Pathogenic Transdifferentiation of Th17 Cells Contribute to Perpetuation of Rheumatoid Arthritis during Anti-TNF Treatment

Abstract

T-helper cells producing interleukin (IL)-17A and IL-17F cytokines (Th17 cells) are considered the source of autoimmunity in rheumatoid arthritis (RA). In this study, we characterized specific pathogenic features of Th17 cells in RA. By using nano-string technology, we analyzed transcription of 419 genes in the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of 14 RA patients and 6 healthy controls and identified 109 genes discriminating Th17 cells of RA patients from the controls. Th17 cells of RA patients had an aggressive pathogenic profile and in addition to signature cytokines IL-17, IL-23 and IL-21, and transcriptional regulators RAR-related orphan receptor gamma of T cells (RORγt) and Janus kinase 2 (JAK2), they produced high levels of IL-23R, C-C chemokine ligand type 20 (CCL20), granulocyte-monocyte colony-stimulating factor (GM-CSF ) and transcription factor Tbet required for synovial homing. We showed that Th17 cells are enriched with Helios-producing Foxp3- and IL2RA-deficient cells, indicating altered regulatory profile. The follicular T-helper (Tfh) cells presented a functional profile of adaptor molecules, transcriptional regulator Bcl-6 and B-cell activating cytokines IL-21, IL-31 and leukemia inhibitory factor (LIF ). We observed that anti-tumor necrosis factor (TNF) treatment had a limited effect on the transcription signature of Th17 cells. Patients in remission retained the machinery of receptors (IL-23R and IL-1R1), proinflammatory cytokines (IL-17F, IL-23, IL-21 and TNF ) and adaptor molecules (C-X-C chemokine receptor 5 [CXCR5] and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]), essential for efficient transdifferentiation and accumulation of Th17 cells. This study convincingly shows that the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of RA patients harbor pathogenic subsets of Th17 and Tfh cells, which may transdifferentiate from Tregs and contribute to perpetuation of the disease.

Figure 1

(A) Clinical characteristics of patients and controls included in the study. (B) Network analysis of differentially expressed genes in PMA-ionomycin–stimulated CCR6⁺CXCR3⁻ CD4⁺ cells from RA patients and healthy controls using the Ingenuity IPA 5.5.1 program. Genes included the following: 1: TLR10, TLR5, CXCL13, IL2, TLR6, TLR7, FASLG; 2: IL-2, CXCR5; 3: IL-21, IL-21R, ICOS, Stat3, CXCR5, IL-6, Bcl-6; 4: TGFBR1, IL-23R, TGFB1, IL-6, TBX21, Rorc, IL-17F, TNF, IL-17A.

Figure 2

(A) Gene expression profiles of PMA-ionomycin–stimulated CCR6⁺CXCR3⁻ CD4⁺ cells from RA patients and healthy controls. Shown are the differential expression levels for genes (rows) in patients or controls (columns). Normalized raw gene expression values from microarray experiments were used to identify differentially expressed genes involved in Th17 differentiation and function. DAS28, disease activity score based on 28 defined joints, is indicated for each RA patient. (B) Cytokines produced by PMA-ionomycin–stimulated CCR6⁺CXCR3⁻ CD4⁺ cells from RA patients (n = 7) and healthy controls (n = 6). Equal amounts of cell culture medium were pooled and subjected to cytokine array analysis. Data shown in the bar graph are the results of densitometric analysis.

Figure 3

(A) Gene expression profiles of PMA-ionomycin–stimulated CCR6⁺CXCR3⁻ CD4⁺ cells from RA patients and healthy controls. The differential expression levels for genes (rows) in patients or controls (columns) are shown. Normalized raw gene expression values from microarray experiments were used to identify differentially expressed genes involved in Th17 differentiation and function. DAS28, disease activity score based on 28 defined joints, is indicated for each RA patient. (B) Gene expression ratios for Th17- and Treg-associated genes.

Figure 4

(A) Representative FACS plots illustrating CCR6/CXCR3-expressing CD4 cells and the CXCR5⁺ population herein. CCR6⁺CXCR3⁻ CD4⁺ cells had a significantly larger subset of CXCR5⁺ cells compared with CCR6⁻CXCR3⁻ and CCR6⁻CXCR3⁺ CD4⁺ cells (n = 4). (B) Gene expression profiles of PMA-ionomycin–stimulated CCR6⁺CXCR3⁻ CD4⁺ cells from RA patients and healthy controls. The differential expression levels for genes (rows) in patients or controls (columns) are shown. Normalized raw gene expression values from microarray experiments were used to identify differentially expressed genes involved in Tfh differentiation and function. DAS28, disease activity score with 28 joint count, is indicated for RA patients. (C) The Bcl6/Blimp1 expression ratio in RA patients and healthy controls. (D) Flow cytometric analysis of CD27⁺CD45RA⁻ memory CD4⁺ T cells’ expression of CXCR5 in RA patients (n = 4) and healthy controls (n = 4). (E) Flow cytometric analysis of CD27⁺CD45RA⁻ memory CD4⁺ T cells’ expression of Bcl-6 in RA patients (n = 14). The size of the Bcl-6⁺CD27⁺ population (within CD45RA⁻CD4⁺) tended to correlate to DAS28 (r = 0.47, p = 0.09) in patients with autoantibodies (RF and/or anti-CCP), but showed no association with the presence of autoantibodies (ab⁺ = patients with RF and/or anti-CCP antibodies).

Figure 5

Fold-change values of gene expression of PMA-ionomycin–stimulated CCR6⁺CXCR3⁻CD4⁺ cells from RA patients in remission (blue, n = 7) and with active disease (red, n = 7), respectively, versus healthy controls (n = 6). (A) Cytokines and transcription factors characteristic for Th17; (B) receptors essential for Th17 differentiation, and IL-2 controlling proliferation; (C) cytokines, transcription factors, and receptors characteristic for Tfh cells. Filled symbols indicate significant difference to controls.