Immunoregulation Of Lycium Barbarum Polysaccharides As A Food-and-Mouth Disease Vaccine Adjuvant To "DC-Tfh Cell-B Cell" Axis

Objective:Lycium barbarum polysaccharide?LBP?is the main component of Chinese wolfberry efficacy,in a number of ways and multiple levels of the immune system to play a regulatory role.In this study,mice were injected intraperitoneally with the recombinant adenovirus?rAd5VP1?expressing VP1 gene of foot and mouth disease virus?FMDV?,and then LBP was given to mice for 7 days by gastric gavage.The proliferation and differentiation of Dendritic cells?DCs?,T follicular helper cells?Tfh?,B cells,regulatory expression of transcription factors,secretion of cytokines and antibodies and the formation of mesenteric lymph node germination center were observed.The role of LBP as an adjuvant for rAd5VP1vaccine in the"DC-Tfh cell-B cell"axis was clarified to study the effect of LBP on immune adjuvant.Methods:Six weeks old BALB/c mice were randomly divided into 5 groups,8 in each group.All mice were injected intraperitoneally with rAd5VP1(1.5x10⁸ TCID₅₀).Mice were given intragastric administration for 7 days using low dose LBP?10mg/kg·d?,medium dose LBP?20mg/kg·d?,high dose LBP?40mg/kg·d?,each 200?L.Negative control group were given the same volume of PBS,and positive control group were lipopolysaccharide LPS?1mg/kg·d?.After 7 days of intragastric administration,do the following:1.Take the mouse spleen,and use density gradient centrifugation extraction to collect mononuclearcells.ThenumberofDC?CD11c⁺MHC?⁺CD86⁺?,Tfhcells?CD4⁺CXCR5⁺ICOS⁺?and GC B cells?B220⁺GL-7⁺?were detected by flow cytometry?FCM?.2.Take the mouse spleen,and use density gradient centrifugation extraction to collect3.mononuclear cells.The expression level of IRF4,IRF8,Bcl6,Blimp1 and AID mRNA were detected by real-time RT-PCR.4.The mesenteric lymph nodes of mice were made paraffin sections.The formation of germinal centers was observed by HE staining and immunofluorescence.5.Take the mouse eye blood,serum was harvested by centrifuge.The amount of IL6,IL21,IL4 and the secretion level of antibody IgG₁ were detected by ELISA.Results:1.Flow Cytometry analysis revealed:?1?The number of DC:the expression of CD11c⁺MHC?⁺DC in LBP?40mg/kg·d?stimulation group was?2.73%±0.18?,and it was significantly higher than PBS negative control group?1.58%±0.15??P<0.05?.The expression was higher than LPS positive control group?2.12%±0.49?,but there was no significant difference between these two groups?P>0.05?.Meanwhile,LBP?20mg/kg·d?and LBP?40mg/kg·d?stimulation groups can enhance DC co-stimulatory molecules the expression of CD86.?2?The number of Tfh cell:the expression of CD4⁺CXCR5⁺Tfh in LBP?40mg/kg·d?stimulation group was?2.93%±0.74?,and it was significantly higher than PBS negative control group?1.06%±0.19??P<0.05?.The expression was higher than LPS positive control group?2.21%±0.17?,but there was no significant difference between these two groups?P>0.05?.Meanwhile,LBP?40mg/kg·d?stimulation group can induce the expression of ICOS which is Tfh cells co-stimulatory molecules.?3?The number of GC B cell:the expression of B220⁺GL-7⁺B cell in LBP?40mg/kg·d?stimulation group was?8.67%±0.87?,and it was significantly higher than PBS negative control group?4.02%±0.79??P<0.05?.The expression was lower than LPS positive control group?9.41%±0.95?,but there was no significant difference between these two groups?P>0.05?.2.real-time RT-PCR analysis revealed:?1?Bcl6 mRNA:the expression of LBP?40mg/kg·d?stimulation group was?1.99±0.42?,higher than PBS negative control group?P<0.05?.And it was higher than LPS positive control group?1.44±0.23?,but there was no significant difference between these two groups?P>0.05?.?2?Blimp1mRNA:theexpressionofLBP?10mg/kg·d?,LBP?20mg/kg·d?,LBP?40mg/kg·d?stimulation groups and LPS positive control group were?0.21±0.03?,?0.15±0.07?,?0.08±0.01?and?0.04±0.01?respectively,which were significantly lower than PBS negative control group?P<0.05?.?3?AID mRNA:the expression of LBP?10mg/kg·d?,LBP?40mg/kg·d?stimulation groups were?2.27±0.21?,?4.57±1.34?,higher than PBS negative control group?P<0.05?.And LBP?40mg/kg·d?group was higher than LPS positive control group?2.58±0.52?,but there was no significant difference between these two groups?P>0.05?.?4?IRF4 mRNA:There was no significant difference among groups?P>0.05?.?5?IRF8 mRNA:There was no significant difference among groups?P>0.05?.3.HE staining and immunofluorescence labeling analysis revealed:LBP?40mg/kg·d?stimulation group compared with the PBS negative control group,could be clearly observed lymphocytes gathered into basophilic colonies,B220 molecules were expressed in a circular shape,and formate a typical follicular germination center structure.This result was not observed in LBP?10mg/kg·d?and LBP?20mg/kg·d?stimulation groups.4.ELISA results showed:?1?IL6 secretion:the expression of LBP?40mg/kg·d?stimulation group was?6.93±0.67?pg/mL,higher than PBS negative control group?4.00±1.07?pg/mL?P<0.05?.The expression was higher than LPS positive control group?4.41±0.37?pg/mL,but there was no significant difference between these two groups?P>0.05?.?2?IL21 secretion:the expression of LBP?20mg/kg·d?and LBP?40mg/kg·d?stimulation groups were?80.53±4.60?pg/mL,?80.97±3.16?pg/mL respectively,and higher than PBS negative control group?43.20±1.19?pg/mL?P<0.05?.The expression were higher than LPS positive control group?67.30±2.61?pg/mL,but there was no significant difference?P>0.05?.?3?IL4 secretion:the expression of LBP?40mg/kg·d?stimulation group was?20.10±1.97?pg/mL,higher than PBS negative control group?11.01±5.14?pg/mL?P<0.05?.This group was consistent with the expression level of LPS positive control group?20.10±1.12?pg/mL,but there was no significant difference?P>0.05?.?4?IgG₁ secretion:the expression of LBP?20mg/kg·d?and LBP?40mg/kg·d?stimulation groups were?12.12±0.70??g/mL,?11.02±0.46??g/mL respectively,and higher than PBS negative control group?8.86±0.55??g/mL?P<0.05?.The expression were higher than LPS positive control group?10.18±0.85??g/mL,but there was no significant difference?P>0.05?.Conclusion:LBP?40mg/kg·d?stimulation group can promote the proliferation and maturation of DC;And can promote the proliferation and differentiation of Tfh cells,promote the expression of transcription factor Bcl6 mRNA,reduce the expression of Blimp1 mRNA;And promote the proliferation of B cells,enhanced the expression of AID mRNA,increase the production of antibody IgG₁,can promote the formation of germinal center;it also can enhance the secretion of IL6,IL21 and IL4,so that enhance the auxiliary role of Tfh cells on B cell,strengthen the humoral immune response.In conclusion,LBP acts as a vaccine adjuvant for FMD and exerts an immune adjuvant by immunoregulation of the“DC-Tfh cell-B cell”axis.