Characterization of bacterial heme oxygenase ChuS of probiotic strain E.coli Nissle 1917 and its potential therapeutic uses
Fecha
2020Autor
PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE
Institución
Resumen
Heme-oxygenase (HO) is an enzyme that catalyzes the metabolism of heme group. Products derived from this reaction are biliverdin (Bv), carbon monoxide (CO), and iron (Fe++). Many properties have been described for this enzyme, such as antioxidant, anti-proliferative, anti-apoptotic, and anti-inflammatory. Most of them are related with CO production, which is able to attenuate the immune response by reducing immunogenicity of dendritic cells and modulating the T cells populations. HO was initially associated with mammal cells (HO-1) specially in humans, but some studies have identified a bacterial heme oxygenase. Some E. coli strains possess chuS gene that encodes bacterial HO ChuS and it is located into an operon that regulates the heme uptake with the subsequent CO production. On the other hand, lately microbiota influence on immune regulation have reached a high importance due to its capability to modulate directly and indirectly the function of immune cells. Indeed, as part of microbiota, probiotics play a key role promoting anti-inflammatory environments, acting against pathogens, modulating barrier function, even ameliorating autoimmune diseases. In that sense, E. coli Nissle 1917 (EcN), is a widely used commercial probiotic, originally used to treat bowel inflammation. Interestingly, chuS gene is present in its genome. The focus of this project is to assess whether anti-inflammatory properties of probiotic strain E. coli Nissle 1917 (EcN) are due to the presence of bacterial heme-oxygenase enzyme (ChuS) produced by this bacterium. For that, we developed a mutant strain which lacks chuS gene. The first part of the project was directed to evaluate both strains (WT and mutant) in DC and macrophages in vitro cultures, infected with different MOI of bacteria. The results of the first part evidenced that both strains were able to activate and induce maturation of DC and macrophages, despite the mutation. Interestingly, WT bacteria induced higher levels of PD-L2, a tolerance marker, than mutant strain. Additionally, IL-10 secretion showed the same pattern. Furthermore, results from antigen presenting assay with DC infected with the probiotic strains showed that T cells were activated and that the production of IL-4 was reduced in the mutant probiotic group. The second part of the project was focused on evaluating ChuS role in vivo. For this, we treated mice with different doses of both probiotics. The first assay evaluated the higher dose tolerated by mice, which was 1*109 CFU. After probiotic treatment, a group of mice were euthanized, and we found that T cells population in liver decreased in mice treated with the mutant strain. Mice were then infected with Salmonella enterica serovar Enteritidis (SEN) to assess whether treatment could affect in some way the outcome of the infection. After 48 h of infection, all mice were euthanized. Interestingly, we found bacterial load in spleen, liver and MLN in mice treated with mutant probiotic and infected with SEN. T cell populations showed differences between groups infected treated with mutant probiotic and WT strain. Also, B cells population in MLN showed to be higher for mice treated with mutant strain. Moreover, 4 additional doses were evaluated for each strain (1*104, 1*105, 1*106, and 1*107 CFU). This assay allowed us to assess differences between strains in inflammation patterns and cytokine profile. For instance, mice from groups of all doses for mutant strain presented inflammation in colon sections and mucus in feces or soft feces. Also, IL-10 production, detected in blood, was higher for mice treated with WT probiotic that those treated with mutant strain. Besides. IL-12p70 remained low for lower doses and increased for higher doses, for groups of mice treated with the WT probiotic. In the case of mice treated with mutant strain IL-12p70 levels were always lower than IL-10. Together, results allowed us to suggest that ChuS apparently has ability to modulate EcN immunomodulatory properties in vitro and in vivo. Which behave similar to HO-1, modulating IL-10 and IL-12p70 profile. Also, effect of EcN and ChuS is not only local but systemic, because we were able to detect differences in spleen, liver, MLN and blood.