Imbalance of Th17, Treg, and helper innate lymphoid cell in the peripheral blood of patients with rheumatoid arthritis

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease involving a variety of immune cells, including adaptive T and B cells and innate lymphoid cells (ILCs). Understanding the pathogenic role of these immune cells in RA provides new insights into the intervention and treatment of RA.

Methods: A total of 86 patients with RA (RA group) and 50 healthy controls (HC) were included in the study. The immune cells of CD4⁺, CD19⁺ B, NK, Th17, Treg, ILCs, and their subsets (i.e., ILC1s, ILC2s, and ILC3s) were characterized in peripheral blood mononuclear cells by flow cytometry. Cytokines (i.e., IFN-γ, IL-4, IL-10, IL-17A, IL-22, and IL-33) in sera were detected using ELISA. The above immune cells and cytokines were analyzed in patients with different disease activity status and positive ( +) or negative ( -) rheumatoid factor (RF)/anti-citrullinated protein antibodies (ACPA).

Results: Patients with RA had higher percentages of CD4⁺ T, CD19⁺ B, Th17, ILC2s, and ILC3s and lower percentages of Treg and ILC1s than HC. Patients with RA had elevated levels of IFN-γ, IL-4, IL-17A, and IL-22 and decreased level of IL-10. Compared with HC, patients with high disease activity had higher percentages of Th17, ILC2s, and ILC3s; lower percentages of ILC1s; and lower level of IL-10. The percentage of Treg cells in remission, low, moderate, and high disease activities decreased, whereas the level of IL-17A increased compared with HC. Furthermore, RF⁺ or ACPA⁺ patients exhibited elevated percentages of CD19⁺ B, ILC2s, and ILC3s and had decreased percentage of ILC1s and Treg cells than HC. The percentage of Th17 cells increased in RF^-/ACPA^- and RF⁺/ACPA⁺ patients. However, the above immune cells between RF or ACPA positive and negative patients were not significantly different.

Conclusion: Th17, Treg, and ILC subset dysregulations are present in patients with RA but may not be associated with conventionally defined seropositive RF and ACPA. Key Points • Th17, Treg, and ILC subset dysregulations are present in patients with RA but may reflect inflammation rather than specific diseases and stages. • No difference for the distribution of Th17, Treg, and ILC subsets between RF⁺ and RF^- patients and between ACPA⁺ and ACPA^- patients. The screening spectrum of RF and ACPA serology should be expanded to elucidate the role of immune cells in RA pathogenesis.

Keywords: Helper innate lymphoid cell; Rheumatoid arthritis; Th17; Treg.

Fig. 1

Flow cytometry analysis of circulating lymphocyte subsets in RA patients and HC. a CD4⁺ T cells, b CD19⁺ B cells, c CD4⁺IL-17⁺ Th17 cells, d CD4⁺CD25⁺Foxp3⁺ Treg cells, e CD3⁻CD56⁺ NK cells, f CD3⁻CD19⁻CD56⁻CD127⁺ ILCs, and g Lin⁻CD294⁻CD117⁻ ILC1s, Lin⁻CD294⁺ ILC2s, and Lin⁻CD294⁻CD117⁺ ILC3s. Data are representative of the experiments. h The percentage of CD4⁺ T cells, i CD19⁺ B cells, j Th17 cells, k Treg cells, l NK cells, m total ILCs, n ILC1s, o ILC2s, and p ILC3s in each group. Results are presented as mean ± SEM. *P < 0.05,**P < 0.01, ***P < 0.001

Fig. 2

Cytokine analysis in sera by ELISA in RA patients and HC. a IFN-γ, b IL-4, c IL-10, d IL-17A, e IL-22, and f IL-33. Results are presented as mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001

Fig. 3

Flow cytometry analysis of circulating lymphocyte subsets in HC and RA patients with different disease activity status. a CD4⁺IL-17⁺ Th17 cells, b CD4⁺CD25⁺Foxp3⁺ Treg cells, c CD3⁻CD19⁻CD56⁻CD127⁺ ILCs, and d Lin⁻CD294⁻CD117⁻ ILC1s, Lin⁻CD294⁺ ILC2s, and Lin⁻CD294⁻CD117⁺ ILC3s. Data are representative of the experiments. e The percentage of Th17 cells, f Treg cells, g total ILCs, h ILC1s, i ILC2s, and j ILC3s in each group. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001

Fig. 4

Cytokine analysis in sera by ELISA in HC and RA patients with different disease activity status. a IFN-γ, b IL-4, c IL-10, d IL-17A, e IL-22, and f IL-33. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01

Fig. 5

Flow cytometry analysis of circulating lymphocyte subsets in HC, RF⁻, and RF⁺ RA patients. a CD4⁺ T cells, b CD19⁺ B cells, c CD4⁺IL-17⁺ Th17 cells, d CD4⁺CD25⁺Foxp3⁺ Treg cells, e CD3⁻CD56⁺ NK cells, f CD3⁻CD19⁻CD56⁻CD127⁺ ILCs, and g Lin⁻CD294⁻CD117⁻ ILC1s, Lin⁻CD294⁺ ILC2s, and Lin⁻CD294⁻CD117⁺ ILC3s. Data are representative of the experiments. h The percentage of CD4⁺ T cells, i CD19⁺ B cells, j Th17 cells, k Treg cells, l NK cells, m total ILCs, n ILC1s, o ILC2s, and p ILC3s in each group. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Flow cytometry analysis of circulating lymphocyte subsets in HC, ACPA⁻, and ACPA⁺ RA patients. a CD4⁺ T cells, b CD19⁺ B cells, c CD4⁺IL-17⁺ Th17 cells, d CD4⁺CD25⁺Foxp3⁺ Treg cells, e CD3⁻CD56⁺ NK cells, f CD3⁻CD19⁻CD56⁻CD127⁺ ILCs, and g Lin⁻CD294⁻CD117⁻ ILC1s, Lin⁻CD294⁺ ILC2s, and Lin⁻CD294⁻CD117⁺ ILC3s. Data are representative of the experiments. h The percentage of CD4⁺ T cells, i CD19⁺ B cells, j Th17 cells, k Treg cells, l NK cells, m total ILCs, n ILC1s, o ILC2s, and p ILC3s in each group. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001