Interleukin 10’s interest in the attempt of developing an efficient therapy against IBD has grown since it was observed that IL-10 knockout (IL-10−/−) mice develop spontaneous enterocolitis when not maintained in germ-free conditions . Although many advances have been made there still does not exist any treatment for IBD, showing that the major drawback to use IL-10 is its correct administration and directioning to the sites of inflammation.
In order to overcome these problems, new strategies based on local delivery of IL-10 to the intestinal mucosa have been developed; high IL-10 concentration at these sites would outweigh the need for high doses and as such avoid the increase of pro-inflammatory cytokine production and other undesired side effects.
One such strategy consists in the use of polymer-based microparticles. These particles encapsulate gelatine nanoparticles containing plasmid DNA expressing murine IL-10 and are capable of releasing these nanoparticles directly at the desired site of action. These IL-10 gene containing particles have shown to successfully reduce the levels of inflammatory cytokines as well as disease activity scores in the TNBS-induced model of colitis . However, despite promising, scale-up to an industrial level appears to be very expensive and complicated.
Another strategy by Yao and collaborators consists in a Bifidobacterium longum strain capable of secreting human IL-10 which was capable of alleviating inflammatory damage of colonic tissue in a DSS-induced mouse model by blocking the colitis-activated NF-κB pathways and upregulating CD4 + CD25 + Foxp3+ Treg in blood and mesenteric lymph nodes .
As such, in order to improve IL-10 delivery to the sites of inflammation using L. lactis, a L. lactis strain expressing FnBPA [15, 28], that has the capacity to efficiently internalize and trigger recombinant DNA expression by human epithelial cells, was used to deliver a eukaryotic expression vector coding for IL-10 of Mus musculus, pValac:il-10, directly to host cells in the GIT for recombinant in situ IL-10 production. This new strategy recently showed to be capable of diminishing inflammation in a TNBS-induced mouse model .
Following these results and in order to evaluate the therapeutic effect of the pValac:il-10 plasmid in a larger context, in the present work we explored the potential of this therapeutic plasmid in a DSS-induced mouse model delivered by two L. Lactis strains as a new therapeutic strategy for the prevention of intestinal inflammation. L. lactis MG1363 FnBPA + expresses FnBPA, which confers the strain the capacity to mediate adhesion to host tissue and bacterial uptake by eukaryotic cells. To evaluate if this characteristic results in improved anti-inflammatory effect, it was compared with the non-invasive L. lactis MG1363 strain. Our results showed that both strains were capable of delivering the eukaryotic expression vector to host cells directly at the sites of inflammation leading to in situ IL-10 production, avoiding strong and undesired side-effects, and diminishing the severity of inflammation by maintaining an anti-inflammatory environment in the gut.
DSS has the intrinsic capacity to disrupt the epithelial cell barrier, causing normal gut substances to activate mucosal macrophages, which in turn produce immunomodulatory cytokines. It is generally accepted that DSS is directly toxic to gut epithelial cells of the basal crypts and affects the integrity of the mucosal barrier . The DSS concentration used in the present model triggered an acute colonic inflammation accompanied by diarrhoea and rectal bleeding and did not lead to mortality. The administration of strains carrying the pValac:il-10 plasmid statistically lowered the macroscopic score compared to the DSS group, regarding diarrhoea and rectal bleeding. No weight loss was observed within the different groups, but this has already been reported earlier . At histological level, administration of strains carrying the pValac:il-10 plasmid were capable of decreasing the severity of inflammation, with tendency to normality, demonstrating the anti-inflammatory effects of this strategy.
IgA is the most abundant immunoglobulin produced, in its secretory form, at mucosal sites. In the luminal mucous layer, sIgA protects the intestinal epithelium against colonization and invasion by pathogens or commensals  and therefore helps modulating and controlling inflammation. In our model, no significant decrease of sIgA was associated with the inflammation observed in mice treated with DSS that did not receive the pValac:il-10. The administration of bacteria carrying the pValac:il-10 plasmid was accompanied by significantly increased levels of sIgA only in the intestinal fluid of mice that received the L. Lactis MG1363 FnBPA + pValac:il-10 strain, compared to the DSS treated mice. Moreover, this strain also induced an increased sIgA production when compared to the L. lactis MG1363 pValac:il-10 strain. We believe that the difference of IgA production induced by these two strains is due to the different cellular entry mechanism these bacteria use after oral administration. In this regard, L. lactis MG1363 FnBPA + pValac:il-10 strain induced increased IgA production because these bacteria invade enterocytes in the epithelium, while L. lactis MG1363 pValac:il-10 are captured by macrophages (as these bacteria have no invasion capacity), which does not directly lead to the induction of IgA. These results showed that the L. Lactis MG1363 FnBPA + pValac:il-10 strain could not only have an anti-inflammatory effect in this model, but also promote the gut immunological barrier by limiting the penetration of bacteria into host tissues and therefore protect mice by helping to modulate inflammation. The expression of FnBPA by this strain might allow for a higher binding capacity and internalization by eukaryotic cells, thus enhancing the production of IL-10 and its anti-inflammatory properties.
Cytokines produced in the gut mucosa greatly influence the resulting immunological outcome; production of anti-inflammatory cytokines induces mucosal tolerance, while high levels of pro-inflammatory cytokines induce inflammation. Since cytokines are major mediators of inflammation and regulatory activity in the gut mucosa, we analysed the ability of L. lactis strains carrying the pValac:il-10 plasmid to modulate the production of cytokines in the colonic tissues of mice. Mice that received the pValac:il-10 plasmid, whether delivered by L. lactis MG1363 or L. lactis MG1363 FnBPA + (pValac:il-10 groups), showed significantly higher IL-10 levels than the control group (healthy mice), probably due to a constant and higher production of IL-10 by the eukaryotic cells of these animals. It is also important to note that the expression of FnBPA by the bacterial strain carrying the pValac:il-10 plasmid was associated to significantly higher IL-10 levels in the intestinal tissues compared to the DSS group, where this cytokine was produced as normal immune response against inflammation. However, these levels of IL-10 were not significantly different from those obtained in the L. lactis MG1363 pValac:il-10 group, nor were associated with significant differences in the levels of the other cytokines tested in both pValac:il-10 groups. The lack of significant difference between both strains carrying the pValac:il-10 plasmid could be explained by the fact that both cellular entry mechanisms used by these bacteria (enterocytes invasion by the invasive strain and capture by macrophages by the non-invasive strains) lead to efficient and similar IL-10 production; however, by different cell types.
Pro-inflammatory cytokines, including TNF-α, IL-6 and IL-17, can be produced by T and B lymphocytes, macrophages and/or neutrophils, which are massively infiltrated in inflammatory lesions in mice with DSS-induced acute colitis. This DSS colitis switches from a Th1-Th17-mediated acute inflammation with increased levels of TNF-α, IL-6 and IL-17, to a predominant Th2-mediated inflammatory response that shows a decrease in TNF-α, IL-6 and IL-17 while increasing levels of anti-inflammatory cytokines IL-4 and IL-10 . In our experimental acute model, at the time of sacrifice, the results only showed higher IL-6 levels in the DSS group and in the group of mice treated with DSS that received the L. lactis MG1363 strain, when compared to the control group (healthy mice). Significant decrease of this cytokine was observed in the intestinal tissues from mice that received the pValac:il-10 plasmid, compared to the DSS group. These results were associated to the increased IL-10 levels obtained in those animals and confirm the anti-inflammatory capacity of both strains carrying the pValac:il-10 plasmid at modulating the gut immune response.