He et al. Cell Biosci (2018) 8:38 Cell & Bioscience https://doi.org/10.1186/s13578-018-0237-z Open Access RESEAR CH Heat shock protein 70 protects mouse against post-infection irritable bowel syndrome via up-regulating intestinal γδ T cell’s Th17 response 1 1 2 2 2 3 1 1 Zhoutao He , Xiaoning Sun , Zhichao Ma , Jiao Fu , Baili Huang , Fujin Liu , Yi Chen , Taozhi Deng , 1 4 1* Xiangyang Han , Deming Sun and Cheng Lan Abstracts Background: This study investigated the role of HSP70 in modulating intestinal γδ T cells’ Th17 response in Trichinella spiralis-induced PI-IBS mice model. Methods: The intestinal HSP70’s expression and mRNA level were measured by Western blot and RT-PCR. The intesti- nal γδ T cell’s morphological changes were analyzed using immunofluorescence staining and confocal laser scanning microscope. The pro-inflammatory cytokines’ level was detected by ELISA. The isolated and purified γδ T cells were pre-incubated with HSP70 and their functions including proliferation, apoptosis, activation and production of IL-17 were also detected. Results: Heat treatment augmented intestinal HSP70 expression and alleviated the clinical presentations in PI-IBS mice. Meanwhile, intestinal γδ T cells and local IL-17 level were increased by pre-induction of HSP70. HSP70 promoted the proliferation of PI-IBS mice’s intestinal γδ T cells, inhibited the apoptosis and stimulated these cells to secret IL-17 rather than IFN-γ. Conclusion: Our results suggest that HSP70 plays a protective role via up-regulating intestinal γδ T cell’s Th17 response in PI-IBS mice. Keywords: Heat shock protein 70, γδ T cells, Post-infectious irritable bowel syndrome, Th17 response, Mice Background of the immune system at the molecular level have been As a kind of clinical syndrome characterized by abdominal shown to be associated with mucosal secretory func- pain, discomfort and bloating accompanied with abnor- tion, smooth muscle and enteric nervous fibers [ 7–9]. In mal defecation, the precise patho-physiological mecha- particular, an imbalance of pro- and anti-inflammatory nism of irritable bowel syndrome (IBS) remains unclear cytokines is found, which may play a key role in the local [1–3]. During the last two decades, abundant clinical and intestinal inflammation. Therefore, experimental infec - experimental research focused on the role of infection and tion with the parasite Trichinella has been widely used inflammation in the pathogenesis of IBS, called as post- to establish models for detecting the pathogenesis of infectious irritable bowel syndrome (PI-IBS) [4–6]. intestinal dysfunction [10, 11]. Infection by Trichinella The pathophysiology of PI-IBS is still not fully under - spiralis larvae induced changes in visceral sensitivity, stood, but low-grade inflammation and chronic alteration alterations of intestinal smooth muscle function, and altered secretion. These abnormalities persisted after *Correspondence: email@example.com animals recovered from infection, suggesting that this is Department of Gastroenterology, Hainan General Hospital, Xiu Hua a suitable model of PI-IBS. Activated immune cells con- Road 19th, Xiu Ying District, Haikou 570311, Hainan Province, China tinue to release various cytokines after an acute intestinal Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. He et al. Cell Biosci (2018) 8:38 Page 2 of 9 infection , for example, T-helper (Th) cells produce hematoxylin–eosin (HE). The HE staining was further used interferon (IFN)-γ and interleukin (IL)-1β to promote the for evaluating inflammatory score basing previous scoring inflammatory response; T-regulatory cells release IL-10 to system . prevent autoimmunity; in contrast, IL-17, which is pro- duced by Th17 cells, can induce autoimmunity . These Abdominal withdrawal reflex (AWR) cytokines may alter the physiology and immunity of the AWR was performed to evaluate the visceral hypersen- host gut to cause symptoms of PI-IBS. The HSP70 family sitivity . The anesthetized animals were inserted via of heat shock proteins consists of molecular chaperones their anus with air chamber and catheter. The air cham - of approximately 70 kDa in size that serve critical roles ber was distended at volume of 0.25/0.35/0.5/0.65 ml × l in survival function in the cell. Recently, it was reported 5 min × 3 times. Between each distending time, the ani- that heat shock protein 70 (HSP70) has a unique capabil- mals were permitted to have a rest for 30 s. The AWR ity of regulating the protein misfolding, aggregation and scoring standard: when stimulated, the animals are in sta- serves critical roles in some diseases [13, 14]. However, ble mood, 0 point; if the animals are in unstable mood, the effects of HSP70 on PI-IBS have not been reported. twisting their heads once in a while, 1 point; slightly con- γδ T cells were proved to participate in inflammatory and tracting their abdomen and back muscles, 2 points; inten- autoimmune disorders [15–17]. Thus, the current study sively contracting their abdomen muscles and uplifting aimed to investigate whether HSP70 regulated the γδ T the abdomen from the ground, 3 points; intensively con- cells’ phenotype and function during PI-IBS. tracting abdomen muscles, bowing abdomen and uplift- ing the abdomen and perineum, 4 points. Methods Animals and study design Colon Transportation Test (CTT ) 96 female 57BL/6 mice (6–8 weeks old and 13–15 g CTT was performed to evaluate the status of the intesti- weigh) were purchased from Medical Animal Center, nal motility. After filled into stomach with 0.4 ml active the Hainan medical College. All animals were housed carbon, the first black stool time was recorded. The total in sterile, pathogen-free, temperature controlled facility stool within 8 h was collected and evaluated by Bristol on normal 12-h light/dark cycle, and standard diet and stool grade : normal shaped stool, 1 point; soft or water were provided ad libitum. The animals were ran - deformed stool, 2 points; water-like stool, 3 points. domly assigned into four groups: control group, PI-IBS group, Heat + PI-IBS group and Heat group (n = 24 in Preinduction of HSP70 each group). In each group, six mice were sacrificed for Expression of HSP70 was induced by heat treatment the detection of the intestinal HSP70 protein level, and according to previous report . The mice were anes - six mice for HSP70 mRNA level. six mice were used for thetized with sodium pentobarbital (50 mg/kg). Rectal the isolation, purification and culture of the intestinal γδ temperature was monitored with a thermistor inserted T cells. The other six mice were examined for the visceral into the rectum in a baking oven with constant tem- hypersensitivity and the intestinal motility. perature 50 centigrade. After the body temperature was maintained at 41 °C form 20 min, the mice were return to Modeling of PI‑IBS their cages at room temperature and allowed water and The mice were infected with T. spiralis (Lanzhou Animal food as libitum. Non-heated mice were only anesthetized Medical Institute, Lanzhou, China) as described previ- but received no hyperthermic stress. ously . Briefly, the parasite larvae were separated from Sprague–Dawley rats 60 days after infection of T. Determination of HSP70 spiralis’ cyst by digestion with 1.5% gastric pepsin (Invit- Western blot rogen Co., CA, USA). The mice were fed with the larvae HSP70 protein level was measured by Western blot in 0.2 ml saline (300 larvae per mouse). The animals in (Wuhan Boster Coporation, Wuhan, China). Briefly, the the control group were fed with only 0.9% saline. ileum tissue sample was grinded and cracked with RIPA. The homogenate was centrifuged for 30 min. The pro - Histopathological study tein concentration in the supernatants was measured The animals were sacrificed at the 8th weeks after infec - by Bradford Assay. 40 µg tissue sample was separated tion and their ileum tissue fixed within 10% formalin in by SDS page gel electrophoresis and transferred to the PBS at 4 °C, dehydrated in a graded series of ethanol, PVDF membrane. The membrane was blotted with TBST and then embedded in paraffin wax. The tissues were for 1 h, then was added with anti-mouse HSP70 multiple sectioned at 5 μm thick and mounted on slides. The clone antibodies (1:1000) (ab2787, abcam, US) and rab- slides were dewaxed, hydrated, and then stained with bit anti-mouse—actin multiple clone antibodies (1:1000) He et al. Cell Biosci (2018) 8:38 Page 3 of 9 (ab8226, abcam, US) at 4 °C for 12 h. One day later, the conjugated anti-FITC mAb staining were used to clear membrane was washed in TBST and autographied by the miscellaneous γδ T cells. The living cells remain up to ECL chemiluminescent assay. The gray-scale value was more than 95%. detected by enhanced chemiluminescence in dark room. The gray-scale value of Hsp70/-actin represented the rel - Determination of γδ T cells’ function in vitro ative expression level of HSP70. Proliferation CFSE proliferation assay was performed to measure the Real‑time quantitative PCR (RT‑PCR) proliferation of γδ T cells. The purified γδ T cells were HSP70 mRNA level was measured by RT-PCR. Total pre-incubated with HSP70 or not. Briefly, 4–8 × 10 γδ T RNA was isolated from the terminal ileum tissue with cells were stained with 3.5 μM CFSE (Sigma-Aldrich, ST. Trizol liquid and treated with DNAase I. Primer was Louis, MO, USA) for 4 min at RT. 1.5 × 10 CFSE-labeled designed according to mouse gene sequence. β-actin γδ T cells/ml were incubated in a 96-well plates for 48 h worked as an internal control. with Hsp70 10 ng/ml, 1.5 × 10 spleen cells irradiated HSP70 gene primer sequence (5′-3′): with γ ray (as antigen presenting cells), T. spiralis excre- tory–secretory-antigen 10 μg/ml (Sigma-Aldrich, ST. F: GAA GGT GCT GGA CAA GTG C, Louis, MO, USA), the total volume per well was 200 μl. R: GCC AGC AGA GGC CTC TAA TC. FACS analysis was performed to measure the CFSE sig- nal/cell with a reduction of intensity as a marker of β-actin gene primer sequence (5′-3′): proliferation. F: AGG CTG TGC TGT CCC TGT ATG, Activation R: GAG GTC TTT ACG GAT GTC AACG. γδ T cells were stained with PE conjugated anti-CD69 mAb and anti-CD62L mAb (100 μl antibodies per 1 × 10 RT-PCR was operated according to following protocol: cells, incubated with IL-23 and TLR4 at 4 °C for 30 min). 1. Pre-denaturation program (5 min at 94 °C); 2. Dena- FACS was used to determine the expression of CD69 and turation program (1 min at 94 °C); 3. Amplification and CD62L on γδ T cells . qualification program, repeated 30 cycles (50 s at 57 °C, 20 s at 60 °C); 4. Prolonging program (7 min at 72 °C). Apoptosis The relative expression was expressed as a ratio of the The purified γδ T cells were pre-incubated with HSP70 target gene to the control gene. and their apoptosis was detected with Annexin V-FITC/ PI Apoptosis Detection Kit (Sigma-Aldrich, St. Louis., Morphological analysis MO, USA) in accordance with the manufacture’s The ultrathin frozen section of the ileum tissue was instruction. treated by immunofluorescence histochemical stain - ing. The primary antibody was florescence- labeled rat Determination of proinflammatory cytokines anti-mouse anti-TCR antibody (GL3 clone). The tissue The tissue sample was ultrasonically shivered and cen - sections were scanned under Laser scanning confocal trifuged at 4 °C for 15 min. The concentration of IL-17 microscope. The intensity of fluorescence was calculated in the supernatants from the intestinal tissue sample and automatically using the image analysis software. the culture of γδ T cells were measured by ELISA. Isolation and purification of γδ T cells Statistics analysis The isolation and purification of γδ T cells were con - Data were analyzed using Student’s t test (SPSS 17.0 soft- ducted as described by Cheng et al. . Briefly, total T ware) and AVNOVA test. Data were expressed as the cells were isolated with collagenase D digestion method mean ± standard error. p < 0.05 was considered as statisti- (Roche, Basel, Switzerland) followed by centrifuging with cally significant difference. lymphocyte separation medium Ficoll (Sigma-Aldrich, ST. Louis, MO, USA). The total T cells were stained with FITC conjugated anti-δ TCR mAb (100 μl antibodies Ethical considerations per 108 cells, incubated at 4 °C for 30 min), followed by The experiment was carried out in accordance with the microbeads conjugated anti-FITC mAb staining (100 μl Chinese guidelines for animal welfare. Experimental pro- antibodies per 108 cells, incubated at 4 °C for 30 min). tocol was approved by the Animal Care and Use Com- MACS was used to select the positive cells for two mittee of Hainan Province. times. FITC conjugated anti-TCR mAb and microbeads He et al. Cell Biosci (2018) 8:38 Page 4 of 9 Table 1 AWR score in heat pretreated PI-IBS mice Results AWR scores Distending air AWR score volume (ml) The anesthetized animals were inserted via their anus 0.25 0.35 0.5 0.65 with air chamber and catheter. The air chamber was dis - Control (n = 7) 0.00 ± 0.00 1.42 ± 0.53 2.67 ± 0.53 3.57 ± 0.51 tended at volume of 0.25/0.35/0.5/0.65 ml × l5 min × 3 PI-IBS (n = 7) 0.00 ± 0.00 2.57 ± 0.52 3.57 ± 0.50 3.72 ± 0.49 times. The AWR score is from 0 to 4 according with the a b Heat + PI-IBS 0.00 ± 0.00 2.14 ± 0.39 2.74 ± 0.49 3.76 ± 0.38 animals’ mood when stimulated. As a result, between (n = 7) 0.35 ml and 0.5 ml, PI-IBS mouse show higher AWR Heat (n = 7) 0.00 ± 0.00 1.24 ± 0.67 1.86 ± 0.69 3.61 ± 0.48 score than control group (p < 0.05). Heat pretreatment Compared with the PI-IBS group, p < 0.05 significantly decreased the AWR score in PI-IBS mouse Compared with the PI-IBS group, p < 0.05 (p < 0.05) but not to the normal level. These results show that hear pretreatment improved the visceral hypersensi- tivity in PI-IBS mouse (Table 1). Table 2 Changes of the intestinal mobility in heat Colon Transportation Test pretreated PI-IBS mice CTT was performed to evaluate the status of the intes- tinal motility. The total stool within 8 h was collected Group First black stool time Bristol stool grade and evaluated by Bristol stool grade 1–3. As shown in (minutes) (Table 2), the first black stool time in PI-IBS mouse obvi - Control (n = 7) 108.29 ± 13.06 0.97 ± 0.11 ously shorten, which was partially prolonged in heat- PI-IBS (n = 7) 68.97 ± 5.96 2.86 ± 0.44 pretreated PI-IBS mouse (p < 0.05). Bristol stool grade a b Heat + PI-IBS (n = 7) 88.74 ± 5.90 2.17 ± 0.18 show the grade in PI-IBS mouse was obviously increased, Heat (n = 7) 102.43 ± 12.18 1.15 ± 0.21 which was partially decreased in heat-pretreated PI-IBS Compared with the PI-IBS group, p < 0.05 mouse (p < 0.05). These results show that heat pretreat - Compared with the PI-IBS group, p < 0.05 ment improved the intestinal motility in PI-IBS mouse (Table 2). Fig. 1 Protein and mRNA expression of HSP 70. a, b Expression of HSP 70 detected by Western blot; c, d Expression of HSP 70 detected by RT-PCR. *p < 0.05 compared with control group; △, p < 0.05 compared with both HSP70 group and IP-IBS group He et al. Cell Biosci (2018) 8:38 Page 5 of 9 Effect of heat treatment on expression of HSP70 in PI‑IBS PI-IBS mice, the fluorescence intensity became the high - mice est, which was similar with the results of HSP70 expres- Both of the protein and mRNA expression of HSP70 was sion (Fig. 2b). significantly higher in HSP group than that in control IL-17 of the intestinal tissue sample was then meas- group (p < 0.05), suggesting that heat treatment upregu- ured by ELISA. The expression of IL-17 was significantly lated HSP70 expression. Meanwhile, T. spiralis infection increased in PI-IBS group compared with control group, in IP-IBS group has also markedly upregulated HSP70 while it was significant decreased by heat treatment expression (p < 0.05). Interestingly, when the T. spiralis (p < 0.05) (Fig. 2c). infected mice were also given heat treatment, the expres- sion of HSP70 became even higher, which was signifi - Heat treatment improved the inflammatory score in PI‑IBS cantly higher than both HSP70 group and IP-IBS group mice (p < 0.05) (Fig. 1). H&E staining of the ileum showed a marked infiltration by T. spiralis infection day 14. Infiltration and edema Intestinal γδ T cells and IL‑17 level were increased gradually reduced from day 14 until day 56, at which in heat‑treated PI‑IBS mice stage no obvious inflammatory infiltrate was observed Stained γδ T cells of different groups were observed (Fig. 3a). However, in the heat treated HSP group, the under confocal microscope (Fig. 2a). The fluorescence inflammatory infiltrate was significantly reduced com - intensity of the γδ T cells was detected. It was shown pared with HSP group (p < 0.05). Furthermore, the that the fluorescence intensity was significantly higher inflammatory score was evaluated. Heat treatment could in PI-IBS group and HSP group compared with control significantly reduce the inflammatory score compared group (p < 0.05). However, after the heat treatment on with HSP group (p < 0.05) (Fig. 3b). Fig. 2 γδ T cells and IL-17 level in PI-IBS mice. a Stained γδ T cells of different groups were observed under confocal microscope; b Fluorescence intensity of γδ T cells in different groups; c IL-17 levels of different groups He et al. Cell Biosci (2018) 8:38 Page 6 of 9 Discussion The precise mechanism underlying IBS remains unclear, as well as effective therapy. AWR scores were altered in response to low or medium pressures. When distention volume was 0.25 ml, the pressure was too low to cause any visceral sensation. But when the distention volume was 0.65 ml (high pressure), the level of stimulation was so high that it resulted in a very intense response in both groups of mice. By either 0.35 or 0.5 ml distention volume, the AWR scores in the model group were higher than those in the control group, and the pain threshold in the model group was lower than that in the control group at the same time point. This suggests an increase of the visceral sensitiv - ity in mice after infection. The irritable bowel syndrome (IBS) is a common disorder characterized by abdominal pain in the setting of altered perception of viscerosensory stimuli. Pain is normally evoked by stimuli that are suf- ficiently intense to activate high-threshold sensory fib - ers, which relay the signal to the spinal cord. However, tissue injury or inflammation may lead to profoundly increased pain sensitivity in which noxious stimuli gener- ate a greater response (hyperalgesia) and stimuli that are normally innocuous elicit pain (allodynia) . Although the pathogenesis of PI-IBS is not well under- stood, increasing evidence suggests that low-grade inflammation and immune activation play pivotal roles Fig. 3 Inflammatory infiltrate in intestine. a H&E staining of the in the occurrence and persistence of its symptoms. ileum; b Inflammation score of different groups. *p < 0.05 compared Recently, development in mucosa immunity research with PI-IBS group provides a potential target for the treatment of IBS. Our previous research reported that the intestinal HSP70 level raised during PI-IBS. Furthermore, Prein- Hsp70 promoted γδ T cells’ Th17 response duction of HSP70 could improve the clinical symptoms After γδ T cells were purified, FITC conjugated anti-TCR of PI-IBS [22, 26]. Several studies also demonstrated mAb and microbeads conjugated anti-FITC mAb staining that HSP70 inhibits the production of proinflamma - were used to clear the miscellaneous γδ T cells (Fig. 4a). tory cytokines in different cell populations . More In the CFSE proliferation assay, the proliferation of γδ T recently, Muralidharan et al. reported that association of cells were significantly promoted by HSP 70 (Fig. 4b). In HSP70 with NF-κB subunit p50 in alcohol-treated mac- the apoptosis assay, HSP 70 could significantly reduce rophages correlates with reduced NF-κB activation and the apoptosis of γδ T cells (Fig. 4c). Moreover, the acti- downstream TNF-α, IL-6 and IL-1β production . vation of γδ T cells was also detected. It was shown that Intestinal mucosal barrier function is the capacity of the the expression of CD62L was significantly reduced by intestine to provide adequate containment of luminal HSP 70, while the expression of CD69 was significantly microorganisms and molecules while preserving the abil- increased, suggesting that the γδ T cells were activated by ity to absorb nutrients. Alteration of the mucosal barrier HSP 70 (Fig. 4d). function with accompanying increased permeability and/ or bacterial translocation has been linked with a variety of conditions, including inflammatory bowel disease . Effect of HSP70 on the IL‑17 production by γδ T cells In the current study, we have found that heat treat- IL-17 in the supernatants from the intestinal tissue sam- ment could induce the increase expression of intestinal ple and the culture of γδ T cells were measured by ELISA. HSP70. It is reported that HSP70 could protect mucosa It was shown that IL-17 was significantly increased by damage and regulate local inflammation via participat - HPS 70 treatment in γδ T cells (p < 0.05) (Fig. 4e). ing in innate immunity, mediating suppression of the He et al. Cell Biosci (2018) 8:38 Page 7 of 9 Fig. 4 γδ T cells’ Th17 response and IL-17 production by γδ T cells. a Purified γδ T cells were stained with Anti-TCRγ/δ-FITC antibodies and runned on FACS; b γδ T cells’ proliferation index was measured; c Annexin V-FITC/PI apoptosis Kit was used to determine the apoptosis rate; d The surface molecular expression of CD62L and CD69 was detected by FACS; e The production of IL17 by γδ T cells stimulated by HSP70 was measured by ELISA intracellular apoptotic pathway, stabilizing and prevent- leading to unknown down-stream events [33–35]. It is ing irreversible aggregation of heat-damaged protein proved that γδ T cell was not the only one participating [30–32]. Heat treated PI-IBS mouse show increased in the sophisticated network in the intestinal inflamma - number of γδ T cells, suggesting that the proliferation tion and immunity. Some other cells could also produce of γδ T cells was increased. Interestingly, it was also IL-17 [36–39]. Thus, HSP70 could utilize other pathways observed that IL-17 level was increased in the intestine of during PI-IBS. In our study, γδ T cells were isolated and PI-IBS mice. These results suggested that HSP70 and γδ purified from PI-IBS mouse’s intestine. HSP70 signifi - T cells maybe simultaneously involved in PI-IBS. cantly promoted the γδ T cells’ proliferation and activa- Local low-grade inflammation and immune activation tion, prevented their apoptosis, and increased the IL-17 are known as important components of the pathophysi- production. These were the potential mechanism how ology of PI-IBS. Levels of interferon γ and interleukin HSP70 exert its protective role in vivo via promotion of (IL)-17 could be significantly increased following T. spi - γδ T cells’ Th17 response. ralis infection, while IL-10 was decreased . The γδ There were also some limitations in our study. The T cells’ Th17 response plays an important role in many type of visceral hypersensitivity in PI-IBS mouse was not diseases, but the behind mechanism remains unclear. investigated. The water content of the bristol stool was They have also displayed dual effect on the progress of not analyzed. the diseases. Moreover, sometimes they act as a trigger He et al. Cell Biosci (2018) 8:38 Page 8 of 9 References In conclusion, IBS seriously impact the patients’ life 1. Soares RL. Irritable bowel syndrome: a clinical review. World J Gastroen- quality, which may result from a synergy of multiple terol. 2014;20:12144–60. etiological factors including the visceral hypersensi- 2. Chey WD, Kurlander J, Eswaran S. 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Cell & Bioscience – Springer Journals
Published: Jun 5, 2018
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