The Research For The Alterations And Its Significance Of Intestinal Dendritic Cells From Septic Mice

Sepsis is a common and serious complication in general surgery patients. Because of its complex pathophysiology, the treatment of septic patients has troubled surgery and intensive care physicians for a long time. Recent studies indicate that immune dysfunction is a major cause of the high mortality rate of septic patients. Among the components of the immune system that may contribute to immune dysfunction in sepsis, dendritic cells (DC) play a key role. Several researchers have reported that profound depletion of DC occurs in both septic patients and septic mice. However, these researches mainly focused on the loss of DC in spleen or other lymph organ. Little insight was provided concerning the alterations of DC present in other organs during sepsis.Today a large body of experimental data have documented that the gastrointestinal tract is not a passive organ for the patients who suffered from endotoxin shock (or sepsis). The ensuing translocation of bacteria and their products gained increasing acceptance as a major contributor to the development of secondary infection and MODS following septic shock. In gut, resident DC has been described in Peyer's patchs and lamina propria. Gut DC can pick up antigen that has been transported across the intestinal epithelium through various different routes. So, they are important for the immunologic and barrier functions of gut. Moreover, because of the needs of specialized immune system of the gut to respond appropriately to the large antigenic load normally present in the form of food antigens and commensal bacteria, gut DC have different properties and functions with those in spleen. Thus, in our present study we carefully characterize the alteration of gut DC during sepsis, and its effect for intestinal inflammation and the treatment of sepsis.Due to its low number, the isolation and examination for gut DC is difficult. In this study, we make some improvements based on methods reported by Sun CM. We digest the intestine of mice with EDAT+DTT and Collagenase IV to get the single suspension of Peyer's patch(PP) or lamina propria(LP).Then the single cell suspension we got was stained with antibody such as CDllc for flow cytometry. The results indicate it is a stable method for the detection of intestinal DC.In the second section, we induce the model of sepsis by intraperitoneal injection of LPS.Then we analyzed the population and MHCII and CD86 expression of DC that present in lamina propria (LP), Peyer's patches (PP), and blood. We also examined whether there was an increase or loss of a specific DC subpopulations. Additionally, according to the levels of TNF-a and IL-10, we assessed the inflammatory state at various time points. Interestingly, we found that the alteration of intestinal DC was different with splenetic DC's. Just after the injection of LPS, the number of intestinal DC significant increased rather than decreased. The loss of intestinal DC was observed until 24h after onset of sepsis. We also noted that the expression of CD86 and MHCII on intestinal DC were down-regulated by 90min in septic mice. Additionally, we found that the increase of LP and PP DC was mostly contributed to the expansion of CD4-CD8+DC. However, the changes of DC in circulating were opposite to gut DC's. Thus we speculated that the recruitment of DC precursors in blood into the intestine might be able to explain this.In the third section, to verify our hypothesis, we examined the alteration of the gene expression of CX3CR1-FKN and CCC6-MIP-3a after LPS stimulation. And to evaluate the effect of the DC migrated from blood on intestinal inflammation induced with LPS, mice were pre-treated with Flt3L or normal saline, and then challenged with or without LPS. After that, occurrence of bacterial translocation to distant organ and inflammatory response were evaluated. Additionally, the population and maturity of DC in LP and circulating was analyzed by flow cytometry. We observed that pretreatment of Flt3L significantly expanded DC in LP and blood, but not induce alteration of their maturity. We also found LPS injection induced up-regulation of CX3CR1-FKN and CCC6-MIP-3a mRNA levels and drastic increase of DC in intestine. However, exacerbation of DC growth induced by Flt3L-pretreatment aggravated intestinal inflammation and increased the mortality of endotoxemic mice rather than enhance resistance to bacterial translocation. The data of this section proved that migration of DC from circulating into intestine was a major mechanism for the increase of gut DC after LPS injection. Moreover, it suggested that DC play a key role in the development of intestinal inflammation induced by LPS. And recent studies have addressed the effect of immune-enhance treatment such as Flt3L, IFN-y and Tal as an immunotherapeutic option to reverses sepsisi-related immunoparalysis. However, the data of the current study show that Flt3L-induced increase in numbers of DC caused serious consequences for the mice after challenged with LPS. Thus down-regulated DC numbers at the pro-inflammatory stage and up-regulated DC numbers when immune was suppressed may be able to help host survival in the campaign to endotoxin shock or sepsis.Thus we verify our hypothesis in last section. Base on the results of above study, we choose 3 hour and 24 hour after LPS injection as the treatment time point. And dexamethasone (DXM) and thymosin alpha-1 (Tal, Zadaxin) that can be directly applied in the clinic were chosen as a means of anti-inflammation or immune-enhance treatment. Septic mice were randomly divided into five treatment groups.Then survival rates, levels of TNF-a and IL-10, the occurrence of bacterial translocation and the ability to clear secondary infections was observed. The behavior of DC over time was also evaluated. Among the five treatment groups, the combined treatment of dexamethasone and thymosin alpha-1 induced the highest survival rate, was associated with a decrease in bacterial translocation to extra-intestinal organs and enhanced the ability to eradicate secondary infections by reversing the change in DC numbers during sepsis. It provides evidence for a possible strategy to modulate numbers of DC to improve outcome of sepsis.