Regulatory Effect Of The Recombinant Lactococcus Lactis Strain Expressing Helicobacter Pylori Neutrophil-activating Protein A On Food Allergy In Mice

Food allergy is a predominant Th2 immune response triggered by innocuous food antigens.Common clinical manifestations of food allergy include hives,wheezing,coughing,nausea,vomiting and diarrhea.Even more,a life-threatening situation,systemic anaphylaxis,could also occur.Recently,it has been a significant public health issue with growing severity,prevalence and limited treatments,which may have great impact on individuals’ life and health.So far,the neutrophil-activating protein A subunit(NapA)of Helicobacter pylori has been shown to be helpful to Th1 polarization of immune response,while the specific effect of NapA on food allergy is unknown.ObjectiveIn this study,the mice model of food allergy induced by ovalbumin(OVA)was used.And Lactococcus lactis(L.lactis),a food-grade probiotics,were adopted as mucosal delivery vehicles of H.pylori NapA.The study sought to determine the regulatory effect of recombinant bacterium(L.lactis NZ3900/p NZ8149-napA)on food allergy,which may provide novel approaches for researches related to the prevention and treatment of food allergy.Methods1.The cultivation and NapA expression analysis of recombinant L.lactis: After resuscitation and cultivation of L.lactis(L.lactis NZ3900/p NZ8149-napA),plasmid was extracted and prepared for gene sequencing.After induction,bacterial protein were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis.2.The establishment of food allergy model in mice: The mice model of food allergy were established with ovalbumin(OVA)by intra-peritoneal sensitization and consecutive intra-gastric challenge.At the meantime,the intervention of L.lactis were applied.3.Evaluation of the regulatory effect of recombinant L.lactis on food allergy in mice: Allergic symptoms were monitored for 30-60 minutes post each challenge.After the last challenge,relevant samples of mice were collected and and processed.Then specific antibodies(OVA-IgE and IgG1,IgG2a),histamine in serum and cytokines(IL4,IL10 and IFN-γ)in spleen homogenate were evaluated by enzyme linked immunosorbent assay(ELISA).The m RNA expression levels of tight junction proteins(Ocln,Tjp1 and JAM1)in duodenum were detected by real-time fluorescence quantitative PCR technique(q PCR).Jejunum sections were cut into thin slices for hematoxylin-eosin(HE)and toluidine blue(TB)staining.HE staining were carried out for the observation of intestinal mucosal integrity and immunocyte infiltration.TB staining were carried out for the observation of the morphology and migration of mast cells.4.Statistical analysis: One-way analysis of variance(ANOVA)followed by Tukey or Games-Howell test were chosen.For diarrheal score,Kruskall-Wallis analysis followed by Dunn’s multiple comparison test were used.Results1.The cultivation and NapA expression analysis of recombinant L.lactis: The gene sequences of NapA in plasmid of Recombinant L.lactis were accordant with those in H.pylori published on Gen Bank(AY366361.1).Recombinant L.lactis could express NapA protein with a molecular weight of 17 KD.And the expression of NapA approximately accounted for 14.0% of total bacterial protein.2.The observation and evaluation of allergic symptoms: Compared with negative control group and LL(8149)group,the scoring curve of LL(8149-NapA)group was milder.After the final OVA challenge,diarrhea score of LL(8149-NapA)group was decreased compared with positive control group(P < 0.05).3.The assessment of antibodies and histamine in serum: The level of OVA-IgG1 and histamine in LL(8149-NapA)group were lower than those in positive control group(P < 0.05).In contrast to positive control group and LL(8149)group,the mice in LL(8149-NapA)group had higher IgG2 a level(P < 0.05).4.The quantification of cytokines in spleen homogenate: Compared with positive control group,the level of IL4 was lower and the levels of IL10,IFN-γ were higher in LL(8149-NapA)group(P < 0.05).5.The detection of intestinal barrier function: Compared with positive control group,the m RNA expression levels of tight junction proteins(Tjp1 and JAM1)were obviously higher in LL(8149-NapA)group and negative control group(P < 0.05).6.The histological analysis of proximal jejunum: According to the results of HE staining,the positive control group showed marked intestinal mucosa and villi destruction,glandular atrophy,submucosa edema and immunocyte infiltration,which are hallmarks of intestinal inflammatory response.As a comparison,intestinal inflammation of the LL(8149-NapA)group were much milder.As for the TB staining,mast cells migrating to the lamina propria of jejunum were observed both in positive control group and LL(8149)group,while no mast cell was observed in LL(8149-NapA)group.Conclusions1.L.lactis were adopted to deliver H.pylori NapA protein to gastrointestinal mucosa through intragastric administration,which could effectively alleviate the food allergic reaction to OVA in mice.2.H.pylori NapA may attenuate food allergy symptoms characterized by acute diarrhea and intestinal inflammation in mice by regulating immune response and/or enhancing intestinal barrier function.3.The engineered L.lactis strain(L.lactis NZ3900/p NZ8149-napA)could express H.pylori NapA,which may be a promising candidate for researches related to the prevention and treatment of food allergy.