Effect Of Lactobacillus Johnsonii On Immune Function Of Monocyte-derived Dendritic Cells From Porcine

Dendritic cells(DCs)are immune cells with a powerful antigen-presenting function.They stimulate the proliferation and differentiation of T cells and B cells.They are the initiator of the adaptive immune response of the body.They play a role in regulating inflammatory immune response and immune tolerance.DCs can regulate the mucosal immune response of the body through its interaction with probiotics in the intestinal tract,such as lactobacillus.Lactobacillus johnsonii(L.johnsonii)as a probiotic in the gut,regulates the host immune response and plays an important role in maintaining the homeostasis of the host gut,regulating the balance of the commensal flora in the gut and maintaining the health of the body.L.johnsonii isolated from the intestine of piglets is often used as feed supplement,which can maintain the balance of intestinal flora,stimulate mucosa immune system development,promote nutrition absorption and improve hematopoietic function.Because of its safety and reliability,it can also be used as an ideal carrier to deliver antigens in oral vaccine.Therefore,the study on the effect of L.johnsonii on the immune function of dendritic cells is of great significance for revealing the mechanism of the activation of intestinal mucosal immunity by lactobacillus.In this study,peripheral blood mononuclear cells(PBMCs)were isolated from peripheral blood of 5-8 weeks old piglets by using Histopaque-1077 lymphocyte separation solution,and the CD172 a monocytes were purified from PBMCs by immunomagnetic labeling of cells using porcine monoclonal antibody(m Abs)CD172a and anti-PE Microbeads.The CD172 a monocytes were cultured for 6 days in RPMI-1640 medium supplemented with 10% fetal bovine serum(FBS),20 ng/m L recombinant porcine IL-4 and 20 ng/ml recombinant porcine GM-CSF,and Mo DCs were obtained after 6 days of culture.The cell culture medium was replenished every 2 days.The cell purity over 90 % by flow cytometry.In order to clarify the activation of porcine Mo DCs by L.johnsonii,in this study,Heat-killed L.johnsonii(HK L.johnsonii)、 L.johnsonii cell-free supernatant(L.johnsonii-CFS)and Live L.johnsonii respectively were used to interact with Mo DCs to analysis of its effect on immune function of Mo DCs.To determine the activation characteristics of L.johnsonii as live oral vaccine vector on dendritic cells.In this study,a recombinant L.johnsonii pPG-T7g10-PPT-ps420/L.johnsonii expressing PEDV protective antigen ps420 protein was constructed,and result showed that recombinant L.johnsonii expressed about59 KDa ps420 protein by western blot.In order to obtain the pure ps420 protein,the recombinantEscherichia coli p ET-28a-ps420/BL21 expressing PEDV ps420 protein was constructed.SDS-PAGE and western blot identification showed that the ps420 protein about 17 KDa was successfully purified.6-day old Mo DCs were treated with Heat-killed L.johnsonii(HK L.johnsonii),L.johnsonii cell-free supernatant(L.johnsonii-CFS),and Live L.johnsonii for 12 h respectively,flow cytometry analysis showed that HK L.johnsonii and L.johnsonii-CFS could not significantly promote up-regulation of Mo DCs surface molecules CD172a、MHCII class molecules and CD80,in contrast,Live L.johnsonii could significantly promote up-regulation of Mo DCs surface molecules.TLRs molecules,cytokines and chemokines were analyzed by q RT-PCR,the results show that HK L.johnsonii did not significantly stimulate the up-regulation of Mo DCs-related TLRs,cytokines and chemokines m RNA levels except IL-1β and IL-10,and L.johnsonii-CFS could only significantly stimulate the up-regulation of IL-10 m RNA levels in Mo DCs,but TLRs,pro-inflammatory cytokines and chemokines were not up-regulated.Live L.johnsonii can not only increase the expression levels of TLR2、TLR6、pro-inflammatory cytokines IL-1β、IL-12p40、TNF-α、IFN-γ and chemokines CCL20,but also promote the up-regulation of anti-inflammatory cytokines IL-10.Unstimulating Mo DCs as control group,Mo DCs stimulated with HK L.johnsonii、L.johnsonii-CFS and Live L.johnsonii were used as stimulator cells and CD4+ T cells were used as responder cells,mixed with 1:10 ratios,and CCK-8 kit was used to detect the proliferation of CD4+ T cells.The results showed that HK L.johnsonii still had no ability to regulate the proliferation of CD4+ T cells,and L.johnsonii-CFS group did not significantly promote the proliferation of CD4+ T cells,but the production of IL-10 cytokines was detected by ELISA in the co-culture system of Mo DCs and CD4+ T cells.Live L.johnsonii group was able to induce the proliferation of CD4+ T cells.In the co-culture system of CD4+ T cells and Mo DCs,it was found that both the production of anti-inflammatory cytokine IL-10 and the presence of pro-inflammatory cytokine IL-12p40,they were in a state of equilibrium.pPG-T7g10-PPT-ps420/L.johnsonii and ps420 protein were used to stimulate Mo DCs,and the results showed that pPG-T7g10-PPT-ps420/L.johnsonii and ps420 protein were up-regulated surface molecules CD172 a 、 CD80 and MHCII of Mo DCs.q RT-PCR analysis showed that pPG-T7g10-PPT-ps420/L.johnsonii and ps420 protein increased the m RNA levels of cytokines such as IL-1β、IL-12p40、TNF-α、IFN-γ、TLR2、TLR6 and CCL20 in Mo DCs,however,the m RNA level of anti-inflammatory cytokine IL-10 was not significantly higher than that of pro-inflammatory cytokines IL-1β 、 IL-12p40 、 TNF-α and IFN-γ,which were related to Th1 cytokines.The CD4+ T cells proliferation experiment results showed that both pPG-T7g10-PPT-ps420/L.johnsonii and ps420 protein could induce CD4+ T cells proliferation,and the cytokine IL-12p40 related to Th1 response was produced in the system co-cultured by Mo DCs and CD4+ T cells.pPG-T7g10-PPT-ps420/L.johnsonii can stimulate Mo DCs to uptake and phagocytize antigen under scanning electron microscope.The above results showed that the constructed lactobacillus johnsonii recombinans can stimulate the maturation and activation of Mo DCs.It promotes the secretion of pro-inflammatorycytokines,and cause the proliferation of CD4+ T cells.Mo DCs activated with pPG-T7g10-PPT-ps420/L.johnsonii skewed CD4+ T cells to Th1 polarization.Live L.johnsonii can also regulate the immune activation of Mo DCs in appropriate extent,causing the proliferation of CD4+ T cells,but to a lower extent than pPG-T7g10-PPT-ps420/L.johnsonii.In addition to this,Live L.johnsonii can also promote the secretion of anti-inflammatory cytokine IL-10,which generally tends to regulate the body’s immune balance.L.johnsonii-CFS can only stimulate Mo DCs to produce IL-10,but has little effect on cell surface molecules and CD4+ T cells proliferation,so it may have the function of regulating immune balance.However,HK L.johnsonii had no immunomodulatory effect on Mo DCs,suggesting that the immunomodulatory effect of L.johnsonii was related to its activity.