Clinical and Experimental Pharmacology and Physiology (2015) 42, 154–161
doi: 10.1111/1440-1681.12330
ORIGINAL ARTICLE
Higher frequency of peripheral blood follicular regulatory T cells in patients with new onset ankylosing spondylitis Yuxing Shan,* Changlin Qi,* Jixue Zhao,* Yijun Liu,* Hui Gao,* Ding Zhao,* Fupeng Ding,* Jing Wang* and Yanfang Jiang*† *Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital of Jilin University, Changchun, and † Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
SUMMARY Follicular helper T (TFH) cells and B cells are linked to the pathogenesis of ankylosing spondylitis (AS). Follicular regulatory T (TFR) cells suppress TFH cell and germinal center B cell numbers in vivo. The role of TFR cells in AS is unknown. The frequency of peripheral blood inducible FOXP3+CXCR5+CD4+TFR cells and CXCR5+CD4+TFH cells were taken from 20 onset AS patients and 10 healthy controls, and were examined by flow cytometry, their disease activity were measured by the Bath Ankylosing Spondylitis Disease Activity Index. The concentrations of serum interleukin (IL)-21, immunoglobulin G, immunoglobulin A, immunoglobulin M and C-reactive protein were examined, and the values of erythrocyte sedimentation rate were measured. The frequency of peripheral blood FOXP3+CXCR5+CD4+TFR cells, CXCR5+CD4+TFH cells, the ratio of FOXP3+CXCR5+ CD4+TFR/CXCR5+CD4+TFH cells and the concentration of serum IL-21 in the AS patients were significantly higher than those in the healthy controls (P < 0.0001, P = 0.0027, P < 0.0001, P = 0.0039, respectively). The frequency of FOXP3+ CXCR5+CD4+TFR cells and the ratio of FOXP3+CXCR5+ CD4+TFR/CXCR5+CD4+TFH cells still significantly rose in those patients after standard treatment (P = 0.0006, P < 0.0001), the concentration of serum IL-21 decreased after treatment (P = 0.0049), accompanied by significantly minimized disease activities. Furthermore, the TFR cells were negatively correlated with serum immunoglobulin A in those patients before treatment (r = 0.582, P = 0.0071), and the frequency of TFR cells was negatively correlated with that of TFH cells and the concentration of serum IL-21 after treatment (r = 0.550, P = 0.046; r = 0.581, P = 0.0371). TFR cells might participate in the pathogenesis of AS, and might be responsible for controlling the autoantibodies, the frequency and function of TFH cells to inhibit the development of AS.
Correspondence: Yan-Fang Jiang, The First Hospital, Jilin University, Changchun 130031, China. Email: [email protected] Received 24 July 2014; revision 26 September 2014; accepted 12 October 2014. © 2014 Wiley Publishing Asia Pty Ltd
Key words: ankylosing spondylitis, follicular helper T cells, follicular regulatory T cell, immunoglobulin A, interleukin21, suppressive function.
INTRODUCTION Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease, and primarily affects the sacroiliac joints and the axial skeleton. It is characterized by inflammatory back pain, enthesitis and specific organ involvement.1 AS occurs predominantly in young adult males with a prevalence rate of 0.2–0.3% in China.2 The pathogenesis of AS is complex, and involves several pathogenetic factors, including infection, environmental triggers, genetic susceptibility, such as human leucocyte antigen (HLA)B27, interleukin (IL)-23R and ERAP1, and in particular, autoimmune disorders.1,3–5 Autoreactive T and B cells, natural killer cells have been reported to contribute to the pathogenesis of AS.6–11 A high frequency of peripheral blood inflammatory Th1 and Th17 cells, B cells are involved in the pathogenesis of AS.6,7,9–11 As a subset of activated CD4+T cells, follicular helper T (TFH) cells have recently been shown to be largely responsible for B cell differentiation, antibody production and humoral immunity.12 Our previous study found a higher frequency of peripheral blood TFH cells associated with higher levels of immunoglobulin (Ig)G and IgA antibodies in AS than healthy controls (HC), and IL-21+TFH cells might be biomarkers for the evaluation of disease severity and therapeutic efficacy in AS patients.13 Therefore, tight control of TFH cells is critical in preventing the secretion of autoantibodies, and might maintain self -tolerance in new onset AS patients.14,15 In autoimmune diseases, such as AS, a breakdown of immunological self-tolerance arises, leading to aberrant immune responses to self-antigen. Ordinarily, regulatory T cells (Treg) – including both natural and induced Treg cells – control these self-reactive cells.16 Derived from natural regulatory T cells, follicular regulatory T (TFR) cells combining phenotypic characteristics with TFH cells and conventional Foxp3+ Treg yet are distinct from both.17 Bcl-6, a transcriptional repressor, is necessary for the development of TFH cells and TFR cells.18,19 Foxp3 is a key factor for the development and function of Treg.18,19
Activated TFR cells in AS patients TFR cells are suppressive in vitro, and limit TFH cell and germinal center B cell numbers in vivo.18 Given the suppressive function of TFR cells on TFH cells and B cells for the control of humoral immune responses, the present study was designed to examine whether TFR cells are involved in the pathogenesis of AS. Currently, standard therapies of patients with AS include non-steroidal anti-inflammatory drugs (NSAIDs), pain killers, steroids, immunosuppressants and tumour necrosis factor-a blockers besides physical therapies. We previously reported that the treatment with standard therapy dramatically controlled the disease progression and significantly reduced the frequency of IL-21+TFH.13 However, there is no information about how the standard treatments affect the frequency of TFR cells in AS patients. In the present study, we examined the frequency of peripheral blood TFR cells, TFH cells and TFR/TFH cells, and the concentration of serum IL-21 in 20 patients with newly diagnosed AS, and sex- and age-matched HC before and 1 month after the standard treatment. We analysed the potential association among the frequency of TFR cells, TFH cells and clinical variations in those patients. Our data showed that a higher frequency of TFR cells, TFH cells and TFR/TFH cells, and a higher concentration of serum IL-21 presented in patients with AS, and the frequency of TFR cells and TFR/TFH cells were still high, whereas the concentration of serum IL-21 decreased after 1 month of standard treatment. The elevated frequency of TFR was negatively correlated with IgA before treatment, the frequency of TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 after treatment. Therefore, TFR cells might have regulatory functions in the pathogenesis of AS.
RESULTS
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(Table 1). In comparison with that in the HC, the majority of AS patients was HLA-B27+, had significantly greater numbers of white blood cells (WBC), and higher values of erythrocyte sedimentation rate (ESR). Furthermore, AS patients showed variable values of Bath AS Disease Activity Index (BASDAI; median 5.1 (range 4–6.3) vs not applicable), and significantly higher concentrations of serum C-reactive protein (CRP), IgA and IgG, but not IgM. In addition, radiological examinations showed that AS patients displayed sacroiliitis with rough and narrowed sacroiliac joint spaces (Fig. 1), supporting the notion of inflammation in the joints of patients. Higher frequency of peripheral blood TFR cells, TFH cells and higher ratio of TFR-to-TFH cells, and elevated concentration of serum IL-21 in patients with AS The frequency of peripheral blood CD4+ and TFR cells was characterized by flow cytometry analysis, and found no significant difference in the frequency of peripheral blood CD4+T cells between the AS patients and HC groups (data not shown). However, the percentages of FOXP3+CXCR5+CD4+TFR cells in AS patients were significantly higher than that in the HC (P < 0.0001; Fig. 2b). The percentages of CXCR5+CD4+TFH cells in AS patients were also significantly higher than that in the HC (P = 0.0027; Fig. 2c). Further analysis showed that the ratio of TFR-to-TFH cells was significantly higher in the AS patients when compared with HC (5.815% (3.31–7.38%) vs 3.015% (2.25–4.01%), P < 0.0001; Fig. 2d). In parallel, the concentration of serum IL-21 was more elevated than that in the HC (P = 0.0039; Fig. 2e). Hence, a higher frequency of TFR cells and TFH cells, a higher ratio of TFR-to-TFH cells, and an elevated concentration of serum IL-21 might be associated with the pathogenesis of AS in this population.
Patient characteristics To determine the frequency of TFR cells, a total of 20 patients with newly diagnosed AS and 10 HC were recruited. As selected, there was no significant difference in the distribution of age and sex between the AS patients and HC groups of participants
Elevated frequency of peripheral blood TFR cells was negatively correlated with the serum IgA in AS patients For testing the importance of peripheral blood TFR cells in AS patients, the potential association of the frequency of TFR cells
Table 1 Demographic and clinical characteristics of patients Parameters
Healthy controls
No. participants Age (years) Male, n (%) HLA-B27+ (%) ESR (mm/h) CRP (mg/dL) IgA (IU/mL) IgG (IU/mL) IgM (IU/mL) BASDAI WBC (109/L)
10 19 8 0 3.5 1.8 1.23 8.6 0.69 NA 5.1
(16–36) (80%) (0%) (1.5–16) (1.3–3.6) (0.8–3.65) (7.2–12.5) (0.46–2.05) (4.2–9.5)
AS (0M) 20 23 16 17 62 32.2 2.89 12.1 1.11 5.1 7.0
(15–38) (80%) (85%) (16–90)* (5.59–89.4)* (1.55–4.63)* (9.5–15.8)* (0.59–2.16) (4–6.3) (4.2–12.4)*
AS (1M) 13 23 9 12 19 6.3 1.83 9.8 0.86 2.4 5.9
(17–29) (69%) (92%) (6–28)** (1.2–16.5)** (1.25–4.13)** (8.6–14.5)** (0.61–2.23) (1.6–3.3)** (4.7–10.8)**
Data shown as median (range) or the real counts of cases. *P < 0.05 versus the healthy controls; **P < 0.05 versus the ankylosing spondylitis (AS) patients (0M). Normal ranges of individual measures are C-reactive protein (CRP) 0–3 mg/dL, erythrocyte sedimentation rate (ESR) 0–15 (mm/h), immunoglobulin (Ig)A 0.7–4 IU/mL, IgG 7–16 IU/mL, IgM 0.4–2.3 IU/mL and white blood cell counts (WBC) (4–10)9109/L. 0M, baseline values before treatment, 1M, 1 month after treatment; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; ESR, erythrocyte sedimentation rate; CRP, Creactive protein; HLA-B27, human leucocyte antigen B27; NA, not applicable; WBC, white blood cell counts.
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Fig. 1 Computed tomography scanning analysis of the bony pelvis. All of the ankylosing spondylitis patients and some healthy controls were subjected to a computed tomography scanning analysis of the sacroiliac joints of the bony pelvis. Data shown are representative horizontal computed tomography images from (a) a healthy control, and (b) a patient before treatment and (c) 1 month (1M) after treatment. Black arrows show the sacroiliac joint areas. 0M, 0, month.
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Fig. 2 Flow cytometry analysis of the frequency of peripheral blood follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH cells, and the concentration of serum IL-21 in ankylosing spondylitis (AS) patients and healthy controls (HC), and the correlation of the frequency peripheral blood TFR cells with the serum IgA in AS patients. Peripheral blood mononuclear cells were isolated from individual patients and HC, and stained with anti-CD4 and anti-CXCR5 or isotype controls. Subsequently, the cells were gated on living CD4+ cells and the percentages of FOXP3+CXCR5+CD4+ TFH cells and FOXP3 CXCR5+CD4+ TFH cells in total CD4+ T cells were determined. (a) Flow cytometry analysis. Quantitative analysis the percentages of (b) FOXP3+CXCR5+CD4+TFR cells, (c) CXCR5+CD4+TFH cells, (d) the ratio of FOXP3+CXCR5+CD4+TFR cellsto-CXCR5+CD4+TFH cells and (e) the concentration of serum interleukin (IL)-21 in 20 patients and 10 HC. (f) The percentages of peripheral blood TFR cells in individual patients were negatively correlated with the serum immunoglobulin A in patients before treatment. Data are representative charts or expressed as individual values from HC and AS patients before treatment. The horizontals indicate the median values of individual groups.
with the values of clinical measures was analysed by the Pearson’s correlation analysis. It was found that the elevated frequency of peripheral blood TFR cells was negatively correlated with the concentration of the serum IgA in those patients before treatment (r = 0.582, P = 0.0071; Fig. 2f). However, there was no significant correlation among other measures tested in this population (data not shown). Standard treatment significantly promoted the peripheral blood TFR cells and ratio of TFR-to-TFH cells in AS patients, and also reduced the concentration of serum IL-21 Currently, AS patients are treated with NSAIDs, steroids, immunosuppressive agents and tumour necrosis factor blocker,
besides physical therapy. Among the 20 patients tested, there were 13 drug-responders and seven non-responders after 1 month post-treatment. Clinical measures and the frequency of TFR cells in the 13 drug-responders were analysed after treatment for 1 month. The treatment significantly relieved clinical symptoms, and reduced the values of WBC, CRP, ESR, IgA, IgG and BASDAI (P < 0.05; Table 1), although there was no significant change in the computer tomography (CT) scanning after treatment (Fig. 1c). The percentage of FOXP3+CXCR5+ CD4+TFR cells (P = 0.0006, Fig. 3b) and the ratio of TFR-toTFH cells (P < 0.0001; Fig. 3d) in drug-responding AS patients were significantly higher as compared with those before treatment. The frequency of CXCR5+CD4+TFH cells decreased after treatment in drug-responders, but this change had no
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Activated TFR cells in AS patients (a)
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Fig. 3 Treatment modulates the frequency of follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH, the levels of serum interleukin (IL)-21 cells in drug-responding patients. (a) Flow cytometry analysis. In all follow up, a total of 13 patients were drug responsive after 1 month (1M) of drug therapy. The frequency of (b) TFR cells, (c) TFH cells in total CD4+ T cells, and (d) the ratio of TFR-to-TFH cells in those patients were determined by flow cytometry analysis before (0 month (0M)) and after treatment (1 month). (e) The levels of serum IL-21 in those patients were determined by enzyme-linked immunosorbent assay.
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Fig. 4b), the ratio of TFR-to-TFH (P = 0.085; Fig. 4c) and the levels of serum IL-21 (P = 0.277, Fig. 4d) in non-responding patients after treatment. Thus, the standard treatment might promote TFR cell numbers and activities in drug-responding AS patients.
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Frequency of peripheral blood TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 in these patients after 1 month of standard treatment
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Fig. 4 The frequency change of follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH, and the levels of serum interleukin (IL)-21 in non-responding patients after treatment. In all follow-up, a total of seven patients were drug nonresponsive after 1 month of drug therapy. The (a) frequency of TFR cells, (b) TFH cells in total CD4+ T cells, and (c) the ratio of TFR-to-TFH cells in those patients were determined by flow cytometry analysis before (0 month (0M)) and after treatment (1 month (1M)). The levels of serum IL-21 in those patients were determined by enzyme-linked immunosorbent assay.
To investigate whether TFR cells limit TFH cell numbers in AS patients, it was found that the frequency of peripheral blood TFR cells was negatively correlated with that of TFH cells after 1 month of standard treatment (r = 0.550, P = 0.046; Fig. 5a), though there was no significant correlation before treatment. Furthermore, we found the frequency of peripheral blood TFR cells was negatively correlated with the concentration of serum IL-21 in drug-responders after treatment (r = 0.581, P = 0.0371; Fig. 5b). However, there was no significant correlation between the frequency of peripheral blood TFR cells and the concentration of serum IL-21 after treatment in non-responding patients (r = 0.666, P = 0.102; Fig. 5c). Thus, the administration of 1 month of standard treatment might promote TFR cells to limit the number and the secretory activity of TFH cells.
DISCUSSION statistical significance (P = 0.1649; Fig. 3c). However, the concentration of serum IL-21 decreased as compared with that before treatment in drug-responding AS patients (P = 0.0049; Fig. 3e). We did not detect significant changes in the frequency of TFR cells (P = 0.406; Fig. 4a), TFH cells (P = 0.110;
In the present study, we found that the frequency of peripheral blood FOXP3+CD4+CXCR5+TFR cells, CD4+CXCR5+TFH cells, the ratio of TFR-to-TFH cells and the concentration of serum IL-21 in the AS patients were significantly higher than in the HC. Our previous study found that the elevated TFH cells
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Fig. 5 The correlation of the frequency peripheral blood follicular regulatory T (TFR) cells with follicular helper T (TFH) cells and the concentration of serum interleukin (IL)-21 in those patients after treatment. (a) The frequency of peripheral blood TFR cells are negatively correlated with TFH cells in drug-responding ankylosing spondylitis (AS) patients after 1 month (1M) of standard treatment. (b) The frequency of peripheral blood TFR cells are negatively correlated with the concentration of serum IL-21 in drug-responding AS patients after 1 month of standard treatment. (c) The frequency of peripheral blood TFR cells are not significantly correlated with the concentration of serum IL-21 in non-responding AS patients after 1 month of standard treatment.
might contribute to the pathogenesis of AS.13 Because of the suppressive function of TFR cells on TFH cells, the higher frequency of TFR cells and ratio of TFR-to-TFH cells in the AS patients suggest that TFR cells might be also involved the pathogenesis of AS. Foxp3+Treg cells are crucial in preventing autoantibody-mediated autoimmunity.16,20 Recent studies have shown that TFR cells are a specialized subset of Foxp3+Treg cells expressing BCL-6 and CXCR5. Foxp3 is a key factor for the development and function of Treg.21–23 The interaction between CXCR5 and CXCL13 mediates homing of lymphocytes to B cell follicle areas, and Bcl-6 also promotes the expression of CXCR5 on TFR cells, as they do in TFH cells.24–27 Several studies of patients with connective tissue diseases have found that reduced or functionally impaired Treg cells, and Treg cells of autoimmune hepatitis patients reducing expression of Fox-P3 and CTLA-4, which might lead to impaired suppressor activity.28–30 Cua et al.31 also found that skewing of responses towards Th17 and away from BMSC or Treg cells could be responsible for the progression of AS. However, the present study found there were significantly elevated TFR cells in AS patients compared with that of HC, although the percentage of BCL-6+ TFR cells had almost no change between the two groups. Chung et al.19 showed that Bcl-6 is essential for the generation of CXCR5+Treg cells in their study. However, only Bcl-6 is not sufficient for CXCR5 expression and the function of TFR cells. For instance, Linterman MA et al. suggested that Bcl-6 was essential for TFR cell formation, and Blimp-1 expression regulates the size of the TFR cell population.32 It shows that multiple signals regulate the function of TFR cells. Different signals of TFR cells and their interaction in AS require further study. In our previous and present study, a higher frequency of peripheral blood TFH cells was detected in AS patients compared with that of HC.13 Treg are crucial for restoring immune tolerance in chronic inflammation. Mills showed that immune responses can result in collateral damage to host tissues, and immune-regulatory mechanisms, including the induction of Treg, are essential to control this immunopathology.33 As infection and autoimmune disorders are
involved in the pathogenesis progress of AS, the similar immuneregulatory mechanisms might exist in the progress of AS. Because of the suppressive function of TFR cells on TFH cells and the participation of TFH cells in AS, the increased frequency of TFR cells might stem from compensative spontaneous regulation of the immune system, so that increased frequency of TFR cells negatively regulates autoimmune responses in AS patients. Thus, TFR cells and TFH cells are both involved in the pathogenesis of AS. To further understand the role of TFR cells and the interrelationship between TFR cells and TFH cells in the development and progression of AS, we characterized the ratio of TFR-to-TFH cells in this population and found that it was significantly higher than that in the HC. TFH cells are a subset of CD4+ T cells, and have been shown to be crucial regulators of B cell differentiation, antibody production and humoral immunity.12 Although the percentages of IL-21+TFH cells in AS patients were positively correlated with BASDI in our previous study, there was no significant correlation between TFR cells and BASDAI.13 However, notably, in the present study, we found that the percentage of TFR cells was negatively correlated with serum IgA. Indeed, Wollenberg et al.17 also found Foxp3+ follicular T cells can limit the amount of secreted Ag-specific IgM, IgG1, IgG2b and IgA in vivo. Therefore, our data suggest that TFR cells might control the autoantibodies and be involved in the pathogenesis of AS. TFR and TFH cells could affect each other in AS, and this activity plays an important role in the pathogenesis of AS. Currently, NSAIDs, pain killers, steroids, immunosuppressant and tumour necrosis factor-a blockers have been used for the standard treatment of AS patients.34–37 In the present study, we examined how the standard therapy could affect TFR cells. Analysis of the available data from 13 drug-responding patients showed that treatment with the standard therapy for 1 month significantly reduced ESR, the WBC counts, and the levels of serum CRP, IgG and IgA, and also significantly minimized the scores of BASDAI. In Fig. 1b, we can find sclerotin under the cartilage is severely invaded, hyperosteogeny and sclerosis are detected, and irregular stenosis exists in sacroiliac joint space.
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Activated TFR cells in AS patients The CT graph in Fig. 1b can be defined as grade 3 according to the 1984 modified New York criteria. After 1 month treatment, the sclerotin invasion, hyperosteogeny and sclerosis, and irregular stenosis in sacroiliac joint space still exist in Fig. 1c. The grade of CT graph in Fig. 1c is also 3. Similar changes were shown in the CT scanning of all the AS patients. Thus, there was no significant change in the CT scanning after treatment. Although we found a positive correlation between the percentage of IL-21+TFH cells and the value of BASDAI after treatment in our previous study, the recent reports have shown the suppressive function of TFR cells on TFH cells in vivo.13,17–19 Notably, we detected a higher frequency of peripheral blood TFR cells and a higher ratio of TFR-to-TFH cells in AS patients after treatment, and found there was a negative correlation between TFR cells and TFH cells. In parallel, the concentration of serum IL-21 decreased compared with that before treatment, and TFR cells were negatively correlated with the concentration of serum IL-21 after treatment. As known, AS is a chronic autoimmune inflammatory disease, but inflammatory stimuli can downregulate the expression of Foxp3 and affect the inhibitory activity of Treg.38 The anti-inflammatory treatments might promote the inhibitory activity of TFR cells to TFH cells by change inflammatory state in AS patients. These data suggested that the administration of 1 month of standard treatment might promote the activity of TFR cells to limit the number and secretory activity of TFH cells for controlling the progression of AS. The interreaction of TFR–TFH cells could take an important part in the development of AS. In conclusion, the present data showed that the percentages of TFR cells and TFH cells, the ratio of TFR-to-TFH cells, and the concentration of serum IL-21 significantly increased in AS patients compared with those of HC, and the frequency of TFR cells was negatively correlated with the serum IgA in those patients. Treatment with 1 month of standard therapy dramatically controlled the disease progression, the treatment significantly elevated the frequency of TFR cells and the ratio of TFR-to-TFH cells, and reduced the concentration of serum IL-21. The frequency of TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 after treatment. TFH cells are involved in the development of AS. Therefore, the TFR cells might be responsible for controlling the autoantibodies, and the frequency and function of TFH cells to inhibit the development of AS. The interaction of TFR–TFH cells might take an important part in the development of AS. We recognized that the present study had limitations of a small sample size, the lack of functional studies of TFR cells and other pro-inflammatory cytokines in AS. Therefore, further studies of TFR cells and other pro-inflammatory cytokines in the pathogenic process with a bigger population are warranted.
METHODS Patients and controls A total of 20 newly diagnosed patients with AS were recruited sequentially at the inpatient service of the First Hospital of Jilin University, Changchun, China. A total of 10 sex-, age- and ethnicity-matched HC were recruited. Written informed consent was obtained from individual patients, and the experimental protocol
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was approved by the ethics committee of the First Hospital of Jilin University. Individual patients with AS were diagnosed, according to the 1984 modified New York criteria.39 None of the patients took steroids or other immunosuppressants before their blood samples were collected. The disease activity of individual patients was evaluated for the BASDAI using a questionnaire with six questions related to: (i) fatigue; (ii) spinal pain; (iii) peripheral joint pain; (iv) enthesitis; and (v) morning stiffness, as well as the intensity and duration, respectively, as described previously.40 The scores for each item ranged from 0 to 10, and high disease activity was defined as a BASDAI score ≥ 4. Individual participants with rheumatoid arthritis, multiple sclerosis, type 1 diabetes, immunodeficiency, chronic inflammatory diseases and recent infection were excluded. Their demographic and clinical characteristics are shown in Table 1. After hospitalization, individual patients were orally treated with 15 mg meloxicam (Boehringer Ingelheim, Shanghai, China) and 50 mg thalidomide daily (Changzhou Pharmaceutical Factory, Changzhou, China), and subcutaneously injected with 25 mg etanercept (Wyeth, Suzhou, China) every 2 weeks. In addition, some patients received symptomatic treatment. Drug responders were classified according to the ASAS20 (response criteria of the Assessment of SpondyloArthritis International Society) after 1 month of treatment.41 Laboratory examinations Fasted venous blood samples were obtained from individual participants before treatment and after 1 month of treatment, and the sera were prepared. The number of WBC, the ESR, and the concentration of serum CRP, IgG, IgA and IgM of individual participants were measured by an analyser (Siemens Healthcare Diagnostics Products, GmbH, Munich, Germany) and enzymespecific immunosorbent assay (ELISA) using specific kits. Individual patients were subjected to CT examination for their sacroiliac joints on a Siemens CT system (Angio CT Miyaby, Berlin, Germany). Flow cytometry analysis The HLA-B27 status in individual participants was characterized by flow cytometry analysis.42 Briefly, peripheral blood mononuclear cells were stained with fluorescein isothiocyanate (FITC)anti-human leococyte antigen (HLA)-B27 and P-phycoerythrin (PE)-anti-CD3, then subjected to flow cytometry analysis on a fluorescence-activated cell sorting (FACS) Calibur (BecktonDickinson, San Diego, CA, USA). Human peripheral blood mononuclear cells at 5 9 105/tube were stained with FITC-antiBCL6, Alexefluor647-anti-CXCR5 and PE-CY7-anti-CD4 (BD PharMingen, San Diego, CA, USA) at dark room temperature for 30 min. Then, the stained cells were fixed, permeabilized and stained intracytoplasmically with PE-anti-Foxp3. Control cells were stained with FITC-anti-IgG1, PE-anti-IgG1, PE-CY7-antiIgG1 and Alexefluor647-anti-IgG1 (BD PharMingen). After washing, the cells were characterized by using a FACS Calibur (Beckton-Dickinson) and FLOWJO software (v7.6.2; TreeStar, Ashland, OR, USA).43 At least 20 000 events of each sample were acquired for analysis.
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160 Measurement of serum IL-21 by ELISA
Serum IL-21 concentrations were measured by a human IL-21 ELISA kit following the manufacturer’s instructions (Roche Diagnostics, Lewes, UK). Briefly, sera were diluted to 1 : 4, and concentrations were calculated with the standard curve generated with the provided recombinant IL-21. The detection limit of the IL-21 ELISA kit was 0.5 ng/L. Statistical analysis Data was expressed as median and range or individual values. The difference between the groups was analysed by Man–Whitney U-nonparametric test using the SPSS 19.0 software (SPSS, Chicago, IL, USA), and differences in individuals before and after treatment were evaluated by using the Wilcoxon matchedpair t-test. The relationship between variables was evaluated by using the Pearson rank correlation test. A two-side P-value of < 0.05 was considered as statistically significant.
ACKNOWLEDGEMENTS This study was supported by grants from the National Natural Science Foundation of China (No. 30972610 and 81273240), Jilin Province Science and Technology Agency (No. 20110716), the Health Department Research Projects in Jilin Province (2009Z054) and Norman Bethune Program of Jilin University (2012206).
DISCLOSURE The authors declare no conflict of interest.
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10. Wright C, Sibani S, Trudgian D et al. Detection of multiple autoantibodies in patients with ankylosing spondylitis using nucleic acid programmable protein arrays. Mol. Cell Proteomics 2012; 11: 1–10. 11. Voswinkel J, Weisgerber K, Pfreundschuh M, Gause A. B-cell involvement in ankylosing spondylitis: The heavychain variable segment gene repertoire of B-cells fromgerminal center-like foci in the synovial membrane indicatesantigen selection. Arthritis Res. 2001; 3: 189–95. 12. Yusuf I, Kageyama R, Monticelli L et al. Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). J. Immunol. 2010; 185: 190–202. 13. Xiao F, Zhang HY, Liu YJ, Zhao D, Shan YX, Jiang YF. Higher frequency of peripheral blood interleukin 21 positive follicular helper T cells in patients with ankylosing spondylitis. J. Rheumatol. 2013; 40: 2029–37. 14. Linterman MA, Rigby RJ, Wong RK et al. Follicular helper T cells are required for systemic autoimmunity. J. Exp. Med. 2009; 206: 561–76. 15. Vinuesa CG, Cook MC, Angelucci C et al. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 2005; 435: 452–8. 16. Vila J, Isaacs JD, Anderson AE. Regulatory T cells and autoimmunity. Curr. Opin. Hematol. 2009; 16: 274–9. 17. Wollenberg I, Agua-Doce A, Hernandez A et al. Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells. J. Immunol. 2011; 187: 4553–60. 18. Linterman MA, Pierson W, Lee SK et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 2011; 17: 975–83. 19. Chung Y, Tanaka S, Chu F et al. Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat. Med. 2011; 17: 983–9. 20. Ochs HD, Ziegler SF, Torgerson TR. FOXP3 acts as a rheostat of the immune response. Immunol. Rev. 2005; 203: 156–64. 21. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol. 2003; 4: 330–6. 22. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299: 1057–61. 23. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+ CD25+ T regulatory cells. Nat. Immunol. 2003; 4: 337–42. 24. Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J. Exp. Med. 2001; 193: 1373–81. 25. Johnston RJ, Poholek AC, Ditoro D et al. Bcl-6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science 2009; 325: 1006–10. 26. Nurieva RI, Chung Y, Martinez GJ et al. Bcl-6 mediates the development of T follicular helper cells. Science 2009; 325: 1001–5. 27. Yu D, Rao S, Tsai LM et al. The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity 2009; 31: 457–68. 28. Banica L, Besliu A, Pistol G et al. Quantification and molecular characterization of regulatory T cells in connective tissue diseases. Autoimmunity 2009; 42: 41–9. 29. Abdulahad WH, Stegeman CA, van der Geld YM et al. Functional defect of circulating regulatory CD4+ T cells in patients with Wegener’s granulomatosis in remission. Arthritis Rheum. 2007; 56: 2080–91. 30. Okumura A, Ishikawa T, Sato S et al. Deficiency of forkhead box P3 and cytotoxic T-lymphocyte-associated antigen-4 gene expressions and impaired suppressor function of CD4(+)CD25(+) T cells in patients with autoimmune hepatitis. Hepatol. Res. 2008; 38: 896– 903.
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Activated TFR cells in AS patients 31. Cua DJ, Sherlock J, Chen Y et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003; 421: 744–8. 32. Linterman MA, Pierson W, Lee SK et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 2011; 17: 975–82. 33. Mills KH. Regulatory T cells: Friend or foe in immunity to infection? Nat. Rev. Immunol. 2004; 4: 841–55. 34. Wolf J, Fasching P. Ankylosing spondylitis. Wien. Med. Wochenschr. 2010; 160: 211–14. 35. Dougados M, Dijkmans B, Khan M et al. Conventional treatments for ankylosing spondylitis. Ann. Rheum. Dis. 2002; 61(Suppl. 3): 40–50. 36. Dagfinrud H, Kvien TK, Hagen KB. The Cochrane review of physiotherapy interventions for ankylosing spondylitis. J. Rheumatol. 2005; 32: 1899–906. 37. Ward MM, Kuzis S. Medication toxicity among patients with ankylosing spondylitis. Arthritis Rheum. 2002; 47: 234–41. 38. Hori S. Lineage stability and phenotypic plasticity of Foxp3⁺ regulatory T cells. Immunol. Rev. 2014; 259: 159–72.
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39. Van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis: A proposal for modification of the New York criteria. Arthritis Rheum. 1984; 27: 361–8. 40. Garrett S, Jenkinson T, Kennedy LG, Whitelock H, Gaisford P, Calin A. A new approach to defining disease status in ankylosing spondylitis: The Bath Ankylosing Spondylitis Disease Activity Index. J. Rheumatol. 1994; 21: 2286–91. 41. Anderson JJ, Baron G, van der Heijde D, Felson DT, Dougados M. Ankylosing spondylitis assessment group preliminary definition of short-term improvement in ankylosing spondylitis. Arthritis Rheum. 2001; 44: 1876–86. 42. Chou SJ, Lai NS, Su JP, Wu JL, Lan JL. Two color analysis of HLA-B27 antigen by flow cytometer—a comparative study by conventional microlymphocytoxicity, DNA genotyping polymerase chain reaction and flow cytometric measurement. J. Clin. Lab. Anal. 1997; 11: 369–73. 43. Jiang Y, Ma Z, Xin G et al. Th1 and Th2 immuneresponse in chronic hepatitis B patients during a long-term treatment with adefovir dipivoxil. Mediators Inflamm. 2010; 2010: 1 43 026.
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doi: 10.1111/1440-1681.12330
ORIGINAL ARTICLE
Higher frequency of peripheral blood follicular regulatory T cells in patients with new onset ankylosing spondylitis Yuxing Shan,* Changlin Qi,* Jixue Zhao,* Yijun Liu,* Hui Gao,* Ding Zhao,* Fupeng Ding,* Jing Wang* and Yanfang Jiang*† *Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital of Jilin University, Changchun, and † Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
SUMMARY Follicular helper T (TFH) cells and B cells are linked to the pathogenesis of ankylosing spondylitis (AS). Follicular regulatory T (TFR) cells suppress TFH cell and germinal center B cell numbers in vivo. The role of TFR cells in AS is unknown. The frequency of peripheral blood inducible FOXP3+CXCR5+CD4+TFR cells and CXCR5+CD4+TFH cells were taken from 20 onset AS patients and 10 healthy controls, and were examined by flow cytometry, their disease activity were measured by the Bath Ankylosing Spondylitis Disease Activity Index. The concentrations of serum interleukin (IL)-21, immunoglobulin G, immunoglobulin A, immunoglobulin M and C-reactive protein were examined, and the values of erythrocyte sedimentation rate were measured. The frequency of peripheral blood FOXP3+CXCR5+CD4+TFR cells, CXCR5+CD4+TFH cells, the ratio of FOXP3+CXCR5+ CD4+TFR/CXCR5+CD4+TFH cells and the concentration of serum IL-21 in the AS patients were significantly higher than those in the healthy controls (P < 0.0001, P = 0.0027, P < 0.0001, P = 0.0039, respectively). The frequency of FOXP3+ CXCR5+CD4+TFR cells and the ratio of FOXP3+CXCR5+ CD4+TFR/CXCR5+CD4+TFH cells still significantly rose in those patients after standard treatment (P = 0.0006, P < 0.0001), the concentration of serum IL-21 decreased after treatment (P = 0.0049), accompanied by significantly minimized disease activities. Furthermore, the TFR cells were negatively correlated with serum immunoglobulin A in those patients before treatment (r = 0.582, P = 0.0071), and the frequency of TFR cells was negatively correlated with that of TFH cells and the concentration of serum IL-21 after treatment (r = 0.550, P = 0.046; r = 0.581, P = 0.0371). TFR cells might participate in the pathogenesis of AS, and might be responsible for controlling the autoantibodies, the frequency and function of TFH cells to inhibit the development of AS.
Correspondence: Yan-Fang Jiang, The First Hospital, Jilin University, Changchun 130031, China. Email: [email protected] Received 24 July 2014; revision 26 September 2014; accepted 12 October 2014. © 2014 Wiley Publishing Asia Pty Ltd
Key words: ankylosing spondylitis, follicular helper T cells, follicular regulatory T cell, immunoglobulin A, interleukin21, suppressive function.
INTRODUCTION Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease, and primarily affects the sacroiliac joints and the axial skeleton. It is characterized by inflammatory back pain, enthesitis and specific organ involvement.1 AS occurs predominantly in young adult males with a prevalence rate of 0.2–0.3% in China.2 The pathogenesis of AS is complex, and involves several pathogenetic factors, including infection, environmental triggers, genetic susceptibility, such as human leucocyte antigen (HLA)B27, interleukin (IL)-23R and ERAP1, and in particular, autoimmune disorders.1,3–5 Autoreactive T and B cells, natural killer cells have been reported to contribute to the pathogenesis of AS.6–11 A high frequency of peripheral blood inflammatory Th1 and Th17 cells, B cells are involved in the pathogenesis of AS.6,7,9–11 As a subset of activated CD4+T cells, follicular helper T (TFH) cells have recently been shown to be largely responsible for B cell differentiation, antibody production and humoral immunity.12 Our previous study found a higher frequency of peripheral blood TFH cells associated with higher levels of immunoglobulin (Ig)G and IgA antibodies in AS than healthy controls (HC), and IL-21+TFH cells might be biomarkers for the evaluation of disease severity and therapeutic efficacy in AS patients.13 Therefore, tight control of TFH cells is critical in preventing the secretion of autoantibodies, and might maintain self -tolerance in new onset AS patients.14,15 In autoimmune diseases, such as AS, a breakdown of immunological self-tolerance arises, leading to aberrant immune responses to self-antigen. Ordinarily, regulatory T cells (Treg) – including both natural and induced Treg cells – control these self-reactive cells.16 Derived from natural regulatory T cells, follicular regulatory T (TFR) cells combining phenotypic characteristics with TFH cells and conventional Foxp3+ Treg yet are distinct from both.17 Bcl-6, a transcriptional repressor, is necessary for the development of TFH cells and TFR cells.18,19 Foxp3 is a key factor for the development and function of Treg.18,19
Activated TFR cells in AS patients TFR cells are suppressive in vitro, and limit TFH cell and germinal center B cell numbers in vivo.18 Given the suppressive function of TFR cells on TFH cells and B cells for the control of humoral immune responses, the present study was designed to examine whether TFR cells are involved in the pathogenesis of AS. Currently, standard therapies of patients with AS include non-steroidal anti-inflammatory drugs (NSAIDs), pain killers, steroids, immunosuppressants and tumour necrosis factor-a blockers besides physical therapies. We previously reported that the treatment with standard therapy dramatically controlled the disease progression and significantly reduced the frequency of IL-21+TFH.13 However, there is no information about how the standard treatments affect the frequency of TFR cells in AS patients. In the present study, we examined the frequency of peripheral blood TFR cells, TFH cells and TFR/TFH cells, and the concentration of serum IL-21 in 20 patients with newly diagnosed AS, and sex- and age-matched HC before and 1 month after the standard treatment. We analysed the potential association among the frequency of TFR cells, TFH cells and clinical variations in those patients. Our data showed that a higher frequency of TFR cells, TFH cells and TFR/TFH cells, and a higher concentration of serum IL-21 presented in patients with AS, and the frequency of TFR cells and TFR/TFH cells were still high, whereas the concentration of serum IL-21 decreased after 1 month of standard treatment. The elevated frequency of TFR was negatively correlated with IgA before treatment, the frequency of TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 after treatment. Therefore, TFR cells might have regulatory functions in the pathogenesis of AS.
RESULTS
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(Table 1). In comparison with that in the HC, the majority of AS patients was HLA-B27+, had significantly greater numbers of white blood cells (WBC), and higher values of erythrocyte sedimentation rate (ESR). Furthermore, AS patients showed variable values of Bath AS Disease Activity Index (BASDAI; median 5.1 (range 4–6.3) vs not applicable), and significantly higher concentrations of serum C-reactive protein (CRP), IgA and IgG, but not IgM. In addition, radiological examinations showed that AS patients displayed sacroiliitis with rough and narrowed sacroiliac joint spaces (Fig. 1), supporting the notion of inflammation in the joints of patients. Higher frequency of peripheral blood TFR cells, TFH cells and higher ratio of TFR-to-TFH cells, and elevated concentration of serum IL-21 in patients with AS The frequency of peripheral blood CD4+ and TFR cells was characterized by flow cytometry analysis, and found no significant difference in the frequency of peripheral blood CD4+T cells between the AS patients and HC groups (data not shown). However, the percentages of FOXP3+CXCR5+CD4+TFR cells in AS patients were significantly higher than that in the HC (P < 0.0001; Fig. 2b). The percentages of CXCR5+CD4+TFH cells in AS patients were also significantly higher than that in the HC (P = 0.0027; Fig. 2c). Further analysis showed that the ratio of TFR-to-TFH cells was significantly higher in the AS patients when compared with HC (5.815% (3.31–7.38%) vs 3.015% (2.25–4.01%), P < 0.0001; Fig. 2d). In parallel, the concentration of serum IL-21 was more elevated than that in the HC (P = 0.0039; Fig. 2e). Hence, a higher frequency of TFR cells and TFH cells, a higher ratio of TFR-to-TFH cells, and an elevated concentration of serum IL-21 might be associated with the pathogenesis of AS in this population.
Patient characteristics To determine the frequency of TFR cells, a total of 20 patients with newly diagnosed AS and 10 HC were recruited. As selected, there was no significant difference in the distribution of age and sex between the AS patients and HC groups of participants
Elevated frequency of peripheral blood TFR cells was negatively correlated with the serum IgA in AS patients For testing the importance of peripheral blood TFR cells in AS patients, the potential association of the frequency of TFR cells
Table 1 Demographic and clinical characteristics of patients Parameters
Healthy controls
No. participants Age (years) Male, n (%) HLA-B27+ (%) ESR (mm/h) CRP (mg/dL) IgA (IU/mL) IgG (IU/mL) IgM (IU/mL) BASDAI WBC (109/L)
10 19 8 0 3.5 1.8 1.23 8.6 0.69 NA 5.1
(16–36) (80%) (0%) (1.5–16) (1.3–3.6) (0.8–3.65) (7.2–12.5) (0.46–2.05) (4.2–9.5)
AS (0M) 20 23 16 17 62 32.2 2.89 12.1 1.11 5.1 7.0
(15–38) (80%) (85%) (16–90)* (5.59–89.4)* (1.55–4.63)* (9.5–15.8)* (0.59–2.16) (4–6.3) (4.2–12.4)*
AS (1M) 13 23 9 12 19 6.3 1.83 9.8 0.86 2.4 5.9
(17–29) (69%) (92%) (6–28)** (1.2–16.5)** (1.25–4.13)** (8.6–14.5)** (0.61–2.23) (1.6–3.3)** (4.7–10.8)**
Data shown as median (range) or the real counts of cases. *P < 0.05 versus the healthy controls; **P < 0.05 versus the ankylosing spondylitis (AS) patients (0M). Normal ranges of individual measures are C-reactive protein (CRP) 0–3 mg/dL, erythrocyte sedimentation rate (ESR) 0–15 (mm/h), immunoglobulin (Ig)A 0.7–4 IU/mL, IgG 7–16 IU/mL, IgM 0.4–2.3 IU/mL and white blood cell counts (WBC) (4–10)9109/L. 0M, baseline values before treatment, 1M, 1 month after treatment; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; ESR, erythrocyte sedimentation rate; CRP, Creactive protein; HLA-B27, human leucocyte antigen B27; NA, not applicable; WBC, white blood cell counts.
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Fig. 1 Computed tomography scanning analysis of the bony pelvis. All of the ankylosing spondylitis patients and some healthy controls were subjected to a computed tomography scanning analysis of the sacroiliac joints of the bony pelvis. Data shown are representative horizontal computed tomography images from (a) a healthy control, and (b) a patient before treatment and (c) 1 month (1M) after treatment. Black arrows show the sacroiliac joint areas. 0M, 0, month.
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Fig. 2 Flow cytometry analysis of the frequency of peripheral blood follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH cells, and the concentration of serum IL-21 in ankylosing spondylitis (AS) patients and healthy controls (HC), and the correlation of the frequency peripheral blood TFR cells with the serum IgA in AS patients. Peripheral blood mononuclear cells were isolated from individual patients and HC, and stained with anti-CD4 and anti-CXCR5 or isotype controls. Subsequently, the cells were gated on living CD4+ cells and the percentages of FOXP3+CXCR5+CD4+ TFH cells and FOXP3 CXCR5+CD4+ TFH cells in total CD4+ T cells were determined. (a) Flow cytometry analysis. Quantitative analysis the percentages of (b) FOXP3+CXCR5+CD4+TFR cells, (c) CXCR5+CD4+TFH cells, (d) the ratio of FOXP3+CXCR5+CD4+TFR cellsto-CXCR5+CD4+TFH cells and (e) the concentration of serum interleukin (IL)-21 in 20 patients and 10 HC. (f) The percentages of peripheral blood TFR cells in individual patients were negatively correlated with the serum immunoglobulin A in patients before treatment. Data are representative charts or expressed as individual values from HC and AS patients before treatment. The horizontals indicate the median values of individual groups.
with the values of clinical measures was analysed by the Pearson’s correlation analysis. It was found that the elevated frequency of peripheral blood TFR cells was negatively correlated with the concentration of the serum IgA in those patients before treatment (r = 0.582, P = 0.0071; Fig. 2f). However, there was no significant correlation among other measures tested in this population (data not shown). Standard treatment significantly promoted the peripheral blood TFR cells and ratio of TFR-to-TFH cells in AS patients, and also reduced the concentration of serum IL-21 Currently, AS patients are treated with NSAIDs, steroids, immunosuppressive agents and tumour necrosis factor blocker,
besides physical therapy. Among the 20 patients tested, there were 13 drug-responders and seven non-responders after 1 month post-treatment. Clinical measures and the frequency of TFR cells in the 13 drug-responders were analysed after treatment for 1 month. The treatment significantly relieved clinical symptoms, and reduced the values of WBC, CRP, ESR, IgA, IgG and BASDAI (P < 0.05; Table 1), although there was no significant change in the computer tomography (CT) scanning after treatment (Fig. 1c). The percentage of FOXP3+CXCR5+ CD4+TFR cells (P = 0.0006, Fig. 3b) and the ratio of TFR-toTFH cells (P < 0.0001; Fig. 3d) in drug-responding AS patients were significantly higher as compared with those before treatment. The frequency of CXCR5+CD4+TFH cells decreased after treatment in drug-responders, but this change had no
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Activated TFR cells in AS patients (a)
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Fig. 3 Treatment modulates the frequency of follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH, the levels of serum interleukin (IL)-21 cells in drug-responding patients. (a) Flow cytometry analysis. In all follow up, a total of 13 patients were drug responsive after 1 month (1M) of drug therapy. The frequency of (b) TFR cells, (c) TFH cells in total CD4+ T cells, and (d) the ratio of TFR-to-TFH cells in those patients were determined by flow cytometry analysis before (0 month (0M)) and after treatment (1 month). (e) The levels of serum IL-21 in those patients were determined by enzyme-linked immunosorbent assay.
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Fig. 4b), the ratio of TFR-to-TFH (P = 0.085; Fig. 4c) and the levels of serum IL-21 (P = 0.277, Fig. 4d) in non-responding patients after treatment. Thus, the standard treatment might promote TFR cell numbers and activities in drug-responding AS patients.
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Frequency of peripheral blood TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 in these patients after 1 month of standard treatment
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Fig. 4 The frequency change of follicular regulatory T (TFR) cells, follicular helper T (TFH) cells, the ratio of TFR-to-TFH, and the levels of serum interleukin (IL)-21 in non-responding patients after treatment. In all follow-up, a total of seven patients were drug nonresponsive after 1 month of drug therapy. The (a) frequency of TFR cells, (b) TFH cells in total CD4+ T cells, and (c) the ratio of TFR-to-TFH cells in those patients were determined by flow cytometry analysis before (0 month (0M)) and after treatment (1 month (1M)). The levels of serum IL-21 in those patients were determined by enzyme-linked immunosorbent assay.
To investigate whether TFR cells limit TFH cell numbers in AS patients, it was found that the frequency of peripheral blood TFR cells was negatively correlated with that of TFH cells after 1 month of standard treatment (r = 0.550, P = 0.046; Fig. 5a), though there was no significant correlation before treatment. Furthermore, we found the frequency of peripheral blood TFR cells was negatively correlated with the concentration of serum IL-21 in drug-responders after treatment (r = 0.581, P = 0.0371; Fig. 5b). However, there was no significant correlation between the frequency of peripheral blood TFR cells and the concentration of serum IL-21 after treatment in non-responding patients (r = 0.666, P = 0.102; Fig. 5c). Thus, the administration of 1 month of standard treatment might promote TFR cells to limit the number and the secretory activity of TFH cells.
DISCUSSION statistical significance (P = 0.1649; Fig. 3c). However, the concentration of serum IL-21 decreased as compared with that before treatment in drug-responding AS patients (P = 0.0049; Fig. 3e). We did not detect significant changes in the frequency of TFR cells (P = 0.406; Fig. 4a), TFH cells (P = 0.110;
In the present study, we found that the frequency of peripheral blood FOXP3+CD4+CXCR5+TFR cells, CD4+CXCR5+TFH cells, the ratio of TFR-to-TFH cells and the concentration of serum IL-21 in the AS patients were significantly higher than in the HC. Our previous study found that the elevated TFH cells
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Fig. 5 The correlation of the frequency peripheral blood follicular regulatory T (TFR) cells with follicular helper T (TFH) cells and the concentration of serum interleukin (IL)-21 in those patients after treatment. (a) The frequency of peripheral blood TFR cells are negatively correlated with TFH cells in drug-responding ankylosing spondylitis (AS) patients after 1 month (1M) of standard treatment. (b) The frequency of peripheral blood TFR cells are negatively correlated with the concentration of serum IL-21 in drug-responding AS patients after 1 month of standard treatment. (c) The frequency of peripheral blood TFR cells are not significantly correlated with the concentration of serum IL-21 in non-responding AS patients after 1 month of standard treatment.
might contribute to the pathogenesis of AS.13 Because of the suppressive function of TFR cells on TFH cells, the higher frequency of TFR cells and ratio of TFR-to-TFH cells in the AS patients suggest that TFR cells might be also involved the pathogenesis of AS. Foxp3+Treg cells are crucial in preventing autoantibody-mediated autoimmunity.16,20 Recent studies have shown that TFR cells are a specialized subset of Foxp3+Treg cells expressing BCL-6 and CXCR5. Foxp3 is a key factor for the development and function of Treg.21–23 The interaction between CXCR5 and CXCL13 mediates homing of lymphocytes to B cell follicle areas, and Bcl-6 also promotes the expression of CXCR5 on TFR cells, as they do in TFH cells.24–27 Several studies of patients with connective tissue diseases have found that reduced or functionally impaired Treg cells, and Treg cells of autoimmune hepatitis patients reducing expression of Fox-P3 and CTLA-4, which might lead to impaired suppressor activity.28–30 Cua et al.31 also found that skewing of responses towards Th17 and away from BMSC or Treg cells could be responsible for the progression of AS. However, the present study found there were significantly elevated TFR cells in AS patients compared with that of HC, although the percentage of BCL-6+ TFR cells had almost no change between the two groups. Chung et al.19 showed that Bcl-6 is essential for the generation of CXCR5+Treg cells in their study. However, only Bcl-6 is not sufficient for CXCR5 expression and the function of TFR cells. For instance, Linterman MA et al. suggested that Bcl-6 was essential for TFR cell formation, and Blimp-1 expression regulates the size of the TFR cell population.32 It shows that multiple signals regulate the function of TFR cells. Different signals of TFR cells and their interaction in AS require further study. In our previous and present study, a higher frequency of peripheral blood TFH cells was detected in AS patients compared with that of HC.13 Treg are crucial for restoring immune tolerance in chronic inflammation. Mills showed that immune responses can result in collateral damage to host tissues, and immune-regulatory mechanisms, including the induction of Treg, are essential to control this immunopathology.33 As infection and autoimmune disorders are
involved in the pathogenesis progress of AS, the similar immuneregulatory mechanisms might exist in the progress of AS. Because of the suppressive function of TFR cells on TFH cells and the participation of TFH cells in AS, the increased frequency of TFR cells might stem from compensative spontaneous regulation of the immune system, so that increased frequency of TFR cells negatively regulates autoimmune responses in AS patients. Thus, TFR cells and TFH cells are both involved in the pathogenesis of AS. To further understand the role of TFR cells and the interrelationship between TFR cells and TFH cells in the development and progression of AS, we characterized the ratio of TFR-to-TFH cells in this population and found that it was significantly higher than that in the HC. TFH cells are a subset of CD4+ T cells, and have been shown to be crucial regulators of B cell differentiation, antibody production and humoral immunity.12 Although the percentages of IL-21+TFH cells in AS patients were positively correlated with BASDI in our previous study, there was no significant correlation between TFR cells and BASDAI.13 However, notably, in the present study, we found that the percentage of TFR cells was negatively correlated with serum IgA. Indeed, Wollenberg et al.17 also found Foxp3+ follicular T cells can limit the amount of secreted Ag-specific IgM, IgG1, IgG2b and IgA in vivo. Therefore, our data suggest that TFR cells might control the autoantibodies and be involved in the pathogenesis of AS. TFR and TFH cells could affect each other in AS, and this activity plays an important role in the pathogenesis of AS. Currently, NSAIDs, pain killers, steroids, immunosuppressant and tumour necrosis factor-a blockers have been used for the standard treatment of AS patients.34–37 In the present study, we examined how the standard therapy could affect TFR cells. Analysis of the available data from 13 drug-responding patients showed that treatment with the standard therapy for 1 month significantly reduced ESR, the WBC counts, and the levels of serum CRP, IgG and IgA, and also significantly minimized the scores of BASDAI. In Fig. 1b, we can find sclerotin under the cartilage is severely invaded, hyperosteogeny and sclerosis are detected, and irregular stenosis exists in sacroiliac joint space.
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Activated TFR cells in AS patients The CT graph in Fig. 1b can be defined as grade 3 according to the 1984 modified New York criteria. After 1 month treatment, the sclerotin invasion, hyperosteogeny and sclerosis, and irregular stenosis in sacroiliac joint space still exist in Fig. 1c. The grade of CT graph in Fig. 1c is also 3. Similar changes were shown in the CT scanning of all the AS patients. Thus, there was no significant change in the CT scanning after treatment. Although we found a positive correlation between the percentage of IL-21+TFH cells and the value of BASDAI after treatment in our previous study, the recent reports have shown the suppressive function of TFR cells on TFH cells in vivo.13,17–19 Notably, we detected a higher frequency of peripheral blood TFR cells and a higher ratio of TFR-to-TFH cells in AS patients after treatment, and found there was a negative correlation between TFR cells and TFH cells. In parallel, the concentration of serum IL-21 decreased compared with that before treatment, and TFR cells were negatively correlated with the concentration of serum IL-21 after treatment. As known, AS is a chronic autoimmune inflammatory disease, but inflammatory stimuli can downregulate the expression of Foxp3 and affect the inhibitory activity of Treg.38 The anti-inflammatory treatments might promote the inhibitory activity of TFR cells to TFH cells by change inflammatory state in AS patients. These data suggested that the administration of 1 month of standard treatment might promote the activity of TFR cells to limit the number and secretory activity of TFH cells for controlling the progression of AS. The interreaction of TFR–TFH cells could take an important part in the development of AS. In conclusion, the present data showed that the percentages of TFR cells and TFH cells, the ratio of TFR-to-TFH cells, and the concentration of serum IL-21 significantly increased in AS patients compared with those of HC, and the frequency of TFR cells was negatively correlated with the serum IgA in those patients. Treatment with 1 month of standard therapy dramatically controlled the disease progression, the treatment significantly elevated the frequency of TFR cells and the ratio of TFR-to-TFH cells, and reduced the concentration of serum IL-21. The frequency of TFR cells was negatively correlated with TFH cells and the concentration of serum IL-21 after treatment. TFH cells are involved in the development of AS. Therefore, the TFR cells might be responsible for controlling the autoantibodies, and the frequency and function of TFH cells to inhibit the development of AS. The interaction of TFR–TFH cells might take an important part in the development of AS. We recognized that the present study had limitations of a small sample size, the lack of functional studies of TFR cells and other pro-inflammatory cytokines in AS. Therefore, further studies of TFR cells and other pro-inflammatory cytokines in the pathogenic process with a bigger population are warranted.
METHODS Patients and controls A total of 20 newly diagnosed patients with AS were recruited sequentially at the inpatient service of the First Hospital of Jilin University, Changchun, China. A total of 10 sex-, age- and ethnicity-matched HC were recruited. Written informed consent was obtained from individual patients, and the experimental protocol
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was approved by the ethics committee of the First Hospital of Jilin University. Individual patients with AS were diagnosed, according to the 1984 modified New York criteria.39 None of the patients took steroids or other immunosuppressants before their blood samples were collected. The disease activity of individual patients was evaluated for the BASDAI using a questionnaire with six questions related to: (i) fatigue; (ii) spinal pain; (iii) peripheral joint pain; (iv) enthesitis; and (v) morning stiffness, as well as the intensity and duration, respectively, as described previously.40 The scores for each item ranged from 0 to 10, and high disease activity was defined as a BASDAI score ≥ 4. Individual participants with rheumatoid arthritis, multiple sclerosis, type 1 diabetes, immunodeficiency, chronic inflammatory diseases and recent infection were excluded. Their demographic and clinical characteristics are shown in Table 1. After hospitalization, individual patients were orally treated with 15 mg meloxicam (Boehringer Ingelheim, Shanghai, China) and 50 mg thalidomide daily (Changzhou Pharmaceutical Factory, Changzhou, China), and subcutaneously injected with 25 mg etanercept (Wyeth, Suzhou, China) every 2 weeks. In addition, some patients received symptomatic treatment. Drug responders were classified according to the ASAS20 (response criteria of the Assessment of SpondyloArthritis International Society) after 1 month of treatment.41 Laboratory examinations Fasted venous blood samples were obtained from individual participants before treatment and after 1 month of treatment, and the sera were prepared. The number of WBC, the ESR, and the concentration of serum CRP, IgG, IgA and IgM of individual participants were measured by an analyser (Siemens Healthcare Diagnostics Products, GmbH, Munich, Germany) and enzymespecific immunosorbent assay (ELISA) using specific kits. Individual patients were subjected to CT examination for their sacroiliac joints on a Siemens CT system (Angio CT Miyaby, Berlin, Germany). Flow cytometry analysis The HLA-B27 status in individual participants was characterized by flow cytometry analysis.42 Briefly, peripheral blood mononuclear cells were stained with fluorescein isothiocyanate (FITC)anti-human leococyte antigen (HLA)-B27 and P-phycoerythrin (PE)-anti-CD3, then subjected to flow cytometry analysis on a fluorescence-activated cell sorting (FACS) Calibur (BecktonDickinson, San Diego, CA, USA). Human peripheral blood mononuclear cells at 5 9 105/tube were stained with FITC-antiBCL6, Alexefluor647-anti-CXCR5 and PE-CY7-anti-CD4 (BD PharMingen, San Diego, CA, USA) at dark room temperature for 30 min. Then, the stained cells were fixed, permeabilized and stained intracytoplasmically with PE-anti-Foxp3. Control cells were stained with FITC-anti-IgG1, PE-anti-IgG1, PE-CY7-antiIgG1 and Alexefluor647-anti-IgG1 (BD PharMingen). After washing, the cells were characterized by using a FACS Calibur (Beckton-Dickinson) and FLOWJO software (v7.6.2; TreeStar, Ashland, OR, USA).43 At least 20 000 events of each sample were acquired for analysis.
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Y Shan et al.
160 Measurement of serum IL-21 by ELISA
Serum IL-21 concentrations were measured by a human IL-21 ELISA kit following the manufacturer’s instructions (Roche Diagnostics, Lewes, UK). Briefly, sera were diluted to 1 : 4, and concentrations were calculated with the standard curve generated with the provided recombinant IL-21. The detection limit of the IL-21 ELISA kit was 0.5 ng/L. Statistical analysis Data was expressed as median and range or individual values. The difference between the groups was analysed by Man–Whitney U-nonparametric test using the SPSS 19.0 software (SPSS, Chicago, IL, USA), and differences in individuals before and after treatment were evaluated by using the Wilcoxon matchedpair t-test. The relationship between variables was evaluated by using the Pearson rank correlation test. A two-side P-value of < 0.05 was considered as statistically significant.
ACKNOWLEDGEMENTS This study was supported by grants from the National Natural Science Foundation of China (No. 30972610 and 81273240), Jilin Province Science and Technology Agency (No. 20110716), the Health Department Research Projects in Jilin Province (2009Z054) and Norman Bethune Program of Jilin University (2012206).
DISCLOSURE The authors declare no conflict of interest.
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