of MHC class II molecules makes them aberrant in T-cell communication by deficient immunological synapse formation. Especially the reduced motility and viability of the tolerigenic CD4- DC could play an important role in initiating a systemic auto-immune response

Insights into spatial configuration of a galactosylated epitope required to trigger arthritogenic T-cell receptors specific for the sugar moiety

The immunodominant epitope of bovine type II collagen (CII256–270) in Aq mice carries a hydroxylysine-264 linked galactose (Gal-Hyl264), the recognition of which is central to the development of collagen-induced arthritis. This study explores the molecular interactions involved in the engagement of T-cell receptors (TCRs) with such epitopes. Responses of three anti-CII T-cell hybridomas and clone A9.2 (all sharing close TCR sequences) to a panel of CII256–270 analogues incorporating Gal-Hyl264 with a modified side chain were determined. Recognition of naturally occurring CII256–270 peptides by either group of T cells depended strictly upon the presence of the carbohydrate and, more precisely, its intact HO-4 group. Modifications of primary amino group on the hydroxylysine side chain eliminated T-cell reactivity, notwithstanding the presence of the galactosyl moiety. Moderate stereochemical changes, such as altered sugar orientation and methylation at the galactose anchor position, were still permissive. Conversely, robust transformations affecting the relative positions of the key elements were detrimental to TCR recognition. To conclude, these data provide strong new experimental evidence that integrity of both galactose HO-4 and hydroxylysine side chain primary amino groups are mandatory for activation of anti-Gal-Hyl264 TCRs. They also indicate that there is a certain degree of TCR plasticity in peptide-TCR interactions

Transgenic rat OX62(+) DCs exhibit multiple cellular deficiencies and the tolerigenic CD4(-) subset suffers reduced viability

Background: Although the association of the MHC class I allele HLA-B27 with Spondyloarthropathy (SpA) has been known for almost 35 years different hypotheses on its relation to disease mechanism still exist in parallel. Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27- transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLAB7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. First, all proteins involved in protein processing and MHC I assembly were upregulated in B27 DCs, illustrating the higher pressure on the ER due to misfolding of the HLA-B27 heavy chain. Second, all proteins directly influencing actin-dynamics were downregulated. We showed earlier that this not only influences motility, but also plays an important role in deficient immunological synapse formation. Third, the key thiol protease Cathepsin S involved in MHC II synthesis was downregulated, which led us to quantify RT1-B and RT1-D surface expression. Downregulation concerned both CD4+ and CD4- OX62+ HLA-B27 DC subpopulations and maturation enlarged differences in both population bias and expression intensity. Deficient actin dynamics could also contribute to this lower MHC II surface expression. Study of sensitivity to MHC class II-mediated apoptosis by antibody stimulation showed that compared to NTG, both B7 and B27 CD4+ DC were more prone to apoptosis but did not mutually differ. In contrast, overnight culturing resulted in a higher cell death in B27 than in control CD4- DC, even without antibody stimulation. Interestingly, decreased actin dynamics could also be involved in DC apoptosis. Conclusions: We have demonstrated that DCs are a very vulnerable cell type in HLA-B27 rats. Deficient cytoskeletal dynamics could immobilize matured DC in the tissue or induce aberrant migration patterns upon activation. On top of that abnormal intracellular trafficking and membrane organization together with a reduced expression of MHC class II molecules makes them aberrant in T-cell communication by deficient immunological synapse formation. Especially the reduced motility and viability of the tolerigenic CD4- DC could play an important role in initiating a systemic auto-immune response

Dendritic Cells from Spondyloarthritis-prone HLA-B27 Transgenic Rat Display Altered Cytoskeletal Dynamics, MHC Class II Expression, and Viability.

Background: Several lines of rats transgenic for HLA-B27 and human β2-microglobulin develop an inflammatory disease that strikingly resembles human SpA. It is hypothesized that disease in HLA-B27-transgenic rats arises as a consequence of interaction between antigen-presenting cells expressing high levels of HLA-B27 and peripheral T lymphocytes, and may result from a rupture of tolerance towards gut bacteria. Methods: We used 2D PAGE and iTRAQ to compare the protein expression profile of HLA-B27 dendritic cells (DCs) to that of healthy HLA-B7 expressing and nontransgenic (NTG) rat DCs. MHC II surface expression and apoptotic sensitivity were quantified using flow cytometry. Results: Three protein sets from the proteome analysis were indicative for aberrant cellular processes. First, all proteins involved in protein processing and MHC I assembly were upregulated in B27 DCs, illustrating the higher pressure on the ER due to misfolding of the HLA-B27 heavy chain. Second, all proteins directly influencing actin-dynamics were downregulated. We showed earlier that this not only influences motility, but also plays an important role in deficient immunological synapse formation. Third, the key thiol protease Cathepsin S involved in MHC II synthesis was downregulated, which led us to quantify RT1-B and RT1-D surface expression. Downregulation concerned both CD4+ and CD4- OX62+ HLA-B27 DC subpopulations and maturation enlarged differences in both population bias and expression intensity. Deficient actin dynamics could also contribute to this lower MHC II surface expression. Study of sensitivity to MHC class II-mediated apoptosis by antibody stimulation showed that compared to NTG, both B7 and B27 CD4+ DC were more prone to apoptosis but did not mutually differ. In contrast, overnight culturing resulted in a higher cell death in B27 than in control CD4- DC, even without antibody stimulation. Interestingly, decreased actin dynamics could also be involved in DC apoptosis

Non-conventional forms of HLA-B27 are expressed in spondyloarthritis joints and gut tissue

AbstractObjectivesHuman leukocyte antigen (HLA)-B27 (B27) is the strongest genetic factor associated with development of Ankylosing Spondylitis and other spondyloarthropathies (SpA), yet the role it plays in disease pathogenesis remains unclear. We investigated the expression of potentially pathogenic non-conventional heavy chain forms (NC) of B27 in synovial and intestinal tissues obtained from SpA patients. We also determined the presence of NC-B27 in joints, lymphoid and gastrointestinal tissue from B27 transgenic (TG1) rats with M.tuberculosis-induced SpA.MethodsExpression of NC-B27 in human SpA joints and gut and in (21-3 × 283-2)F1 HLA-B27/Huβ2m rat tissue was determined by immunohistochemistry, flow cytometry and confocal microscopy analysis using HC10 and HD6 antibodies.ResultsBoth HC10- and HD6-reactive HLA molecules were present in synovial tissue from SpA patients. Both NC-B27 and KIR3DL2, a ligand for NC-B27, were expressed in inflamed terminal ileal tissues in patients with early SpA. Infiltrating cells in inflamed joint tissues isolated from B27 TG1 rats expressed high levels of NC-B27. NC-B27 were also expressed in joint-resident cells from ankle and tail joints of B27 TG1 rats prior to clinical arthritis. The expression of NC-B27 on B27 TG1 rat CD11b/c+, CD8α+, cells from spleens and LNs increased with animal age and disease progression.ConclusionsNon-conventional HLA class 1 molecules are expressed on resident and infiltrating cells in both synovial and GI tissues in human SpA. NC-B27 expression in joints and lymphoid tissues from B27 TG1 rats prior to the onset of arthritis is consistent with the hypothesis that they play a pathogenic role in SpA

Extracellular traps formation following cervical spinal cord injury

International audienceSpinal cord injuries involve a primary injury that can lead to permanent loss of function and a secondary injury associated with pathologic and inflammatory processes. Extracellular traps are extracellular structures expressed by immune cells that are primarily composed of chromatin, granular enzymes and histones. Extracellular traps are known to induce tissue damage when overexpressed and could be associated in the occurrence of secondary damage. In the present study, we used flow cytometry to demonstrate that at 1 day following a C2 spinal cord lateral hemisection in male Swiss mice, resident microglia form vital microglia extracellular traps, and infiltrating neutrophils form vital neutrophil extracellular traps. We also used immunolabelling to show that microglia near the lesion area are most likely to form these microglia extracellular traps. As expected, infiltrating neutrophils are located at the site of injury, though only some of them engage in post-injury extracellular trap formation. We also observed the formation of microglia and neutrophil extracellular traps in our sham animal models of durotomy, but formation was less frequent than following the C2 hemisection. Our results demonstrate for the first time that microglia form extracellular traps in the spinal cord following injury and durotomy. It remains however to determine the exact mechanisms and kinetics of neutrophil and microglia extracellular traps formation following spinal cord injury. This information would allow to better mitigate this inflammatory process that may contribute to secondary injury and to effectively target extracellular traps to improve functional outcomes following spinal cord injury