Much research in the last decades has focused in unveiling the etiology and pathogenesis of IBDs to develop therapies for their treatment, as no cure for them exists. Many strategies have based their research on the use of IL-10 due to its anti-inflammatory properties and key role in the development of the disease, as evidenced by IL-10−/− mice, which develop enterocolitis, a severe disease in the colon characterized by weight loss, anemia and strong inflammatory lesions, apparent already at 4 weeks of age and lethal up to the age of 3 months, when maintained under conventional conditions [4].
The research and development of therapies using IL-10 are based on its delivery directly at the intestinal mucosa, as its correct administration and targeting to the sites of inflammation has been the major bottleneck for successful results. These strategies have included the use of polymer-based microparticles [20] and the use of bacterial strains capable of secreting human IL-10 [21,22,23].
As a new and innovative approach for IL-10 production at inflammation sites, our research group developed a new strategy that involved the use of a L. lactis strain expressing FnBPA [17, 24], that has the capacity to efficiently internalize and trigger recombinant DNA expression by human epithelial cells, as delivery vehicle of a eukaryotic expression vector coding for IL-10, pValac:il-10, directly to the host’s cells in the GIT for recombinant IL-10 in situ production. This strategy does not only ensure a more effective and direct delivery of the therapeutic plasmid but consequently a higher and more efficient IL-10 production. This strategy showed to be capable of preventing inflammation in the TNBS-and DSS-induced mouse models [18, 19].
In this research, 2 weeks old mice received the pValac:il-10 plasmid during six consecutive weeks to evaluate its therapeutic effect. Macroscopic and histological analysis showed that non-treated mice presented an overall lower weight gain during this period while those under treatment showed a rapid weight gain equaling that of the healthy control group. This growth retardation in IL-10−/− mice could be explained by the severe lesions in the GIT that led to disturbed nutrient absorption, contrary to the animals that received the therapy and consequently produced IL-10. Regarding the inflammatory damage in the AC and DC, mice from the KO and FnBPA groups presented higher macroscopic damage scores and important lesions in the colon while IL-10−/− mice that received the pValac:il-10 plasmid showed an attenuated pathology’s development and as such a much lower macroscopic damage scores, showing that IL-10 administration does prevent a worse onset of inflammation and therefore disease development.
Other important parameters measured during this period were the levels of pro-inflammatory and anti-inflammatory cytokines. The normal gastrointestinal inflammatory response is tightly regulated, and the balance of pro-inflammatory and anti-inflammatory cytokines produced by CD4+ Th1 and Th2 cells is an important part of this regulatory process [25]. Intestinal inflammation in IL-10−/− mice is characterized by dysregulation of the immune system with regulatory T cells incapable of developing or functionally impaired in the absence of IL-10, leading to activation of Th1 cells and overproduction of pro-inflammatory cytokines resulting in chronic inflammation [5]. However, administration of the pValac:il-10 plasmid led to IL-10 production by mice naturally incapable of producing this cytokine, which, as a consequence, lowered the disease development when compared to IL-10−/− mice that did not receive any treatment. As already mentioned, IL-10 is essential to control intestinal immune responses. Moreover, some evidence suggests that IL-10 can inhibit the translocation of nuclear factor-kB, inhibiting the immediate-early pro-inflammatory response [26] and downregulate acute inflammatory responses.
This IL-10 production altered the production of pro-inflammatory cytokines, such as IL-6, which presented lower levels in all tissues of treated mice. IL-6 is produced by T and B lymphocytes, which infiltrate inflammatory lesions in IL-10−/− mice, and which presents high levels during IBDs, therefore playing a functional role in their pathogenesis [4]. Moreover, in IL-10−/− mice, IL-6 can also be considered an inflammatory mediator responsible for maintaining/increasing the intestinal inflammation [5] and its levels were therefore measured. On the other hand, IFN-γ, identified as a major mediator initiating colitis in IL-10−/− neonates [5, 27], showed increased levels in IL-10−/− mice as a result of failed regulatory T cells production or even functionality in the absence of IL-10, suggesting that Th1 cells are activated very early in the disease process [27]. A higher IFN-γ production is related to a more severe colitis [28].
To confirm that the genetically modified L. lactis strain is indeed transient through the gastrointestinal tract and does not colonize it, bacterial translocation to the spleen and liver was evaluated. No bacterial translocation was observed in these organs at any of the evaluated times (2 h, 4 h, 6 h, 12 h and 24 h) after oral administration. This is a particularly important safety parameter regarding human use.
The work here presented represents the last step in validating this DNA delivery strategy using the L. lactis MG1363 bacterial strain. When first constructing the pValac:il-10 plasmid, this was tested for its transfection efficiency and subsequent IL-10 production and secretion by eukaryotic cells through ELISA, confocal microscopy and FACS [18]. Following, this L. lactis MG1363 FnBPA+ (pValac:il-10) strain was tested in two animal models to evaluate not only the success of the strategy in delivering the pValacil-10 plasmid and following in situ IL-10 production and secretion by the animals cells, but also its immunomodulatory potential in diminishing the onset and development of intestinal inflammation due to increased IL-10 production, when compared with the control groups. In 2013, del Carmen and co-workers demonstrated that the severity of TNBS-induced intestinal inflammation decreased in mice after administration of this strain as a result of an increase in IL-10 levels and decrease in IFN-γ and IL-17 levels, when compared to the not treated groups [18]. Later, Zurita-Turk et al. demonstrated that administration of this L. lactis strain resulted in decreased severity of DSS-induced intestinal inflammation, probably also due to increased IL-10 levels and decreased IL-6 levels [19]. These studies showed that direct IL-10 production at inflammation sites produced an anti-inflammatory environment, as well as downregulated Th1-and Th17-mediated inflammation, capable of diminishing the onset and severity of the disease.
Thus, the main interest when testing this bacterial strain in the IL-10−/− model was to evaluate if animals would produce IL-10 and consequently diminish the development of the intestinal inflammation and as such, validate the efficiency of this DNA delivery strategy. The results here presented are very exciting, as they demonstrate that our strategy leads to IL-10 production by mice that are naturally not able to, as well as higher production when compared to healthy mice capable of normally producing this cytokine, however, at a lower rate.
However, this current study presents one research limitation: FACS analyses were not performed. FACS analyses would have brought better knowledge regarding the mechanism of pValac:il-10 delivery and uptake/invasion into eukaryotic cells, as well as which are the IL-10-producing cell populations. From the obtained results it can be stated that the proposed strategy was successful, as the pValac:il-10 plasmid was delivered to eukaryotic cells in IL-10−/− mice and subsequent IL-10 production was responsible diminish the development of intestinal inflammation. Nevertheless, the exact mechanism and which cells populations were involved, were not elucidated. In this context, future studies are required to answer these current research limitations.