Aberrant alteration of follicular T helper cells in ulcerative colitis patients and its correlations with interleukin-21 and B cell subsets

Abstract Patients with ulcerative colitis (UC) are at increased risk of developing colitis-associated colon cancer. Accumulating evidence suggests that follicular T helper (TFH) cells play a crucial role in the pathogenic process of autoimmune diseases. However, little is known about the role of TFH cells in the development of UC. To investigate the role of TFH cells in the development of UC, the number of TFH cells, the level of interleukin-21 (IL-21), the numbers of B cell subsets, and clinical parameters were detected in peripheral blood from 31 UC patients and 29 healthy controls. TFH cells and the level of IL-21 were significantly higher in UC patients than in the healthy controls. A positive correlation between TFH and IL-21 cells was found in UC patients. Moreover, aberrant frequencies of different subsets of B cells were observed in UC patients, and a positive correlation was found between CD38+CD19+ B cells and TFH cells and between CD86+CD19+ B cells and TFH cells. A high number of TFH cells were positively associated with Mayo score, serum C-reaction protein (CRP) and serum IgG in UC patients. Our data indicate that TFH cells and IL-21 are involved in the pathogenesis of UC.


Introduction
Ulcerative colitis (UC) is one of the major forms of inflammatory bowel diseases (IBDs) in humans. Patients with ulcerative colitis have an increased risk of developing colon cancer (CAC), [1] and the risk rank is correlated with the duration and severity of the disease. [2] Although the etiology and pathogenic process of UC remain unclarified, recent studies suggest that the abnormal interplay between T cells and other immune and nonimmune cells is pivotal in driving pathologic processes of UC, [3,4] and T cellderived cytokines are essential mediators of this cross-talk. [5] The primary function of CD4 + T H cells is to help the immune system fight infections. This function relies on the flexibility of naïve CD4 + T cells, which differentiate into diverse T H cell subsets with specialized effector function by distinct pathogens. [6] Under pathogenic conditions, aberrant levels of some cytokines promote the differentiation or function of specific effector T cell populations, which may unbalance the intestinal immune system and bring about a state of inflammation. [3,4] Moreover, the differentiation of B cells to antibody-secreting cells requires the assistance of T H cell subsets, and then, the production of neutralizing antibodies provides lasting protection against most pathogen infections. In this interplay, follicular T helper (T FH ) cells play a key role in the development of antigen-specific B cells. [7,8] T FH cells are a specialized subset of CD4 + helper T cells located in germinal centres (GC) and are classically identified by their combined surface expression of inducible costimulator (ICOS), C-X-C chemokine receptor type 5 (CXC-R5), and programmed cell death-1 (PD-1), as well as the intracellular expression of the transcription factor Bcl-6. [9][10][11][12] Notably, as shown recently, T FH cells secrete IL-21 to modulate both humoural and cell-mediated immune responses in different murine and human autoimmune diseases, including systemic lupus erythaematosus (SLE), Sjögren's syndrome (SS), and rheumatoid arthritis (RA). [13][14][15] However, little is known about how these different subsets of T FH cells act in UC patients and whether T FH cells are involved in the pathogenesis of UC. IL-21 is a pleiotropic cytokine synthesized not only by T FH cells but also by T H 1 and T H 17 cells in the germinal centres. [14] IL-21 regulates the activation, proliferation, and survival of both CD4 + T cells and B cells, as well as the functional activity of CD8 + T cells and NK cells, and it limits the differentiation of inducible regulatory T cell. [16] Clearly, IL-21 is involved in the regulation of central functions of the immune system. Elevated IL-21 has been observed in the gut of patients with IBD, especially in Crohn's disease (CD), which is thought to be caused by an exaggerated Th1 response against the luminal flora, [5,17] whereas there are limited data about IL-21 in patients with UC.
In this context, to further investigate the potential relationship between the T FH cells and clinical parameters of UC, we measured the proportions of circulating T FH cells and activated B cells in patients with UC compared to those of healthy controls and analysed the correlation between the population ratio of T FH cells and the IL-21 level, the proportion of B cell subsets, Mayo score, and other clinical-pathological features.

Patients
Patients gave informed consent for their samples to be analyzed in accordance with the Declaration of Helsinki. The study was approved by the Human Ethics Committee of Jiujiang First People's Hospital (Jiangxi, China). A total of 31 untreated UC patients and 29 sex-and age-matched healthy controls were recruited from our hospital. The diagnosis and Mayo score of 31 UC patients had been established by clinical, endoscopic, histological, and/or radiological criteria. Infection or the presence of parasites was excluded by stool culture and microscopic examination. The disease activity of active UC was determined using a grading scale including clinical and para-clinical variables. Another 29 subjects without prior history of chronic disease, autoimmune disease, cancer or recent infection were included as a healthy control group. Peripheral blood samples were collected from participants for in vitro cell culture and clinical analysis.

Blood sampling and analyses
Fasting venous blood samples were collected from individual healthy controls and UC patients. A portion of blood was used to isolate peripheral blood mononuclear cells (PBMCs) by densitygradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare Bio-science, Sweden), and the surplus blood samples were centrifuged for preparing to examine serum IL-21, IgA, IgM, and IgG.

Enzyme-linked immunosorbent assay (ELISA) for measurement of serum IL-21
The concentration of serum IL-21 in individual UC patients and HC was determined by ELISA using a human IL-21 ELISA kit (eBioscience) in accordance with the manufacturer's instructions. Individual serum at 1:4 dilutions were subjected to ELISA, and the concentrations of serum IL-21 in individual samples were calculated according to the standard curve established using the recombinant IL-21 provided.

Scattering nephlometry for measurement of serum IgA, IgM, and IgG
The concentrations of serum IgA, IgM, and IgG in individual UC patients and HC were examined by scattering nephlometry using an IMMAGE800 biochemical analyzer and matched reagents (Beckman Coulter) in accordance with the manufacturer's instructions.

Statistical analysis
Statistical analyses were performed by the GraphPad Prism 6.0 software (La Jolla, CA). The data are expressed as median and range unless specified. The difference between two groups was analysed by the Mann-Whitney U test. The relationship between variables was evaluated by Pearson's rank correlation test. A twosided P value of <.05 was considered statistically significant.

Patient characteristics
To assess the potential role of T FH cells in the pathogenesis of UC, 31 Chinese patients with UC before treatment and 29 sex-and  , between these 2 groups. As expected, the concentrations of serum C-reactive protein (CRP) and IgG and the erythrocyte sedimentation rate (ESR) were significantly higher in the UC patients than those in the HCs ( Table 1), suggesting that those patients were in a state of immune imbalance.

Increased proportions of circulating T FH and T H 17 cells in UC patients
The numbers of T FH cells and T H 17 cells were analysed by flow cytometry (Fig. 1A). As shown in Figure 1B to D, the numbers of peripheral blood CD3 + CD4 + IL-17A + T H 17 cells, CD3 + CD4 + CXCR5 + T FH cells and CD3 + CD4 + CXCR5 + ICOS + T FH cells were significantly higher in the UC patients than those in the HCs (p<0.01, Fig. 1b-d). The higher numbers of CD4 + CXCR5 + ICOS + T FH cells and CD4 + IL-17A + T H 17 cells may contribute to the development of UC.

Elevated level of serum IL-21 is correlated to T FH cells in UC patients
To explore the correlation between the level of IL-21 and the number of T cell subsets in UC, we detected their serum concentrations via ELISA, and correlation analysis was performed with Pearson's rank correlation. The level of IL-21 was significantly higher in UC patients than that in HCs (P < .01, Fig. 2A). There was no correlation between the level of IL-21 and the CD4 + CXCR5 + T FH cell proportion (data not shown). Interestingly, we observed a positive correlation between the level of IL-21 and the percentage of CD4 + CXCR5 + ICOS + T FH cells in UC patients (P = .0002, r = 0.6130, Fig. 2C) but no correlation between the level of IL-21 and the percentage of CD4 + IL-17A + T H 17 cells (P > .05, r = 0.0990, Fig. 2B). Our data indicate that IL-21 may be predominantly secreted by CD4 + CXCR5 + ICOS + T FH cells in UC patients, and this T FH subset and IL-21 may participate in the development of UC.

Aberrant frequencies of different subsets of B cells in UC patients
We next characterized the frequencies of different stages of B cells by flow cytometry (Fig. 3A) to assess the impact of B cells in the pathogenic process of UC. The percentages of CD38 + CD19 + B cells and CD86 + CD19 + B cells in the CD19 + B cell population of the UC patients were significantly higher than those in the HCs (P < .01, Fig. 3B and C). Interestingly, the percentage of CD27 + CD19 + B cells in the UC patients was reduced compared to that in the healthy controls (P < .01, Fig. 3D). These data indicate that different subpopulations of B cells may contribute to the pathogenesis in UC patients.

The relationship between T FH cells and B cell subsets in patients with UC
T FH cells can provide help for B cells by secreting cytokines and stimulating their surface molecules. [7,9] We found that the percentage of CD38 + CD19 + B cells in the CD19 + B cell population was positively correlated with the percentage of CD4 + CXCR5 + ICOS + T FH cells in the CD4 + T H cell population of UC patients (P < .0001, r = 0.6188 Fig. 4A), and the percentage of CD86 + CD19 + B cells was positively correlated with the percentage of CD4 + CXCR5 + ICOS + T FH cells in UC patients (P = .0044, r = 0.4973, Fig. 4C). The percentage of CD86 + CD19 + B cells was negatively correlated with that of CD4 + CXCR5 + I-COS + T FH cells in UC patients (P = .0080, r = À0.4674, Fig. 4B).

The relationship between CD4 + CXCR5 + ICOS + T FH cells and clinicopathologic parameters of UC patients
Having validated the relationship between T FH cells and the clinical-pathological features of UC patients, we examined the correlations of abnormal clinical laboratory parameters with the number of T FH cells in UC patients. As shown in Figure 5, the concentrations of serum CRP and IgG and the ESR of UC patients were significantly increased compared to those of the HCs (P < .0001, Fig. 5A, B, and E). No significant difference was found in the concentration of serum IgA (P = .0790, Fig. 5C) or IgM between groups (P = .5245, Fig. 5D). Importantly, compared

Discussion
Increasing evidence shows that the pathogenesis of IBD is associated with immunological abnormalities. [4,5,18] T FH cells, as a specific subset of CD4 + T helper cells, play an important role in immune balance. [11] T FH cells are involved in the pathogenesis of common autoimmune diseases; [15] however, study about T FH cells in UC disease is lacking. In the present study, we found that T FH cells and T H 17 cells were significantly more abundant in the peripheral blood of UC patients than in the HCs. The data suggest T FH cells and T H 17 cells may take part in the development of UC.
In addition, T FH cells express high levels of CXCR5, which provides essential help for B cells to produce antibodies and for GCs to produce plasma and memory B cells. [9,19] We found that levels of CD4 + CXCR5 + T FH cells were significantly elevated in the blood of UC patients, but there was no correlation with the level of IL-21. ICOS is expressed in activated T FH cells, which stimulates the differentiation of activated T FH cells through enhanced IL-21 expression and facilitates B cell antibody production. [20] ICOS deletion leads to a profound decrease in memory B cell formation and to a switched antibody response in humans. [21] Our data show that circulating CD4 + CXCR5 + ICOS + T FH cell and serum IgG levels were higher in UC patients than those in HCs, and a linear positive correlation existed between them, which indicated that ICOS may stimulate B cells to produce IgG to induce the development of UC.
IL-21 is produced mostly by activated CD4 + T cells and controls the differentiation and functional activity of effector T H  cells, [14] whereas a high level of IL-21 triggers the inflammatory pathway and promotes tissue damage in some autoimmune diseases. [22][23][24][25] A study by Ge et al [26] reported that elevated intracellular IL-21 was correlated to the T H 17 cell proportion in UC patients, suggesting that IL-21 is mainly produced by T H 17 cells in UC. Nevertheless, our results demonstrate that the IL-21 level was significantly higher in the UC patients than in the HCs, and no correlation was found between serum IL-21 levels and T H 17 cells in UC patients, while a significant positive correlation between IL-21 and CD4 + CXCR5 + ICOS + T FH cells was observed. Yu et al [27] reported elevated levels of T FH cells and IL-21 in tissues of UC patients and that IL-21-deficient mice were resistant to dextra sulfate sodium (DSS)-induced colitis and produced fewer T FH cells. In combination with our data, these results indicate CD4 + CXCR5 + ICOS + T FH cells may be more closely related to the level of IL-21 than to other IL-21-synthesizing cells, and the secretion of IL-21 could be mainly through T FH cells in UC patients. Certainly, in this regard, the main source of IL-21 in the UC pathogenic process necessitates further investigation, and more research is required to reveal the underlying mechanism of interaction between T FH cells and IL-21 in UC. A dysregulated frequency of B cell subsets has been implicated in UC, [3,4] but the precise mechanism remains unclarified. There is general agreement that activated B cells highly express the CD86 costimulatory molecule, and CD86 + B cells can differentiate into CD38 + plasma B cells and then produce antibodies, such as IgM, IgG and IgA. Memory B cells express CD27 and can rapidly respond to antigens and generate immunoglobulin during secondary immune responses. In this study, the levels of circulating CD86 + CD19 + activated B cells and CD38 + CD19 + plasma B cells in UC patients were significantly higher than those in HCs, and levels of peripheral blood CD27 + CD19 + memory B cells in UC patients were reduced compared to those of healthy controls. These data are consistent with previous studies, [3,28] suggesting that inflammation may promote the development and redistribution of these three subsets of B cells. Alternatively, memory B cells may not play a major role in the pathogenesis of UC. Furthermore, through correlation analysis between T FH cells and B cell subsets, we found that the proportions of activated B cells and plasm B cells were positively correlated that of T FH cells and that the proportion of memory B cells was negatively correlated with that of T FH cells. The basal level of IgG is highly dependent on T H cells, [29] and IgG is reduced markedly in the serum of ICOS -/mice. [30] Similarly, we found that elevated serum IgG was correlated with the proportion of CD4 + CXCR5 + ICOS + T FH cells. These findings indicate that T FH cells may represent a better biomarker to assess the dysregulation of B cell response in UC.
CRP is a nonspecific marker of systemic inflammation. A high level of CRP has been observed in IBD. [31] We found that elevated CRP was positively correlated with elevated T FH cells in UC patients, implying that the T FH cells may have the potential to be a specific marker to estimate the inflammation degree of UC patients. Furthermore, the Mayo score, as a common disease activity index, provides a unified and standard assessment for the severity of UC, including stool frequency, rectal bleeding, mucosal appearance and physician rating of disease activity. However, measuring the Mayo score requires invasive examination and specialized calculation, which is subjective and slightly inaccurate. Our present study found that the Mayo score was well correlated with T FH cell proportion, suggesting that T FH cell percentage may have potential as a noninvasive biomarker to evaluate the activity of UC. Much more exploration is required to verify this hypothesis.
Collectively, our work demonstrates that circulating T FH cells, activated B cells and plasma B cells may participate in the pathogenesis and development of UC, and IL-21 may be a crucial cytokine in the dysimmunity of UC. In UC, the level of IL-21 was strongly correlated with the level of T FH cells, but not with T H 17 cells. T FH cells might become a novel noninvasive biomarker to assess immune state and the disease activity of UC. Further evidence on the mechanism and aetiology of UC await further study.