The Mechanism Of TLR2/4 And NLRP3 Mediate Inflammatory Response In Mice Infected By Escherichia Coli

Escherichia coli is a gram-negative bacterium,which is an important part of normal intestinal flora of humans and other mammals,nevertheless,it is also a highly diverse and even deadly pathogen that can cause a range of diseases.Pattern recognition receptors?PRRs?,including toll-like receptors?TLRs?and node-like receptors?NLRs?,recognize specific pathogen-associated molecular patterns?PAMPs?or damage-associated molecular patterns?DAMPs?in the early response of hosts to invading pathogens.TLR4 of immune cells such as macrophages and dendritic cells is mainly activated by lipopolysaccharide?LPS?,a component of the cell wall of gram-negative bacteria,and then activates the host inflammatory response.Substantial evidence supports a broad role for TLR2 as a PRR for a variety of microbes and microbial structures.Thus,both TLR2 and TLR4 play important roles in the host response to E.coli infection.The NLRP3 inflammasome is currently the most fully characterized inflammasome,which responds to numerous physically and chemically diverse stimuli,and leads to a series of diseases when out of control.However,whether NLRP3 inflammasome is involved in innate immune responses to E.coli infection remains unclear.In the present study,based on the above background,the following questions were raised.Whether NLRP3 inflammasome,like TLR2 and TLR4,plays a role in the inflammatory response caused by E.coli infection?Detailed researchcontents are as follows:First section,the role of TLR2,TLR4 and NLRP3 in innate immunity of E.coliinfected peritoneal macrophage was investigated:?1?NLRP3 expression of LPS?BLP?E.coli DH5? and E.coli JE5505 stimulated WT?TLR2^-/-and TLR4^-/-macrophage was investigated using Flow cytometry,the results indicated that in WT,TLR2^-/-and TLR4^-/-macrophages NLRP3 expression was significantly increased after LPS,BLP,E.coli DH5a and E.coli JE5505 stimulated compared with the unstimulated group.?2?The secretion of inflammatory cytokines?pro-inflammatory cytokines TNF-a and IL-1?,anti-inflammatory cytokines IL-10 and chemokines RANTES?in LPS?BLP?E.coli DH5? E.coli E5505 stimulated WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-macrophages was investigated using ELISA,the results indicated that the secretion of TNF-? and IL-1? in TLR2V^-/-,TLR4^-/-and NLRP3^-/-macrophages after LPS,BLP,E.coli DH5a and E.coli JE5505 stimulated was significantly lower than that in WT macrophages.?3?The activation of MAPK,NF-?B and caspase-1 signaling pathways in WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-macrophages stimulated by LPS,BLP,E.coli DH5a and E.coli JE5505 was investigated using Western bloting,The results showed that the activation of the MAPK,NF-?B and caspase-1 signaling pathways in the TLR2^-/-,TLR4^-/-and NLRP3^-/-macrophages was decreased compared with that in WT macrophages.?4?The expression of COX-2 in WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-macrophages after LPS,BLP,E.coli DH5a and E.coli JE5505 respectively stimulated was investigated using real-time PCR and Western bloting,The results showed that the expression of COX-2 in LPS,BLP,E.coli DH5a and E.coli JE5505 respectively stimulated TLR2^-/-,TLR4^-/-macrophages was significantly reduced,in BLP,E.coli DH5a and E.coli JE5505 respectively stimulated NLRP3^-/-macrophages was significantly reduced compared with WT macrophages.Secondly,the role of TLR2,TLR4 and NLRP3 in innate immunity of E.coli infected mice was investigated.LPS,BLP,E.coli DH5a and E.coli JE5505 were injected into the abdominal cavity of WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-mice,respectively.?1?The secretion of inflammatory cytokines?pro-inflammatory cytokines TNF-a and IL-1?,anti-inflammatory cytokines IL-10 and chemokines RANTES?in LPS?BLP?E.coli DH5a ? E.coli JE5505 stimulated WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-serum was investigated using ELISA,the results indicated that LPS,BLP,E.coli DH5a and E.coli JE5505 stimulated TLR2^-/-,TLR4^-/-and NLRP3^-/-mice serum exhibited impaired TNF-a and IL-1? secretion compared with that in WT serum;?2?the expression of HMGB1 in LPS,BLP,E.coli DH5a and E.coli JE5505 stimulated WT,TLR2^-/-,TLR4^-/-and NLRP3^-/-mice liver was investigated using real-time PCR and Immunofluorescence assays,the results indicated that HMGB1 expression levels in livers were significantly impaired in TLR2^-/-,TLR4^-/-and NLRP3^-/-mice after LPS,E.coli DH5a or E.coli JE5505 stimulation,compared with that in WT mice,TLR2^-/-and NLRP3^-/-mice exhibited impaired HMGB1 expression level compared with WT mice after BLP stimulation;?3?Levels of AST and ALT in sera and morphological examination of liver from mice injected intraperitoneally with E.coli DH5a were analyzed by a bench-top dry chemistry analyzer and hematoxylin and eosin?H&E?staining,the results indicated that the levels of serum AST and ALT and liver damage were reduced in TLR2^-/-,TLR4^-/-and NLRP3^-/-mice compared with those in WT mice.The above results suggest that TLR2,TLR4 and NLRP3 play an important role in the secretion of inflammatory cytokines,the activation of relevant signaling pathways and the expression of COX-2 in macrophages of mice infected with E.coli;TLR2,TLR4 and NLRP3 play an important role in the secretion of inflammatory cytokines in serum,the expression of HMGB1 in liver and liver injury after mice infected by E.coli.In conclusion of this present study:in addition to TLR2 and TLR4,NLRP3 participates in E.coli-induced inflammatory responses in mice.