| The bacterial translocation(BT),defined as translocation of bacteria or bacterial products from the gut to the mesenteric lymph nodes(MLNs)and/or other extraintestinal organs.An increasing amount of evidence indicates that BT is intimately associated with the development of liver cirrhosis and its complications,such as hepatic encephalopathy,hepatopulmonary syndrome,hepatorenal syndrome,and liver failure.Indeed,BT-associated infections and spontaneous peritonitis are major causes of death in patients with liver cirrhosis.Although bacterial overgrowth into the small intestine and increased intestinal permeability have been observed in patients with liver cirrhosis,the mechanism of the intestinal immune changes that are closely related to BT had not previously been fully explained.Several types of intestinal CD4+ T cells are critical to host defences against BT.Th17 cells are distributed primarily in the intestinal lamina propria,especially in the small intestine,and are important for maintaining the integrity of the intestinal mucosal barrier and therefore crucial for preventing BT.Treg cells accumulate in the intestine lamina propria,where they play key roles in gut homeostasis and thereby affect the occurrence and development of BT.In this report,a rat model of cirrhotic rats with ascites were induced by CCl4 and phenobarbital,and the profiles of Treg(CD3 + CD4 + CD25 + Foxp3 +),Th17(CD3 + CD4 + IL-17 +),and Thl(CD3+ CD4 + IFN-?+)cells in the intestinal lamina propria,liver and blood were determined by flow cytometry and their relationships with BT was investigated.Additionally,we explored the mechanism of interaction between BT and alterations in Treg,Th17 and Thl cells in liver cirrhosis.MethodsA rat model of cirrhotic rats with ascites were induced by CCl4 and phenobarbital.Liver function was measured using a standard clinical automated analyser.The profiles of Treg,Th17,and Th1 cells in the intestinal lamina propria,liver and blood were determined by flow cytometry.Plasma LBP level was measured using an enzyme-linked immunosorbent assay(ELISA)kit according to the manufacturer's instructions.To explore the cause and effect relationship between BT and the changes in the proportions of Treg,Th17,and Th1 cells that occurred specifically in BT rats,as described above,we investigated the changes that occurred in these cells in rats with liver cirrhosis that were prevented from developing BT by intestinal decontamination,which was ensured using cocktails of antibiotics.And the level of LBP in plasma was measured by ELISA.ResultsTo determine if cirrhosis affects BT,MLN tissues were isolated for bacteria culture.The results showed that there was no BT in the normal control group,and the incidence of BT in CCl4-induced cirrhosis ascites rats was 47.8%.The concentration of LBP was significantly higher in cirrhotic rats with BT than in normal control rats or cirrhotic rats without BT,and the plasma concentration of LBP was correlated with colony number,with a correlation coefficient as high as 0.918(P<0.001).Cirrhotic rats with BT have worse liver functions,lower body weight,and higher spleen weight and ascites volume than those without BT.The concentration of LBP in cirrhotic rats with BT was negatively associated with the albumin concentration(p =-0.745;P<0.01)and the albumin/globulin ratio(p =-0.769,P<0.01)but was positively associated with the levels of AST(p = 0.655,P<0.05),TBil(p = 0.688,P<0.05),and DBil 0.692(P<0.05).The concentration of LBP in cirrhotic rats with BT was negatively associated with body weight(p =-0.893,P<0.001)but positively associated with spleen weight(p =0.665;P<0.05)and the amount of ascites(p = 0.878,P<0.001).Some changes in the percentage of Treg,Th17,and Th1 cells in liver cirrhotic rats with BT than those without BT have obvious differences.These changes included:in the proximal small intestine,the percentage of Tregs increased significantly(28.4±10.0 vs.13.2±2.9,P<0.001),the percentage of Th17 and Thlcells decreased significantly(14.9± 2.8 vs.31.9±9.0,P<0.001;9.7 ± 3.2 vs.21.8±3.7,P<0.001);in the distal small intestine,the percentage of Tregs increased significantly(29.5 ± 9.2 vs.15.3 ± 3.1,P<0.001),the percentage of Th17 and Th1cells decreased significantly(11.7 ± 4.3 vs.24.1 ± 6.2,P<0.001;7.3±2.8 vs.19.7 ± 4.7,P<0.001);in the cecum,the percentage of Th17cells decreased significantly(10.7±2.9 vs.27.4±9.0,P<0.001);in the colon,the percentage of Tregs increased significantly(21.2 ± 4.7 vs.14.5 ± 1.9,P<0.01),the percentage of Th17 and Thlcells decreased significantly(3.6 ±2.2 vs.8.3±2.8,P<0.001;5.0±2.8 vs.18.1±6.1,P<0.001);in the liver,the percentage of Tregs and Th17 cells increased significantly(32.5±4.0 vs.19.2±4.0,P<0.001;14.4 ± 3.3 vs.9.4±0.8,P<0.01),the percentage of Thl cells decreased significantly(8.4 ± 4.1 vs.13.3±3.0,P<0.05);in the blood,the percentage of Th17 and Thlcells decreased significantly(1.7±0.5 vs.3.2±0.4,P<0.01;2.0±0.2 vs.4.0±0.6,P<0.001).Furthermore,we investigated the association between BT and cellular immunity changes in cirrhotic rats with BT.The percentage of Treg cells was positively correlated with the serum concentration of LBP in the jejunum,ileum,and liver;the correlation coefficients were 0.691(P<0.05),0.636(P<0.05),and 0.760(P<0.05),respectively.The percentage of Th17 cells was negatively associated with the serum concentration of LBP in the jejunum,ileum,and cecum;the correlation coefficients were-0.836(P<0.01),-0.861(P<0.01),and-0.675(P<0.05),respectively.Of the Thl cells,the percentage of Th1 cells was also negatively associated with the blood concentration of LBP in the jejunum,ileum,and liver;the correlation coefficients were-0.809(P<0.01),-0.802(P<0.01),and-0.836(P<0.05),respectively.In summary,the levels of Treg,Th17,and Th1 cells were all significantly associated with the LBP concentrations in the proximal small intestine,and the distal small intestine,suggesting that these organs are primary sites for host defense against BT.To explore the cause and effect relationship between BT and the changes in the proportions of CD4 + T cells that occurred specifically in BT rats,as described above,we investigated the changes that occurred in these cells in rats with liver cirrhosis that were prevented from developing BT by intestinal decontamination,which was ensured using cocktails of antibiotics.None of the cirrhotic rats treated with antibiotics showed BT.gut decontamination resulted in a significantly(P<0.05)lower concentration of LBP in the antibiotic-treated than in the placebo-treated cirrhotic rats(4.9 ± 0.8 vs.11.2±5.5;P<0.05)and final body weight was significantly higher,while spleen weight and ascites volume were significantly lower.Gut decontamination significantly(P<0.05)increased the frequencies of Thl7 in intestinal lamina propria of antibiotic-than placebo-treated cirrhotic rats.Interestingly,antibiotics did not significantly amend the proportions of Treg and Thl in the intestinal lamina propria,liver and blood of antibiotic-than placebo-treated cirrhotic rats,except Treg in the proximal small intestine.However,the percentage of these cells was similar between cirrhotic rats with antibiotics and normal rats with either placebo or antibiotics.These data indicate that BT caused the changes observed in these cells.ConclusionIn conclusion,we found that BT may cause an increase in the number of Treg cells in the proximal small intestine and a decrease in the number of Th17 cells in the whole intestine and blood in cirrhotic rats.It may also aggravate the CCl4-induced decrease in the number of Thl cells in the proximal and distal small intestine,liver,caecum,and blood and the CC14-induced increase in the number of Th17 cells in the liver and in the number of Tregs in the distal small intestine,colon,and liver.Our data suggest that the intestinal decreasing Th17 cell,and increasing Treg cell seems to predispose to BT in cirrhotic rats.Immune changes were organ-specific and associated with BT.Cirrhosis results in BT;BT likely contributes to liver cirrhosis progression;cirrhosis and BT form a vicious circle.Understanding the gut-liver axis and the underlying mechanisms could lead to new avenues for liver cirrhosis treatment. |
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