The Role Of The Oligopeptide/histidine Transporters In The Innate Immune Responses

Proton-coupled oligopeptide/histidine transporters(POTs,SLC15A)are membrane proteins that utilize the proton gradient and negative membrane potential for the transmembrane transport of di-/tripeptide and peptide-mimetic molecules.PepTl(SLC15A1)and PepT2(SLC15A2)are mainly expressed in the intestine,kidney and brain,respectively.PepTl is responsible for the absorption of small peptides from the digestion of dietary proteins into intestinal epithelial cells.PepT2 is involved in the reabsorption of peptide substrates from glomerular filtration into renal epithelial cells.PepT2 is also expressed in the apical membrane of the choroid plexus epithelial cells,and is important for maintaining the neuropeptides homeostasis and removal of neurotoxins from the brain.The other two members,PhTl(SLC15A4)and PhT2(SLC15A3)are mainly expressed in the lysosomal membrane of the immune cells,including dendritic cells and macrophages.PhTl and PhT2 are responsible for the transport of histidine,di-/tripeptides and peptidomimetic from inside the lysosomes to the cytosol,and play a role in the intracellular trafficking of short chain peptides and innate immune responses.Several studies have shown that SLC15A can transport bacterial peptidoglycan components,such as muramyl dipeptide(MDP)and tripeptide(Tri-DAP)to the cytosol,then lead to the activation of NOD-dependent(nucleotide oligomerization domain)signal pathway,which will induce the transcription of various inflammatory cytokine genes.However,the route of bacterial peptidoglycan components into the cytosol remains to be controversial.Therefore,how the bacterial peptides enter into the cells and the role of SLC15A in the innate immune responses need to be further characterized.As PepTl is not responsible for transporting MDP into the cytosol of mouse bone marrow derived macrophages(BMDM),but PepT2 is expressed in the progenitor cells from mouse bone marrow.So we detected the mRNA and protein expression of SLC15A in mouse bone marrow by real-time PCR and western blot.The results showed that only PepT2,PhTl and PhT2 was abundant in mouse bone marrow.To further characterize the distribution of SLC15A in cells within the bone marrow,we isolated bone marrow macrophages,differentiated mature or immature immune cell populations.We found that PepT2 was mainly expressed in the differentiated mature cells and BMDM,which was localized in the outer cell membranes of BMDM.In order to elucidate the role of PepT2 and PhTl in MDP mediated NOD2-dependent innate immune responses,we isolated BMDM from wild-type,PepT2 or PhTl knockout mice and compared the production of proinflammatory cytokines.The results showed that PepT2 or PhTl deletion influenced the intracellular accumulation of MDP-rhodamine in BMDM.And also decreased the production of proinflammatory cytokines,such as IL-6 and TNF-a.Moreover,mRNA expression of IL-6 induced by MDP was significantly higher in Hela cells overexpressed with PepT2 and PhTl than transfected with PepT2 or PhTl transporter.To investigate whether MDP and Tri-DAP were the substrates of PhT1,we constructed the mutant human PhT1 plasmid and stably transfected to MDCK cells,which will resort the human PhT1 protein from subcellular to outer cell membrane.Using this model,the functional activity of human PhT1 was evaluated by cellular uptake studies.The results showed that MDP,Tri-DAP,histidine and GlySar were all substrates of PhT1.Human PhT1 showed high affinity for histidine and low affinity for GlySar,with the Km values of 16.3 ± 1.9 ?M and 1.60 ± 0.30 mM,respectively.In addition,the uptake of MDP by human PhT1 could be inhibited by di-/tripeptides and peptide-like molecules,but not by glycine and acyclovir.PhT2 can be induced by TLR4 ligand lipopolysaccharide at the mRNA level,and PhT2 is existed in mouse bone marrow.Based on these data,we hypothesized that PhT2 plays a role in TLR-mediated innate immune responses.The data showed that PhT2 was upregulated by TLR2,TLR4,TLR7 and TLR9 ligands in BMDM and mouse peritoneal macrophages(PM)at both of the mRNA and protein levels,which via the activation of NF-?B,MAPK and IRF3 signal pathway.A reporter gene assay also showed that the mouse PhT2 promoter contained potential NF-?B binding sites.Furthermore,knockdown of PhT2 in human acute monocyte leukemia cell line THP-1 decreased the TLR4-triggered expression of IL-6 and TNF-a.And overexpression of PhT2 in human lung cancer epithelial cell line A549 increased LPS-induced production of proinflammatory cytokines.Additionally,PhT2 expression was increased and positively related to inflammation in mice with bacterial peritonitis.Taken together,our study characterized the regulation and role of the proton-coupled oligopeptide/histidine transporters in NOD-and TLR-mediated innate immune responses.These finding would provide a useful theoretic basis in better understanding the development of inflammation and inflammation-related diseases,and also could be useful for the treatment of inflammation-related diseases.