Differences in IDO1+ dendritic cells and soluble CTLA-4 are associated with differential clinical responses to methotrexate treatment in rheumatoid arthritis

Abstract

Objective: Antigen-presenting dendritic cells (DCs) and monocytes play an essential role in rheumatoid arthritis (RA) pathogenesis, however, their tolerogenic potential remains unclear. Herein, the tolerogenic profiles of DCs are characterized in treatment-naïve RA patients to determine their role to inflammatory arthritis management.

Methods: Thirty-six treatment-naïve RA patients were enrolled, of which 62% were non-responders to methotrexate (MTX) monotherapy based on disease activity score (DAS) after 6-months of therapy. DC and monocyte subset frequencies, activation (CD40, CD86, CD209 expression), and tolerogenic profile (intracellular indoleamine-2,3-dioxygenase [IDO1] and cytotoxic T lymphocyte antigen 4 [CTLA-4] expression) were examined in the baseline peripheral blood by multicolor flow-cytometry. Soluble CTLA-4 (sCTLA-4) levels in plasma were measured.

Results: DC subsets were decreased in RA compared to healthy controls (HC), and the frequency of conventional DCs (cDC) inversely correlated with inflammatory markers and improvement in disease activity. CD141⁺ cDC1s were the major IDO1-expressing cells. IDO1⁺cDC1s were reduced in RA patients compared to HC. The baseline frequency of IDO1⁺cDC1s inversely correlated with improvement in disease activity. CTLA-4 expression in CD1c⁺ cDC2s and monocytes was lower in RA patients compared to HC. Moreover, MTX-responders had a significantly lower frequency of IDO1⁺cDC1 cells and higher level of sCTLA-4 in the plasma compared to MTX non-responders. There was a strong predictive association of low IDO1⁺cDC1 cells, low sCTLA-4 and non-response to MTX.

Conclusions: Our findings reveal altered DC and monocytes immunophenotypes that are associated with RA pathology and treatment response. The frequencies of tolerogenic IDO1⁺cDC1s and the low level of sCTLA-4 are strongly associated with MTX non-responsiveness and therapeutic outcome. These results suggest that investigation of the association IDO1⁺cDC1 and sCTLA-4 with response to treatment may be more generalizable to other autoimmune diseases.

Keywords: CTLA-4; IDO1; dendritic cells; methotrexate; rheumatoid arthritis; therapy response; tolerogenic.

Figure 1

Decreased frequency of dendritic cells in the blood of RA patients. (A), Frequency of monocyte subsets based on monocyte gate and (B), Frequency of CD141⁺ cDC1, CD1c⁺ cDC2, and CD123⁺ pDC subsets based on the mononuclear cell gate (MNC) in the blood of healthy controls (HC), treatment-naïve rheumatoid arthritis (RA) patients, MTX-responders (R), and non-responders (NR) at baseline. Symbols represent individuals. Data are summarized using median and interquartile range. *=P < 0.05; **=P < 0.01; ***=P < 0.001; ****=P < 0.0001 by Mann-Whitney test. Correlations of DC subsets with erythrocyte sedimentation rate (ESR) (C, E, G), and with disease activity measured by Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR) (D, F, H) at baseline. Associations between variables were analyzed by Spearman’s rank correlation test.

Figure 2

The CD209⁺ cDC2 subset is more abundant in RA patients. (A), Frequency of the CD209⁺ DC subsets and CD14⁺ monocytes in healthy controls (HC), treatment naïve RA patients, MTX-responder (R) and non-responder (NR) patients at baseline. (B), Geometric mean of surface CD209 expression on cDC2 cells. Data are summarized using median and interquartile range. **=P < 0.01 by Mann-Whitney test.

Figure 3

Frequency of tolerogenic IDO1⁺ cDC1 cells is decreased in RA and may be associated with MTX therapy response. (A), Intracellular indoleamine-2,3-deoxygenase (IDO1) expression in DC subsets. Data are summarized using median and interquartile range. *=P < 0.05; **=P < 0.01; ***=P < 0.001; ****=P < 0.0001 (B), Geometric mean of intracellular IDO1 expression on cDC1 cells. Data are summarized using median and interquartile range. (C), Receiver operating characteristic curve showing area under the curve (AUC) for the frequency of IDO1⁺ cDC1 subset applied to distinguish MTX non-responders from responders. (D), Intracellular IDO1 expression in DC subsets following 5h incubation with poly (I:C) vs. medium. Data are the individual matched values (connecting line) in 7–14 individuals. *=P < 0.05; **=P < 0.01; ***=P < 0.001; ****=P < 0.0001 by Wilcoxon matched-pairs signed rank test or Mann-Whitney test, based on normality.

Figure 4

The frequency of CTLA-4⁺ cDC2 cells and monocyte subsets is lower in RA. (A), Frequency of intracellular CTLA-4⁺ cells among DC subsets. (B), Frequency of intracellular CTLA-4⁺ cells among indicated monocyte subsets. Data are summarized using median and interquartile range. **=P < 0.01; ***=P < 0.001 by Mann-Whitney test.

Figure 5

The level of soluble CTLA-4 in the plasma may predict MTX response at baseline. (A), Soluble CTLA-4 (sCTLA-4) concentration was measured in the plasma of healthy controls (HC) and rheumatoid arthritis (RA) patients at baseline. Data are summarized as median and interquartile range. *=P < 0.05, **=P < 0.01 by Mann-Whitney test. (B), Correlation of the plasma sCTLA-4 concentration with the improvement of DAS28-ESR disease activity index after 6 months of methotrexate treatment. Associations between variables were analyzed by Spearman’s rank correlation test. (C), Receiver operating characteristic curve showing area under the curve (AUC) for the concentration of sCTLA-4 applied to distinguish MTX non-responders from responders.