MHC class II alleles associated with Th1 rather than Th17 type immunity drive the onset of early arthritis in a rat model of rheumatoid arthritis

Abstract

Polymorphisms in the MHC class II (MHCII) genes are strongly associated with rheumatoid arthritis, supporting the importance of autoreactive T helper (Th) cells for the development of this disease. Here, we used pristane-induced arthritis (PIA), induced by the non-antigenic hydrocarbon pristane, to study the impact of different MHCII alleles on T-cell activation and differentiation. In MHCII-congenic rats with disease-promoting MHCII alleles, pristane primarily induced activation of Th1 cells, whereas activated T cells were Th17 biased in rats with protective MHCII alleles. Neutralization of IFN-γ during T-cell activation abrogated the development of disease, suggesting that Th1 immunity is important for disease induction. Neutralization of IL-17, by contrast, suppressed arthritis only when performed in rats with established disease. Adoptive T-cell transfers showed that T cells acquired arthritogenic capacity earlier in strains with a prevailing Th1 response. Moreover, upon pristane injection, these strains exhibited more Ag-primed OX40+ and proliferating T cells of polyclonal origin. These data show that T cells are polarized upon the first encounter with peptide-MHCII complexes in an allele-dependent fashion. In PIA, the polyclonal expansion of autoreactive Th1 cells was necessary for the onset of arthritis, while IL-17 mediated immunity contributed to the progression to chronic disease.

Keywords: Animal models; Autoimmunity; Immune responses; MHC; Rheumatoid arthritis; T helper (Th) cells.

Figure 1

Tcs2 controls pathogenicity and expansion of T cells after pristane administration. (A) Physical map of the rat MHC‐II region. Asterisked genes are located within the flanking borders of T cell selection QTL 2 (Tcs2) while shaded genes are excluded. Congenic strains are depicted as vertical bars with dashed lines representing intervals of unknown genotype. Numbers next to fragments represent genetic markers and have been described previously 12. (B‐C) Clinical arthritis scores (B) and weight change (C) in DA and Tcs2‐congenic strains after pristane administration (max score = 60). Inset table shows day of disease onset (± standard deviation, S.D.) and p‐value versus DA. (D) Individual transfer T cells were adoptively transferred (one donor to one recipient) on day 3, 5, 8, or 10 after administration of pristane. Numbers in brackets depict recipients with arthritis out of total. (E) Individual transfer between Tcs2‐congenic strains. T cells were transferred on day 5 after pristane administration. Pc = p value for cumulative incidence. (F) Expression of Ki67 in CD4+ (upper row) and CD8+ (lower row) activated/memory T cells (CD90‐ CD45RClo Foxp3‐) in pristane dLNs at indicated time‐points after disease induction. Counter plots show gating strategy and adjacent histograms representative examples of Ki67 expression in DA (black) and DA.1HR10 (shaded); n = 5 per group. A–F: *p<0.05, **p<0.01 (by Mann–Whitney). (B–E) Data shown are representative of at least three independent experiments. (F) Data shown are pooled from five independent experiments. Error bars in B, C (graph), D, E, and F (graphs) represent standard error of the mean (SEM).

Figure 2

The TCR Vβ repertoire on CD4 T cells is largely unaffected by pristane administration. CD4+ T cells were analyzed for the relative frequency of Vβ10, Vβ16.1, Vβ8.5, and Vβ8.2/8.4+ subsets in dLNs before (day 0) and 5 days after pristane injection. Stacked bars show Vβ frequencies among total CD4+ T cells (no significant differences were observed between Foxp3+ and Foxp3‐ CD4 T cells). Numbers within bars represent mean frequencies. Data are representative of at least three independent experiments. n = 6 rats/ group; *p<0.01 compared to DA day 0, ^† p<0.01 compared to same strain day 0 (Mann–Whitney U).

Figure 3

Variation in OX40+ Treg cells and T effector cells among DA and Tcs2‐congenic strains. (A) Frequency of Treg cells (CD25+ Foxp3+) among total CD4+ T cells in pristane dLNs. Dot plots show representative examples of Treg cells from dLN on day 4 after pristane administration in DA and DA.1HR10. (B) Expression of OX40 on Foxp3+ and Foxp3‐ T effector/memory cells (CD45RClo CD90‐ CD4+; see Fig. 1F for gating strategy). Counter plots show representative examples of OX40 expression at different time‐points after pristane administration in DA (top) and DA.1HR10 (bottom). Plots are representative of —three to five independent experiments. (C) Ratio of OX40+ Treg cells to OX40‐ Treg cells (gated as shown in (B)). (D) Frequency of Ag‐primed (OX40+) Foxp3‐ effector/memory T cells. (E) Ratio of total Treg cells to OX40+ Foxp3‐ effector/memory T cells at day 5 and 8 after pristane injection. n = 5 rats/group; *p<0.05, **p<0.01 (by Mann–Whitney); data shown are pooled from three to five independent experiments (A–E). Error bars represent standard error of the mean (SEM) (A, C, and D). In (E) each box represents the 25th to 75th percentiles; lines inside the boxes represent the median; lines outside the boxes represent the 10th and the 90th percentiles.

Figure 4

Administration of pristane drives Th1‐differentiation in rats with disease promoting MHCII alleles. (A) Messenger RNA expression (as determined by qPCR) of cytokine and Th‐lineage‐defining transcription factors from isolated CD4 T cells harvested on day 8 after pristane injection. Horizontal bars show mean values ±SEM. Variation is expressed as fold‐change differences between individuals, n = 9–10 per group. (B) Cytokine levels after in‐vitro stimulation of dLN cells from DA and DA.1HR10 with anti‐CD3/CD28. Cells were harvested 8 days after pristane administration. (C) Frequencies of IFN‐γ+ (Th1) and IL‐17+ (Th17) cells among non‐RTE (CD90‐) CD4+ T cells. Dot plots show DA (upper row) and DA.1HR10 (lower row). Data are summarized in adjacent line chart. n = 5 rats/group. (D) Total number of CD90‐ Th1 and Th17 cells in dLNs. AU = arbitrary units. (A–D) *p < 0.05; **p < 0.01; ***p < 0.001 (by Mann–Whitney). Results shown are representative of —two to three independent experiments (A and B). Data shown in (C and D) are pooled from five independent experiments. Error bars represent SEM (A, C, and D); vertical line represents mean (B).

Figure 5

Pristane‐induced arthritis is dependent on IFN‐γ for T‐cell priming. (A‐B) Antibodies to INF‐γ (DB‐1) (A) and IL‐17 (17F3) (B) were administrated s.c. on day 2, 4, and 6 after pristane administration (control rats were injected with PBS; since, DB‐1 and 17F3 are both of mouse IgG1 isotype). (C) Day of disease onset for rats treated with DB‐1 (left panel) and 17F3 (right panel). n = 7–8 rats/group as indicated in figure; *p < 0.05; **p < 0.01; ***p < 0.001 (Mann–Whitney). Data are shown as mean + SEM and are pooled from two independent experiments with similar results.

Figure 6

IL‐17 more than IFN‐γ is required for the progression of arthritis in PIA. (A–D) Neutralizing antibodies to IFN‐γ (DB‐1) and IL‐17 (17F3) were injected i.v. into rats at day 8, 10, 12, and 14 after pristane administration using MOPC‐21 as an isotype control. Disease development (left) and weight change (% versus day of onset, right) are shown for DA (A), DA.1FR9 (B), DA.1HR10 (C) and DA.1UR10 (D); n = 8–11 per group as indicated in figures, *p < 0.05; **p < 0.01; ***p < 0.001 (compared to isotype); p‐values for trend in (B) and (D) represent differences between DB‐1 and MOPC‐21 (All data in A–D was analyzed by Mann‐Whitney). (E) Frequencies (gates in dot plots) and numbers (scatter plot) of CD11 b/c+ CD8a‐ polymorphonuclear leukocytes (PMNs) in blood were determined in rats shown in (A) at day 16 post pristane injection; ***p < 0.001 (compared to isotype and DB‐1, Mann–Whitney). Data shown in A–D are pooled from two independent experiments with similar results. Error bars in (A–D) represent SEM; vertical line and error bars in (E) represent mean and standard deviation, respectively.