IL-22-producing CD4+T cells in the treatment response of rheumatoid arthritis to combination therapy with methotrexate and leflunomide

Abstract

T cells are key players in immune-mediated rheumatoid arthritis (RA). We previously reported that interleukin (IL)-22⁺CD4⁺T helper (IL-22⁺ Th) cells and IL-22 critically control the pathogenesis of RA. Here we monitored circulating levels of different IL-22⁺ Th cell subsets and measured plasma levels of IL-22, IL-17, and interferon (IFN)-γ in 60 patients with active RA following 12-week combination methotrexate (MTX) and leflunomide (LEF) therapy (MTX+LEF) and 20 healthy individuals. We found the frequencies of circulating IFN-γ^-IL-17^-IL-22⁺ (Th22), IFN-γ^-IL-17⁺ (total Th17), IFN-γ⁺IL-17^-IL-22⁺ (IL-22⁺Th1) cells, and IFN-γ^-IL-17⁺IL-22⁺ (IL-22⁺Th17) cells, as well as the plasma levels of IL-22, IL-17 and IFN-γ to be significantly reduced in RA patients that responded to treatment, but not in non-responders. Reductions in plasma IL-22 level significantly correlated with percentage of circulating Th22 cells and the decrease of plasma IL-22 level correlated with the reduction of DAS28 in responders. Our data suggests that circulating Th22 cells and plasma IL-22 level play a detrimental role in RA. The combination MTX+LEF therapy, by targeting Th22 cells and reducing IL-22 level, relieves the immune defects and ameliorates symptoms of RA. This study provides novel mechanistic understanding of the pathogenesis of RA, which may promote a design of better therapies for RA.

Figure 1. DAS28 scores were significantly improved in RA responders (n = 40) but not in RA non-responders (n = 20) after the combination methotrexate (MTX)+ leflunomide (LEF) therapy.

DAS28 values were compared between pre- and post-treatment in each RA responder (A) or non-responder (B).

Figure 2. The percentages of circulating Th22, total Th17, and IL-22⁺Th17, IL22⁺Th1, but not total Th1 cells decreased following MTX+LEF therapy only in patients that exhibited clinical improvement (n = 40).

Peripheral blood mononuclear cells (PBMCs) were collected from RA patients responsive to treatment at baseline (before treatment, 0 week) and after treatment (12 weeks) with MTX+LEF, and analyzed by flow cytometry for the percentage of different Th subsets, including IFN-γ⁻IL-17⁻IL-22⁺ (Th22), IFN-γ⁻IL-17⁺ (total Th17), IFN-γ⁻IL-17⁺IL-22⁺ (IL-22⁺Th17), IFN-γ⁺IL-17⁻ (total Th1), and IFN-γ⁺IL-17⁻IL-22⁺ (IL-22⁺Th1) cells. (A) The gating strategy for detecting different subsets of Th cells and representative flow images on samples from RA patients at 0 week (0 W), 12 week (12 W), or from healthy controls (HC; n = 20). R1, lymphoctyes; R2, IFN-γ⁻CD4⁺T cells; R3, IL-17⁻CD4⁺T cells; I, Th22 cells; II, IL-22⁺Th17 cells; III, IL-22⁻Th17 cells; IV; IL-22⁺Th1 cells; V; IL-22⁻Th1 cells.

Figure 3. The percentages of circulating Th22, total Th17, IL-22⁺Th17, IL22⁺Th1, or total Th1 cells did not decrease following the combination MTX+ leflunomide LEF therapy in non-responding RA patients (n = 20).

PBMCs were collected from RA patients resistant to treatment at baseline (before treatment, 0 week) and after treatment (12 weeks) with MTX+LEF, and analyzed by flow cytometry for the percentage of different Th subsets, including IFN-γ⁻IL-17⁻IL-22⁺ (Th22), IFN-γ⁻IL-17⁺ (total Th17), IFN-γ⁻IL-17⁺IL-22⁺ (IL-22⁺Th17), IFN-γ⁺IL-17⁻ (total Th1), and IFN-γ⁺IL-17^-IL-22⁺ (IL-22⁺Th1) cells.

Figure 4. Plasma levels of IL- 22, IL-17 and IFN-γ decreased following MTX+LEF treatment in RA responders.

The plasma levels of IL-22 (A), IL-17 (B), and IFN-γ (C) were measured by ELISA in RA responders and non-responders before treatment (0 W), after treatment (12 W), and in HC. R, response group; NR, non-response group. *P < 0.05 versus before treatment in response group. ^#P < 0.05 versus healthy control. ^△P < 0.05 versus non-response group at the same time point.

Figure 5. Plasma levels of IL-22 and IL-17 positively correlate with the percentage of circulating Th22 and total Th17 cells, respectively in RA responders while the plasma levels of IL-22 positively correlated with the percentage of circulating Th22 cells in RA non-responders.

The correlation between the plasma IL-22 level and the percentage of circulating Th22 cells (A), the percentage of circulating IL-22⁺Th1 cells (B), or the percentage of circulating IL-22⁺Th17 cells (C) the correlation between the plasma level of IL-17 and the percentage of total circulatingTh17 cells (D), the correlation between the plasma level of IFN-γ and the percentage of total circulating Th1 cells (E) in RA responders; the correlation between the plasma IL-22 level and the percentage of circulating Th22 cells (F), the percentage of circulating IL-22⁺Th1 cells (G), or the percentage of circulating IL-22⁺Th17 cells (H) the correlation between the plasma level of IL-17 and the percentage of total circulatingTh17 cells (I); the correlation between the plasma level of IFN-γ and the percentage of total circulating Th1 cells (J) in RA non-responders after treatment were analyzed using Spearman’s rank correlation test.

Figure 6. DAS28 positively correlated with plasma IL-22, IL-17 levels, and IL-22-producing Th cells in untreated RA, only with plasma IL-22 level in RA responders, and with plasma IL-22 level as well as the percentage of circulating Th22 cells in RA non-responders after treatment.

The correlation between DAS28 and the plasma IL-22 level (A), the plasma level of IL-17 level (B), the percentage of circulating Th22 cells (C), the percentage of circulating IL-22⁺Th1 cells (D), or the percentage of circulating IL-22⁺Th17 cells (E) in untreated RA; the correlation between the decrease of DAS28 and the decrease of the plasma level of IL-22 (F) in RA responders after treatment; the correlation between the DAS28 and the plasma level of IL-22 (G), the percentage of circulating Th22 cells (H) in RA non-responders after treatment were analyzed using Spearman’s rank correlation test.