Establishment Of A HFA Mouse Model For Evaluating The Effects Of Fecal Microbiota On The Pathogenesis Of Cow’s Milk Protein Allergy

Objective: To explore the effects of fecal microbiota on cow’s milk protein allergy(CMPA)through observing the reaction of germ free mice to cow’s milk protein stimulation after transplantation with fecal microbiota from CMPA infants and healthy control infants.Methods: The fecal microbiota of the CMPA infants(I-CMPA group)and healthy control infants(I-NC group)that were collected in Children’s Hospital of Chongqing Medical University were respectively transplanted into the germ free mice to establish the cow’s milk protein allergy group(M-CMPA group)and healthy control group(M-NC group).Two groups of the transplanted mice were feeding with formula for 14 days.The colonization and variation of the fecal microbiota in the transplanted mice were detected by 16 S rRNA gene sequencing.The clinical symptoms of the 2 transplanted groups were observed,and then the morphological changes of intestinal tissue were examined by HE staining.The percentage of CD4+CD25+Foxp3+ regulatory T cells(CD4+CD25+Foxp3+ Treg cells)in spleens were measured by flow cytometry.Results:(1)The 16 S rRNA sequencing results showed that the advantage bacterium between I-CMPA group and I-NC group were different,and the diversity of fecal microbiota in I-CMPA group was lower than that in I-NC group.The main components in fecal microbiota of M-CMPA group mice were similar to I-CMPA group.The overall microbiota structure of M-CMPA and M-NC group were obviously different,the two groups showed a clear separation in samples distances heatmap 、 principal component analysis(PCA)and hierarchical clustering tree.The wilcoxon rank-sum test bar plot on Genus level showed that the proportion of Bifidobacterium in I-CMPA group and M-CMPA group were both higher than that in their control groups;(2)After feeding with formula,the M-CMPA group had more obvious clinical symptoms than the M-NC group,such as swollen perianal and loose stools;(3)HE staining showed that the M-CMPA group had more serious pathological changes than the M-NC group,such as the focal necrosis,the shedding of the epithelial cells in intestinal villi and the inflammatory cells infiltration.(4)The percentage of CD4+CD25+Foxp3+ Treg cells was significantly decreased in spleen cell suspension of M-CMPA group than M-NC group.Conclusions:(1)HFA mice are able to partly imitate the fecal microbiota of CMPA infants.The HFA mice model is a new tool for investigating the relationship between fecal microbiota and the pathogenesis of CMPA.(2)Different fecal microbiota components could affect the sensitivity of the transplanted mice on cow’ milk protein,which suggested that the variation of fecal microbiota may be the initiating factor in CMPA,and the sensitivity to CMPA could be transmitted through fecal microbiota transplantation.(3)There might be some specific species in bifidobacterium which influenced the pathogenesis of food allergy.