Expression Of Toll-like Receptor 2/4 On Rat Macrophages And Its Regulation By Neuroendocrine Hormones Following Trauma

Immunosuppression is a common complication after trauma, and it may lead to the development of infection, sepsis, and multiple organ dysfunction syndrome (MODS),which eventually influence outcome. Innate immunity is the first line to defend body against infection and injury through rapid initiation of non-specific cellular and fluid response. Innate immunity plays an important role in the occurrence and development of inflammation after trauma. Macrophages are important resident cells to mediate innate immunity. Toll-like receptor(TLR)2 and TLR4 are two major members of TLRs, mainly expressed on monocytes and macrophages. TLR4 is the pattern recognition receptors(PRRs) of LPS, and recently it was found that it can bind to many damage-associated molecular patterns(DAMPs). As a sentinel to detect tissue injury TLR4 mediate sterile inflammation. TLR2 can recognize a wider variety of pathogens, and it also can participate in infla- mmation after injury. After TLR2/4 bind to their respective ligands, they can cause a similar casade of signal transduction, resulting in the NF-κB activation and finally the production of inflammatory cytokines, chemokines, and costimulatory molecules, which are considered to be the switch to innate immune response to pathogenic microorganisms and endogenous damage-associated molecules. With the changes of innate immunity trauma often sets off neuroendocrine changes, which is charactrized by excited sympathetic nervous system and activated hypothalamic-pituitary-adrenal(HPA) axis to some extent. Norepinephrine(NE), epinephrine(E), and glucocorticoids are produced in large quantity. Neuroendocrine hormones may influence immune functions to keep homeostasis through the neuro-endocrine-immune network. Previous research has shown that following trauma peritoneal or spleen macrophages often could display impaired responsiveness to LPS, but the mechanism remains unclear. As two important receptors, the levels of TLR2/4 expression are closely related with the sensitivity to their ligands. Although extensive research is focused on TLRs and their signaling, how to regulate TLR2/4 expression remains unknown. To study the expression of TLR2/4 and its regulation by neuroendocrine hormones after trauma is of importance, it will provide valuable knowledge to explore the mechanism behind immune dysfunction and prevent and treat its complications to improve survival rate.Based on the above knowledge, the present research established a rat trauma model, peritoneal macrophages were collected from rats and the following studies were done: the level of TLR2/4 mRNA expression on rat macrophages was evaluated dynamically after trauma by RT-PCR; tumor necrosis factor-α(TNF-α) secretion of macrophages stimulated by different concentrations of Pam3CSK4 and LPS respectively was observed 24h after trauma to analyze receptors reactivity; changes of serum E and NE were determined by HPLC after trauma for 24 hours; at the mean time, the levels of serum adrenocorticotrophic hormone (ACTH), cortisol,T3,T4,TSH,β-EP,and PRL were determined by radioimmunity assay; following study in vitro is done to observe the effects of corticosterone (CORT), E, and NE on TLR2/4 mRNA expression on macrophages by real-time quantitative PCR and semi-quantitative RT-PCR in different concentrations and for different durations; finally, transection of the cervical sympathetic trunk(TCST) was performed to observe its effect on the changes of neuroendocrine hormone after trauma.Following results were made from the research:1. The expression of TLR2/4 mRNA could be downregulated by trauma, which continued from 2h to 24h following trauma. Trauma resulted in impaired responsiveness to both LPS and Pam3CSK4 on macrophages.2. Following trauma, the elevation of serum levels of E, NE, ACTH, and cortisol continued from 2h to 24h. Although the levels of T3 and T4 were decreased, those of TSH remained instant following trauma. The serum levels ofβ-EP and PRL not from pituitary-adrenal axis were also increased.3. After macrophages exposure to CORT, its down regulation of TLR2 mRNA expression was time-dosedependent, but no significant influence on TLR4 mRNA expression. The down regulation of TLR2 mRNA and TLR4 mRNA expression by E was also time-dosedependent in macrophages. After macrophages exposure to NE, it(0-10000ng/ ml) could not downregulate the expression of TLR2/4 mRNA significantly, and neither did NE(1000ng/ml) 48h after exposure.4. Although TCST can not attenuate remarkable changes of neuroendocrine hormones 2h following trauma, the levels of serum NE and cortisol were decreased significantly 24h following trauma.In conclusion, TLR2/4 expression is downregulated, which may help to prevent excessive inflammatory response following trauma and means that the organism has less ability to cope with pathogens then. Trauma results in impaired macrophages responsiveness, which may be related with the down regulation of TLR2/4 expression. Following trauma remarkable neuroendocrine changes occur, which is mainly characterized with excited sympathetic nervous system and activated hypothalamic-pituitary-adrenal axis. The pituitary-thyroid axis is changed, and the serum hormones levels not from pituitary-adrenal axis are increased, which cooperate to regulate body functions to keep homeostasis. After macrophages exposure to higher concentrations of CORT and E for longer durations, they may downregulate the levels of TLR2 mRNA or TLR4 mRNA expression . TCST can attenuate neuroendocrine hormones changes following trauma, which may be a useful measure.The present study provided valuable laboratorial and theoretical evidence for the research on the expression and regulation of TLR2/4 following trauma.