Lactobacillus Reuteri-Derived Extracellular Vesicles Mediate Intestinal Inflammation Protection And Immune Regulation In Broilers

Probiotics can improve host health by shaping intestinal microecological balance and immune homeostasis.They are not only commonly used to prevent and treat human intestinal inflammatory diseases,but also have great application potential in livestock and poultry production.Lactobacillus reuteri is one of the most abundant symbiotic Lactobacillus in the chicken gut,which can be used as a candidate to develop probiotic for chicken.In order to improve the application effect,the potential mechanism of communication between probiotics and their host should be clarified.In addition to interacting with host cells in direct contact,probiotics can also establish contact with the host through the input of functional molecules.However,the mechanism by which symbiotic/probiotics deliver effector molecules into the host remains unclear.In recent years,it has been found that extracellular vesicles（EVs）released by bacteria play an important role in intercellular communication as the carrier of bacterial molecules.In this study,the highly immunoreactive symbiotic L.reuter strain in the gut of Black-Bone chickens was targeted to explore whether this strain could release EVs and whether these EVs could mediate immune regulation and inflammatory protection in broilers.Moreover,the mechanism associated with EVs-mediated immunomodulation was further studied using in vitro experiments.The obtained results are as follows:Experiment 1:Screening of L.reuteri isolate with high immunoregulatory activity from the chicken gutIn this experiment,four L.reuteri isolates were identified from Lactobacillus spp.isolated from the gut of Black-Bone chickens in our laboratory by using a molecular biology method based on 16S r DNA sequencing.A L.reuteri BBC3 isolate with good probiotics and strong immunomodulatory attributes was screened out through assays of the tolerance to gastrointestinal conditions,antibacterial assays,adhesion assays and the evaluation of an immunoregulatory activity.The results showed that L.reuteri BBC3 isolate show good tolerance to p H 3.0 artificial gastric juice with 0.6%（w/v）pepsin and grew well in a culture medium containing 0.25%bile salts.This isolate also had significant inhibitory activities on three pathogenic bacteria（E.coli,S.Enteritidis and S.aureus）and a high adhesion rate to Caco-2 cells.Furthermore,L.reuteri BBC3 isolate significantly enhanced the gene expression of antigen-presenting molecule BLB2,co-stimulating molecule CD80,and immunoregulatory cytokines（IL-6 and IL-12）in chicken HD11 macrophages.Based on the above results,L.reuteri BBC3 was selected as the subsequent target strain.Experiment 2:Isolation,purification and proteomic analysis of L.reuteri BBC3-derived extracellular vesicles（LrEVs）The purpose of this experiment was to establish a standard procedure for the isolation and purification of LrEVs from culture supernatants of L.reuteri BBC3 based on the ultrafiltration method.Then the purified LrEVs were identified and characterized by scanning electron microscopy,transmission electron microscopy and nanoparticle tracking analysis（NTA）.Finally,the protein composition of LrEVs was analyzed by proteomics,and the identified proteins were analyzed functionally.The results showed that L.reuteri BBC3could release nanoscale membrane vesicles.The method of ultrafiltration concentration combined with ultracentrifugation can be used to obtain a considerable amount of LrEVs.The purification of LrEVs by Opti Prep gradient density centrifugation showed excellent performance and could meet the needs of subsequent experiments.The diameters of most LrEVs ranged from 50 nm to 150 nm,and the densities ranged from 1.127 g/m L to 1.199g/m L.Electron microscopy and NTA analysis further confirmed that the characterization of purified LrEVs obtained in this experiment was consistent with the previously reported characteristics of bacterial extracellular vesicles.Proteomics results showed that LrEVs contained higher protein content and less DNA and RNA content of 354μg,2.9μg and 12.2μg(all were converted to 1×10¹¹ particles),respectively.A total of 92 proteins were identified in LrEVs,which were mainly from the cytoplasm and membrane,and were mainly involved in physiological processes such as metabolism,protease hydrolysis,stress,nucleic acid biosynthesis and regulation,and transport.Besides,some homologous proteins that are associated with immunomodulatory or probiotic effects in other probiotics or symbionts also observed in LrEVs.These results suggest that LrEVs carry functional molecules from parental bacterium,which may mediate the bacteria-host interaction.Experiment 3:The regulatory effect of LrEVs on intestinal inflammatory responses induced by lipopolysaccharide（LPS）in broilersThis experiment aimed to explore whether L.reuteri BBC3 and LrEVs have immunomodulatory and protective effects against LPS-induced intestinal inflammation in vivo.Healthy broilers（Arbor Acres）with consistent body weight were given by gavage the cultured L.reuteri BBC3（5×10⁹ CFU/bird）and purified LrEVs（200μg/bird）in 200μL protectant（5%skim milk）,respectively.At 12,14 and 18 days of age,the birds in the LPS-challenged groups were intraperitoneally injected LPS（500μg/bird）in 100μL PBS to establish a model of intestinal inflammation.At the same time,a negative control group was set with PBS administration and PBS stimulation,and a positive control group with PBS administration and LPS stimulation.The results showed that administration of L.reuteri BBC3 significantly attenuated the reduced growth performance,the occurrence of intestinal injury and inflammatory responses caused by LPS challenge in broilers.LrEVs could reproduce the protective effects on the LPS-induced intestinal injury and inflammation.Both LrEVs and L.reuteri BBC3 inhibited the expression of pro-inflammatory genes（TNF-α,IL-1β,IL-6,IL-8,IL-17A and MIP-1β）and enhanced the expression of anti-inflammatory genes（IL-10 and TGF-β）in the jejunum of broilers challenged with LPS.But,LrEVs were different from L.reuteri BBC3 in regulating the immune response,indicating that the immunoregulatory activities between LrEVs and L.reuteri BBC3 were not completely equivalent.Nevertheless,the above results suggest that LrEVs can reproduce the protective effect of L.reuteri BBC3 on LPS-induced intestinal inflammation in broilers,and this protective effect is accompanied by the enhancement of anti-inflammatory gene expression and inhibition of pro-inflammatory gene expression.Experiment 4:Study on immunoregulatory mechanisms of LrEVs-mediated inflammation protectionIn this study,the innate immune and adaptive immune regulation mechanisms of LrEVs-mediated inflammation protection were explored through macrophages assays and macrophage-spleen lymphocyte co-culture assays,respectively,and the role of vesicular protein and nucleic acid in LrEVs-mediated immunoregulation was preliminarily explored through intestinal tissue culture assays.Macrophages assays were performed as the following:we first used confocal microscopy to study the internalization of LrEVs in chicken HD11 macrophages,then HD11 cells were stimulated with LrEVs for 12 h and then stimulated with LPS for 12 h to establish an inflammatory model of in vitro macrophages.After culture,the survival rate of macrophages,immune gene expression and NF-κB activity were measured.Macrophage-spleen lymphocyte co-culture assays were carried out as the following:HD11 cells were prestimulated with LrEVs for 12 h.After culture,the culture medium was discarded,the cells were washed and transferred to the bottom compartment of the Transwell system.The spleen lymphocytes of LPS-challenged broilers were then inoculated into a chamber above the Transwell system and co-cultured for 16 h to detect the expression level of immune genes in spleen lymphocytes.Jejunal tissue culture assays were carried out as the following:LrEVs were treated with DNase I and RNase I（DR）and proteinase K-agarose（PK）to remove nucleic acids and proteins,respectively.The enzymes were inactivated at 75°C for 1 h and proteinase K-agarose was additionally removed by centrifugation.Then,LrEVs treated with these enzymes were used to pre-stimulate jejunal explants for 6 h,followed by LPS stimulation for 6 h and MPO activity and immune gene expression levels were detected in cultured jejunal tissues.The results showed that LrEVs could be effectively internalized by HD11 cells.An appropriate dose of LrEVs did not affect the viability of HD11 cells and activated the expression of anti-inflammatory genes（IL-10,TGF-β）and pro-inflammatory genes（TNF-αand IL-1β）.LrEVs could inhibit the expression of NF-κB-dependent pro-inflammatory genes（TNF-α,IL-1βand IL-6）and enhanced the expression of anti-inflammatory genes（IL-10 and TGF-β）in LPS-activated HD11 cells.The expression of pro-inflammatory genes（IFN-γand IL-17）was significantly down-regulated,the expression of anti-inflammatory genes（IL-10 and TGF-β）was significantly up-regulated and the expression of genes associated with immunosuppression（CD25,CTLA-4 and LAG-3）was also significantly enhanced in splenic lymphocytes of LPS-challenged broilers after co-culture with LrEVs-pretreated HD11 cells.Although enzymatic treatments significantly reduced the content of vesicular DNA,RNA or proteins to some extent,they did not remove these components as much as expected,implying that the signals delivered by EVs are highly protected against exogenous proteases.During ex vivo jejunum explant culture with LPS challenge,DR treatment significantly decreased the suppression of native LrEVs on the expressions of TNF-α,IL-6 and IL-17,and PK treatment significantly decreased the suppression of native LrEVs on the expressions of TNF-α,IL-6,IFN-γand IL-17.Additionally,DR-and PK-treated LrEVs showed a lower ability to induce the expressions of IL-10 and TGF-βcompared to native LrEVs.Finally,DR and PK treatments also markedly reduced the ability of native LrEVs in the inhibition the MPO activity,indicating that these two treatments significantly decreased the suppressive activity of native LrEVs to inflammatory responses.The above results indicated that LrEVs could be internalized by chicken macrophages and induced innate immune responses,and had inhibitory effects on the inflammatory responses of chicken macrophages induced by LPS.LrEVs could enhance the anti-inflammatory effect mediated by spleen lymphocytes through macrophages,thus playing an adaptive anti-inflammatory immune regulation role.Both vesicle protein and nucleic acid participated in the anti-inflammatory immune regulation process mediated by LrEVs.In summary,this study screened a probiotic L.reuteri BBC3 with good probiotics and strong immune activity from the chicken intestines and confirmed that it could produce and secrete extracellular vesicles containing active molecules of the parental bacteria,and proved that these vesicles could reproduce the regulatory effects of its parental bacteria on the intestinal inflammation of broilers.The stud further elucidated that these vesicles could maintain immune homeostasis by inhibiting the inflammatory responses involved by macrophages and enhancing the anti-inflammatory responses involved by spleen lymphocytes and that both vesicular protein and nucleic acid were involved in the immune regulation process mediated by LrEVs.These results provided a theoretical basis for screening probiotics for chicken and also provided new insights into the potential mechanism of probiotic-host communication.