Responsive Outcomes Of Mast Cells And Macrophages To Food-and-Mouth Disease Virus-Like Particles

Foot and mouth disease(FMD),caused by foot and mouth disease virus(FMDV),is a high contact infection disease of cloven-hoofed animals,which can infect more than 70 cloven-hoofed animals including cattle,swine,goats and sheep,livestock with potential devastating consequences.FMDV invades the body mainly via the mucosa of both respiratory and digestive tracts,which has a serious impact on the health,growth of animals and the trade of animal and animal products worldwide.There is still no safe and effective vaccine for the prevention of FMD.Mast cells and macrophages are important immune cells of the innate immune system,predominantly distributed in the skin and mucosa,and together with dendritic cells forming the first line of defense against infection.Mast cells and macrophages can recognize pathogens and pathogen-related products inside and outside the cell via the pattern-recognition receptors(PRRs),and act as effector cells to kill pathogens via phagocytic or non-phagocytic pathways,thus playing an important role in the body's antimicrobes.Therefore,they are referred to as "sentinel cells" of the body.Mast cells recognize pathogens through PRRs,trigger degranulation and release a variety of active substances to modulate the vascular permeability,initiate inflammatory response and regulate other immune cells.However,the role of mast cells in anti-FMDV infection remains elusive at present.The scavenger receptor(SR)is a phagocytic receptor and PRRs expressed by macrophages,which can recognize a variety of viruses and bacteria,resulting in macrophage activation and secretion of TNF-?,IL-6,and IFN-?.Our studies revealed that macrophages recognized recombinant FMDV VP1-VP4 via SR,and SR played a negative regulatory role in dendritic cells presenting FMDV VP1-VP4 antigen,but the immune response of SR-mediated macrophages against FMDV is unclear.In this study,the mouse peritoneal mast cells(pMCs)and the degranulation inhibitor,tanshinone?A-pretreated pMCs were pulsed with the FMDV virus-like particles(FMDV-VLPs)respectively,with pMCs alone as a control.The supernatants were collected from the pMCs culture wells after 6 h for lymphocyte proliferation assay by CCK-8 kit,the lymphocytes alone as control,and concanavalin A(ConA)-treated lymphocytes as control.The pMCs adherent to coverslips were identified by the toluidine blue staining.The results showed that the number of undegranulated pMCs in the pMCs pulsed with FMDV-VLPs were significantly lower than the pMCs alone(P<0.01)and the tanshinone?A-pretreated pMCs pulsed with the FMDV-VLPs(P<0.01).This indicates that the pMCs' degranulation was significantially induced by FMDV-VLPs.The proliferation of splenic lymphocyte pool was steadily triggered by the supernatants of FMDV-VLPs-activated pMCs.Of noting,the degranulation of pMCs was significantly inhibited by tanshinone?A,resulting in a remarkable decrease of splenic lymphocyte proliferation.Importantly,the splenic T lymphocyte proliferation was significantly inhibited by the supernatants collected from na?ve pMCs and FMDV-VLPs-activated pMCs.Furthermore,T lymphocyte proliferation was very significantly inhibited by the supernatant from tanshinone?A-pretreated pMCs,indicating that granular components released from pMCs pulsed with FMDV-VLPs are capable of stimulating the proliferation of splenic na?ve lymphocytes in vitro.However,the secretory components of pMCs loaded with FMDV-VLPs significantly inhibit splenic na?ve lymphocyte proliferation.Our data reveal an unappreciated role of pMCs in modulation of adaptive immune responses to FMDV-VLPs.To explore the potential role of macrophages in the immune response to FMDV immunogen,mouse peritoneal macrophages(pM?),the shikonin and sennoside B-treated pM? were pulsed with FMDV-VLPs respectively,with na?ve pM? as a control.The supernatants were collected from the pM? culture wells after 6 h for lymphocyte proliferation assay by the CCK-8 kit,and the splenic na?ve lymphocytes alone,the splenic na?ve lymphocytes pulsed with FMDV-VLPs and the the splenic na?ve lymphocytes-pM? pulsed with FMDV-VLPs were used as the control group.And concanavalin A(ConA)-treated lymphocytes as control.The results show that FMDV-VLPs alone stimulated the splenic na?ve lymphocytes proliferation(P<0.01),but could not stimulate T lymphocytes proliferation.The pM? pulsed with FMDV-VLPs significantly stimulated the proliferation of splenic na?ve lymphocytes and T lymphocytes(P<0.01),and FMDV-VLPs-initiated proliferation splenic na?ve lymphocytes via the presentation of pM? were stronger than direct stimulation of splenic na?ve lymphocytes proliferation.But the supernatant of pM? pulsed with FMDV-VLPs could not trigger the proliferation of splenic na?ve lymphocytes and T lymphocytes.The supernatant of na?ve pM? also failed to initiate the proliferation of splenic na?ve lymphocytes and T lymphocytes.The supernatant of shikonin(inhibitor of the maturation of TNF-? mRNA)and sennoside B(SR inhibitor)-treatment pM? pulsed with FMDV-VLPs significantly inhibited splenic na?ve lymphocyte and T lymphocyte proliferation(P<0.01).These results show that FMDV-VLPs can initiate B cell activation and proliferation via specific recognition by B cell receptor(BCR),without the processing of antigen presenting cells(APC).Activation of B lymphocytes requires the simultaneous presence of antigenic signals and costimulatory signals.Although pMCs and pM? can be activated by FMDV-VLPs and secrete a variety of protein molecules(such as G-CSF,IL-13,IL-15,IL-17,IL-17 BR,et al.),in comparison with pMCs,pM? pulsed with FMDV-VLPs can activate lymphocytes only when pM? was kept contact with lymphocytes.The biological effects mediated by TNF-? and SR of splenic macrophages can promote the establishment of splenic immunity.The pM?,the wognin(NF-?B inhibitor)and sennaine B(NF-?B inhibitor)-treated pM? were pulsed with FMDV-VLPs,designated as FMDV-VLPs group and inhibitor treatment group,respectively.The pM? pulsed with OVA and/or recombinant FMDV protein VP1-VP4 respectively were used as control group,i.e.,OVA group and VP1-VP4 group,and the no-treatment pM? was as a blank group(pM? group).The pM? of each group were harvested after 4 hours for the binding activity of NF-?B to TNF-? promoter assayed with chromatin immunoprecipitation(ChIP).Compared with the % Input value of TNF-? promoter fragment captured with rabbit anti-mouse NF-?B1 p105/p50 mAb and rabbit IgG,which is against an irrelevant antigen,the percentage of Input value of the TNF-? promoter fragment captured from the control group increased 3.28 times(P<0.01),the OVA group increased 11.27 times(P<0.01),the VP1-VP4 group increased 14.54 times(P<0.01),the FMDV-VLPs group increased 4.65 times(P<0.01),and the inhibitor treatment group increased 3.53 times(P<0.01).In comparison with the levels of NF-?B precipitated by rabbit anti-mouse NF-?B1 p105/p50 antibody,the pM? group was remarkable lower than the OVA group(P<0.001),the VP1-VP4 group(P<0.001)and the FMDV-VLPs group(P<0.05).The OVA group was significantly higher than the FMDV-VLPs group(P<0.01)and the inhibitor-treated group(P<0.001).The VP1-VP4 group was significantly higher than the FMDV-VLPs group(P<0.05)and the inhibitor-treated group(P<0.001).These results show that the rabbit anti-mouse NF-?B1 p105/p50 specifically precipitated the NF-?B-TNF-? promoter complex.NF-?B can be activated by FMDV-VLPs and FMDV VP1-VP4,and activated NF-?B can productively bind to the TNF-? promoter.The binding activity of NF-?B to TNF-? promoter was highest in VP1-VP4 group,followed by OVA and FMDV-VLPs.We can deduce that using VP1-VP4 as FMD vaccine antigen may aggravate tissue inflammation at the injection site of the vaccinated animals,while FMDV-VLPs may alleviate tissue inflammation at the injection site.Wogonin reduces the expression of TNF-? by inhibiting the binding activity of NF-?B to the TNF-? promoter,thereby alleviating inflammation at the injection site and relieving the pain of vaccinated animals.These data mentioned above indicate that pMCs could recognize FMDV-VLPs,and the pMCs' degranulation was significantially induced by FMDV-VLPs.The granular components released from pMCs pulsed with FMDV-VLPs can promote the proliferation of splenic na?ve lymphocytes.By contrast,the secretory components of pMCs loaded with FMDV-VLPs significantly inhibit the proliferation of splenic na?ve T lymphocytes.Macrophages could also recognize FMDV-VLPs and initiated lymphocyte proliferation via antigen presentation,and need macrophages directly contact with lymphocytes.The biological effects mediated by TNF-? and SR of splenic macrophages can promote the establishment of splenic immunity.Both FMDV-VLPs and FMDV VP1-VP4 could significantly increase the binding activity of NF-?B bind to TNF-? promoter in pM?,but the the binding ability of NF-?B to TNF-? promoter in pM? pulsed with VP1-VP4 was more stronger than that of pM? pulsed with FMDV-VLPs.This indicates that VP1-VP4 stimulates the production of TNF-?,which may cause inflammatory reaction at the injection site,while FMDV-VLPs are weak,do not cause inflammatory reactions,and can alleviate the pain of vaccination.Wogonin can reduce the TNF-? secretion via inhibiting the binding activity of NF-?B,alleviating inflammation at the injection site and relieving the pain of vaccinated animals,and thus may be used as an adjuvant component of a foot-and-mouth disease vaccine.