The Innate Immune Response Mechanism Of TLR2, TLR4and RP105in Mouse Macrophages Infected By Staphylococcus Aureus

Staphylococcus aureus (S. aureus) is a common Gram-positive pathogen. Variousinfectious diseases are caused by S. aureus in human and animals, such as pneumonia,endocarditis, sepsis and bovine mastitis. Sometimes, serious S. aureus infection cancause death in both human and animals. As soon as the infection occurs, recognitionof invading pathogens by immune system is the first step of host defense. Thisrecognition process is mediated and depended on pattern recognition receptors (PRRs),such as Toll-like receptors (TLRs). So far, thirteen TLRs have been identified inhumans and mice, which named simply TLR1to TLR13. TLRs are type Itrans-membrane proteins and express on the cell membranes of both immune cells andnon-immune cells. Among these TLRs, it is demonstrated that TLR2serves a crucialrole in host responses to S. aureus infection. Previous studies indicated thatlipoteichoic acid (LTA) and peptidoglycan (PGN) from S. aureus were TLR2ligandsand induced cytokine and chemokine secretion by immune cells in a MyD88signalingpathway-dependent manner. However, more subsequent studies indicated thatlipoproteins, not LTA and PGN, were the dominant immunobiologically activecompounds in S. aureus that activated immune cells via TLR2. It has been reportedthat TLR2cooperates with TLR1, TLR6and forms heterodimeric complexes in hostresponse to S. aureus lipoproteins, but no detailed analysis has addressed whetherother TLRs are involved and cooperate with TLR2in this process. The aim of thisstudy is to investigate whether other TLR family proteins cooperate with TLR2inhost innate immune response to S. aureus, if they did, what roles they play in thisprocess.Lipoproteins from pathogens such as mycobacteria, spirochetes and mycoplasma,in addition to those from S. aureus, have been identified as TLR2agonists in previousstudies. Blumenthal et al. recently reported that a TLR2agonist,19kDa lipoprotein ofMycobacterium tuberculosis (Mtb), activated mouse macrophages not only in a TLR2-dependent manner. Another member of the TLR family of proteins, RP105,was also involved in this process. In mouse macrophages, TLR2signaling wascooperated with and optimized by RP105in response to19kDa lipoprotein of Mtb,and then induced cytokine and chemokine secretion by macrophages. Similar to thatof Mtb, lipoproteins are the dominant immunobiologically active compounds in S.aureus that activate immune cells through TLR2signaling. We predict that RP105also involved in innate immune response to S. aureus in macrophages. It had beenconfirmed that RP105/MD-1complex physical interacted with TLR4/MD-2complexin antigen presenting cell (APC), such as in macrophages and DCs, andproinflammatory cytokine secretion via TLR4signaling was inhibited by RP105. In Bcells, recognition of lipopolysaccharide (LPS) through TLR4signaling was optimizedby RP105. The function of RP105related closely to TLR4in immune cells. It wasnecessary to determine TLR4is also involved in the host macrophages activated by S.aureus in this study.In our work conditions, mouse peritoneal macrophages were infected with S.aureus SA113(ATCC35556) wild type (WT), SA113lgt::ermB (Δlgt) or SA113lgt::ermB+pRBlgt (+pRB). Our data indicated that in comparison with the other two S.aureus strains, SA113lgt::ermB (Δlgt) could not induce an abundant proinflammatorycytokine, anti-inflammatory cytokine and chemokine secretion by macrophages.SA113lgt::ermB (Δlgt) also could not induce an abundant activation of IκB-α-NFκBand MAPK signaling pathway in mouse macrophages. These results suggested thatwithout lipoproteins expression, S. aureus could not be recognized or trigger animmune response in macrophages. We also found that deletion of lipoproteinsexpression in S. aureus had no effect on costimulatory molecules CD80and CD86surface expression in macrophages after infection. These results indicated that theactivation of macrophages by S. aureus lipoproteins could be mainly depended onTLRs signaling pathway and lipoproteins had no effect on macrophages costimulatorymolecules surface expression.The data demonstrated that TLR2, TLR4, RP105and MD-1surface expressionwere increased in macrophages after S. aureus infection. The data also exhibited thatTLR2gene expression was increased but TLR4, RP105and MD-1gene expression was decreased in this process. In order to explain these results, TLR4and RP105expression in total cellular protein was analyzed in further studies. The data indicatedthat total cellular TLR4and RP105expression by macrophages were decreased after S.aureus infection. These results are well supported by TLR4and RP105geneexpression in our experiments, which suggests that the transfer efficiency of TLR4and RP105protein onto cell membrane could be changed after S. aureus infection.TLR2, TLR4and RP105can form heterodimeric complexes in macrophages, whichplay their roles in innate immune system in host resistance against S. aureus infection.After function blockage on macrophages surface, we find that TLR2, TLR4andRP105all play their unique roles in cytokine secretion and in activation ofIκB-α-NFκB and MAPK signaling pathway by macrophages after S. aureus infection,demonstrate that TLR2, TLR4and RP105are involved in activation of macrophagesby S. aureus.In order to investigate these results, further experiments were carried out todetermine the effect of RP105on TLR2and TLR4signaling. In our work conditions,TLR4ligand LPS and TLR2ligand Pam3CSK4were used in stimulation of mousemacrophages. The data indicated that LPS induced NFκB p65localization into cellnuclei and proinflammatory cytokine secretion by macrophages were inhibited byRP105. But RP105had no effect on Pam3CSK4induced NFκB p65localization intocell nuclei and proinflammatory cytokine secretion. We also found that LPS andPam3CSK4induced anti-inflammatory cytokine and chemokine secretion bymacrophages was RP105dependent, which suggested that similar to other TLRs,RP105could also have its own signaling pathway.In conclusion, not only TLR2, but also TLR4and its homolog RP105wereinvolved in macrophages innate immune response to S. aureus infection and playedcrucial roles during this process. We also suggested that the function of RP105beyondregulating of TLR4signaling. RP105could also play important roles in host innateimmune resistance against pathogens infection. This hypothesis will be supportedwith more evidences in our future studies.