An HLA-DRB1-coded signal transduction ligand facilitates inflammatory arthritis: a new mechanism of autoimmunity

Abstract

Particular alleles of HLA contribute to disease susceptibility and severity in many autoimmune conditions, but the mechanisms underlying these associations are often unknown. In this study, we demonstrate that the shared epitope (SE), an HLA-DRB1-coded sequence motif that is the single most significant genetic risk factor for erosive rheumatoid arthritis, acts as a signal transduction ligand that potently activates osteoclastogenesis, both in vitro and in vivo. The SE enhanced the production of several pro-osteoclastogenic factors and facilitated osteoclast (OC) differentiation in mouse and human cells in vitro. Transgenic mice expressing a human HLA-DRB1 allele that code the SE motif demonstrated markedly higher propensity for osteoclastogenesis and enhanced bone degradation capacity ex vivo. In addition, the SE enhanced the differentiation of Th17 cells expressing the receptor activator for NF-κB ligand. When the two agents were combined, IL-17 and the SE enhanced OC differentiation synergistically. When administered in vivo to mice with collagen-induced arthritis, the SE ligand significantly increased arthritis severity, synovial tissue OC abundance, and bone erosion. Thus, the SE contributes to arthritis severity by activating an OC-mediated bone-destructive pathway. These findings suggest that besides determining the target specificity of autoimmune responses, HLA molecules may influence disease outcomes by shaping the pathogenic consequences of such responses.

Figure 1. Activation of signaling and OC differentiation by the SE in pre-OCs

(A) RAW 264.7 cells (3 × 10⁴ /well) were plated in flat-bottom 96-well plates and treated with or without 50 µg/ml of the SE ligand 65-79*0401, or a SE-negative control peptide 65-79*0402. Data are expressed as the mean ± SEM fluorescence units (FU) in triplicate wells. P values were calculated using a paired t-test. (B) Rates of NO and ROS production (FU/min) in RAW 264.7 cells, cultured as in A. (C) Flow cytometric analysis of CRT surface expression in RAW 264.7 cells. Cells were labeled first with either rabbit anti-mouse CRT antibody (solid line), or rabbit anti-mouse control antibody (dashed line), followed by FITC anti-rabbit IgG Ab. The gray-colored histogram represent second-stage antibody only. (D) RAW 264.7 cells were cultured as in A, in the presence or absence of rabbit anti-mouse CRT antibodies (1:100), or rabbit anti-mouse, BCL – X s/l (S-18) polyclonal Ig (1:100) as control. NO and ROS production rates were calculated as above. (E) RAW 264.7 cells were cultured with M-CSF and RANKL in the presence of 50 µg/ml of the SE ligand 65-79*0401, a SE-negative control peptide 65-79*0402, or PBS for different periods of time. (F) RAW 264.7 cells, cultured with M-CSF and RANKL were treated with various concentrations of the SE ligand 65-79*0401, SE-negative control peptide 65-79*0402, or with PBS. (G and H) RAW 264.7 cells, cultured with M-CSF in the presence or absence of RANKL were treated with the SE ligand, a control peptide, or PBS for 6 days. In all experiments, TRAP-positive multinucleated OCs (>3 nuclei) were counted microscopically. (H) Representative microscopic views of the experiment shown in G.

Figure 2. Osteoclastogenic effects of SE in primary cells

(A) BMCs harvested from DBA/1 mice were cultured with M-CSF and RANKL in the presence of 50 µg/ml of the SE ligand 65-79*0401, a SE-negative control peptide 65-79*0402, or PBS for 6 days, and TRAP-positive cells counted. (B) OCs were differentiated from human PBMCs for 7 days in the presence or absence of the SE ligand 65-79*0401, or a SE-negative control peptide 65-79*0403. (C) BMCs from DR4*0401 Tg and DR4*0402 Tg mice in medium without growth factors were allowed to adhere to culture wells overnight and the number of TRAP-positive mononuclear pre-OC cells (MNCs) per well was determined. (D) BMCs from DR4*0401 Tg and DR4*0402 Tg mice were cultured for 6 days OC-differentiating conditions, and TRAP-positive multinucleated cells were counted. (E) BMCs from DR4*0401 Tg and DR4*0402 Tg mice were cultured atop bone matrices in OC-differentiating conditions and matrix resorption was quantified at different time points. Black areas represent intact matrix; white areas correspond to matrix resorption. Right hand side shows quantitative data of mean ± SEM of matrix resorption. (F) BMCs from DR4*0401 Tg and DR4*0402 Tg mice were cultured atop bone slices in OC-differentiating medium. On day 10, slices were stained for TRAP (upper panel). Arrowheads point at representative OCs. Lower panel shows toluidine blue (TB)-stained bone slices after removal of attached BMCs. Arrowheads point at representative resorption pits. White horizontal bars = 100 µM. Bar graphs in the right hand side represent mean ± SEM events from quintuplicate cultures in each of the two respective panels.

Figure 3. The SE stimulates pro-osteoclastogenic factors

(A) RAW 264.7 cells, cultured under OC differentiating conditions as above were treated with 50 µg/ml of the SE ligand 65-79*0401, SE-negative control peptide 65-79*0402, or with PBS. Supernatants were collected at different time points and assayed for cytokines by ELISA. (B) BMC from DBA/1 mice, cultured under OC differentiating conditions were treated and cytokines were quantified in the supernatants as in A. (C and D) BMCs from DBA/1 mice were cultured under OC-differentiation conditions as above in the presence of 50 µg/ml of the SE ligand 65-79*0401, or a SE-negative control peptide 65-79*0402. Rat anti IL-6 (10µg/ml) (C) or anti-TNF-α (5 µg/ml) (D) monoclonal antibodies were added to respective wells. Rat monoclonal IgG1 was used as a control antibody. After 6 days, cells were stained and OCs were counted. (E) RAW 264.7 cells were cultured under OC-differentiating conditions as above in the presence or absence of 50 µg/ml of the SE ligand 65-79*0401, a SE-negative control peptide 65-79*0402, or with PBS. After 6 days, cells were harvested, and RANK surface expression was determined by flow cytometry. (F) DBA/1 splenocytes were cultured under Th17-differentiating conditions in the presence or absence of 50 µg/ml of the SE ligand 65-79*0401, a SE-negative control peptide 65-79*0402, or PBS for 5 days. Expression levels of surface RANKL and intracellular IL-17A were determined by flow cytometry. Left panels, Bar graphs show the data as percentage (mean ± SEM). Right panels, Dot plots of representative experiments. (G) BMCs harvested from DBA/1 mice were isolated and cultured for 6 days with M-CSF (10 ng/ml) and a sub-optimal concentration of RANKL (5 ng/ml), in the presence or absence of 50 µg/ml of the SE ligand 65-79*0401, a SE-negative control peptide 65-79*0402, or PBS, in the presence or absence of low concentrations of rmIL-17. TRAP-positive OCs were counted microscopically.

Figure 4. The SE facilitates CII-specific Th17 polarization in CIA mice

Popliteal and inguinal lymph nodes from CIA mice (DBA/1 CII-TCR Tg) treated intraperitoneally with the SE ligand 65-79*0401 or a SE-negative control peptide 65-79*0402. (A and B) Freshly isolated lymph node cells were cultured for 6 h with PMA, ionomycin and Brefeldin A, and then stained with anti-mouse CD4, IL-17A and Foxp3. (C and D) Lymph node cells were re-stimulated ex-vivo for 5 days with chicken CII (100 µg/ml) or PBS (NIL). Left panels, Representative dot plots show percentages of IL17A⁺ and Foxp3⁺ cells in gated CD4+ cells. Right panels, Bar graphs in B and D show compiled results as mean ± SEM (n = 8).

Figure 5. The SE ligand enhances arthritis and bone damage in vivo

CIA mice (DBA/1 CII-TCR Tg) were injected intraperitoneally with the SE ligand 65-79*0401, a SE control peptide 65-79*0402 or PBS. Data in A, and B represent compiled results from 2 experiments, n = 28. (A) Mean day of arthritis onset. (B) Arthritis scores. P value represents analysis of 65-79*0401 versus 65-79*0402 in a paired Student t test. A comparable level of significance was found in the analysis of 65-79*0401 versus PBS groups. (C) Radiological scores of rear and front paws (n = 6) of CIA mice treated with the SE ligand 65-79*0401, or the SE-negative control peptide 65-79*0402. (D) Representative ankle and wrist micro-CT images of CIA mice as in C. (E) Micro-CT alpha blend images showing bone mineral content (green = none, blue = lowest, red = highest). (F) Quantitative measurement of tissue mineral density (TMD) ± SEM in the two CIA groups (n = 5 per group).

Figure 6. The SE activates osteoclastogenesis in vivo

SE ligand 65-79*0401 or control treatments were administered intraperitoneally to CIA mice (DBA/1 CII-TCR Tg). (A) Quantification of TRAP-positive cells in CIA joint tissues. (B) Representative views of TRAP and H&E-stained knee joint tissue sections of CIA mice. Magnification: × 10. (C) Fresh bone marrow cells harvested from CIA mice were analyzed for the abundance of pre-OCs. (D) Bone marrow cells harvested from CIA mice were cultured for 6 days under OC-differentiating conditions and OCs were quantified as above.