Divergent patterns of colonization and immune response elicited from two intestinal Lactobacillus strains that display similar properties in vitro

Abstract

Lactobacilli derived from the endogenous flora of normal donors are being increasingly used as probiotics in functional foods and as vaccine carriers. However, a variety of studies done with distinct strains of lactobacilli has suggested heterogeneous and strain-specific effects. To dissect this heterogeneity at the immunological level, we selected two strains of lactobacilli that displayed similar properties in vitro and studied their impact on mucosal and systemic B-cell responses in monoxenic mice. Germfree mice were colonized with Lactobacillus johnsonii (NCC 533) or Lactobacillus paracasei (NCC 2461). Bacterial loads were monitored for 30 days in intestinal tissues, and mucosal and systemic B-cell responses were measured. Although both Lactobacillus strains displayed similar growth, survival, and adherence properties in vitro, they colonized the intestinal lumen and translocated into mucosal lymphoid organs at different densities. L. johnsonii colonized the intestine very efficiently at high levels, whereas the number of L. paracasei decreased rapidly and it colonized at low levels. We determined whether this difference in colonization correlated with an induction of different types of immune responses. We observed that colonization with either strain induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that both strains were able to activate mucosal B-cell responses. However, clear differences in patterns of immunoglobulins were observed between the two strains in the mucosa and in the periphery. Therefore, despite similar in vitro probiotic properties, distinct Lactobacillus strains may colonize the gut differently and generate divergent immune responses.

FIG. 1.

Adhesion of L. johnsonii and L. paracasei to differentiated Caco-2 cells in vitro. Results represent the mean ± standard deviation from six individual experiments. The adhesion value for L. johnsonii in each experiment was set at 100%. Each experiment was performed in triplicate, and the percentages were calculated from the means of these triplicate cultures.

FIG. 2.

Kinetics of bacterial translocation in monoassociated mice. Germfree C3H/n mice received a single gavage with 10⁹ CFU of L. johnsonii or L. paracasei at weaning. Bacterial loads were counted in fecal pellets extracted from the rectum (A), the luminal contents of the small intestine (B), and the colon (C). Bacteria were also counted in Peyer's patches (D) and mesenteric lymph nodes (E). Counts were made by gavage at days 4, 15, and 30 after colonization. This is one representative experiment out of three performed individually. Results are expressed as mean (± standard error of the mean) CFU per gram for feces, small intestine, and colon or as CFU per organ for Peyer's patches and mesenteric lymph nodes for four mice per group. The horizontal dashed line indicates the limit of detection due to the initial dilution of the samples (see Materials and Methods), and data points below that line represent negative cultures. *, P < 0.05 between the two groups.

FIG. 3.

Hematoxylin- and eosin-stained histological section of Peyer's patches of monoassociated mice. After 30 days of colonization, the jejunum was taken from germfree and monoassociated mice, and 5-μm-thick paraffin-embedded tissue sections were made from Peyer's patches of germfree controls (A), L. johnsonii-colonized mice (B), and L. paracasei-colonized mice (C).

FIG. 4.

Increased number of IgA⁺ cells in the lamina propria of monoassociated mice. After 30 days of colonization, the jejunum was taken from germfree and monoassociated mice, and 5-μm-thick paraffin-embedded tissue sections from intestinal tissue of germfree controls (A), L. johnsonii-colonized mice (B), and L. paracasei-colonized mice (C) were stained with biotinylated goat anti-mouse IgA followed by horseradish peroxidase-labeled streptavidin.

FIG. 5.

Levels of Lactobacillus-specific IgA in Peyer's patch whole-organ culture supernatants and intestinal contents of monoxenic mice. After 30 days of colonization, Peyer's patches were collected, washed, and incubated for 10 days in culture. The whole small intestine was taken to extract intestinal contents. Culture supernatants from Peyer's patch cultures (A and B) and intestinal contents (C and D) were assessed for L. johnsonii-specific (A and C) and L. paracasei-specific (B and D) IgA by ELISA. Results are expressed as optical density at 405 nm of specific IgA antibodies and represent means of duplicates (A and B) and of eight mice per group (C and D) (± standard error of the mean). The data show results from one representative experiment out of three. *, significantly increased above values for germfree controls (P < 0.05).

FIG. 6.

Lactobacillus-specific IgG2a and IgG1 isotypes in the serum of monoassociated mice. After 30 days of colonization, serum was collected from L. johnsonii-associated mice and assayed for L. johnsonii-specific IgG2a (A) and IgG1 (B). Serum from L. paracasei-colonized mice was assayed for L. paracasei-specific IgG2a (C) and IgG1 (D). Results were extrapolated from a positive serum standard from mice immunized systemically with L. johnsonii or L. paracasei crude antigens and represent means for eight mice per group (± standard error of the mean). The data show results from one representative experiment out of three. *, significantly increased above values for germfree controls (P < 0.05).