Intervention Of Diet On Different Phenotype Of Asthma Modulated By Lactobacillus Paracasei

Background and objectives:Asthma is a common chronic lower respiratory inflammation disease characterized by cough,wheezing,chest tightness,and bronchial hyperresponsiveness.It is often accompanied by rhinitis and gastroesophageal reflux,and other comorbidities.Avoiding allergic triggers and using corticosteroids are effective controller strategies in asthma management,but the prevalence of uncontrolled asthma is still high,maybe for the resistance to long-term administration of high-dose steroids.The asthma patients had a considerable economic burden.Based on distinct mechanistic pathways and severity,asthma is categorized into eosinophilic asthma and non-eosinophilic asthma.Neutrophilic asthma has the worst asthma control and more significant symptoms among non-eosinophilic asthma.The heterogeneity of asthma pathogenesis has similar symptoms that are difficult to diagnose with low recoveries.Mounting evidence suggests that probiotics can be applied to treat asthma.It is estimated that the gut contains about 1014 known bacteria,with the colon harboring the majority of bacteria and the most diversity.Gut microbiota is vital for human health by exerting metabolic,nutrition,and immunological activities.Probiotics attenuated the dysbiosis implicated with asthma,but some studies have conflicting results.Therefore,it is unclear whether the probiotics could correct the imbalance responses of immunity caused by asthma.Excluding the asthma phenotype,dietary is suggested to be an environmental factor affecting the effect of probiotics.Different diet results in microbiota alteration.The high-fat diet increases Firmicutes and Proteobacteria,while the high-fiber diet increases Bacteroidetes and Actinobacteria,supporting a vital role in the immune response.One therapeutic potential of probiotics is to provide metabolic benefits to the host,such as short-chain fatty acids production,downregulating the inflammation and improving the intestinal barrier.Therefore,in this study,we assess the diet intervention on different phenotype asthma modulated by Lactobacillus paracasei DMLA16017,which was previously suggested to prevent allergic diseases effectively.Materials and Methods:Specific pathogen-free(SPF)three-week-old male Wistar rats were purchased from the Animal Center of Dalian Medical University.L.paracasei DMLA16017 was provided by the Department of Microecology of Dalian Medical University.The eosinophilic asthma model was established by ovalbumin(OVA),and the neutrophilic asthma model was established by OVA combined with lipopolysaccharide(LPS).Rats were gavaged with probiotics only or combined with high-fat chow or high-fiber chow starting from the day before the challenge.The role of diet on L.paracasei-modulated asthma was identified by recording the frequencies of nasal rubs and sneezes,ELISA,and hematoxylin-eosin staining.Subsequently,DNA isolation and 16 S r DNA sequencing described the microflora composition in the feces of rats,and the SCFAs levels were detected by gas chromatography.Pearson’s correlation was computed between intestinal microbiota,cytokines,or SCFAs.For further study,the cytokines and transcription factors were measured by quantitative polymerase chain reaction(q PCR)and Western blotting on the levels of m RNA or protein,respectively,to discuss the mechanisms of diet intervention on L.paracasei-modulated asthma.Results:1.Intervention of diet on different phenotypes of asthma modulated with L.paracasei.(1)Nasal rubs and OVA-Ig E level were increased than those in control after OVA combined with LPS.The same situations are in using OVA only.Specifically,the IL-8level increases dramatically higher in the former treatment.Both the eosinophilic asthma model and neutrophilic asthma model were established.(2)The effect of L.paracasei on modulating different asthma phenotypes is distinct.After L.paracasei modulating,the allergic symptoms of eosinophilic asthma were relieved,the level of OVA-Ig E and the total number of WBCs decreased,infiltration of eosinophilic asthma was suppressed,IL-4 level decreased,IFN-γ and TGF-β increased,but no significant difference in the level of IL-17.On the other hand,the allergic symptoms in neutrophilic asthma were relieved more,no significant change in OVA-Ig E level,the WBCs decreased,infiltration of neutrophils was suppressed,and the IL-17 level dropped significantly,the levels of TGF-β and IL-4 increased apparently,but the IFN-γ had no significant change.(3)High-fat diet intervented the effect of L.paracasei on modulating different phenotypes of asthma.High-fat diet intervention caused more allergic severe symptoms in all asthma rats.With a more robust inflammatory response,the OVA-Ig E level and WBC counts increased significantly in L.paracasei-modulated eosinophilic asthma.The ratio IFN-γ/IL-4 decreased significantly.In the L.paracasei-modulated neutrophilic asthma,the serum OVA-Ig E level and WBC counts had no change,but the neutrophils infiltrated more.Both the ratio IFN-γ/IL-4 and IL-17/TGF-β increased.(4)High-fiber diet intervented the effect of L.paracasei on modulating different phenotypes of asthma,but not signigicantly.The high-fiber diet intervention slightly improved the L.paracasei-modulated eosinophilic asthma compared with L.paracasei-modulated only,and it also improved L.paracasei-modulated neutrophilic asthma,the alveoli structure is recovered,and the ratio IL-17/TGF-β decreased.2.The impact of diet on intestinal flora in different phenotypes of asthma modulated by L.paracasei.(1)The intestinal flora changed for the diet intervention.After the high-fat diet intervention,the diversity of gut flora decreased in L.paracasei-modulated eosinophilic asthma,while it increased in L.paracasei-modulated neutrophilic asthma.After the high-fiber diet intervention,the diversity of gut flora increased in L.paracasei-modulated eosinophilic asthma.However,it decreased in L.paracasei-modulated neutrophilic asthma.(2)Biomarkers in different communities.After the high-fat diet intervention,the Erysipelotrichaceae,Enterobacteriaceae,Allobaculum,Escherichia-Shigella,and Escherichia coli were enriched in L.paracasei-modulated eosinophilic asthma;the Firmicutes,Lachnospiraceae,and Oscillospiraceae were enriched in L.paracasei-modulated neutrophilic asthma.After the high-fiber diet intervention,abundances of Verrucomicrobiota,Lachnospiraceae,Akkermansiaceae,Erysipelatoclostridiaceae,Akkermansia muciniphila,and Erysipelatoclostridium ramosum were enriched in L.paracasei-modulated eosinophilic asthma;biomarkers in L.paracasei-modulated neutrophilic asthma included Proteobacteria,Verrucomicrobiota,Actinobacteria,Enterobacteriaceae,Akkermansiaceae,Erysipelatoclostridiaceae,Clostridiaceae,Escherichia-Shigella,Akkermansia,Erysipelatoclostridium,Clostridium sensu stricto 1,Escherichia coli,Akkermansia muciniphila,Erysipelatoclostridium ramosum.(3)SCFAs profiling was intervented by diets.After high-fat diet intervention,the production of SCFAs decreased in the L.paracasei-modulated eosinophilic asthma,espcecially the acetic acid.There is no significant change in L.paracasei-modulated neutrophilic asthma but the butyric acid decreased significantly.After the high-fiber diet intervention,the SCFAs levels increased in all asthma phenotypes.(4)Microbiota composition changed with the environmental factors.The abundance of Erysipelotrichaceae was positively correlated with the level of IL-17 and ROR-γt in BALF.The Firmicutes abundance was positively correlated with the OVA-Ig E level,whereas it was negatively correlated with SCFAs levels.Strong correlations between IFN-γ and Escherichia-Shigella and Escherichia coli,respectively,were found.3.The expression of cytokines and transcription factors were differentially in the L.paracasei-modulated asthma intervented by diet.(1)The m RNA relative expression.After high-fat diet intervention,in the L.paracasei-modulated eosinophilic asthma,the ratio IFN-γ/IL-4 and Tbet/Gata3 m RNA relative expressions decreased,IL-17/TGF-β and ROR-γt/Foxp3 increased.On the other hand,the ratio IFN-γ/IL-4,IL-17/TGF-β,Tbet/Gata3,and ROR-γt/Foxp3 m RNA were of increased expressions in L.paracasei-modulated neutrophilic asthma,but it showed a significant higher ratio of IL-17/TGF-β.After the high-fiber diet intervention,all m RNA relative expressions were not significantly different from the L.paracasei-modulated asthmas but improved.(2)The relative expression of the proteins.After high-fat diet intervention,the IFN-γ,TGF-β,and Foxp3 relative expressions decreased,and the Gata3 and ROR-γt expression increased in L.paracasei-modulated eosinophilic asthma.The IFN-γ,Gata3,ROR-γt,and Foxp3 relative expressions increased,but the TGF-β decreased in L.paracasei-modulated neutrophilic asthma.After the high-fiber diet intervention,the relative expression levels of TGF-β,Gata3,ROR-γt,and Foxp3 increased but IFN-γ in L.paracasei-modulated eosinophilic asthma.The protein extracted from the lungs of L.paracasei-modulated neutrophilic asthma has an increased expression of IFN-γ,TGF-β,Gata3,ROR-γt,and Foxp3,especially the IFN-γ,TGF-β,and Foxp3 increased significantly.Conclusions:1.The effect and mechanism of L.paracasei on the modulation of different asthma phenotypes are distinct.L.paracasei reduced the level of OVA-Ig E in eosinophilic asthma.It suppressed the eosinophilic airway inflammation by enriching the abundance of Ruminoccoccus,which is responsible for the decreased level of Ig E,rebalancing the Th1/Th2 cells.L.paracasei relieved the allergic symptoms and decreased the systemic inflammation in neutrophilic asthma.In addition,it skews the Th17/Treg imbalance by decreasing the ratio of Bacteroides to Prevotella.2.High-fat diet influences the effect of L.paracasei on modulating asthma.The high-fat diet enhanced the allergic symptoms in L.paracasei-modulated eosinophilic asthma by enriching the abundance of E.coli,which is involved with a low IFN-γ expression and high Gata3 expression,disturbed the Th1/Th2 balance to Th2 cells.Firmicutes was predominant in L.paracasei-modulated neutrophilic asthma after the high-fat diet intervention resulting in higher expressions of proinflammation cytokines and transcription factors resulting in the Th17/Treg imbalance.3.The high-fiber diet influences the effect of L.paracasei on modulating asthma.In L.paracasei-modulated eosinophilic asthma,the high-fiber diet enriched the butyric acid-producing bacteria Lachnospiraceae and promoted the expression of TGF-βand Foxp3 for exerting immune regulation.The Akkermansiaceae was predominant in L.paracasei-modulated neutrophilic asthma after the high-fiber diet intervention,which is correlated with butyric acid positively,and an up-regulated expression of TGF-β and Foxp3,but the difference is not significant.4.The strategy for L.paracasei modulating different phenotypes of asthma.The results above suggest that the appropriate diet intervention strategy for L.paracasei modulating asthma works according to the asthma phenotypes.Especially,L.paracasei administered in eosinophilic asthma should avoid taking a high-fat diet but a proper high-fiber diet.