Abstract

Previously, we performed a detailed characterization of the probiotic properties of Lactobacillus rhamnosus UCO-25A originally isolated from human stomach. We demonstrated that the UCO- 25 strain has high hydrophobicity properties and is able to form biofilms on glass and cell surfaces. Moreover, we demonstrated that L. rhamnosus UCO-25A is a strain with immunomodulatory activity. Using a murine model, we observed that the oral administration of UCO-25A strain is able to increase the activity of peritoneal macrophages and intestinal antigen presenting cells as well as the levels of intestinal IgA (Ilabaca et al., 2018, submitted for publication). Then, our findings provided a scientific rationale for the use of L. rhamnosus UCO- 25A in the development of new immunobiotic products. One of the major challenges in the production of new food or pharmaceutical products is to preserve the viability of immunobiotic strains in order they can exert their beneficial effects. Encapsulation is an interesting alternative to achieve this goal. Encapsulation can protect microorganisms from external environments and induce the release of them in a metabolically active state in the intestine. The aim of this work was to evaluate whether L. rhamnosus UCO-25A, administered in an encapsulated form, does preserve its immunomodulatory activities. Moreover, the immunomodulatory capacities of its planktonic o biofilm states were comparatively studied. Immunobiotic capsules were prepared with carob tree-gum and alginate through an extrusion process in the B-390/B395Pro Encapsulator. Capsules were then covered with low molecular weight chitosan (Type I). Female 5-week-old BALB/c mice were used for in vivo studies. L. rhamnosus UCO-25A encapsulated in planktonic (P25A) or biofilm (B25A) forms were administered at a concentration of 109 cells/mouse/day to different groups of mice for two consecutive days by gavage. Mice treated with PBS solution were used as controls. One day after the last treatment we evaluated: a) peritoneal macrophages phagocytic and microbicidal activities; b) IgA levels in intestinal fluid (IF); c) levels of IFN-γ and TNF-α in IF and serum, and d) immune cell populations in peritoneal cavity, Peyer´s patches and spleen by flow cytometry. Although P25A or B25A did not significantly change the number of peritoneal macrophages (F4/80+ cells) both treatments improved their phagocytic and microbicidal activity (% of phagocytosis: control=4.2±1.2, P25A=19.8±2.4, B25A=16.8±2.1) as well as their MHC-II expression. In addition, enhanced levels of IF and serum TNF-α and IFN-γ were detected, indicating a potent systemic stimulation as well (serum IFN-γ (pg/mL): control=65.2±4.3, P25A=187.3±4.7, B25A=145.6±3.9). It should be noted that P25A was more efficient to increase those parameters when compared to B25A. We also observed changes in antigen presenting cells of mice Peyer´s patches. Higher percentages of CD11b+CD86+ as well as CD11c+CD86+ cells were detected in mice treated with P25A or B25A when compared to controls. Both treatments were equally effective to modulate antigen presenting cells (% of CD86+ in CD11c+ cells: control=72.4±3.7, P25A=88.1±4.1, B25A=85.6±3.3). P25A or B25A were also able to stimulate intestinal humoral immunity as observed by the improved levels of IF IgA (IgA (ug/mL): control=2.1±0.2, P25A=3.1±0.1, B25A=2.7±0.1) and the percentages of B220+ and CD24+ cells in Peyer´s patches (% of B220+ cells: control=18.3±2.3, P25A=32.5±3.1, B25A=23.4±2.7). Once again, the P25A treatment was more efficient to increase those parameters when compared to B25A. No significant influence of P25A or B25A was observed on T cells as demonstrated by studies of CD4+ and CD8+ cells in Peyer´s patches and spleen. The results of this work show that the UCO-25A strain, encapsulated in a planktonic state, has optimal immunomodulatory properties demonstrated by its ability to stimulate immune cells in both intestinal and systemic compartments. These findings open the possibility to develop prototype immunobiotic products using encapsulated planktonic L. rhamnosus UCO-25A intended for use in humans.