Levels of Pathogenic Th17 and Th22 Cells in Patients with Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune condition characterized, among others, by tissue damage and activation/differentiation of proinflammatory lymphocytes. Accordingly, several studies have concluded that type 17 T helper (Th17) cells seem to have an important role in the pathogenesis of this condition. However, the strategy for the identification and analysis of proinflammatory Th17 cells in those studies has not been consistent and has usually been different from what was originally described. Therefore, we decided to evaluate the levels of Th17 cells in patients with RA employing an extended immune phenotype by using an eight-color multiparametric flow cytometry analysis. For this purpose, blood samples were obtained from 30 patients with RA and 16 healthy subjects, and the levels of Th17 and type 22 helper (Th22) lymphocytes were analyzed as well as the in vitro differentiation of peripheral blood mononuclear cells into Th17 lymphocytes induced by interleukin-23 (IL-23) and IL-1β. We found significant enhanced levels of total Th17 lymphocytes (defined as CD4⁺IL-17⁺) as well as enhanced numbers of their pathogenic (defined as CD4⁺CXCR3⁺IL-17⁺IL-22⁺CD243⁺CD161⁺IFN-γ ⁺IL-10^-) and nonpathogenic (CD4⁺CXCR3⁺IL-17⁺IL-22^-CD243^-CD161^-IFN-γ ^-IL-10⁺) cell subsets in patients with RA. Likewise, the number of Th22 (CD4⁺CXCR3^+/-IL-17^-IL-22⁺) was also increased in these patients compared to healthy controls. However, the in vitro induction/differentiation of pathogenic Th17 cells showed similar results in controls and patients with RA. Likewise, no significant associations were detected in patients with RA between the levels of Th17 or Th22 cells and clinical or laboratory parameters. Our data indicate that patients with RA show enhanced blood levels of the different subsets of Th17 cells and Th22 lymphocytes tested in this study and suggest that these levels are not apparently associated with clinical or laboratory parameters.

Figure 1

Flow cytometry strategy for the analysis of Th17 and Th22 cells. (a) PBMC were labeled with MoAbs against CD4, CXCR3, IL-17, IL-22, CD243 (MDR-1), CD161, IFN-γ, and IL-10 and analyzed by multiparametric flow cytometry, as indicated. This analysis allowed to detect the cell subsets of interest, pathogenic Th17 cells, nonpathogenic Th17 cells, and Th22 lymphocytes. (b) Flow cytometry images of a representative analysis of a blood sample from a healthy donor are shown. The type of fluorochrome coupled to each antibody is indicated, and the negative stain controls were selected according to the fluorescence minus one (FMO) strategy.

Figure 2

Levels of Th17 and Th22 lymphocytes in patients with RA. Blood samples from 30 patients with rheumatoid arthritis (RA) and 16 healthy controls (Control) were obtained and PBMC were isolated, stained, and analyzed by multiparametric flow cytometry, as stated in Materials and Methods and shown in Figure 1. (a) Percentages of CD4⁺IL-17⁺ cells, referred to total lymphocytes. (b) Percentages of pathogenic Th17 (pTh17) cells referred to total lymphocytes. (c) Absolute numbers of pTh17 cells/ml. (d) Percentage of nonpathogenic Th17 (npTh17) cells, referred to total lymphocytes. (e) Percent of Th22 cells (CD4⁺CXCR3^+/-IL-17⁻IL-22⁺), referred to total lymphocytes. (f) Absolute numbers of Th22 lymphocytes/ml. Horizontal lines correspond to the median and Q₁-Q₃ interquartile range. ^∗p < 0.05, ^∗∗∗p < 0.005 (Mann–Whitney U test).

Figure 3

Association analysis between the levels of Th17 lymphocytes or Th22 cells and clinical parameters in RA patients. The correlation analysis between the percentages of pathogenic Th17 (pTh17) and Th22 cells (a), the percentage of pTh17 cells and time of evolution of disease (b), the percentage of pTh17 cells and the Disease Activity Score 28 (DAS28) (c), the percentage of Th22 cells and time of evolution of disease (f), or DAS28 (g) is shown. r and p values (Spearman sum rank test) are shown. The comparisons between the levels of pTh17 or Th22 cells in patients with low and high disease activity ((d) and (h), respectively), or with the presence or not of rheumatoid factor ((e) and (i), respectively), are also shown. Horizontal lines (in (d), (e), (h), and (i)) correspond to the median and Q₁-Q₃ interquartile range. RF: rheumatoid factor; ns: nonsignificant.

Figure 4

In vitro induction of pathogenic Th17 cells. PBMC from healthy controls and patients with rheumatoid arthritis were incubated in the presence or not of IL-23 or IL-1β plus IL-23 and then the percent of pathogenic Th17 (pTh17) cells, referred to total lymphocytes, and were analyzed by flow cytometry, as described in Material and Methods. Data correspond to the arithmetic mean and SD of ten separate experiments. ^∗p < 0.05. ns: nonsignificant (Kruskal-Wallis test with Dunn's posttest).