The role of pili on Lactobacillus rhamnosus GG in the interaction with macrophages and other immune cells

The human gastrointestinal tract is colonized by large numbers of microbes, referred to as the gut microbiota. This microbiota establishes a complex and bilateral interplay with local host cells such as epithelial and immune cells. Functions including contribution to digestion, pathogen exclusion, enhancement of the epithelial barrier functioning and immune system modulation have been attributed to these gut microorganisms. Their described beneficial effects in human health have led to the selection of strains that are used as probiotics, which are defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host'. During the last decades, the prevalence of allergic diseases has been dramatically increased in industrialized countries, likely due to a reduced exposure to microorganisms and parasites that help to educate and train the regulatory arm of the immune system. The potential use of probiotics in the prevention and/or treatment of atopic disorders has thus received a lot of attention. Lactobacillus rhamnosus GG is one of the best clinically documented probiotic strains. Its reported beneficial effects include enhancing certain immune functions and decreasing the development of atopic diseases in susceptible individuals. However, not all reported health effects of L. rhamnosus GG are univocal. Health organizations highlight the importance of better molecular-based research techniques for unraveling the underlying molecular mechanisms of action of probiotics including L. rhamnosus GG. Recently, pili which are proteinaceous extracellular appendages, were discovered on the cell surface of L. rhamnosus GG. There is extensive evidence suggesting that pili are the major structures for adhesion to intestinal epithelial cells and human mucus and that can even induce anti-inflammatory responses.

In this PhD thesis, we aimed to investigate the interaction and possible immunomodulatory role of the SpaCBA pili of L. rhamnosus GG in immune cells. First, we chose a simplified model with murine macrophage-like (Raw 264.7) and human monocytic (THP-1) cell lines. By using a unique collection of isogenic single mutants of L. rhamnosus GG, we demonstrated that SpaCBA pili of L. rhamnosus GG are crucial for its adhesion capacity to macrophages and that this interaction stimulates bacterial uptake which is partially mediated by complement receptor 3. In contrast, exopolysaccharide molecules were shown to prevent bacterial internalization by these phagocytic cells. Furthermore, Our findings suggest that SpaCBA pili mediate anti-inflammatory effects by induction of interleukin-10 mRNA in macrophages; however, . The pili do not appear to be directly involved ininduce this induction IL-10 expression, because similar levels of IL-10 mRNA were observed for the SpaCBA pilus mutant and the wild-type. Instead, enhanced exposure of SpaCBA pili and/or other molecules in the mutant lacking the galactose-rich exopolysaccharide layer may be responsible for the increased expression of IL-10. Moreover, SpaCBA pili seem to act as a negative modulator of IL-6 mRNA expression in macrophages. In addition, the protective role of exopolysaccharides by shielding and indirectly preventing the immunostimulatory function of other bacterial molecules in macrophages was also demonstrated.

Secondly, we aimed to assess the immunomodulatory capacity of the SpaCBA pili of L. rhamnosus GG in a more complex cellular model, i.e. using freshly isolated human peripheral blood mononuclear cells from donors with different backgrounds. Interestingly, pili also showed to be (directly or indirectly) involved in the induction of IL-10 mRNA and inhibition of IL-6 expression in PBMCs from healthy donors, again pointing towards an anti-inflammatory role of the SpaCBA pili of L. rhamnosus GG. In contrast, the galactose-rich exopolysaccharide molecules seem to have an immunostimulatory role. Furthermore, cytokine profiles of PBMCs from allergic and healthy subjects after stimulation with L. rhamnosus GG cells were evaluated and our results suggest that PBMCs from allergic patients may be less responsive than those from healthy individuals. This might have serious consequences for successful probiotic treatments since patients may not respond as well as healthy subjects.

In the last part of this PhD study, we used a murine antigen presenting cell/T cell co-culture system to investigate whether SpaCBA pili could have an impact on T cell polarization, since differentiation of particular subsets of T cells (i.e. T helper 2 cells) and the impaired function of regulatory T cells have been linked to allergy. By studying T cell polarization, We could obtain extra information about the differentiation of specific T cell subsets after stimulation of antigen presenting cells previously treated with the different L. rhamnosus GG cells. Although the production of IFN-γ, IL-4, IL-17, IL-10 and expression of Foxp3 in T cells studied in this work was very low, our findings indicate that galactose-rich exopolysaccharide molecules of L. rhamnosus GG may be involved in T helper 1 and regulatory Foxp3+ T cell differentiation. Unfortunately, no role could be attributed to the pili in T cell polarization, at least under these tested conditions in murine cells.

Taken together, we have demonstrated in this PhD that SpaCBA pili, but also galactose-rich exopolysaccharide molecules, of L. rhamnosus GG are crucial for the immunomological interactions with immune cells, although various indirect (merely physical) effects are involved and many details thus remain to be unraveled. The exact role of pili and exopolysaccharide molecules seems to be highly dependent on the host cell type and the genetic background of the host, but also on the way these large surface molecules are presented on live L. rhamnosus GG cells. This study also gives the basis for future research orientated to the use of L. rhamnosus GG in the treatment of allergic diseases, in the sense that indications were found that immune cells of allergic individuals are less responsive to L. rhamnosus GG cells than healthy subjects.