JEADV
DOI: 10.1111/jdv.13027
ORIGINAL ARTICLE
Increased circulating follicular helper T cells and activated B cells correlate with disease severity in patients with psoriasis J. Niu, Z. Song, X. Yang, Z. Zhai, H. Zhong,* F. Hao* Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing, China *Correspondence: H. Zhong, E-mail: [email protected], and F. Hao, E-mail: [email protected]
Abstract Background Follicular Helper T (TFH) Cells are a population of recently discovered CD4+ T cells involved in autoimmune diseases. However, the contribution of TFH cells in patients with psoriasis remains unknown. Objective We aimed to investigate the levels of TFH cells, B cells and their clinical relevance in patients with psoriasis. Methods Using multi-colour flow cytometry, we detected different subsets of TFH cells and B cells in the peripheral blood of 27 patients with psoriasis and 13 healthy donors. Serum IL-21 levels were measured by ELISA. The relationship between the levels of TFH cells, IL-21, B cells and disease severity were analysed. Results Compared with healthy donors, higher levels of circulating CD3+CD4+CXCR5+ cells, CD3+CD4+CXCR5+ICOS+, CD3+CD4+CXCR5+PD-1+, CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells and CD19+IgD+CD27
naive B,
CD19+CD86+ activated B, but lower levels of CD19+IgD+CD27+ preswitch and CD19+IgD-CD27+ postswitch memory B cells, were observed in patients with psoriasis. In addition, serum IL-21 levels in patients with psoriasis were significantly higher than those in healthy donors, and showed to be positively correlated with the levels of different subsets of TFH cells, and the level of CD19+CD86+ B cells was also correlated with TFH cells and IL-21 levels. Furthermore, a significant correlation was found between the levels of CD3+CD4+CXCR5+ICOS+ TFH cells, CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells, CD19+CD86+ B cells and IL-21 with Psoriasis Area and Severity Index scores. Conclusion The levels of TFH cells and activated B cells were increased in the peripheral blood of patients with psoriasis, and positively correlated with disease severity. These results suggest that TFH cells and activated B cells may play a role in the pathogenesis of psoriasis. Received: 6 August 2014; Accepted: 13 January 2015
Conflicts of interest The authors declare that they have no conflict of interest.
Funding sources This work was supported by grants from the National Natural Science Foundation of China (NSFC, No.81271754) and (NSFC, No.81101200)
Introduction Psoriasis is a chronic inflammatory skin disease and affects about 2% of the population worldwide.1 Generally, patients with psoriasis develop erythematous, inflamed and scaly plaques. Although many agents, including small molecules, are widely used for treating psoriasis, the resolution of psoriasis remains difficult.2 It has been long recognized that CD4+ T cells orchestrate the adaptive immune response in psoriasis, especially the Th1-cell-mediated inflammatory response. However, Th17 cells, a novel subset of CD4+ T cells expressing IL-17A, but not IFN-c, are found to accumulate in psoriatic skin,3 suggesting a critical role of Th17 cells in human psoriasis. Currently, biological drugs
JEADV 2015, 29, 1791–1796
targeting IL-17A are undergoing clinical trials for the treatment of psoriasis.4 Follicular Helper T (TFH) Cells have been recently identified as a separate CD4+ T-cell subset with distinct developmental programming and effector functions. TFH cells are characterized by the expression of CXCR5, ICOS, PD-1 and the transcription factor Bcl-6 and the secretion of IL-21.5 CXCR5 is not only the most widely utilized surface marker for the identification TFH cells but it is also critical for TFH-cell function.6 ICOS is required for the optimal differentiation of TFH cells and PD-1 appears to be important for regulating TFH-cell function.7,8 IL-21, which is secreted by
© 2015 European Academy of Dermatology and Venereology
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Figure 1 The different subsets of circulating TFH cells were analysed by flow cytometry in patients with psoriasis and healthy donors. Human PBMCs from patients with psoriasis and healthy donors were stained with anti-CD3, anti-CD4, anti-CXCR5, anti-ICOS and antiPD-1 antibodies or isotype controls. The cells were gated on lymphocytes (top left) and then on CD3+CD4+ T cells (top right) for the analysis of the proportion of CD3+CD4+CXCR5+ cells. Subsequently, the levels of CD3+CD4+CXCR5+ICOS+ cells (ICOS+ TFH cells), CD3+CD4+CXCR5+PD-1+ cells (PD-1+ TFH cells) and CD3+CD4+CXCR5+ICOS+PD-1+ cells (ICOS+PD-1+ TFH cells) in the CD3+CD4+CXCR5+ population were analysed. Data were expressed as the mean SEM. Each plot represents a single donor.
TFH cells, can also regulate TFH-cell development and function.9 TFH cells have been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis.10,11 In these studies, the levels of TFH cells are increased in inflammatory skin lesions and the peripheral blood of patients, displaying a positive correlation with disease severity. However, the prevalence of
JEADV 2015, 29, 1791–1796
TFH cells in human psoriasis remains poorly understood. In addition, TFH cells can regulate the activation and function differentiation of B cells.12 Activated B cells up-regulate CD86 expression, and the expression of IgD and CD27 distinguish the distinct subsets of B cells. However, the activation and maturation states of B cells in human psoriasis remain unknown.
© 2015 European Academy of Dermatology and Venereology
Follicular helper T cells and B cells in psoriasis
In this study, we examined the levels of TFH cells, IL-21 and B cells in the peripheral blood of patients with psoriasis, evaluated their relevance with the severity of disease, and attempted to elucidate the contribution of TFH cells, as well as B cells, in patients with psoriasis.
Materials and methods
1793
bated with surface antibodies for 30 min at 4°C, and then fixed for 20 min with 4% paraformaldehyde. Next, PBMCs were washed twice with PBS containing 1% FCS and then cells were collected by flow cytometry with a FACSCantoII (BD Biosciences, San Diego, CA, USA). Isotype-matched antibodies were used to enable correct compensation and confirm antibody specificity. Data were analysed with FlowJo software (Stanford University, San Francisco, CA, USA).
Subjects and samples
A total of 27 patients with psoriasis (male/female = 15/12; average age 43.6 2.9 years) and disease duration 9.3 1.4 years were taken from the Southwest Hospital of Third Military Medical University. The patients had no other autoimmune or systemic disease and not received any anti-psoriatic treatment for at least 1 month before enrolment in the study. Thirteen normal healthy donors (male/female = 7/6; average age 42.4 3.3 years) without any autoimmune or systemic disease were enrolled as the controls. The study was approved by the Ethics Committee of Southwest Hospital, Third Military Medical University. Written informed consent was obtained from each subject. With the approval of the Ethics Committee of the Third Military Medical University, fresh peripheral blood (15 mL) was collected from each study subject in heparinized blood collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. Serum samples were collected and stored at 80°C until further use. The disease activity of patients with psoriasis was measured using Psoriasis Area and Severity Index (PASI) scores as described previously.13 Antibodies and flow cytometry analysis
The following labelled antibodies were used to stain single-cell suspensions: CD3-APC-H7, CD4-PE, CXCR5-PercP-Cy5.5, ICOS-FITC, PD-1-APC, CD19-APC, CD86-PE, IgD-FITC and CD27-PE-Cy7 (Biolegend, San Diego, CA, USA). After PBMCs were obtained from each study subject, PBMCs were washed twice with PBS containing 1% fetal calf serum (FCS) and incu(a)
Enzyme-linked immunosorbent assay (ELISA)
Serum from patients with psoriasis and healthy donors were collected and the level of IL-21 was determined using an ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Statistical analysis
All results were summarized as the mean standard error of the mean (SEM). Statistical analyses were performed with the Prism 5.0 Software (GraphPad software Inc., San Diego, CA, USA). Differences between groups were determined by Mann–Whitney U-test. Correlation analysis between two groups of data was measured by Spearman’s correlation test. P < 0.05 was considered statistically significant.
Results Circulating TFH cells are increased in patients with psoriasis
To study whether TFH cells are involved in the pathogenesis of psoriasis, we first determined the levels of TFH cells in the peripheral blood of healthy donors and patients with psoriasis using multi-colour flow cytometry (Fig. 1). We detected a substantial amount of CD3+CD4+CXCR5+ cells that was significantly increased in patients with psoriasis when compared with healthy donors (16.06% 1.13%, vs. 9.62% 0.87%, P = 0.001). Further analysis revealed that the levels of CD3+CD4+CXCR5+ICOS+ TFH cells (ICOS+ TFH cells), CD3+CD4+CXCR5+PD-1+ TFH cells (PD-1+ TFH cells) and
(b)
Figure 2 Increased IL-21 levels correlated with the levels of circulating TFH cells in patients with psoriasis. (a) The concentrations of serum IL-21 in 27 patients with psoriasis and 13 healthy donors were detected by ELSIA. (b) The correlation between the level of IL-21 with the levels of ICOS+ TFH cells, PD-1+ TFH cells and ICOS+PD-1+ TFH cells was analysed. Data were expressed as the mean SEM. Each plot represents a single donor.
JEADV 2015, 29, 1791–1796
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CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells (ICOS+PD-1+ TFH cells) in the CD3+CD4+CXCR5+ population were also significantly increased in patients with psoriasis when compared with healthy donors. These results suggested that increased levels of circulating TFH cells were present in patients with psoriasis.
Niu et al.
Increased serum IL-21 levels correlated with the levels of TFH cells in patients with psoriasis
Because IL-21 is produced by TFH cells and regulates the B-cell humoural response, we measured the levels of IL-21 in healthy donors and patients with psoriasis by ELISA (Fig. 2). The concentrations of IL-21 were significantly increased in
Figure 3 The different subsets of B cells were analysed by flow cytometry. Human PBMCs from patients with psoriasis and healthy donors were stained with anti-CD3, anti-CD19, anti-IgD, anti-CD27 and anti-CD86 antibodies or isotype controls. The cells were gated on lymphocytes (top left) and then on CD3 CD19+ B cells (top right). Subsequently, the levels of CD86+, IgD+CD27 IgD+CD27+, IgD-CD27+ and IgD-CD27 in CD3 CD19+ B-cell populations were analysed, and the correlation between the level of CD3 CD19+CD86+ B cells with the levels of ICOS+ TFH cells, ICOS+PD-1+ TFH cells and IL-21 was further analysed. Data were expressed as the mean SEM. Each plot represents a single donor. P values of
DOI: 10.1111/jdv.13027
ORIGINAL ARTICLE
Increased circulating follicular helper T cells and activated B cells correlate with disease severity in patients with psoriasis J. Niu, Z. Song, X. Yang, Z. Zhai, H. Zhong,* F. Hao* Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing, China *Correspondence: H. Zhong, E-mail: [email protected], and F. Hao, E-mail: [email protected]
Abstract Background Follicular Helper T (TFH) Cells are a population of recently discovered CD4+ T cells involved in autoimmune diseases. However, the contribution of TFH cells in patients with psoriasis remains unknown. Objective We aimed to investigate the levels of TFH cells, B cells and their clinical relevance in patients with psoriasis. Methods Using multi-colour flow cytometry, we detected different subsets of TFH cells and B cells in the peripheral blood of 27 patients with psoriasis and 13 healthy donors. Serum IL-21 levels were measured by ELISA. The relationship between the levels of TFH cells, IL-21, B cells and disease severity were analysed. Results Compared with healthy donors, higher levels of circulating CD3+CD4+CXCR5+ cells, CD3+CD4+CXCR5+ICOS+, CD3+CD4+CXCR5+PD-1+, CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells and CD19+IgD+CD27
naive B,
CD19+CD86+ activated B, but lower levels of CD19+IgD+CD27+ preswitch and CD19+IgD-CD27+ postswitch memory B cells, were observed in patients with psoriasis. In addition, serum IL-21 levels in patients with psoriasis were significantly higher than those in healthy donors, and showed to be positively correlated with the levels of different subsets of TFH cells, and the level of CD19+CD86+ B cells was also correlated with TFH cells and IL-21 levels. Furthermore, a significant correlation was found between the levels of CD3+CD4+CXCR5+ICOS+ TFH cells, CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells, CD19+CD86+ B cells and IL-21 with Psoriasis Area and Severity Index scores. Conclusion The levels of TFH cells and activated B cells were increased in the peripheral blood of patients with psoriasis, and positively correlated with disease severity. These results suggest that TFH cells and activated B cells may play a role in the pathogenesis of psoriasis. Received: 6 August 2014; Accepted: 13 January 2015
Conflicts of interest The authors declare that they have no conflict of interest.
Funding sources This work was supported by grants from the National Natural Science Foundation of China (NSFC, No.81271754) and (NSFC, No.81101200)
Introduction Psoriasis is a chronic inflammatory skin disease and affects about 2% of the population worldwide.1 Generally, patients with psoriasis develop erythematous, inflamed and scaly plaques. Although many agents, including small molecules, are widely used for treating psoriasis, the resolution of psoriasis remains difficult.2 It has been long recognized that CD4+ T cells orchestrate the adaptive immune response in psoriasis, especially the Th1-cell-mediated inflammatory response. However, Th17 cells, a novel subset of CD4+ T cells expressing IL-17A, but not IFN-c, are found to accumulate in psoriatic skin,3 suggesting a critical role of Th17 cells in human psoriasis. Currently, biological drugs
JEADV 2015, 29, 1791–1796
targeting IL-17A are undergoing clinical trials for the treatment of psoriasis.4 Follicular Helper T (TFH) Cells have been recently identified as a separate CD4+ T-cell subset with distinct developmental programming and effector functions. TFH cells are characterized by the expression of CXCR5, ICOS, PD-1 and the transcription factor Bcl-6 and the secretion of IL-21.5 CXCR5 is not only the most widely utilized surface marker for the identification TFH cells but it is also critical for TFH-cell function.6 ICOS is required for the optimal differentiation of TFH cells and PD-1 appears to be important for regulating TFH-cell function.7,8 IL-21, which is secreted by
© 2015 European Academy of Dermatology and Venereology
1792
Niu et al.
Figure 1 The different subsets of circulating TFH cells were analysed by flow cytometry in patients with psoriasis and healthy donors. Human PBMCs from patients with psoriasis and healthy donors were stained with anti-CD3, anti-CD4, anti-CXCR5, anti-ICOS and antiPD-1 antibodies or isotype controls. The cells were gated on lymphocytes (top left) and then on CD3+CD4+ T cells (top right) for the analysis of the proportion of CD3+CD4+CXCR5+ cells. Subsequently, the levels of CD3+CD4+CXCR5+ICOS+ cells (ICOS+ TFH cells), CD3+CD4+CXCR5+PD-1+ cells (PD-1+ TFH cells) and CD3+CD4+CXCR5+ICOS+PD-1+ cells (ICOS+PD-1+ TFH cells) in the CD3+CD4+CXCR5+ population were analysed. Data were expressed as the mean SEM. Each plot represents a single donor.
TFH cells, can also regulate TFH-cell development and function.9 TFH cells have been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis.10,11 In these studies, the levels of TFH cells are increased in inflammatory skin lesions and the peripheral blood of patients, displaying a positive correlation with disease severity. However, the prevalence of
JEADV 2015, 29, 1791–1796
TFH cells in human psoriasis remains poorly understood. In addition, TFH cells can regulate the activation and function differentiation of B cells.12 Activated B cells up-regulate CD86 expression, and the expression of IgD and CD27 distinguish the distinct subsets of B cells. However, the activation and maturation states of B cells in human psoriasis remain unknown.
© 2015 European Academy of Dermatology and Venereology
Follicular helper T cells and B cells in psoriasis
In this study, we examined the levels of TFH cells, IL-21 and B cells in the peripheral blood of patients with psoriasis, evaluated their relevance with the severity of disease, and attempted to elucidate the contribution of TFH cells, as well as B cells, in patients with psoriasis.
Materials and methods
1793
bated with surface antibodies for 30 min at 4°C, and then fixed for 20 min with 4% paraformaldehyde. Next, PBMCs were washed twice with PBS containing 1% FCS and then cells were collected by flow cytometry with a FACSCantoII (BD Biosciences, San Diego, CA, USA). Isotype-matched antibodies were used to enable correct compensation and confirm antibody specificity. Data were analysed with FlowJo software (Stanford University, San Francisco, CA, USA).
Subjects and samples
A total of 27 patients with psoriasis (male/female = 15/12; average age 43.6 2.9 years) and disease duration 9.3 1.4 years were taken from the Southwest Hospital of Third Military Medical University. The patients had no other autoimmune or systemic disease and not received any anti-psoriatic treatment for at least 1 month before enrolment in the study. Thirteen normal healthy donors (male/female = 7/6; average age 42.4 3.3 years) without any autoimmune or systemic disease were enrolled as the controls. The study was approved by the Ethics Committee of Southwest Hospital, Third Military Medical University. Written informed consent was obtained from each subject. With the approval of the Ethics Committee of the Third Military Medical University, fresh peripheral blood (15 mL) was collected from each study subject in heparinized blood collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. Serum samples were collected and stored at 80°C until further use. The disease activity of patients with psoriasis was measured using Psoriasis Area and Severity Index (PASI) scores as described previously.13 Antibodies and flow cytometry analysis
The following labelled antibodies were used to stain single-cell suspensions: CD3-APC-H7, CD4-PE, CXCR5-PercP-Cy5.5, ICOS-FITC, PD-1-APC, CD19-APC, CD86-PE, IgD-FITC and CD27-PE-Cy7 (Biolegend, San Diego, CA, USA). After PBMCs were obtained from each study subject, PBMCs were washed twice with PBS containing 1% fetal calf serum (FCS) and incu(a)
Enzyme-linked immunosorbent assay (ELISA)
Serum from patients with psoriasis and healthy donors were collected and the level of IL-21 was determined using an ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Statistical analysis
All results were summarized as the mean standard error of the mean (SEM). Statistical analyses were performed with the Prism 5.0 Software (GraphPad software Inc., San Diego, CA, USA). Differences between groups were determined by Mann–Whitney U-test. Correlation analysis between two groups of data was measured by Spearman’s correlation test. P < 0.05 was considered statistically significant.
Results Circulating TFH cells are increased in patients with psoriasis
To study whether TFH cells are involved in the pathogenesis of psoriasis, we first determined the levels of TFH cells in the peripheral blood of healthy donors and patients with psoriasis using multi-colour flow cytometry (Fig. 1). We detected a substantial amount of CD3+CD4+CXCR5+ cells that was significantly increased in patients with psoriasis when compared with healthy donors (16.06% 1.13%, vs. 9.62% 0.87%, P = 0.001). Further analysis revealed that the levels of CD3+CD4+CXCR5+ICOS+ TFH cells (ICOS+ TFH cells), CD3+CD4+CXCR5+PD-1+ TFH cells (PD-1+ TFH cells) and
(b)
Figure 2 Increased IL-21 levels correlated with the levels of circulating TFH cells in patients with psoriasis. (a) The concentrations of serum IL-21 in 27 patients with psoriasis and 13 healthy donors were detected by ELSIA. (b) The correlation between the level of IL-21 with the levels of ICOS+ TFH cells, PD-1+ TFH cells and ICOS+PD-1+ TFH cells was analysed. Data were expressed as the mean SEM. Each plot represents a single donor.
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CD3+CD4+CXCR5+ICOS+PD-1+ TFH cells (ICOS+PD-1+ TFH cells) in the CD3+CD4+CXCR5+ population were also significantly increased in patients with psoriasis when compared with healthy donors. These results suggested that increased levels of circulating TFH cells were present in patients with psoriasis.
Niu et al.
Increased serum IL-21 levels correlated with the levels of TFH cells in patients with psoriasis
Because IL-21 is produced by TFH cells and regulates the B-cell humoural response, we measured the levels of IL-21 in healthy donors and patients with psoriasis by ELISA (Fig. 2). The concentrations of IL-21 were significantly increased in
Figure 3 The different subsets of B cells were analysed by flow cytometry. Human PBMCs from patients with psoriasis and healthy donors were stained with anti-CD3, anti-CD19, anti-IgD, anti-CD27 and anti-CD86 antibodies or isotype controls. The cells were gated on lymphocytes (top left) and then on CD3 CD19+ B cells (top right). Subsequently, the levels of CD86+, IgD+CD27 IgD+CD27+, IgD-CD27+ and IgD-CD27 in CD3 CD19+ B-cell populations were analysed, and the correlation between the level of CD3 CD19+CD86+ B cells with the levels of ICOS+ TFH cells, ICOS+PD-1+ TFH cells and IL-21 was further analysed. Data were expressed as the mean SEM. Each plot represents a single donor. P values of
