Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent

Sreeram Udayan,Janaki Velmurugan,David Sancho,Ludovica F Buttó,Paul W O'Toole,Silvia Melgar,Ken Nally,Valerio Rossini,Maria Martinez-Lopez

doi:10.1038/s41598-021-85347-7

Abstract

The mechanisms through which cells of the host innate immune system distinguish commensal bacteria from pathogens are currently unclear. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) expressed by host cells which recognize microbe-associated molecular patterns (MAMPs) common to both commensal and pathogenic bacteria. Of the different TLRs, TLR2/6 recognize bacterial lipopeptides and trigger cytokines responses, especially to Gram-positive and Gram-negative pathogens. We report here that TLR2 is dispensable for triggering macrophage cytokine responses to different strains of the Gram-positive commensal bacterial species Lactobacillus salivarius. The L. salivarius UCC118 strain strongly upregulated expression of the PRRs, Mincle (Clec4e), TLR1 and TLR2 in macrophages while downregulating other TLR pathways. Cytokine responses triggered by L. salivarius UCC118 were predominantly TLR2-independent but MyD88-dependent. However, macrophage cytokine responses triggered by another Gram-positive commensal bacteria, Bifidobacterium breve UCC2003 were predominantly TLR2-dependent. Thus, we report a differential requirement for TLR2-dependency in triggering macrophage cytokine responses to different commensal Gram-positive bacteria. Furthermore, TNF-α responses to the TLR2 ligand FSL-1 and L. salivarius UCC118 were partially Mincle-dependent suggesting that PRR pathways such as Mincle contribute to the recognition of MAMPs on distinct Gram-positive commensal bacteria. Ultimately, integration of signals from these different PRR pathways and other MyD88-dependent pathways may determine immune responses to commensal bacteria at the host-microbe interface.

Highlights

Lactobacillus salivarius (L. salivarius) is a widely studied Gram-positive commensal bacteria of the phylum Firmicutes, one of the dominant phyla of the human gut m icrobiome[1,2]
Live cells of thirty-three L. salivarius strains from different environmental sources (Table 1), control probiotic and pathogenic bacteria and individual Toll-like receptors (TLRs) ligands were screened using MSD multi-plex cytokine assays for their ability to stimulate WT, TLR2−/− and MyD88−/− BMDMs to produce a panel of cytokines (TNF-α, IL-6, IL-10, IL-12p70, IL-1β and IFN-γ) and the chemokine KC/GRO
We found that macrophage cytokine responses to these bacteria were TLR2 independent yet completely MyD88 dependent and associated with the upregulation of Tlr[1], Tlr[2] and Clec4e pattern recognition receptors (PRRs) genes in these cells

Summary

Introduction

Lactobacillus salivarius (L. salivarius) is a widely studied Gram-positive commensal bacteria of the phylum Firmicutes, one of the dominant phyla of the human gut m icrobiome[1,2]. Recent research has focused on understanding the beneficial effects of L. salivarius as a candidate probiotic because of its ability to induce tolerogenic T cell responses[3,4], ameliorate c olitis[5], translocate from the maternal gut to breast m ilk[6], modulate immune-related functions of host intestinal epithelial cells[7,8], induce antimicrobial activity[9,10], induce anti-tumour activity[11], maintain gastro-intestinal barrier integrity[12] and induce anti-inflammatory a ctivity[7,13] While some of these properties have been attributed to the production of a well characterized bacteriocin Abp[118] (an antibacterial peptide), the host signaling pathways required for mediating recognition and responses to L. salivarius are poorly characterised[10,14,15]. Since MAMP-PRR interactions contribute to the establishment and regulation of commensal-host homeostasis, it is important to identify PRRs involved in recognition of commensal strains with purported immunomodulatory properties such as L. salivarius UCC118 To address this question we screened a panel of well characterised Gram-positive L. salivarius strains isolated from different environmental sources for their effects on macrophage cytokine and chemokine responses. Macrophages co-cultured with L. salivarius upregulated the expression of Clec4e/Mincle, a C-type lectin receptor widely associated with recognition of glycolipids on Mycobacterium tuberculosis and Candida albicans[34]

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Results

Conclusion