Modulation Of Endoplasmic Reticulum Stress In Immunostimulatory Effects Of Glucocorticoids And Their Molecular Mechanisms

Infection is a common complication after severe trauma. there are still no effective therapeutic strategies so far. Therefore, the prevention and treatment of injury-induced organ dysfunction is very important at the early stage of injury. Major trauma results in massive impairment of immunologic reactivity, which has been demonstrated to correlate clinically with increased susceptibility to serious infection. It has been found to elicit neuroendocrine responses immediately after trauma. There is increasing evidence revealing that the neuroendocrine system is important in the regulation of immune function. The key neuroendocrine response to stress is activation of the hypothalamic-pituitary-adrenal (HPA) axis, The glucocorticoids (GCs) are the final effectors of the HPA axis. These GCs play an important role in the modulation of the immune system. GCs are regarded widely as being immunosuppressive and are used clinically as antiinflammatory agents. A growing body of literature suggests that, far from being suppressive, endogenous or natural GCs are also known to exert immunostimulatory effects. Previous study showed that GCs can enhance the immune functions of peritoneal macrophages, specially at low concentrations. However, the precise cellular mechanisms underlying the immunostimulatory effects of GCs have not been fully elucidated.The endoplasmic reticulum (ER) is a critical organelle involved in intracellular calcium regulation and protein folding, which are crucial for cellular homeostasis. Once ER homeostasis is perturbed by various stressors, newly synthesized unfolded proteins accumulate in the ER, resulting in ER stress (ERS). To cope with accumlated unfolded ER proteins, mammalian cells trigger a specific adaptive response called the unfolded protein response (UPR). UPR is crucial both for cell recovery under condition of ERS and for the function and survival of active secretory cells, such as immune cells. Macrophages are ubiquitous in mammalian tissues and play a central role in both innate and acquired immune responses. Recently, emerging evidence indicates that ERS in immune cells has been shown to play an important role in the regulation of the cellular functions, such as antigen presentation, plasma cell differentiation and antibody production, and T cell response to antigen. In addition, there is also increasing evidence indicating a strong link between ERS and immune inflammatory response. ERS-induced transcription factors, such as X-box binding protein 1 (XBP1), activating transcription factor 6 (ATF6) have been demonstrated to play an essential role in ERS-induced changes in cellular functions. However, the effects of GCs on macrophage ERS and its relationship with the immunostimulatory effects have yet to be defined.Based on our previous findings and current researches, the aim of this work was to investigate whether the immunostimulatory effects of GCs mediated by ERS and their potential molecular mechanisms, which includes the following four aspects: (1) To observe the expression changes of ERS-associated molecules GRP78, XBP1 and ATF6 in mice peritoneal macrophages treated with various concentrations of endogenous GCs (corticosterone). In order to further elucidate the relationship between ERS and altered immune functions, we investigated the effects of thapsigargin, an ERS inducer, on immune functions of mice peritoneal macrophages. Furthermore, to investigate the relationship the immunosuppressive effects of GCs between ERS in vitro. (2) We used CRH knockout (CRH-/-) mice, to observe in vivo the effects of the HPA axis activation on ERS and immune functions of immune cells of mice exposed to acute restraint stress. (3) Using lentiviral-mediated RNAi (RNA interference) technology, selectively silenceing of the XBP1 gene in mice, to investigate the role of XBP1 in the immunostimulatory effects of low concentration of corticosterone in vitro. (4) To use the GR antagonists RU486 (mifepristone) to explore the relationship between low concentration of corticosterone-induced ERS and GR. The purpose of this study is to further elucidate the molecular mechanisms of the immunostimulatory effects of GCs, and then provide more evidence for deepening the understanding of the regulation of the neuroendocrine system on the immune function.The main results are shown as follows:1. Previous works indicate that low concentrations of GCs exert immunostimulatory effects on macrophages function. In order to test the hypothesis that GCs induces ERS in mice peritoneal macrophages, and to elucidate the role of ERS in low concentrations of GCs-induced immunostimulation. We treated mice peritoneal macrophages with various concentrations of corticosterone, a major type of endogenous GCs in rodents, to observe the GCs-induced ERS. Meanwhile, we further examined the expression of XBP1 and ATF6, two key transcription factors of UPR signaling pathway. In parallel to induction of ERS, low concentrations of corticosterone (10 ng/ml and 50 ng/ml) could also activate the UPR. Splicing of XBP1 mRNA and ATF6 cleavage were observed in mice peritoneal macrophages. In order to further elucidate the relationship between ERS and altered immune functions of macrophages, we investigated the effects of thapsigargin, an ERS inducer, on macrophage function. Similar to the immunostimulatory effects of low concentrations of corticosterone, thapsigarin could enhance macrophages chemotaxis, phagocytosis and TNF-αproduction. However, high concentrations of corticosterone (1000 ng/ml) exert immunosuppressive effects on mouse macrophages (RAW264.7 cells) following stimulation with lipopolysaccharide (LPS), and inhibit LPS-induced ERS of RAW264.7 cells.2. Corticotropin-releasing hormone (CRH) is the most proximal element of the HPA axis, and it acts as central coordinator for neuroendocrine response to stress. To further examine the effects of HPA axis activation on ERS of immune cells and its relationship with altered immune functions in vivo, we investigated the expression of GRP78, XBP1 and ATF6 and altered immune functions of immune cells of CRH knockout mice exposed to the psychological stress (restraint stress) for 1 h. Our results show that following acute restraint stress, compared to CRH-/- mice, the mRNA and protein expression levels of GRP78 were significantly increaed in immune cells of CRH+/+ or CRH+/- mice. Meanwhile, IRE1/XBP1 and ATF6 signaling pathway were activated. Furthermore, we show here that the immune functions of primary peritoneal macrophages of CRH+/+ or CRH+/- mice was greatly enhanced, compared to those of CRH-/- mice.3. To explore the role of XBP1 in modulation of macrophage functions by GCs, after construction and screening of lentiviral vector of RNA interference of mouse XBP1 gene, we selectively inactivated the XBP1 gene in mice peritoneal macrophages infected with XBP1-siRNA lentivirus. We found that low concentration of corticosterone (50 ng/ml) did not increase the cellular phagocytosis and TNF-αproduction.4. The activities of GCs are mostly mediated by the glucocorticoid receptor (GR). Thus, we investigated the possible role of GR in the induction of ER stress in macrophages by low concentration of corticosterone. Our results showed that pretreatment of macrophages with the classical GR antagonist RU486 (mifepristone) could significantly inhibit the increased expression of GRP78 and XBP1 at both mRNA and protein levels induced by low concentration of corticosterone. Furthermore, blocking of the GR by RU486 partly abolished the immunostimulatory effects of low concentration of corticosterone.Taken together, we concluded that:1. In parallel to induction of ERS, low concentrations of corticosterone could also activate the UPR. Similar to the immunostimularoty effects of low concentrations of corticosterone, thapsigarin could enhance macrophages chemotaxis, phagocytosis and TNF-αproduction. high concentrations of corticosterone exert immunosuppressive effects related to ERS. Together, these results suggest that the immunostimulatory effects of low concentrations of corticosterone on macrophages might be related to its induction of intracellular ERS.2. HPA axis activation could induce ERS and enhance immune functions of immune cells, and also highlight a mechanistic association between the moderate activation of the HPA axis elicited immunostimulatory effects and induced ERS of immune cells.3. XBP1 might play an important role in modulation of the immunostimulatory effects of low concentration of corticosterone.4. GR was at least partly responsible for the immunostimulatory effects of macrophages induced by low concentration of corticosterone. Furthermore, low concentration of corticosterone induces ERS via GR in macrophages.