Disengaging bacterial recognition patterns:

Analyzing the combined effect of multiple signalling through different TLRs

FB Velhote1, J Rodríguez-Ruiz2, E Barboza2, P Barbarroja2, J Gálvez3, J Xaus3, M Comalada2,3. 1University of Sao Paulo (USP), Pharmacology Department, Brazil, 2Institute for Research in Biomedicine (IRB), Macrophage Biology Group, 08028, Spain, 3CIBER-ehd, University of Granada, Pharmacology Department, Spain

Background: Intestinal bacteria are differently recognized by innate immune cells in the gut allowing the induction of immune tolerance or inflammatory response when required. However, relatively little is known about the capacity of these cells, including macrophages, to discriminate between beneficial/commensal and potential pathogen bacterial antigens. Bacteria express diverse components that can stimulate different Toll-like receptors (TLRs) expressed in macrophages and dendritic cells. Therefore, it is very possible that the activation of different combination of TLRs by each bacterium in macrophages might be responsible for the final immune response: inflammation or tolerance.

Aims: The objective of this work is to evaluate the response induced in macrophages after recognition of two particular gram positive bacterial strains which exhibit different inflammatory profile and to compare them with the response obtained after simultaneous TLR stimulation using different TLRs agonist.

Methods: For the experiments, we used primary cultures of bone marrow-derived macrophages from male balb/c mice (8-wk-old). Starved macrophages were stimulated with 5x107 cfu/ml of heat-inactivated bacterial strains (Lactobacillus fermentum (LF) and Lysteria monocytogens (LM)) for 5 h (for RNA) or 24 h (cytokine production). In parallel experiments, macrophages were stimulated with several agonists of specific TLRs (TLR2, TLR4, TLR5) or agonists that activate combinations of different TLRS (TLR2/1 and TLR2/6). In this sense, lipopolissacharide (LPS) from Escherichia coli (10 ng/ml) was used as a TLR4 agonist, LTA-SA (1μg/ml) as a TLR2 agonist and FLA-ST (10 ng/ml) as a TLR5 agonist. For a combination of different TLRs activation, the Pam2CSK4 (100 ng/ml) was used as an agonist of TLR2/1 and TLR2/6 heterodimers and FSL-1 (100 ng/ml) as an agonist of simple TLR2/TLR6 heterodimer. RNA was extracted to perform Real Time-PCR to analyze gene expression while supernatants of the cultures were analyzed for cytokine expression by Cytometric Bead Assay (CBA). Student’s t-test was used for statistical analysis.

Results: The results obtained suggest that immune response induced in macrophages depends on the bacterial strain recognized. In this sense, the activation of macrophages with LF induces high amount of TNFα while reduced levels of IL-10 (TNFα/IL-10 ratio: 0.98±0.04). In contrast, LM induces higher amounts of IL-10 and moderate levels of TNFα (TNFα/IL-10 ratio: 0.04±0.005; p<0.05 vs LF). Moreover, TLR agonist combinations using Pam2CSK4 and FSL generate a TNFα/IL-10 ratio similar to that observed with LF while FLA has a similar anti-inflammatory ratio as LM. Interestingly, the addition of LPS to the bacteria strains change the inflammatory profile of LF, increasing IL-10 and reducing TNFα (TNFα/IL-10 ratio: 0.03±0.004; p<0.05 vs LF w/o LPS) suggesting that simultaneous stimulation of TLR2 and TLR4 exerts an antagonistic effect on the expression of some inflammatory markers.

Conclusion: The combined repertoire of stimulated-TLR induced by a particular bacterial strain determines the final response induced in macrophages. This fact is clearly observed in the opposite effects induced by some probiotic strains depending on the inflammatory state of the gut, acting as immune-enhancers in resting conditions but as suppressive agents on inflammatory conditions.