Study On The Association And Mechanisms Of Early Life Gut Microbiota And The Allergic Diseases In The Later Life Of The Host

Background and ObjectiveIn recent years,the prevalence of allergic diseases in children has risen sharply both in developed and developing countries.Children’s allergic diseases often last a lifetime and are extremely difficult to cure.There is no way to completely eradicate them.The resulting socio-economic burden is huge.Therefore,it is particularly important to understand the pathogenesis of childhood allergic diseases and develop new and effective early preventive measures.Studies have put forward the hypothesis that the occurrence of allergic diseases is related to the defects of the intestinal microbiota in the early life,and targeted interventions for the intestinal microbiota(such as Probiotics)may play a role in the early prevention of allergic diseases.However,it is not clear how the intestinal microbiota in early life participates in the occurrence and development of children’s allergic diseases,and the preventive effect of probiotics on children’s allergic diseases is strain-specific.Therefore,this study aims to use animal experiments to observe the impact of antibiotic-induced early life intestinal microbiota defects on the host immune system and allergic diseases and to explore the underlying mechanisms,and to observe the possible protective effects of early life probiotic strain Bifidobacterium bifidum TMC3115 supplements at the strain level and expect to further provide new ideas for the early prevention of children’s allergic diseases.MethodsThis study was consisted of four parts.In the first part,antibiotics were used to induce the early intestinal microbiota disorder of newborn mice after birth,and its influence on the intestinal development and immune function of the mice were observed;in the second part,antibiotics and Bifidobacterium bifidum TMC3115 were used to alter the formation of early intestinal microbiota in newborn mice after birth,and allergy were induced after that,which aimed to explore the relationship and mechanism between early intestinal microbiota construction after birth and allergic diseases of the host in adulthood;in the third part,antibiotics were used to induce the intestinal microbiota disorder of the mother during pregnancy and observe its influence on the placental microbiota and the immune function of the offspring;in the fourth part,antibiotics and Bifidobacterium bifidum TMC3115 were used to alter the intestinal microbiota of the mother during pregnancy,and then allergy were induced in the offspring,which aimed to explore the relationship and mechanism between maternal intestinal microbiota and allergic diseases in offspring.The detailed methods used in each part were listed as follows:1.Neonatal mice were orally administered with saline,ceftriaxone or vancomycin from PND 0 to PND 21 and sacrificed on PND21,feces,cecal contents,the whole intestine and spleen were collected.The diversity and abundance of the intestinal microbiota were determined by PCR-DGGE,depth of villus and crypts and the expression of KI67,MUC2 and ZO-1 of intestinal tract were detected by HE and IHC,and the splenic CD4~+CD25~+Foxp3~+T cells were investigated by flowcytometry.2.Neonatal mice were administered with saline,ceftriaxone,vancomycin,ceftriaxone+Bifidobacterium bifidum TMC3115(TMC3115),vancomycin+TMC3115,TMC3115 for 21 days.Then the gavage was discontinued after weaning,and the weaned mice were sacrificed partly at PND21.Afterwards,the left mice were sensitized with OVA for a total of four sensitizations.The left mice were sacrificed at PND56,and serum,feces,cecal contents and organs were collected.ELISA was used to detect the serum IgE and OVA-IgE;Luminex was used to detect the serum cytokine level;16S rRNA sequencing was used to detect the intestinal microbiota diversity and community;HE,IHC and IF staining were used to observe the depth of intestinal villus and crypts and the expression of KI67,MUC2 and ZO-1;flow cytometry was used to detect the content of CD4~+T cells in the spleen of mice;GC-MS was used to detect the content of SCFAs in the cecum.3.Pregnant mice were gavaged with saline and ceftriaxone respectively until their C-section.The microbiota in the feces and placenta of the dams were comprehensively analyzed using16S rRNA sequencing.Furthermore,viable bacteria in the placentas and blood of pups were also isolated by plate cultivation then taxonomically identified in detail by clone sequencing.Serum cytokines collected from dams and pups were quantitatively profiled using Luminex.4.Pregnant mice were gavaged with saline,ceftriaxone,and Bifidobacterium bifidum TMC3115 suspension until their vaginal delivery.After delivery,the feces and serum of the mother and the blood of the newborn mice were collected immediately.At PND21,the mother and part of 21-day young mice were sacrificed,after that,OVA stimulation was performed for a total of 3 sensitizations.At PND49,the left mice were sacrificed,and serum,feces and organs were collected.16S rRNA sequencing was used to detect the diversity and structure of fecal intestinal microbiota;GC was used to detect fecal SCFAs levels;HE staining was used to detect the depth of ileal villus and colonic crypts;RT-q PCR was used to detect colonic KI67,MUC2,ZO-1,Claudin-1,Claudin-2 and Occludin and splenic cytokine and transcription factor mRNA expression;ELISA was used to detect serum IgG,IgM,IgA,sIgA,IgE and OVA-specific IgE/IgG1and colonic sIgA contents;Luminex was used to detect serum cytokine levels.Results1.The gut microbiota and intestinal tissue were damaged,and the numbers of KI67-and ZO-1-positive cells were significantly decreased,and the MUC2 expression were significantly altered in the antibiotic treatment groups during lactation.Furthermore,the administration of ceftriaxone,but not vancomycin,led to a significant reduction in the abundance of splenic CD4~+CD25~+Foxp3~+T cells.2.Ceftriaxone and vancomycin administration during lactation,but not TMC3115,significantly damaged the intestinal microbiota and the morphology and barrier function of the intestinal epithelium,reduced SCFAs level and affected serum total IgE level in the neonatal mice.In the adult stages,the diversity and composition of the intestinal microbiota were significantly different in the antibiotic-treated mice,and ceftriaxone-treated mice exhibited significantly higher serum total IgE,OVA-specific IgE levels and splenic CD4~+T cells percentage.TMC3115 significantly mitigated the intestinal microbiota perturbation and abnormally elevated IgE caused by ceftriaxone.3.The spleen organ index of dams was significantly decreased and the offspring serum interleukin-6 levels were significantly increased in ceftriaxone-treated mice.The maternal fecal microbiota community was drastically altered in ceftriaxone-treated mice with significantly decreased diversity,depletion of Bacteroidetes and the blooming of Tenericutes.However,the placenta microbiota was dominated by Proteobacteria especially characteristically by Ralstonia,which was distinct from the maternal gut microbiota,regardless of whether ceftriaxone treatment or not.In addition,viable bacteria have been found in placenta and blood cultures.4.Oral administration of ceftriaxone during pregnancy significantly destroyed the intestinal microbiota of the mother and offspring and reduced their SCFAs levels,and the damaged microbiota could last to PND21 or even in adulthood.Serum IgE,IgM and IL-12p70,and the expression of the IFN-γand IL-10 mRNA in spleens were significantly decreased in the sensitized offspring of ceftriaxone-treated mothers.Oral administration of probiotic during pregnancy did not significantly alter the mother’s intestinal microbiota and SCFAs levels at the end of delivery.However,at PND21,the relative abundance of Firmicutes and Lactobacillus were significantly increased both in the mother and her offspring and were accompanied by the recovery of SCFAs,and serum IgE was significantly decreased.The expression of colonic KI67,MUC2 and tight junction protein mRNA were significantly increased in offspring at PND21,even in adulthood.After immunized by OVA,the serum IgM,the relative abundance of Firmicutes and Lactobacillus and the production of fecal SCFAs were significantly increased,and the serum OVA-specific IgE,IgG1 and Th2-type cytokines IL-4,IL-5,IL-13 and transcription factor GATA-3 mRNA were slightly decreased.Conclusions1.Antibiotics exposure during lactation caused intestinal dysbiosis and characteristically influenced the regeneration of intestinal epithelial cells,formation of the intestinal mucus layer and tight junctions,and differentiation of splenic Foxp3~+Treg cells of the neonatal mice before any clinical side effects were observed.The above-mentioned destructive effect of ceftriaxone is stronger than that of vancomycin,indicating that the potent ability of each antibiotic to affect the makeup of intestinal commensal microbiota may be a key determinant of its influence on the health of the host animal,at least partly.2.During lactation,ceftriaxone-induced intestinal dysbiosis and abnormal development of intestinal tissues and immunity could partly last into adulthood.This might be associated with the enhancement of host susceptibility to IgE-mediated allergies and related immune responses in the later stage of the host,TMC3115 might protect against the side effects of antibiotic treatment,at least partly.3.Ceftriaxone exposure during pregnancy could induce the maternal intestinal microbiota dysbiosis and regulate the immune system development of the newborn offspring,however,it could not significantly influence the placental microbiota;The uterus was not a sterile environment,the placenta might harbor its own unique microbiota,and these microbes were significantly different from the maternal intestinal microbiota,which should be paid attention to in future study.4.Pregnancy exposure of ceftriaxone could dramatically alter intestinal microbiota of the mother and offspring,and inhibit the normal immune development of the offspring,resulting in its reduced ability to respond and antagonize IgE-mediated allergic reactions;while supplementing with probiotics during pregnancy could improve the development of intestinal tract and intestinal barrier function and enhance the immune function in offspring through significantly increasing abundance of intestinal beneficial bacteria(such as Lactobacillus)and SCFAs production both in mothers and offspring.Maternal intestinal microbiota could play a certain regulatory role on offspring’s microbiota construction and immune system development through vertical transmission from mother to infant.The specific transmission patterns and microbiota involved in the vertical transmission and their roles in the health of host need to be further studied.