The Molecular Mechanisms Of Rif Regulating Host Innate Immune Response Induced By PAMPs

The Rho subfamily of small G proteins plays critical roles in cell morphology,movement,and adhesion by dynamic regulation of actin cytoskeleton.In the meanwhile,they can also affect cell proliferation and immune responses by regulating gene expression.It is known that the small Rho GTPase Rif(Rho in filopodia)can regulate the formation of filopodia structure,but it is still unclear whether it can participate in the intracellular immune regulation processes.We found that Rif displays a restricted expression pattern in immune cells,indicating its potential function in immune response.Pathogen-associated molecular patterns(PAMPs)are essentially different components from bacteria or viruses,which can be recognized by corresponding PRRs(pattern recognition receptors)and cause immune responses in the body.This study revealed for the first time the regulatory mechanism of the small G protein Rif on pattern recognition receptor-mediated innate immune responses.We found that LPS,upon recognition by TLR4,rapidly caused the activation of Rif through Src tyrosine kinase phosphorylation and induced NF-κB activation and pro-inflammatory factor production by activating downstream PKCα;we also revealed that HKSA transmitted signals through TLR2-Src-Rif-PKCα-NF-κB axis and induced the expression of pro-inflammatory cytokines;meanwhile,ds RNA was found to induce NF-κB-dependent gene transcription through the TLR3-Src-Rif axis.In addition,Rif activation can also activate the expression of related cytokines and other genes by enhancing the p38 MAPK-mediated signaling pathway.Using Rif knockout mice,the function of endogenous Rif in innate immune cells was further explored.It was found that LPSTLR4 or HKSA-TLR2 mediated activation of NF-κB signaling pathway was directly related to the presence of Rif,but the activation of TLR2-mediated NF-κB signaling pathway in BMDCs is Rifindependent.Moreover,this study observed the effect of Rif knockout on GM-CSF-induced BMDCs differentiation,and found that after Rif knockout,the proportion of BMDCs differentiated from Bone Marrow cells decreased,that is,the proportion of cells expressing CD11 c decreased,and the number of cells expressing CD80 costimulatory molecules in BMDCs also decreased.After inducing the complete maturation of BMDCs,Rif knockout significantly reduced the proportion of CD86 molecules expressed on the cell surface compared with wild-type cells,indicating that Rif knockout affects the maturation and differentiation of BMDCs.In summary,this study found that externally stimulated PRR regulates multiple different downstream signaling pathways by activating Rif GTPase,thereby activating transcription factors NF-κB,in turn controlling the transcription of different pro-inflammatory cytokine genes,enabling host cells to generate appropriate innate immune responses in response to exogenous stimuli.This study is the first time to elucidate the role of the small GTPase Rif in the innate immune response mediated by pattern recognition receptors under pathogenic microorganism infection.Rif possess broad anti-pathogenic effect and understanding of the molecular mechanisms by which this small Rho GTPase interferes with innate immune system will be beneficial to develop therapies against infectious agents.