CD4+CXCR5+PD-1+ T Follicular Helper Cells Play a Pivotal Role in the Development of Rheumatoid Arthritis

Abstract

BACKGROUND T follicular helper (Tfh) cells are a subgroup of activated CD4+ T cells in the germinal centers of secondary lymphoid organs, they play critical roles in the development of many chronic autoimmune inflammatory diseases. The aim of this study was to investigate whether circulating Tfh cells contribute to the development of rheumatoid arthritis (RA). MATERIAL AND METHODS Thirty patients fulfilled the diagnosis criteria that was established by the American College of Rheumatology and 30 healthy controls were recruited. The frequency of Tfh cells in patients and collagen-induced arthritis (CIA) in DBA/1J mice were analyzed by flow cytometry. The serum IL-21 level was examined by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of Blimp-1 and Bcl-6 were detected by qRT-PCR. RESULTS RA patients had more CD4⁺PD-1⁺CXCR5⁺ Tfh cells in peripheral blood compared with healthy controls, and CIA in DBA/1J mice showed similar results. Higher mRNA expression of Bcl-6 and lower Blimp-1 mRNA expression were observed in patients with RA compared to healthy controls, and the expression level of IL-21 was higher in RA patients, which was also seen in CIA mice. Furthermore, the spleen CD4⁺ICOS⁺CXCR5⁺ Tfh cells in CIA mice show significantly higher frequency than that in the control mice. The percentage of CD4⁺PD-1⁺CXCR5⁺ Tfh cells was correlated positively with the values of erythrocyte sedimentation rate (ESR) (r=0.968, P<0.001), rheumatoid factor (RF) (r=0.962, P<0.001), C-reactive protein (CRP) (r=0.953, P<0.001), and anti-cyclic citrullinated peptide antibodies (ACPA) (r=0.966, P<0.001), and the level of serum interleukin (IL)-21 in RA patients showed positive correlation with ESR (r=0.982, P<0.001), RF (r=0.959, P<0.001), CRP (r=0.951, P<0.001), and ACPA (r=0.971, P<0.001) as well. CONCLUSIONS The activated Tfh cells in the peripheral blood may be responsible for the development of RA.

Figure 1

Frequency of circulating follicular helper T (Tfh) cells was increased and associated positively with the levels of autoantibodies and inflammation markers in new-onset rheumatoid arthritis (RA) patients. Peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls were stained with labelled antibodies as previously described, and analyzed by flow cytometry. (A) Percentage of CD4+ T lymphocytes in RA patients and healthy controls. (B) Percentage of CD4+ CXCR5+ PD-1+ T cells in RA patients and healthy controls (* P<0.01). (C) The percentage of Tfh cells was correlated positively with the level of serum RF (rheumatoid factor) in new-onset RA (r=0.962, P<0.0001). (D) The percentage of Tfh cells was correlated positively with the level of serum ACPA (anti-cyclic citrullinated peptide antibodies) in new-onset RA (r=0.966, P<0.0001). (E) The percentage of Tfh cells was correlated positively with the value of ESR (erythrocyte sedimentation rate) in new-onset RA (r=0.968, P<0.0001). (F) The percentage of Tfh cells was correlated positively with the level of serum CRP (C-reactive protein) in new-onset RA (r=0.953, P<0.0001).

Figure 2

Elevated interleukin (IL)-21 concentration in new-onset rheumatoid arthritis (RA) patients. (A) IL-21 expression by enzyme-linked immunosorbent assay (ELISA), * P<0.05 compared with healthy controls. (B) The level of IL-21 was correlated positively with the level of serum RF (rheumatoid factor) in new-onset RA (r=0.971, P<0.0001). (C) The level of IL-21 was correlated positively with the level of serum ACPA (anti-cyclic citrullinated peptide antibodies) in new-onset RA (r=0.959, P<0.0001). (D) The level of IL-21 was correlated positively with the value of ESR (erythrocyte sedimentation rate) in new-onset RA (r=0.982, P<0.0001). (E) The level of IL-21 was correlated positively with the level of serum CRP (C-reactive protein) in new-onset RA (r=0.951, P<0.0001).

Figure 3

Assessment of collagen-induced arthritis in DBA/1J mice. (A) Paw swelling (mean ± standard deviation (SD), n=10 per group) were significant increase in collagen-induced arthritis (CIA) model. (B) Arthritic scores (mean ±SD, n=10 per group) were significant increase in CIA model. (C) Hematoxylin and eosin (H&E) staining of paw sections in normal mice (10×20, arrow). (D) H&E staining of paw sections in CIA model mice (10×20, arrow). Histological joint destruction in paw sections revealed remarkable synovial hyperplasia, inflammatory cell infiltration into the joint capacity in CIA mice compared with the normal mice. * P<0.05, compared with the normal mice.

Figure 4

Increased frequency of the circulating Tfh cells and high serum concentration of interleukin (IL)-21 in collagen-induced arthritis (CIA) mice. (A) Percentage of CD4+ CXCR5+ ICOS+ T cells in CIA model mice. (B) Serum concentration of IL-21 in CIA mice. * P<0.05, compared with the normal mice.

Figure 5

Decreased Blimp-1 mRNA expression and elevated Bcl-6 mRNA expression lead to increased Tfh cells in new-onset rheumatoid arthritis patients. (A) Bcl-6 mRNA expression by qRT-PCR. (B) Blimp-1 mRNA expression by qRT-PCR. * P<0.05 compared with healthy controls.