Immunomodulation of dendritic cells by Lactobacillus reuteri surface components and metabolites

Abstract

Background: Lactic acid bacteria are commensal members of the gut microbiota and are postulated to promote host health. Secreted factors and cell surface components from Lactobacillus species have been shown to modulate the host immune system. However, the precise role of L. reuteri secreted factors and surface proteins in influencing dendritic cells (DCs) remains uncharacterized.

Hypothesis: We hypothesize that L. reuteri secreted factors will promote DC maturation, skewing cells toward an anti-inflammatory phenotype. In acute colitis, we speculate that L. reuteri promotes IL-10 and dampens pro-inflammatory cytokine production, thereby improving colitis.

Methods & results: Mouse bone marrow-derived DCs were differentiated into immature dendritic cells (iDCs) via IL-4 and GM-CSF stimulation. iDCs exposed to L. reuteri secreted factors or UV-irradiated bacteria exhibited greater expression of DC maturation markers CD83 and CD86 by flow cytometry. Additionally, L. reuteri stimulated DCs exhibited phenotypic maturation as denoted by cytokine production, including anti-inflammatory IL-10. Using mouse colonic organoids, we found that the microinjection of L. reuteri secreted metabolites and UV-irradiated bacteria was able to promote IL-10 production by DCs, indicating potential epithelial-immune cross-talk. In a TNBS-model of acute colitis, L. reuteri administration significantly improved histological scoring, colonic cytokine mRNA, serum cytokines, and bolstered IL-10 production.

Conclusions: Overall these data demonstrate that both L. reuteri secreted factors and its bacterial components are able to promote DC maturation. This work points to the specific role of L. reuteri in modulating intestinal DCs.

New & noteworthy: Lactobacillus reuteri colonizes the mammalian gastrointestinal tract and exerts beneficial effects on host health. However, the mechanisms behind these effects have not been fully explored. In this article, we identified that L. reuteri ATTC PTA 6475 metabolites and surface components promote dendritic cell maturation and IL-10 production. In acute colitis, we also demonstrate that L. reuteri can promote IL-10 and suppress inflammation. These findings may represent a crucial mechanism for maintaining intestinal immune homeostasis.

Keywords: Lactobacillus; cytokines; dendritic cells; inflammation; metabolites.

FIGURE 1

L. reuteri ATCC PTA 6475 adheres to human MUC2. a. Scanning electron microscopy (SEM) image of L. reuteri on a membrane (left) and L. reuteri adhered to mucus in human mucus‐producing HT29‐MTX cells. Scale bar =5 μm. b. Representative images of CFDA‐SE tagged L. reuteri (pink) co‐localizing with human HT29‐MTX MUC2 (green) after 1 hr incubation. Nuclei are marked with Hoechst dye (blue). Scale bar =100 μm. n = 3 replicates

FIGURE 2

Modulation of mouse bone‐marrow dendritic cell surface markers by L. reuteri. a. Representative light microscopy images of DCs exposed to 25% uninoculated LMD4 (Media), 100 ng/mL of LPS (LPS), 25% L. reuteri LDM4 conditioned media (LR CM) or 10⁶ UV‐irradiated L. reuteri (LR bacteria). Scale bar =100 μm. b. Representative images of flow cytometry gating for mouse bone marrow‐derived dendritic cells. Flow cytometry was performed by gating CD11c^hi DC populations and examining CD80 (C) and CD86 (D) abundance on DCs exposed to 25% uninoculated LMD4 (Media), 100 ng/mL of LPS (LPS), 25% L. reuteri LDM4 conditioned media (L. reuteri CM) or 10⁶ UV‐irradiated L. reuteri (L. reuteri Bacteria). Surface marker abundance was expressed by % abundance for (c) CD80, (d) CD86 positive populations. e. qPCR analysis of CCR7 mRNA expression in treated DCs. n = 3 technical replicates, 4 mice/group. ANOVA, *p < 0.05

FIGURE 3

Modulation of mouse bone marrow‐derived dendritic cell cytokines by L. reuteri. a. DCs exposed to 25% uninoculated LMD4 (Media), 100 ng/mL of LPS (LPS), 25% L. reuteri LDM4 conditioned media (LR CM) or 10⁶ UV‐irradiated L. reuteri (LR bacteria). Scale bar =100 μm. Viability analysis of resazurin assay, measured by resorufin fluorescence (Ex:560 nm/Em:600 nm). Secreted cytokines from treated DCs after 16 hrs of treatment measuring: (b) KC (mouse IL‐8 homolog), (c) TNF, (d) IL‐12, (e) IL‐6, and (f) IL‐1β. n = 3 technical replicates, 4 mice/group. ANOVA, *p < 0.05

FIGURE 4

L. reuteri promotes IL‐10 production in vitro. a. Representative light microscopy images of mouse DCs and ileal organoids. Scale bar =100 μm. b. IL‐10 levels from treated DCs or DC/organoid co‐cultures after 16 hrs of treatment. c. IL‐10 to IL‐12 ratio for DCs treated with 25% uninoculated LMD4 (Media), 100 ng/mL of LPS (LPS), 25% L. reuteri LDM4 conditioned media (LR CM) or 10⁶ UV‐irradiated L. reuteri (LR bacteria). n = 3 technical replicates, 4 mice/group. ANOVA, *p < 0.05

FIGURE 5

L. reuteri promotes IL‐10 production and suppresses inflammation in vivo. a. Representative Giemsa stains of mouse colon 3 days following TNBS administration in mice treated with either PBS (control) or 10⁹ live L. reuteri. Scale bar =100 μm. b. Histological scoring performed by a blinded board‐certified pathologist. qPCR analysis of mRNA of pro‐inflammatory cytokines (c) KC (IL‐8 homolog) and (d) TNF, as well as anti‐inflammatory (e) IL‐10. mRNA analysis of colonic (f) MUC2. Serum anti‐inflammatory IL‐10 (g) and pro‐inflammatory cytokines (h) as assessed by Luminex MAGPIX (n = 8 per group; 4 males, 4 females). Student's t‐test, * p < 0.05