Prevention of allergic airway and gut inflammation in humanized mice by lactobacilli, bifidobacteria, and butyrate

Probiotic bacteria and their metabolites, particularly the short chain fatty acid butyrate, possess beneficial immunomodulatory and anti-inflammatory properties and have been shown to protect against gastrointestinal and respiratory diseases including IgE-mediated allergic inflammation.1-3 However, underlying mechanisms such as modulation of dendritic cell maturation and induction of regulatory T (Treg) cells are complex and not fully understood.2, 4 Moreover, to achieve a strong immune-modulatory response, it is crucial to provide multiple bacteria in optimal concentration and simultaneously. Here, we investigated for the first time a well-defined commercially available probiotic formulation (BactoFlor® 10/20, BF), consisting of 10 large and small intestine-specific strains of Lacto- and Bifidobacteria and the prebiotic carbohydrate inulin, in comparison to butyrate in a well-established and valid humanized mouse model of allergen-induced gut and lung inflammation.5 Nonobese diabetic severe-combined-immunodeficient γc−/− (NSG) mice were intraperitoneally injected with PBMC from highly-sensitized birch or grass pollen allergic human donors together with the respective allergen or PBS as control. During the whole experiment, mice were orally treated with BF or butyrate as described in the Appendix S1. In this model, sufficient numbers of human cells are detectable 3 weeks after PBMC transfer, and inflammation of the gut and lungs can be measured by high-resolution video mini-endoscopy, evaluating the parameters translucency, granularity, fibrin production, vascularity, and stool, or by invasive body plethysmography after rectal or intranasal allergen challenge, respectively.5 Figure 1A,B shows that the strong inflammation of the gut in PBMC plus allergen-treated mice was completely prevented by oral administration of BF or butyrate. In addition, disturbed intestinal barrier integrity, as measured by FITC-dextran assay, was significantly restored (Figure 1C). Analysis of lung allergic inflammation further revealed reduced lung airway resistance, reduced mucosal hypertrophy, and reduced mucus-producing goblet cells in BF- or butyrate-treated mice (Figure 1D,E). Interestingly, despite equal numbers of human CD45+ cells in all organs, an increased migration of FoxP3+ Treg cells into the lung and gut tissue was found in BF- as well as butyrate-treated mice after respective allergen challenge (Figure 1F). In the spleen, a slight enhancement of CD4+FoxP3 expression was also detected without reaching significance (Figure 1G). Engraftment of BF- or butyrate-pretreated PBMC also significantly reduced gut and lung inflammation, but less pronounced as compared to oral treatment (Figure S1A–D). This superior protective effect of orally applied probiotics/butyrate suggests that besides their immunomodulatory effect on PBMC, they also directly affect the mucosal epithelium and barrier. Moreover, a slight but not significant increase in butyrate concentration was observed in stool of BF-treated mice (Figure S2). This implicated that other microbiota-derived metabolites than butyrate, possibly tryptophan metabolites or retinoid acid, might also be involved in the induction of the regulatory immune response.2, 6 To analyze whether Treg cells are responsible for the protective effect of BF and butyrate, CD25high-expressing cells were depleted from the PBMC prior to injection (Figure S3). In these CD25negPBMC engrafted NSG mice, gut and lung inflammation was only marginally reduced by BF or butyrate (Figure 2A–D). This is in accordance with former studies demonstrating the strong Treg cell dependency in prevention of allergic diseases.5 Notably, neutralization of allergy promoting group 2 innate lymphoid cells (ILC2), which are known to be controlled by Treg,1 by anti-ST2 mAbs restored the protective effect of BF and butyrate in Treg-depleted mice (Figure S4). Other cell types might also be involved which is currently under investigation. Taken together, our study support the relevance of Treg cells regarding the combined effect of defined beneficial intestinal microbial species and their products on allergic intestinal and airway inflammation. Hence, it advances our current understanding of the previously suspected adjuvant effect of certain probiotics in allergic desensitization. Our results may also be applicable to other inflammatory diseases of the gastrointestinal and respiratory tract. DS, JS, and IB generated the idea and supervised the work; RK, BW, FS, SS, and IB performed laboratory assays and contributed to data analysis; RK, DS, and IB wrote the manuscript; all authors have read and approved the manuscript. The authors would like to thank Mrs. Claudia Braun and Mrs. Anke Heinz for their excellent technical assistance. Open Access funding enabled and organized by Projekt DEAL. This work was supported by Deutsche Forschungsgemeinschaft (DFG), grants BE 4504/3-3 (I.B.), SFB1181-B02 (B.W.), SCHU 646/20-3 and CRC TR355/1 project B08 (D.S.). The authors have no conflicting financial interests to disclose. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.