GRP78 Impairs Production Of Lipopolysaccharide-induced Cytokines By Interaction With CD14

[Background]The 78 kDa glucose-regulated protein(GRP78),also referred to as Bip or HSPA5,belongs to the heat shock 70kDa protein(HSP70)family.GRP78 has highly conserved structure and versatile functions and can participate in the protein folding and assembling as the ER molecular chaperone.GRP78 can also be released at times of cellular stress as stress-inducible chaperone in order to prevent cells from stress-inducible apoptosis,such as hypoxia,nutritional deprivation,pathogenic microbial infection,physical and chemical stimulation.GRP78 has recently been described to be released into extracellular microenvironment as having immunomodulatory properties and thus has been defined as one of resolution-associated molecular patterns(RAMPs).Our previous work confirmed that GRP78 over-expression could protect insulinoma cells from cytotoxic T cell-mediated lysis and raise the frequency of molecules with immunomodulatory properties.We also reported that GRP78 could induce myeloid antigen presenting cells to maintain tolerogenic signature upon LPS stimulation and increase the frequency of regulatory B cells producing IL-10 and highly expressed PD-L1 and FasL,whose mechanisms remain unclear.On the basis of previous studies,we continued to explore the membrane molecules interacting with GRP78 and their signaling pathways in vitro in order to provide scientific theoretical basis and experimental data for further study of its role in induction of immune tolerance and tumor immune escape.[Methods]1.Preparation of recombinant mouse GRP78The recombinant mouse GRP78 prokaryotic expression vector(pGEX-4T-3-GRP78)was transformed into E coli expression strain BL21(DE3).Then isopro-pyl-D-thiogalactopyranoside was added into the medium to induce expression of the recombinant protein.The soluble GRP78 protein with GST tag was collected by centrifugation and purified using the Pierce(?)Glutathione Spin Columns.After digested by thrombin,endotoxins were removed by the Pierce High-Capacity Endotoxin Removal Resin.The endotoxin of the protein samples was detected by limulus test and its concentration was below 10EU/ml.After its concentration was detected using the BCA Protein Assay Kit,the rmGRP78 protein was identified by SDS-PAGE and WB and stored at-80?.2.Induction and culture of bone marrow-derived dendritic cells(BMDCs)C57BL/6 wild type female mice and C57BL/6 background TLR4 deficient and CD 14 deficient female mice were utilized at 6-8 weeks of age.Red blood cells were removed from bone marrow mononuclear cells from femur and tibia.Mouse BMDCs were generated by culturing bone marrow cells in RPMI 1640 medium containing 10%FBS with GM-CSF(10ng/ml),IL-4(lOng/ml)and Penicillin/streptomycin(100U/ml)for 7 days.Flow cytometry analysis showed the positive percentage of CD11c expression was more than 90%.3.Detection of cytokines and mRNA expression in differently-treated BMDCs from WT mouseCell total RNA was extracted 2 hours later from negative control group,LPS treated group,GRP78 at different concentrations + LPS treated group,and qPCR was carried out to detect the mRNA expression of IL-1?,IL-6,TNF-? and IFN-(3.Four hours later,concentrations of IL-6,TNF-a and IFN-? in the culture supernatants from the same groups above were determined by ELISA.4.TLR4 expression and localization analysis in GRP78-treated DCsWT mouse BMDCs and DC2.4 cell line were treated with GRP78 at different concentrations at 37? for 30 minutes before the expression of surface TLR4 was detected by FCM.TLR4 expression and co-localization with LAMP1(early lysosomal marker)in cells were examined with laser confocal microscopy.The influence of GRP78 on the expression of TLR4 in the presence of Puromycin(protein translation inhibitor)and Chloroquine(lysosomal inhibitor)was measured by western blotting.5.The labeling of GRP78 protein with Alexa Fluor(?)488The labeling of purified GRP78 protein with Alexa Fluor(?)488(AF488-GRP78)was done by Protein Labeling Kit according to manufacturer's instructions and AF488-BSA was used as a negative control in the analysis of DCs functioning mode.6.Detection of interactive mode between GRP78 and DCsBMDCs and DC2.4 cell line were respectively incubated with AF488-GRP78 or AF488-BSA(1?M)at 4? for 1 hour before the binding of GRP78 to membrane of DCs was examined by FCM and laser confocal microscopy.DC2.4 cell line was incubated with AF488-GRP78 or AF488-BSA(1?M)at 4? for 1 hour and then at 37? for 30 minutes before the endocytosis of GRP78 was detected by laser confocal microscopy.7.Co-localization analysis between GRP78 and membrane molecules of DCsBMDCs and DC2.4 cell line were respectively incubated with AF488-GRP78 or AF488-BSA(1?M)at 4? for 1 hour and then fixed with paraformaldehyde.TLR4 and CD14 were respectively labeled with Alexa Fluor(?)555 before co-localization analysis between GRP78 and TLR4/CD14 was carried out by laser confocal microscopy.8.Analysis of interactions between GRP78 and membrane molecules of DCsRecombinant fusion proteins GST-GRP78 were constructed and purified before incubation with DC2.4 cell membrane protein lysates overnight at 4? and the GST-GRP78-protein complexes were precipitated by GST binding resin.The nonspecific binding protein was washed off by PBS and the precipitates were analyzed by western blotting using antibody specific for GST,GRP78,TLR4 and CD 14.Recombinant CD 14-hlgG1-Fc fusion proteins were constructed and purified before incubation with purified mouse GRP78 proteins overnight at 4?and the CD14-Fc-protein complexes were precipitated by protein G agarose beads.The nonspecific binding protein was washed off by PBS and the precipitates were analyzed by western blotting using GRP78 specific antibody.HEK293T cells were transfected with pcDNA3.1-CD14 plasmids or empty vector before the binding of AF488-GRP78 with cells was examined by laser confocal microscopy.9.Analysis of interactive functional domain between GRP78 and CD14Prokaryotic expression plasmids of truncated protein GST-GRP78(19-392/393-655/19-280/281-655aa,none contains the 1-18aa signal peptide)were constructed and purified,which respectively contains ATP enzyme domain and the substrate binding domain.Truncated GST-GRP78 fusion proteins were incubated with DC2.4 cell membrane protein lysates overnight at 4? and the protein complexes were precipitated by GST binding resin.The nonspecific binding protein was washed off by PBS and the precipitates were analyzed by western blotting using CD 14 specific antibody.10.Interaction and effect analysis of GRP78 on BMDCs from CD14KO and TLR4KO mouseBMDCs from WT mouse and CD14KO/TLR4KO mouse were incubated with AF488-GRP78 or AF488-BSA(1?M)at 4? for 1 hour before the binding of GRP78 to the three types of BMDCs was examined by FCM.BMDCs from WT mouse and CD14KO mouse were incubated with GRP78 at 37? for 30 minutes before the expression of TLR4 on the membrane of DCs was detected by FCM.BMDCs from WT mouse and CD14KO/TLR4KO mouse were treated as the negative control group,LPS treatment group,GRP78 at different concentrations +LPS treatment group and the concentration of TNF-a in the culture supernatants was determined by ELISA 4 hours later.[Results]1.Effect of GRP78 on the inflammatory cytokines induced by LPSCompared with the negative control group,LPS stimulated BMDCs to release large quantities of inflammatory cytokines,like TNF-a,IL-6 and IFN-?.However,such promotion could be attenuated by the supplement of GRP78 at concentration dependent manner.Even as little as 1?g/ml of GRP78 could significantly reduce the production of IFN-?.GRP78 treatment at the concentration of 40?g/ml leaded to a nearly complete abolishment of those cytokines.The results of qRT-PCR corroborated the ELISA data above,suggesting that GRP78 could inhibit the production of inflammatory cytokines induced by LPS in BMDCs.2.Effect of GRP78 on the expression and localization of TLR4(1)ELISA assay manifested that LPS couldn't induce BMDCs from TLR4KO mouse to produce TNF-a.FCM assay and laser confocal imaging showed that GRP78 treatment at 37? for 30 minutes could obviously reduce TLR4 expression on the DC2.4 membrane and that surface TLR4 decreased dramatically as the concentrations of GRP78 increased.GRP78 treatment at the concentration of 40?g/ml for 30 minutes decreased the mean fluorescent intensity(MFI)of TLR4 by 75%.The results suggested that GRP78 exerted its modulatory effects probably through decreasing TLR4 expression on the DCs membrane since TLR4 is the key receptor for LPS to exert pro-inflammatory function(2)Confocal imaging manifested that TLR4 was evidently endocytosed after GRP78 treatment in DC2.4 and had fluorescence co-localization with the early lysosomal marker LAPM-1.Western blotting assay showed that the expression of TLR4 was gradually reduced in the presence of both Puromycin(protein translation inhibitor)and GRP78,but it was gradually enhanced in the presence of both Chloroquine(lysosomal inhibitor)and GRP78.These data suggested that GRP78 could inhibit TLR4 signaling through promoting TLR4 endocytosis into lysosomes to be degraded and reducing its expression on the membrane of DCs.3.Interactive function mode between GRP78 and DCsFCM analysis showed that BMDCs and DC2.4 cells were stained by AF488-GRP78.Laser confocal imaging revealed that at 4?(to block vesicular trafficking),90-95%of AF488-GRP78 stained cells emitted green fluorescence while cells treated with AF488-BSA were null for staining.The fluorescence of AF488 was only localized at the plasma membrane even when such staining was conducted at 37?.The results suggested it was through binding with cell surface structures but not via being endocytosed that GRP78 exerted its modulatory effects.4.Interactive function mode between GRP78 and molecules on the membrane of DCs(1)Laser confocal imaging revealed no co-localization between AF488-GRP78 green fluorescence at the DC2.4 plasma membrane and TLR4 red fluorescence.Furthermore.in glutathione S-transferase(GST)precipitation assays,GST-GRP78 failed to precipitate TLR4.Therefore,GRP78 probably interacted not with TLR4 but with other partners to decrease the TLR4 expression.(2)Laser confocal imaging showed that AF488-GRP78 green fluorescence at the DC2.4 plasma membrane was almost identical to that of CD14 red fluorescence Furthermore,AF488-GRP78 was found to stain the plasma membrane of 293T cells transfected with CD 14,but failed to stain 293T mock control.The results of GST-pull down assay and IP assay revealed that CD 14 was precipitated via binding to GRP78 directly,indicating that GRP78 could probably interact with CD 14 to reduce TLR4 expression.5.Construction of truncated GRP78 fragments and their interaction with CD14GST-pull down assay showed that none of the truncated GRP78 fragments 19-392/393-655/281-655aa,among which 19-280aa plasmid had no soluble protein expression in BL21,interacted with CD14,suggesting that only full length GRP78 retained the ability to bind to CD 14.6.Effect of GRP78 interacting with CD14 on DCs function(1)FCM analysis revealed that AF488-GRP78 could bind with BMDCs from WT mouse but not from CD14KO mouse,further indicating that GRP78 could interact with CD14 to regulate function of DCs.(2)FCM analysis revealed that no alterations were found in surface level of TLR4 in CD14KO BMDCs which previously was evidently downregulated by GRP78 in WT BMDCs at a concentration dependent manner.WB assay suggested that GRP78 didn't induce TLR4 into lysosomes to be degraded in CD14KO-BMDCs.ELISA assay showed that CD14KO BMDCs exhibited no changes in TNF-?production upon treatment with GRP78 and LPS.These results indicated the immunomodulatory property of GRP78 in LPS-induced production of inflammatory cytokines was CD14-dependent.(3)To explore whether endocytosis of TLR4 induced by GRP78 activates TRIF-IRF3-IFN?/? signaling,western blotting assay was carried out to examine the IRF3 phosphorylation of BMDCs from WT mouse and CD14KO mouse in the negative control group,LPS treatment group,GRP78 + LPS treatment group.We found GRP78 didn't induce IRF3 phosphorylation of WT-BMDCs and inhibited the activation of IRF3 phosphorylation triggered by LPS.Moreover,none of the groups in CD14KO-BMDCs could induce IRF3 phosphorylation.These data suggested that IRF3 phosphorylation in BMDCs was CD 14-dependent and GRP78 induced non-inflammatory endocytosis of TLR4.[Conclusions]This work explored the function of GRP78 in LPS-induced production of inflammatory cytokines and the interactive function mode between GRP78 and DC and studied the mechanisms underlying the relative molecules.The results suggested that:?GRP78 could inhibit production of inflammatory cytokines(eg.TNF-??IL-6?IFN-?)induced by LPS in DCs.?GRP78 could reduce surface TLR4 expression in DCs while TLR4 is the key receptor for LPS to exert pro-inflammatory function.?GRP78 could reduce TLR4 expression on the surface of DCs through interacting with surface CD 14 to induce TLR4 endocytosis into lysosomes to be degraded.?GRP78 induced non-inflammatory endocytosis of TLR4 in a CD 14-dependent manner and thus impaired inflammatory reaction induced by LPS as TLR4 agonist.?CD 14,as the co-receptor for TLR4,had a dual function in TLR4 mediated inflammation:pro-inflammatory induced by LPS and anti-inflammatory induced by GRP78.