| ObjectSalmonella typhi is a gram-negative enteric bacillus, which is bacterial pathogen able to cause a spectrum of diseases in an array of different hosts including human and domestic animals. Salmonella is a leading cause of human fever gastroenteritis, but liver as a major organ for the accumulation of bacteria is considered to play an important role in the replication of Salmonella inside the host and is also available to regulate the clearance of pathogens.Functional characterization of Toll-like receptors (TLRs) has established that innate immunity is a skillful system that detects invasion of microbial pathogens. Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune responses and further instruct development of antigen-specific acquired immunity. Recent aspects of TLR biology show that although TLRs are crucial for an efficient immune response, certain pathogens use TLR-based strategies to evade the host defense. Therefore, specific roles of different TLRs had become a focus of research in immunology. In our study, a mouse model for virulent Salmonella typhimurium was established by oral infection for observation of the different type and severity of Salmonella hepatitis in wide-type, TLR2-/-, TLR4-/-, and TLR9-/-mice.And a study on macrophages was designed to identify the mechanism of TLRs involved in Salmonella hepatitis. MethodsFirstly we observed the difference in survival among wide-type, TLR2-/-,TLR4-/-and TLR9-/-mice after orally infected with S. typhimurium. Cytokines in serum were detected by cytometric bead array.Secondly, Recruitment of numerous lymphocytes in liver from infected mice was detected by H&E. And then, the extent of liver injury was evaluated by the bacterial burden and ALT levels in liver. Cytokines in supernatant of liver homogenate were detected by CBA after S. typhimurium infection.At last, different ability of S. typhimurium elimination in macrophages were detected in wide-type, TLR2-/-, TLR4-/-, and TLR9-/-mice. And flow cytometry was used to explore the crosstalk between macrophages and NK cells following S. typhimurium infection.Results1. A mouse model for S. typhimurium infection.Genotypes of TLR-/-mice were detected by PCR. And then, standard curve between optical density (OD) and bacterial number was established by plate count method. On these basis, a mouse model for virulent Salmonella typhimurium was established by oral infection2. TLR2, TLR4and TLR9play different roles in the mouse model forS.typhimurium infection.We found that survival of C57BL/6wild-type, TLR2-/-TLR4-/-and TLR9-/-mice were significantly different following infection with S. typhimurium. TLR9-/-mice were significantly more susceptible to S. typhimurium infection than that of wide-type (WT) mice; by contrast, TLR2-/-mice showed stronger resistibility to S. typhimurium infection than that of WT mice; and the clinical course and survival of TLR4-/-mice were similar to that of control mice. A cytokine storm was induced in TLR9-/-mice, and a weak cytokine response was observed in TLR2-/-mice3. Different degrees of salmonella hepatitis in WT, TLR2-/-, TLR4-/-and TLR9-/-infected mice.We next assessed the histopathology, bacteria burden, and cytokine levels in liver from WT and TLR knockout mice following S. typhimurium infection. S.typhimurim infection would lead to an acute hepatitis in WT mice, and TLR9deficiency would aggravate this consequence, whereas TLR2or TLR4deficiency, especially TLR2deficiency, would relieve S. typhimurium infection-mediated hepatic inflammation.4. TLR2, TLR4and TLR9involved in the bactericidal effect of macrophages.The expression of TLR2, TLR4and TLR9on peritoneal macrophages were different in wide-type, TLR2-/-, TLR4-/-, and TLR9-/-mice during S. Typhimurium infection. And a little number of live bacteria was detected in TLR2-/-macrophages, companied by an increased inducible nitric oxide synthase (iNOS) activity following5. typhimurium infection. Meanwhile, the production of reactive oxygen species (ROS) was decreased in TLR9-/-macrophages compared to WT, TLR2-/-or TLR4-/-macrophages following S. typhimurium infection5. TLR2, TLR4and TLR9deficiency inhibited NK cell activation in response to S. typhimurium infection.Flow cytometry analysis showed that S. typhimurium infection augements the proportion of liver lymphocyte. And TLR2, TLR4and TLR9deficiency affect the expression of CD69, NKG2D,CD107a, and IFN-y of hepatic NK cells.6. TLR2, TLR4and TLR9deficiency disrupted the cytotoxicity of NK against S. typhimurium infected macrophages.A study of the crosstalk betwween NK cells and macrophages showed that TLR2, TLR4and TLR9deficiency on macrophages not only affect the activation of hepatic NK cells, but also inhibited NK cell activation in response to S. typhimurium infection.7. TLR2, TLR4and TLR9deficiency inhibited the expression of RAE-1on S. typhimurium infected macrophages.The expression of RAE-1was increased on macrophages from both wild type and TLR deficient mice by S. typhitnurium-infection, the rising of RAE-1was much higher in WT mice compared with TLR deficient mice.In the case of Qa-1,the ligand inhibiting NK cytotoxicity was lower in TLR deficient macrophages than that in wild type macrophages, but it was downregulated in all phenotypes of macrophages and reached a similar level. Conclusion1. Severity of Salmonella hepatitis affect the survival of wide-type, TLR2"’ TLR4-/-, and TLR9-/-mice during S. typhimurium infection.2. Hepatic macrophages play critical roles in anti-S. typhimurium immune response, and TLR2deficiency enhanced the clearance ability of macrophages, while TLR9deficiency disrupted the clearance ability of macrophages, and TLR4deficiency affected little on the clearance of S. typhimurium.3. TLR2, TLR4and TLR9deficiency will all inhibit the cytotoxicity of NK cells against S. typhimurium-infected macrophages, which is due to the down-regulated of RAE-1on TLR2-/-, TLR4-/-, and TLR9-/-macrophages. |
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