The Mechanism Of Monomeric C-Reactive Protein Activating Macrophages

C-reactive protein(CRP)is a typical acute phase protein of the pentamer protein family,and its plasma concentration could increase rapidly when human body gets infected or suffers damage.For this reason,it is common treated as a non-specific marker of inflammatory response in clinical practice.As a soluble pattern recognition receptor(PRR),CRP plays a significant role in inflammatory response and host defense.There have been more and more studies showing that CRP is not only a non-specific inflammatory marker,but also directly involved in the development of some diseases,such as atherosclerosis and rheumatoid arthritis.Furthermore,it has been discovered in our lab and by other groups that pentamer CRP can be depolymerized into monomeric CRP(mCRP)after the interaction with pathogens or damaged cell membrane surface ligands,thus enhancing ligand recognition and stimulation efficiency.As an important part of innate immunity,macrophages play a vital role in inflammation and host defense.When tissue damage or pathogen infection occurs,macrophages are enriched in lesions to play a crucial role in the initiation,development,and regression of inflammation and tissue repair.Otherwise,plasma levels of CRP would be significantly elevated during tissue damage and pathogen infection.Besides,the mCRP dissociated fromCRP is selectively produced and located in lesions,which may affect inflammation by regulating macrophage responses.Despite some studies revealing that CRP can activate macrophages,it remains unclear whether the effect is caused by endotoxin pollution or conformational heterogeneity(mCRP doping).To begin with,we prepared the CRP and mCRP with high purity and uniform conformation.Then,the effect of CRP and its conformational changes on macrophages was explored using these proteins not subjected to endotoxin pollution.High-purity CRP and mCRP were applied to stimulate macrophages from different sources.It was discovered that mCRP,rather than CRP,could promote the secretion of inflammatory cytokines in macrophages,suggesting that the effect of CRP on macrophages is conformation dependent.The use of polymyxin B(PMB)as an endotoxin neutralization reagent eliminated the effect of residual endotoxin.As suggested by the transcriptome analysis,mCRP may play its role through the same downstream signaling pathway as LPS.Furthermore,the experiment conducted through a concentration gradient combination of mCRP and LPS to stimulate macrophages revealed a competitive inhibition between mCRP and LPS,which suggests a possibility that mCRP and LPS could compete for the same receptor.It is also known that the receptor of LPS is Toll-like receptor 4(TLR4).For this reason,the interaction between mCRP and TLR4 was investigated.According to the results of SPR,mCRP have a strong ability to bind to TLR4.mCRP was found capable to promote TLR4-MD2 dimerization in the flow cytometry experiment.These two experiments suggested mCRP could not only bind to TLR4 but also activates downstream signal pathways.It was confirmed that mCRP does play its role via the TLR4 receptor by using the cell lines with TLR4 signaling pathway key protein knockout and mice with TLR4 knockout.The knockout of TLR4 and its key downstream proteins could contribute to eliminating the pro-inflammatory effect of mCRP.The peptide competitive ELISA revealed the potential of aa35-47 being the mCRP-TLR4 binding motif.The binding efficiency of aa35-47 deletion mCRP to TLR4 is significantly lower compared to wild-type mCRP,confirming that aa35-47 is the binding motif of mCRP to TLR4.It was discovered in the subsequent experiments that the endosome signaling pathway,as the other downstream of the TLR4 signal pathway,was not activated by mCRP,nor were there any significant changes in IFN-α/β or its key downstream proteins,Viperin and p-STAT1.In summary,the interaction between mCRP and TLR4,as well as the regulatory mechanism of mCRP on macrophage response,were elaborated in this study.Additionally,it was found out that CRP,known as a PRR,could be transformed into a ligand of another PRR,TLR4,through dissociation into mCRP,which is conducive to the construction of a new PRR coupling interaction model.By improving the understanding as to the role of CRP in host defense,this study is expected to promote the development of potential targeted interventions in related inflammatory diseases.