Abstract
Purpose: This study aimed to investigate immune-related gene expression in rat small intestine after heat stress. Materials and methods: Twelve Sprague Dawley (SD) rats were randomly divided into control and heat-stressed groups. Rats in both groups were housed at 25 °C with 60% relative humidity. The heat-stressed group was subjected to 40 °C for 2 h/day for 3 days. After heat stress, the mRNA expression profile of small intestine epithelial tissue was evaluated by microarray analysis. Results: A total of 23 genes related to immune responses were significantly altered, of which 12 genes were up-regulated and 11 genes were down-regulated. Conclusions: Microarray analysis demonstrated the JAK-STAT pathway had a potentially important role in the regulation of inflammation in the small intestine, and changes in antigen presentation might reduce intestinal immune responses after heat stress.
Introduction
The effect of heat stress on the immune response is being increasingly investigated [Citation1,Citation2]. Heat stress can reduce the immune function of intestinal mucosa in rats and suppress innate host immunity [Citation3,Citation4]. Microarray analysis revealed that differentially expressed genes in rat small intestine are related to immune regulation [Citation5]. Previous studies on the effect of heat stress on immune function mostly focused on cancer therapy, examining cytokine expressions or humoral immune responses [Citation4,Citation6–8]. However, little is known about the mechanisms of molecular regulation of intestinal immune responses. Therefore, this study analysed the microarray profile of immune-related genes following the heat stress.
After adaptation to the environmental conditions of the animal experimental room for 7 days, 12 male Sprague Dawley (SD) rats weighing 210 ± 10 g were randomly divided into two groups: control group and heat-stressed group (each group contained six rats). Rats in the heat-stressed group were then subjected to 40 °C for 2 h daily (11:00–13:00) for 3 successive days. All experimental protocols were approved by the Committee for the Care and Use of Experimental Animals, China Agricultural University, and all the studies met Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines. mRNA was isolated from the small intestine epithelial tissue and used for microarray analysis. From the microarray data, the profile of immune-related genes was selected for further bioinformatics analysis, which contained gene ontology (GO) terms and Kegg pathway analysis.
Twenty-three immune-related genes in the small intestine were altered significantly in response to heat stress (), of which 12 genes (including HSPA1A, ZBTB16, CSF2, HPX, IL22RA2, PTDSR, LOC301133, BCL6, MAdCAM1, PPARA, CDK6, and VEGFA) were up-regulated (log2 ratio > 1, p < 0.01) and 11 genes (including TNFRSF6, MS4A2, CCL5, SPN, CCR5, MMP9, IRF4, TLR2, CCL4, S100A9, and RT1-Bb) were down-regulated (log2 ratio <−1, p < 0.01). The main GO categories (functional categories) for altered genes were the regulation of immune system processes, leucocyte migration, positive regulation of immune system processes, immune responses, lymphocyte co-stimulation, immune system development, T cell selection, leucocyte activation, immune effector processes and negative regulation of immune systems. These differentially expressed genes were involved in such pathways as cytokine–cytokine receptor interaction, antigen processing and presentation, cell adhesion molecules (CAMs), chemokine signalling pathways, graft-versus-host disease, allograft rejection and Leishmania infection.
Table 1. Differentially expressed genes related to immune responses.
The small intestine plays a vital role in protecting the host against harmful pathogens [Citation9], and severe heat stress results in the induction of inflammatory responses [Citation10]. As the coordinators of immune and inflammatory responses, cytokines transmit multiple signalling pathways by interacting with cytokine receptor superfamily members [Citation11–13]. Several differentially expressed genes (such as IL17A, IL1A and IL8RB) of the heat stress response in cattle blood participated in the cytokine–cytokine receptor interaction pathway [Citation14]. CCL5 is an inflammatory mediator, and CCL4 is crucial for immune responses to infection and inflammation, both of which interact with their receptor CCL5 [Citation15,Citation16]. Interactions with CCL5 activate distinct Janus kinase/signal transducers and activators of transcription JAK-STAT proteins to mediate immune responses. Furthermore, the JAK-STAT pathway transmits signals for development and homeostasis, and regulates all cell types involved in the initiation, propagation, and resolution of inflammation [Citation17–20]. In mouse 3T3-F442A preadipocytes and hamster cells in vitro, the responsiveness of cytokine receptors was reduced, and the JAK-STAT signalling response was diminished at an elevated temperature of 40 °C [Citation21]. The phosphorylation of extracellular signal-regulated kinases (ERK) in the rat small intestine was increased after three days of heat stress at 40 °C, while ERK down-regulated JAK-STAT signalling in human melanoma cells [Citation22,Citation23]. In the current study we found that genes involved in the cytokine–cytokine receptor interaction pathways were CCR5, CCL4, CSF2, TNFRSF6, LOC301133, IL22RA2, CXCR3, VEGFA, and CCL5. CCL4, CCL5 and CCR5, also involved in chemokine signalling pathways were down-regulated, indicating they may participate in inflammation through JAK-STAT signalling.
Intestinal epithelial cells and dendritic cells (DCs) function as antigen-presenting cells (APCs) to regulate T-cell responses in intestinal mucosa [Citation24,Citation25]. As the most potent APCs, DCs stimulate strong T-cell and immune responses [Citation25,Citation26]. Immature DCs in the lamina propria sample pathogens from the intestinal lumen [Citation27], migrate to the mesenteric lymph nodes (MLNs) to present processed antigen to naive T cells [Citation25,Citation28], while mature DCs in MLNs initiate primary immune responses through the expression of major histocompatibility complex (MHC) [Citation27,Citation29]. In the current study we found that HSPA1A (Hsp70) was up-regulated significantly and RT1-Bb (MHC II) was significantly down-regulated after heat stress. Hsp70 chaperones misfolded or newly synthesised polypeptides, the overexpression of which is the result of the protection against heat stress [Citation30–33]. Hsp70 promotes DC functions and is involved in the presentation of antigenic peptides via MHC class II, and enhances antigen-specific CD4+ T cell proliferation [Citation34,Citation35]. RT1-Bb encodes the B beta chain of MHC class II, whose main function is to present processed antigens. MHC class II molecules are critical for the initiation of antigen-specific immune responses, and the antigen-specific proliferation of CD4+ T cells is dependent on MHC class II [Citation36,Citation37]. We found that the expression of Hsp70 and RT1-Bb, both of which were involved in antigen presentation and antigen-specific CD4+ T cell proliferation, was altered. The number of CD3+ CD4+ CD8− cell (Th cells) in rat MLNs was significantly decreased after heat stress [Citation3], which may be caused by decreased expression of RT1-Bb in the small intestine. Although thermoseed inductive heating at a high temperature for a short time improves immune response in tumour treatment [Citation38,Citation39], heat stress at 40 °C for 3 days induces the reduction of the intestinal mucosal immune function in rats and the translocation of bacteria from the intestine [Citation3], and chronic heat stress suppresses humoral and cellular immune responses of rats to H5N1 vaccination through regulating CD4+CD25+FOXP3+TREGS [Citation4,Citation40]. Different hyperthermia location, stimulus intensity and stimulus duration would cause different immune response.
Overall, all these findings indicate the JAK-STAT pathway has a potentially important role in the regulation of inflammation, changes in the functions of APCs and interactions between APCs and T cells in the gut-associated lymphoid tissue and might explain the mechanism of molecular regulation of the intestinal immune responses after heat stress.
Declaration of interest
This work was supported by grants from the National Nature Science Foundation of China (31272478); the Ministry of Agriculture, Public Service Sectors Agriculture Research Projects (201003060-9/10), and the Twelve-Five National Technological Support Plan of China (2011BAD34B01).
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. The authors alone are responsible for the content and writing of the paper.
Acknowledgements
We are thankful for the help of the members of CAU-BUA TCVM teaching and research team.
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