Characteristics Of The Intestinal Flora And Its Relationship With Innate Immunity In Mice With Endotoxemia:An Experimental Study

Objective:Sepsis-induced intestinal injury increases the mortality of sepsis but lacks effective therapeutic targets and measures.Intestinal flora and intestinal innate immune play an important role in maintaining intestinal homeostasis.However,their changes and their correlation during the development of sepsis are not clear.Lipopolysaccharide(LPS)-induced symptoms of endotoxemia appear similar to the reactions of sepsis.This experiment intends to explore the changes in the intestinal flora and innate immune cells of mice during the onset and development of endotoxemia and their correlation,to provide a new therapeutic target and theoretical basis for the prevention and treatment of the sepsis-induced intestinal injuryMethods:Part 1:Establish and evaluate the models of endotoxemiaTwenty-four male C57BL/6J mice,8-10 weeks,were randomly divided into Control group(n=6)and Endotoxemia group(n=18).The mice in Endotoxemia group were given a single intraperitoneal injection of lipopolysaccharide(LPS,10mg/kg),and those in Control group were given 0.9%Na Cl in the same amount,and the changes of clinical symptoms,diarrhea symptoms,weight and survival rate were observed within 1 week after the mice were modeled.Another eighteen male C57Bl/6J mice,8-10 weeks,were randomly divided into Day0 group,Day2 group,and Day7 group,n=6 per group.The mice from the Day2 group and the Day7 group received a single intraperitoneal injection of LPS(10 mg/kg)and were sacrificed on the 2nd and 7th days after injection.The mice in the Day0 group were injected with an equal amount of 0.9%Na Cl and immediately sacrificed after injection.Their serum and large intestine tissue were collected.Cytometric Bead Array(CBA)was used to detect the changes of inflammatory factors(TNF-?,IL-1?,IL-6,IL-10)in peripheral blood.Hematoxylin-Eosin(HE)staining was used to detect the pathology of intestinal tissue.Real-time quantitative gene amplification fluorescence detection system(RT-q PCR)was used to detect the m RNA expression of intestinal inflammatory factors(TNF-?,IL-1?,IL-6,IL-10).Part 2:Characteristics of the intestinal flora and its relationship with innate immunity in mice with endotoxemiaTwenty-four male C57Bl/6J mice,8-10 weeks,were randomly divided into Day0group,Day2 group,and Day7 group,n=8 per group.The modeling method and sample collection time were the same as Part 1.The large intestine was collected and its lamina propria immune cells were isolated.The number of MNPs and ILCs and their cytokines(MNPs:IL-10,TNF-?,ILCs:IL-22)were analyzed by flow cytometry.After the cecum and large intestine stool were collected,16S r RNA gene sequencing technology was used to analyze the intestinal flora diversity and species composition.Finally,Spearman correlation analysis was performed to explore the relationship between intestinal flora and innate immunity in mice with endotoxemia.Results:Part 1:Establish and evaluate the models of endotoxemiaCompared with the Control group,the 1-week survival rate of the Endotoxemia group mice was significantly reduced(P<0.05).After the LPS treatment,the clinical score and diarrhea score were significantly increased on the 2nd day and reached the highest level(both P<0.001),bodyweight was significantly reduced(P<0.001),peripheral blood inflammatory factors(TNF-?,IL-1?,IL-6,IL-10)were significantly increased(all P<0.05),and the expression of colorectal inflammation genes was significantly increased(IL-1?,P<0.05),the pathological damage of the large intestine was obvious;by the 7th day,the clinical score was close to Day0,and the diarrhea disappeared,the bodyweight increased obviously,the peripheral blood inflammatory factors recovered to the baseline,the pathological damage of the large intestine was getting better.It is determined that Day0 is the period of steady-state,Day2 is the period of clinic manifestation,and Day7 is the period of clinic recovery.Part 2:Characteristics of the intestinal flora and its relationship with innate immunity in mice with endotoxemia1.Characteristics of intestinal flora in mice with endotoxemiaPCo A analysis showed there were significant changes in the intestinal flora of mice during endotoxemia(P=0.001).At the Phylum level,compared to the period of steady-state(Day0),the abundance of Firmicutes decreased at the period of clinic manifestation(Day2)and then recovered at the period of clinic recovery(Day7),the abundance of Proteobacteria increased first and then decreased significantly(P<0.05),the abundance of Saccharibacteria continued to decrease(P<0.01).At the Genus level,the abundance of norank?f??Bacteroidales?S24-7?group and Lachnospiraceae decreased during the period of clinic manifestation(Day2),then recovered at the period of clinic recovery(Day7).The abundance of Enterococcus and Escherichia-Shigella increased significantly at the period of clinic manifestation(Day2)(both P<0.01),but all disappeared at the period of clinic recovery(Day7).2.Characteristics of intestinal innate immunity in mice with endotoxemiaDuring the endotoxemia,intestinal inflammatory macrophages(IMs)continued to increase(P<0.01),and resident macrophages(RMs)continued to decrease(P<0.05).Compared with the period of steady-state(Day0),the intestinal DCs at the period of clinic manifestation(Day2)were significantly reduced(P<0.01),and its subgroups proinflammatory DCs(CD103~-DCs)were significantly increased(P<0.001),the regulatory DCs(CD103~+DCs)decreased significantly(P<0.001),but all returned to baseline at the period of clinic recovery(Day7).At the period of clinic manifestation(Day2),the TNF-?expressed by IMs and the IL-10 expressed by RMs increased significantly(both P<0.05),and then recovered basically at the period of clinic recovery(Day7).At the period of clinic manifestation(Day2),The expression of IL-22 in ILC3 was significantly reduced(P<0.01),and it could not return to normal entirely at the period of clinic recovery(Day7).3.Correlation analysis between intestinal flora and innate immunity in mice with endotoxemiaSpearman correlation analysis found that among the top 50 species in the intestinal flora,22 bacterial species were significantly correlated with innate immune factors.The number of innate immune factors significantly related to norank?f??Bacteroidales?S24-7?group was the most(6/12),followed by Enterococcus and Escherichia-Shigella(both 5/12).The norank?f??Bacteroidales?S24-7?group were significantly positively correlated with ILC3,IL-22~+ILC3,and CD103~+DCs(all P<0.05),and significantly negatively correlated Escherichia-Shigella were significantly negatively correlated with IL-22~+ILC3 and CD103~+DCs(both P<0.05),but with CD103~-DCs,TNF-?~+IMs,IL-10~+RMs showed a significantly positive correlation(all P<0.05).Among all innate immune factors,the number of bacterial species related to TNF-?+IMs was the most(12/22),followed by CD103~+DCs,CD103~-DCs,IMs,and IL-10~+RMs(all 6/22),while ILC2,IL-10~+IMs,and TNF-?~+RMs have no significant correlation with intestinal flora.TNF-?~+IMs was significantly negatively correlated with the abundance of norank?f??Bacteroidales?S24-7?group and Lachnospiraceae(both P<0.05),while was significantly positively correlated with the abundance of Enterococcus and Escherichia-Shigella(both P<0.05).Conclusions:1.The composition of the intestinal flora changed significantly during the endotoxemia in mice.The main characteristic was that the norank?f??Bacteroidales?S24-7?group decreased when during intestinal injury,and increased in quantity when the intestinal injury improved,the Enterococcus and Escherichia-Shigella only appeared and increased in quantity during intestinal injury,but could not be detected at the period of steady-state and when intestinal injury improved.2.The intestinal innate immunity changed significantly during the endotoxemia in mice.The main manifestation was that TNF-?expressed by IMs increased in quantity during intestinal injury and decreased significantly when intestinal injury improved,but there was no significant change in TNF-?expressed by RMs.IL-22 expressed by ILC3significantly decreased during intestinal injury,and was still suppressed when the intestinal injury improved.3.There was a significant correlation between the intestinal flora and innate immunity during the endotoxemia in mice.The number of bacterial species significantly related to TNF-?~+IMs was the most.It was significantly negatively correlated with the abundance of norank?f??Bacteroidales?S24-7?group and Lachnospiraceae and positively correlated with the abundance of Enterococcus and Escherichia-Shigella.The number of innate immune factors significantly related to norank?f??Bacteroidales?S24-7?group was the most.Its abundance was significantly positively correlated with ILC3,IL-22~+ILC3,and CD103~+DCs,and significantly negatively correlated with CD103~-DCs,TNF-?~+IMs,and IL-10~+RMs.4.The results of this study are expected to provide inspiration for the study of the pathological mechanism and prevention measures of sepsis-induced intestinal injury.