Lactobacillus reuteri modulates dendritic cells and the immune response in vitro and in vivo
Authors: Engevik Melinda Anne , Ganesh Bhanu Priya , Visuthranukul Chonnikant , Versalovic James 
Published: November 2023

Background

Lactic acid bacteria (LAB) are commensal members of the gut microbiota which reside in the small and large intestine. Lactobacillus species have been shown to extensively modulate the host immune system. Our group has previously demonstrated that Lactobacillus reuteri is capable of ameliorating inflammation in TNBS‐ and AOM‐DSS‐induced colitis and cancer models. However, the role of L. reuteri bacterial components or secreted factors in direct modulation of dendritic cells (DCs) remains uncharacterized. DCs are key antigen‐presenting cells (APCs) which present antigens to T cells, thereby mediating inflammation or tolerance. Recent studies have shown that autophagy, a conserved intracellular degradation pathway, regulates DC antigen presentation.

Hypothesis

We hypothesize that L. reuteri secreted factors will promote DC maturation and dampen pro‐inflammatory cytokine production. We speculated that bacterial membrane proteins may stimulate autophagy, thereby regulating DC‐T cell interaction and promoting homeostasis.

Methods & Results

Human monocytoid THP‐1 cells were differentiated into immature dendritic cells (iDCs) via IL‐4 and GM‐CSF stimulation. iDCs were exposed to 30% L. reuteri strain 6475 secreted factors and DC maturation markers were examined by flow cytometry. iDCs stimulated with L. reuteri secreted factors had increased CD86, a co‐stimulator of T cell activation. L. reuteri stimulated DCs exhibited extensive dendrite formation and increased metabolic activity as measured by resazurin. L. reuteri matured DCs also upregulated the C‐C chemokine receptor 7 (CCR7) as denoted by enhanced chemotaxis to the ligand CCL21. Importantly L. reuteri matured DCs did not secrete pro‐inflammatory TNF, INFγ, IL‐6, IL‐1β or IL‐12. However, L. reuteri secreted factors were able to decrease LPS‐driven TNF production by 11‐fold, indicating that even in the presence of a pro‐inflammatory stimulus, L. reuteri has immunosuppressive effects. iDCs exposed to UV‐irradiated L. reuteri increased autophagy related genes ATG5 and LC3, while no changes were observed in Beclin1, ATG12, or ATG16L1. Direct co‐culturing of L. reuteri with DCs increased dendrite formation and metabolic activity of DCs. To assess the role of L. reuteri in modulating the immune system in vivo, Swiss Webster germ free mice were mono‐associated with L. reuteri for 23 days. L. reuteri colonization was demonstrated by standard bacterial plating and 16S fluorescent in situ hybridization (FISH). L. reuteri mono‐associated mice showed reduced pro‐inflammatory serum IL‐6 levels compared to germ free controls. In addition, L. reuteri colonization increased anti‐inflammatory IL‐10 mRNA gene expression in the colon. Consistent with mRNA, IL‐10 protein levels were elevated in the sera of L. reuteri mono‐associated mice compared with germ free controls.

Conclusions

Overall these data demonstrates that L. reuteri is able to promote DC maturation and dampen pro‐inflammatory cytokines. These data demonstrates a specific role of L. reuteri in modulating intestinal DCs maturation. This may represent a crucial mechanism for maintaining intestinal immune homeostasis.