3.1. DC are able to capture L. casei through the intestinal epithelium
To evaluate the uptake of lactic acid bacteria (LAB) by DC through a human intestinal epithelium, DC from healthy or allergic donors were incubated with FITC-labelled L. casei for 24 or 48 hours in the Transwell coculture system. DC adherent and nonadherent to the filter were harvested and analyzed separately. The number of DC adherent to the filter was slightly increased with DC from healthy donors compared to allergic patients after L. casei stimulation (59800±28200 and 33900±2200 adherent cells per well, resp.), whereas unstimulated DC from healthy and allergic donors similarly adhere to epithelium (20417±4641 and 17000±5778, resp.). Both in allergic and healthy donors, the capture of FITC-bacteria by DC directly adherent to the epithelium increased between 24 and 48 h. In contrast, nonadherent DC captured fewer bacteria and only at 48 h. For the analyzed donors, the uptake of L. casei by DC was lower for allergic compared to healthy donors (Figure 2). Higher percentages of FITC-positive cells were detected when DC were directly incubated with bacteria, without epithelium (data not shown).
The bacteria uptake through the intestinal epithelium was further confirmed by confocalmicroscopy. Different sections of the same adherent DC detected with an anti-CD11c antibody (Figure 1(a)) clearly showed a colocalization of FITC-labelled L. casei into the DC cytoplasm (Figure 1(b)).
3.2. DC acquire amoderatematuration status upon L. casei stimulation through the intestinal epithelium
To determine whether maturation of DC by L. casei, in the presence of the epithelium, is associated with phenotypic changes; the expression of CD80, CD86, HLA-DR, and CD54 (intercellular adhesion molecule (ICAM)-1) was assessed. L. casei induced a low increase in the expression of the costimulatory molecules CD86 (P
3.3. Cytokine production by L. casei-stimulated DC
The production of IL-10 and IL-12, involved in the orientation of the immune response, was analyzed. DC fromhealthy donors incubated with L. casei significantly produced IL-10 and IL-12 48 h after the stimulation, with similar levels in the presence or in the absence of the intestinal epithelium (Figure 4(a)). In contrast, DC from allergic patients incubated with L. casei only slightly increased IL-10 and IL-12 production in the absence of the epithelium, whereas the secretion of these two cytokines was amplified in the presence of the epithelial layer (P
3.4. Cytokine production by naive CD4+ T cells
To evaluate the effect of L. casei on the orientation of the response induced by DC from allergic and healthy donors, the production of IFN-γ (Th1 cytokine), IL-5 (Th2 cytokine), and IL-10 (cytokine produced by some regulatory T cells) by autologous naive T cells was analyzed. As no differences were detected in the maturation status between adherent and nonadherent DC and as L. casei capture was more efficient in adherent DC, adherent DC were chosen for T cell stimulation. Without epithelium, L. casei mainly increased the IFN- γ production by naive T cells instructed by DC from healthy donors (62.40±34.88 compared to 39.45±26.13 formedium condition). IL-5 and IL-10 production by T cells was less increased. No increase in these cytokine levels was detected with L. casei-activated DC from allergic donors. In healthy donors, the sole presence of Caco2 epithelial layer (without L. casei stimulation) decreased the IFN-γ production by DC-instructed T cells (5.35 ± 5.58 ng/ml with the epithelium compared to 39.45 ± 26.13 ng/ml without the epithelium). In contrast, for all cytokine production, the increase induced by L. casei-stimulated DC from healthy or allergic donors was further enhanced if DC were conditioned by the epithelium. The increase in cytokine production for T cells activated by L. casei-stimulated DC was more pronounced for IFN-γ (26.75±8.02 ng/ml for the L. casei condition compared to 9.72 ± 10.41 ng/ml for the medium condition for allergic patients; 48.60 ± 16.68 ng/ml for the L. casei condition compared to 5.35 ± 5.58 ng/ml for healthy donors). The increase in cytokine production for allergic donors reached similar levels to healthy donors for IL-5 and IL-10, whereas IFN-γ levels remained higher for healthy donors (Figure 5).