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CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

Paulissen SM, van Hamburg JP, Davelaar N, Vroman H, Hazes JM, de Jong PH, Lubberts E - Arthritis Res. Ther. (2015)

Bottom Line: Similar proportions of CCR4(+) and CCR10(+) Th cells were found.In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions.This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

View Article: PubMed Central - PubMed

Affiliation: Departments of Rheumatology and Immunology, Erasmus MC, University Medical Center, Rotterdam, P.O. Box 2040, 3000, CA, Rotterdam, The Netherlands. s.paulissen@erasmusmc.nl.

ABSTRACT

Introduction: Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.

Methods: We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.

Results: ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA(-) patients (R(2) = -0.35; p < 0.01) but not in ACPA(+) (R(2) < 0.01; p = 0.94) patients.

Conclusions: These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

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Memory CCR6+CD4+ T cell subpopulations are increased in ACPA+ patients compared to matched ACPA− patients. a Gating strategy for the identification of peripheral blood Th17, Th17.1, Th22 and CCR4/CXCR3 DP cell subpopulations. CCR6+ cells were gated on CD4+CD45RO+CD25− T cells. b Real-time PCR expression analysis for IL-17, IFNγ, RORC and TBX21 in sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells obtained from patients with RA (8–10 patients per population). Prior to RNA isolation cells were stimulated with antiCD3/CD28 and cultured for 3 days. c-d Proportions of the indicated CD4+ T cell subpopulations within the total memory CD4+ T cell population (c) and memory CCR6+CD4+ T cell population (d) of 27 ACPA+ and 27 ACPA− patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).
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Fig2: Memory CCR6+CD4+ T cell subpopulations are increased in ACPA+ patients compared to matched ACPA− patients. a Gating strategy for the identification of peripheral blood Th17, Th17.1, Th22 and CCR4/CXCR3 DP cell subpopulations. CCR6+ cells were gated on CD4+CD45RO+CD25− T cells. b Real-time PCR expression analysis for IL-17, IFNγ, RORC and TBX21 in sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells obtained from patients with RA (8–10 patients per population). Prior to RNA isolation cells were stimulated with antiCD3/CD28 and cultured for 3 days. c-d Proportions of the indicated CD4+ T cell subpopulations within the total memory CD4+ T cell population (c) and memory CCR6+CD4+ T cell population (d) of 27 ACPA+ and 27 ACPA− patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).

Mentions: T cells co-express different chemokine receptors on their surface. Specific combinations of CCR4, CCR6, CCR10 and CXCR3 are expressed by human Th cell populations. We applied a chemokine receptor gating strategy to identify memory CD4+ T cells with a Th1, Th2, Th17, Th22 or a Th17.1 profile. Within the CD4+CD45RO+CD25− T cell population, cells positive for CCR6 expression were gated. Within this CCR6+ population, Th17 cells were gated as CXCR3−CCR4+CCR10− and Th22 cells as CXCR3−CCR4+CCR10+ [33–35]. Th17.1 cells were gated as CXCR3+CCR4−. Using this gating strategy (Fig. 2A), an unclassified subpopulation was identified, that was double-positive (DP) for the expression of CCR4 and CXCR3. Recently we validated the gating strategy for Th17 and Th22 cells of patients with RA [35]. To validate the gating strategy for the other CCR6+ subpopulations we sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells from patients with RA and analyzed their Th17 and Th1 profile by the transcription levels of IL-17A, IFN-γ, RORC and TBX21. These analyses confirmed the expression profile of Th1, Th17 and Th17.1 as reported previously [36–38]. The CCR4/CXCR3 CCR6+ DP were IL-17A low and RORC+ with intermediate IFN-γ and TBX21 levels (Fig. 2b). This gating strategy was applied to PBMCs of ACPA+ and ACPA− early RA patients. Proportions of the CCR6+ Th cell subpopulations Th22, Th17.1 and CCR4/CXCR3 DP Th cells were significantly higher in ACPA+ than in ACPA− patients. No statistical significant (p = 0.10) difference was reached for the distribution of Th17 cells between ACPA+ and ACPA− patients (Fig. 2c).Fig. 2


CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

Paulissen SM, van Hamburg JP, Davelaar N, Vroman H, Hazes JM, de Jong PH, Lubberts E - Arthritis Res. Ther. (2015)

Memory CCR6+CD4+ T cell subpopulations are increased in ACPA+ patients compared to matched ACPA− patients. a Gating strategy for the identification of peripheral blood Th17, Th17.1, Th22 and CCR4/CXCR3 DP cell subpopulations. CCR6+ cells were gated on CD4+CD45RO+CD25− T cells. b Real-time PCR expression analysis for IL-17, IFNγ, RORC and TBX21 in sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells obtained from patients with RA (8–10 patients per population). Prior to RNA isolation cells were stimulated with antiCD3/CD28 and cultured for 3 days. c-d Proportions of the indicated CD4+ T cell subpopulations within the total memory CD4+ T cell population (c) and memory CCR6+CD4+ T cell population (d) of 27 ACPA+ and 27 ACPA− patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663738&req=5

Fig2: Memory CCR6+CD4+ T cell subpopulations are increased in ACPA+ patients compared to matched ACPA− patients. a Gating strategy for the identification of peripheral blood Th17, Th17.1, Th22 and CCR4/CXCR3 DP cell subpopulations. CCR6+ cells were gated on CD4+CD45RO+CD25− T cells. b Real-time PCR expression analysis for IL-17, IFNγ, RORC and TBX21 in sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells obtained from patients with RA (8–10 patients per population). Prior to RNA isolation cells were stimulated with antiCD3/CD28 and cultured for 3 days. c-d Proportions of the indicated CD4+ T cell subpopulations within the total memory CD4+ T cell population (c) and memory CCR6+CD4+ T cell population (d) of 27 ACPA+ and 27 ACPA− patients with RA. For statistical analysis Wilcoxon matched-pairs signed-ranks test was performed (* = p < 0.05).
Mentions: T cells co-express different chemokine receptors on their surface. Specific combinations of CCR4, CCR6, CCR10 and CXCR3 are expressed by human Th cell populations. We applied a chemokine receptor gating strategy to identify memory CD4+ T cells with a Th1, Th2, Th17, Th22 or a Th17.1 profile. Within the CD4+CD45RO+CD25− T cell population, cells positive for CCR6 expression were gated. Within this CCR6+ population, Th17 cells were gated as CXCR3−CCR4+CCR10− and Th22 cells as CXCR3−CCR4+CCR10+ [33–35]. Th17.1 cells were gated as CXCR3+CCR4−. Using this gating strategy (Fig. 2A), an unclassified subpopulation was identified, that was double-positive (DP) for the expression of CCR4 and CXCR3. Recently we validated the gating strategy for Th17 and Th22 cells of patients with RA [35]. To validate the gating strategy for the other CCR6+ subpopulations we sorted Th1, Th17, Th17.1 and CCR4/CXCR3 DP CCR6+ Th cells from patients with RA and analyzed their Th17 and Th1 profile by the transcription levels of IL-17A, IFN-γ, RORC and TBX21. These analyses confirmed the expression profile of Th1, Th17 and Th17.1 as reported previously [36–38]. The CCR4/CXCR3 CCR6+ DP were IL-17A low and RORC+ with intermediate IFN-γ and TBX21 levels (Fig. 2b). This gating strategy was applied to PBMCs of ACPA+ and ACPA− early RA patients. Proportions of the CCR6+ Th cell subpopulations Th22, Th17.1 and CCR4/CXCR3 DP Th cells were significantly higher in ACPA+ than in ACPA− patients. No statistical significant (p = 0.10) difference was reached for the distribution of Th17 cells between ACPA+ and ACPA− patients (Fig. 2c).Fig. 2

Bottom Line: Similar proportions of CCR4(+) and CCR10(+) Th cells were found.In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions.This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

View Article: PubMed Central - PubMed

Affiliation: Departments of Rheumatology and Immunology, Erasmus MC, University Medical Center, Rotterdam, P.O. Box 2040, 3000, CA, Rotterdam, The Netherlands. s.paulissen@erasmusmc.nl.

ABSTRACT

Introduction: Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.

Methods: We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.

Results: ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA(-) patients (R(2) = -0.35; p < 0.01) but not in ACPA(+) (R(2) < 0.01; p = 0.94) patients.

Conclusions: These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

Show MeSH
Related in: MedlinePlus