Regulatory Effect of Sishen Pill on Tfh Cells in Mice With Experimental Colitis

The T follicular helper T (Tfh) cells play a significant role in the pathogenesis of inflammatory bowel disease (IBD), which is regulated by the Bcl-6/Blimp-1 pathway. Some studies have suggested that regulating activation of the Bcl-6/Blimp-1 pathway should be an effective method to treat IBD. Sishen Pill (SSP) has been used frequently to treat chronic colitis. Its mechanism is related to the downstream proteins in the Bcl-6/Blimp-1 pathway. However, it is unknown whether SSP regulates the function and differentiation of Tfh cells to treat IBD. In the present study, chronic colitis was induced by dextran sodium sulfate and treated with SSP for 7 days. SSP effectively treated chronic colitis, regulated the balance between Tfh10, Tfh17 and T follicular regulatory cells, while SSP increased the Blimp-1 level, inhibited expressions of Bcl-6, T-cell costimulator, programmed death (PD)-1 and PD-ligand 1 on the surface of Tfh cells. SSP inhibited activation of BcL-6, phosphorylated signal transducer and activator of transcription (p-STAT)3, signal lymphocyte activation molecule (SLAM)-associated protein but improved Blimp-1 and STAT3 expression in colonic tissues. The results indicated that SSP regulated the differentiation and function of Tfh cells to treat IBD, which was potentially related with inhibiting the Bcl-6/Blimp-1 pathway.


INTRODUCTION
Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), are characterized by idiopathic chronic intestinal inflammation (Sairenji et al., 2017). Their pathogenic factors are closely related to the environment, microorganisms, genes, and immunity (Zhang and Li, 2014;Kelsen and Sullivan, 2017). In recent years, a large number of studies have found that abnormal function and differentiation of T follicular helper (Tfh) cells can lead to autoimmune diseases including IBD (Mesquita et al., 2016;Ueno, 2016).
Ten years ago, many researchers found a new subpopulation of CD4 + T cells, Tfh cells, localized to B cell follicles and germinal centers (GCs) (Breitfeld et al., 2000;Schaerli et al., 2000). Tfh cell differentiation is mainly divided into three phases (Crotty, 2014). The first phase is early Tfh cell differentiation, which is mainly performed by dendritic cells (DCs).
Many studies have shown that high levels of T-cell receptor can lead to continuous interaction between Tfh cells and DCs (Tubo et al., 2013). The second phase is the finish of Tfh cells differentiation, which is located at the T cell and B cell accumulation area by chemokine CXC receptor (CXCR)5 expression (Webb and Linterman, 2017). The third phase is mainly located at the GC, which can highly express CXCR5 and programmed death (PD)-1, while other markers mainly include CXCR5, GL7, and inducible T-cell costimulator (ICOS). Tfh cells are located at the follicular area of the mutant B cells, secrete interleukin (IL)-21, induce expression of transcription factor Bcl-6, and promote high-affinity B cell clones (Weinstein et al., 2016). Functionally, Tfh cells are divided into Tfh1, Tfh2, Tfh10, Tfh17, Tfh21, and Tfr cell lineages, which respectively produce or express interferon (IFN)-γ, IL-4, IL-10, IL-17, IL-21, and forkhead box (FOX) P3, and these are closely related to colitis (Fazilleau et al., 2009;Crotty, 2014;Jandl et al., 2017).
It has been shown that high expression of Tfh-cell-related genes leads to development of IBD . Clinical trials have shown that the balance of Tfr and Tfh cells is closely related to IBD (Wang et al., 2019b). In IBD patients, FOXP3 and IL-10 levels decrease while IL-21 level increases, which are positively correlated with the decreased ratio of Tfr/ Tfh cells (Wang et al., 2017). Liu et al. demonstrated that Tfh cell population was increased in mice with dextran sulfate sodium (DSS)-induced colitis, and then reduced after the colonic damage was alleviated by probiotics . The results suggest that Tfh cells may become a new target for the treatment of IBD.
In China, Sishen Pill (SSP) is the classic prescription of traditional Chinese medicine (TCM) for 1,000 years, and frequently-used to treat IBD, allergic colitis, chronic colitis, irritable bowel syndrome (IBS), and so on (Wang et al., 2019a). In clinic, SSP is widely accepted by IBD patients for definite therapeutic effect, little adverse reaction, and lower recurrence rate (Lu et al., 2011). In previous studies, SSP was found to be effectively treat colitis in rats by inhibiting signal transducer and activator of transcription (STAT)3, IFN-γ, IL-1β, IL-2, IL-6, and IL-17 expressions, and increase IL-4 and transform growth factor (TGF)-β expression Zhao et al., 2019). These cytokines are associated with Tfh cells, but it is not known whether SSP can regulate Tfh cells to treat IBD. In the present study, the mechanism underlying the anti-colitis effect of SSP was investigated in DSS-induced colitis in mice.

Animals
Male BALB/C mice weighing 18-22 g (Animal Certificate No. SCXK 2006-0008) were purchased from Hunan Slake Jing da Experimental Animal Co., Ltd. (Changsha, China). All animals were housed in specific-pathogen-free conditions, with standard laboratory diet, 12-h light/dark cycle and constant room temperature, and had free access to standard diet and tap water according to the guidelines of the Animal Center. This Protocol (License No.: JZ2018-105) was approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangxi University of Traditional Chinese Medicine. The mice were acclimated for 3 days according to the IACUC Animal Welfare Guidelines before formal experiments were performed. Thirty-two mice were divided into two groups: eight mice were in the normal group and the remaining mice were treated by DSS to induce colitis. The twenty-four mice were observed to have bloody stools on the fourth or fifth day after DSS treatment, which hinted the colitis was successfully induced. Twenty-four colitis mice were randomly divided into three groups: DSS: colitis without treatment; DSS + SSP: colitis treated with SSP; and DSS + 5-ASA: colitis treated with 5-aminosalicylic acid (ASA). Eight colitis mice were in every group.

DSS-Induced Colitis
According to the previous study (Yoshihara et al., 2006), colitis was induced in BALB/C mice with 3% (w/v) DSS (molecular weight: 36,000-50,000 kDa) dissolved in deionized water drunk ad libitum (days 1-7). Fresh 3% DSS solutions were made every morning in deionized water. Control mice were given tap water.

Therapeutic Protocols
On day 8, the DSS + SSP group was administered 2.5 g kg −1 SSP dissolved in physiological saline by oral gavage for 7 days; the DSS + 5-ASA group was administered 300 mg kg −1 mesalazine by oral gavage for 7 days; and mice in DSS and normal groups were treated with the same volume of physiological saline by oral gavage for 7 days. On day 15, all animals were sacrificed under sodium pentobarbital (50 mg/kg ip) anesthesia.

Macroscopic Evaluation
The mice were weighed before anesthesia, and the colons were quickly removed. The colon length and weight were measured, and the colon weight index (CWI), CWI = colon weight/body weight × 100 was calculated.

Enzyme-Linked Immunosorbent Assay
The colon tissue of the mice was collected, and the RIPA tissue lysate was added at a ratio of 1:10, incubated at 4°C for 1 h, homogenized by ultrasonic homogenizer for 20 min, centrifuged at 4°C at 19,375 g for 30 min, and the supernatant was obtained and analyzed. The levels of , , , , and IL-23 (No. 88-7230-88) (eBioscience, San Diego, CA, USA) and TGF-β1 (No. 555052) (BD Biosciences, Franklin Lakes, NJ, USA) were measured by commercial ELISA kits, and absorbance at 450 nm was read using a microplate reader (Bio-Rad, Hemel Hempstead, UK).

Flow Cytometry
For isolation of peripheral lymphocytes, 500 μl peripheral blood were collected from each mouse and lysed by treatment with 1 ml lysing Solution (BD Biosciences, Franklin Lakes, NJ, USA) to clear red blood cells. The obtained lymphocytes were incubated with fluorescence-conjugated monoclonal antibodies in staining buffer. Eight-color flow cytometry analysis (n = 8) was performed on a FACSCalibur device (Becton Dickinson, Mountain View, CA, USA). The frequency of positive cells was analyzed using the program Cell Quest in two regions. The lymphocyte region was determined using granularity (SSC) and size (FSC) plots. Tfh cells were identified as a CD4 + lineage + (CXCR5 + , ICOS + , FOXP3 + , IL-10 + , IL-17 + , and BCL-6 + ) population, and within this group, the CD4 + population was assessed. The following monoclonal antibodies were used: APC-H7 anti-mouse CD4 (

Statistical Analysis
All statistical analyses were performed using GraphPad Prism software, using one-way analysis of variance followed by a Tukey's test of multiple comparisons. p < 0.05 indicated that the difference was significant.

SSP Ameliorated DSS-Induced Colitis
DSS-induced colitis is a classic model to develop new drugs for treatment of IBD. After 4-5 days' administration of 3% DSS, bloody stools were observed via the naked eye, body weight decreased, and hair was sparse and lusterless. Mice in the DSS group had significantly reduced body weight compared to the normal group ( Figure 1A), while their colon length was shorter (Figures 1D,E), and the colonic weight and colon weight index were significantly increased (Figures 1B,C). Pathological observation showed ulcer formation, serious hyperemia and edema, and many inflammatory cells in the colonic mucosa ( Figure 1F). The pathological damage score was increased compared with that in the normal group ( Figure 1G). After 7 days' treatment with SSP or 5-ASA, compared with the DSS group, the body weight was increased ( Figure 1A) in the third day to eighth day, colonic length was restored (Figures 1D,E), and colonic weight and colonic weight index were reduced (Figures 1B,C). Pathological sections of colonic tissues from colitis mice treated with SSP and 5-ASA showed fewer ulcers, decreased inflammatory cell infiltration, and colonic epithelial cell hyperplasia ( Figure 1F). The pathological damage scores of mice treated with SSP and 5-ASA were lower than in the DSS group ( Figure 1G). The results showed that SSP ameliorated pathological colonic damage in DSS-induced colitis.

SSP Controlled Activation of BCL-6/ Blimp-1 Signaling Pathway in Colitis Mice
Through Western bolting assay, we verified the key proteins in the Bcl-6/Blimp-1 signaling pathway and found that SSP significantly inhibited expression of Bcl-6 ( Figures 5A,C), STAT3 (Figures 5A,D), and p-STAT3 (Figures 5A,E) proteins and improved expression of Blimp-1 (Figures 5A,B) in colonic tissues of colitis mice. It was not obvious that SSP regulated signal lymphocyte activation molecule (SLAM)-associated protein (SAP) expression (Figures 5A,F). The results indicated that SSP inhibited BCL-6/Blimp-1 signaling pathway in colitis mice. Frontiers in Physiology | www.frontiersin.org

DISCUSSION
IBD is a nonspecific colonic inflammation, whose common clinical manifestations include abdominal pain, diarrhea, fecal occult blood, and hematochezia. Colonoscopy clearly shows colonic mucosal hyperemia, edema, petechia, and ulcer formation. In the present study, the colitis model was induced in BALB/C mice by DSS. The body weight of the mice began to decrease on the second day after drinking 3% DSS, and bloody stools were observed on the day 5. After SSP treatment, body weight, colon weight, and colon length of the mice were restored. Pathological observation showed crypt epithelial cell dropout, crypt destruction, and variable lymphocytic infiltration of epithelium and lamina propria in untreated colitis mice. However, after 7-days' treatment with SSP, there was evidence of improvement in pathological colonic damage, with fewer inflammatory cells, gland irregular hyperplasia, fewer and smaller ulcers in the mucosa, and decreased colonic weight index and pathological damage score, which indicated that SSP effectively treated DSS-induced chronic colitis. At the same time, IL-6, IL-17A, IL-23, TNF-α, and TGF-β1 expressions were significantly decreased in colitis mice treated by SSP. As pro-inflammatory factors, IL-6, IL-17A, IL-23, and TNF-α are classical hallmarks of colitis (Xiao et al., 2016;Allocca et al., 2018), which induce the occurrence of colitis. And IL-17A is regard as a main product of Tfh17 cells, so high-expressed IL-17A in colitis mice hinted that elevated level of Tfh17 cells is possible phenomenon in the pathogenesis of colitis. As a classic anti-inflammatory factor, IL-10 secretion was decreased for the downregulated function of Tfh10 cells in DSS-induced colitis. TGF-β1 has both anti-inflammatory and pro-inflammatory effects in different diseases. In the present study, elevated levels of TGF-β1 in DSS-induced colitis were positively correlated with disease progression. Vitamin D can treat colitis by inhibiting TGF-β1 (Stadnicki et al., 2009;Tao et al., 2015). The elevated level of TGF-β1 in the DSS group may play a pro-inflammatory role, and was decreased after treatment, which proves that SSP inhibited expression of pro-inflammatory factors to alleviate colonic mucosal damage in experimental colitis. Mesalamine, or 5-aminosalicylic acid (5-ASA), is a mainstay of therapy for individuals with inflammatory bowel disease. In the present study, 5-ASA was selected to evaluate the therapeutic effect of SSP as control drug. As a classic chemical drug in anti-inflammatory therapy of IBD, 5-ASA can inhibit NF-κB signaling pathway to decrease inflammatory secretion, or restrain prostaglandin or leucotriene to eliminate inflammatory injury. In anti-inflammatory action, it is obvious that 5-ASA is better than SSP. However, it is fortunate that SSP has an analogous anti-inflammatory action as downregulating pro-inflammatory factors (as IL-6, IL-17A, IL-23, and TNF-α and so on) or upregulating anti-inflammatory factor (IL-10). Certainly, the effect of SSP are realized by regulating immune or apoptosis and so on. In recent years, it has been found that Tfh cells are closely related to colitis. Due to their heterogeneity, Tfh1, Tfh2, Tfh10, Tfh17, Tfh21, and Tfr cells have different immunological functions and can be differentiated (Fazilleau et al., 2009). Therefore, in this experiment, CD4 + CXCR5 + IL-10 + (Tfh10), CD4 + CXCR5 + IL-17 + (Tfh17), and CD4 + CXCR5 + Foxp3 + (Tfr) cells were measured. The numbers of Tfh10 and Tfr cells were decreased and the number of Tfh17 cells was increased in the DSS group, which was reversed after treatment with SSP. CD4 + Foxp3 + Treg cells play a major role in immune homeostasis (Jung et al., 2017), and their expression is decreased (Ohno et al., 2017) in colitis mice. Tfr cells are a specialized subset of Treg cells with high expression of CXCR5 + and FOXP3 + , which are mainly located in the GCs Fonseca and Graca, 2019). Tfr cells can secrete IL-10 that acts as an immunosuppressant and maintains immunological tolerance. IL-10 also inhibits stimulation of B cells by Tfh cells . As an anti-inflammatory factor secreted by Tfh10 cells, IL-10 can effectively maintain intestinal balance. Cardoso et al. (2018) found that IL-10 can protect mice with DSS-induced colitis. Tfh17 cells produce IL-17, which induces autoimmune diseases and was increased in IBD, Guillain-Barré syndrome, Hashimoto's thyroiditis, RA, systemic lupus erythematosus, and other diseases (Che, et al., 2016;Zhao et al., 2018;Sun et al., 2019). In the present study, the increase in Tfh17 cells was synchronous with the pathological colonic damage. It is important to note that SSP reduced the quantity of Tfh17 cells. Therefore, the balance between Tfr and Tfh10/Tfh17 cells is critical in the pathogenesis of autoimmune diseases . Higher numbers of Tfh17 or lower numbers of Tfh10 and Tfr cells can lead to inflammatory immunological damage in the colonic mucosa . As shown in the present study, SSP can inhibit the decrease in Tfr and Tfh10 cells and the increase in Tfh17 cells to restore the balance of Tfr to Tfh10/Tfh17cells. These results suggest that SSP alleviates the pathological colonic injury induced by DSS, which is achieved by regulating the balance of Tfr and Tfh10/Tfh17 cells. However, the pathway regulating the balance is unclear.
Tfh cells, also known as CD4 + CXCR5 + ICOS + T cells (Choi et al., 2011), have a regulatory effect on ICOS signaling during initiation of DCs in early Tfh development (Wikenheiser et al., 2016). ICOS can regulate activation of CD4 + T cells and support formation of GCs. Compared with ICOS +/+ mice, Tfh cells are reduced in the spleen of ICOS −/− mice, which indicates that Tfh cell expansion is dependent on ICOS (Marriott et al., 2015;Wong et al., 2019). Furthermore, expression of ICOS is regulated by IL-6. In IL-6 receptor (IL-6R) knockout mice, expression of ICOS in Tfh cells is decreased, and sufficient IL-6R can provide an advantage for Tfh cell accumulation (Leavenworth et al., 2015). ICOS can induce differentiation of Tfh cells through the interaction of the p85α-OPN-i axis and Bcl-6 (Kolenbrander et al., 2018).
Recent studies have shown that the Bcl-6/Blimp-1 signaling pathway regulates Tfh cell differentiation and does not produce Tfh cells in the absence of Bcl-6 signaling. It is known that Bcl-6 is the regulatory transcription factor of Tfh cells (Wu et al., 2018). Just as Blimp-1 is an antagonist of Bcl-6, CD4 + T cells lacking Blimp-1 can preferentially develop into Tfh cells. High expression of Blimp-1 inhibits activation of Bcl-6, thereby inhibiting differentiation of Tfh cells (Linterman et al., 2010). Similarly, IL-21R can promote the expression of Bcl-6 (Choi et al., 2013;Lahmann et al., 2019). TGF-β, IL-6, and IL-6R can mediate STAT3-induced Bcl-6 expression and promote Tfh differentiation in human (Wu et al., 2015). In STAT3 deficiency, Tfh cells develop at a slower rate, inhibit IL-21, and increase expression of IFN-γ and IL-4, leading to decreased helper activity in B cells (Park et al., 2017;Deng et al., 2018). PD-1 is a co-inhibitory molecule that is highly expressed on Tfh cells, whereas PD-1 + Tfh cells play an important role in normal immune responses Shi et al., 2018). As a major marker of Tfh cells in GCs, PD-1 can inhibit T cells from entering follicles, allowing these T cells to concentrate in the GC and, together with PD-ligand 1 (PD-L1), optimize the competitiveness and affinity of B cells (Rajabian et al., 2019). The relative gene expression levels of PD-L1 and PD-L2 in UC patients are higher than those in the control group (Hu et al., 2013;Zhao et al., 2018). While SAP binding to SLAM family receptors increases cytokine secretion and regulates the duration of T and B cell interaction, enhancing Tfh cells' help to GC and B cells, and leading to GC response defects in the case of SAP loss (Hu et al., 2013(Hu et al., , 2016Wang et al., 2015). In the present study, the percentages of ICOS + , BCL-6 + , PD-1 + , and PD-L1 + Tfh cells were downregulated in the peripheral blood of colitis mice treated by SSP, while the number of Blimp-1 + Tfh cells was upregulated. Synchronously, expression of BCL-6, p-STAT3, and SAP proteins in colonic mucosa was markedly decreased, and Blimp-1 activation was inhibited after 7-days' treatment with SSP. Combined with the above analysis, we found that the BCL-6/Blimp-1 signaling pathway played an important role in the pathogenesis of experimental colitis induced by DSS; SSP inhibited BCL-6, p-STAT3, and SAP protein activation; and activated Blimp-1 expression to regulate Tfh cell differentiation. SSP restrained ICOS, Bcl-6, PD-1, and PD-L1 expression and activated Blimp-1 on the Tfh cell surface to limit the differential direction of Tfh cells, which improved the numbers of Tfh10 and Tfr cells and suppressed the number of Tfh17 cells to restore the balance of Tfh10 to Tfr/Tfh17 cells. All the above results combined with pathological analysis show that SSP eliminated the pathogenic immunocomplex in colonic mucosa by controlling the balance of Tfh10-Tfr/Tfh17 cells to restore immune stability and attenuate pathological colonic injury. However, it is regretful that we cannot analyze the levels of these protein molecules in the Tfh cells for absent fluorescence-activated cell sorting (FACS) Flow Cytometer. The results hinted SSP perhaps could regulate the expression of these protein molecules to create the environment of the differentiation of tissue resident Tfh cells from mesenteric lymph nodes and/or lamina propria to indirectly influence subpopulation of Tfh cells in peripheral blood.
In conclusion, the present study indicated that SSP regulated differentiation and function of Tfh cells to treat IBD, which was potentially related with inhibiting the Bcl-6/Blimp-1 pathway. To explore the explicit mechanism of SSP regulated differentiation of Tfh via BCL-6/Blimp-1 signaling pathway in the next studies, we will induce colitis animal model in the BCL-6 knock-in mice or Blimp-1 knock-out mice, separate tissue resident Tfh cells from mesenteric lymph nodes, and analyze the activation of Bcl-6/Blimp-1 signaling pathway after colitis mice were treated by Sishen Pill or Bcl-6 inhibitor or siRNA technology. As a traditional Chinese medicine, the effective constituents of SSP are extraordinarily complex. It is unknown which effective constituents of SSP optimize the function and differentiation of Tfh cells. In future work, we aim to establish the effective target for SSP regulation of Tfh cells to treat IBD by network pharmacology, metagenomics, and bioinformatics.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ supplementary material.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of Jiangxi University of Traditional Chinese Medicine.