Probiotic Bacteria and Their Cell Walls Induce Th1-Type Immunity Against Salmonella Typhimurium Challenge

Abstract

Probiotics have been associated with a variety of health benefits. They can act as adjuvant to enhance specific immune response. Bacterial cell wall (CW) molecules are key structures that interact with host receptors promoting probiotic effects. The adjuvant capacity underlying this sub-cellular fraction purified from Lactobacillus casei CRL431 and L. paracasei CNCMI-1518 remains to be characterized. We interrogated the molecular and cellular events after oral feeding with probiotic-derived CW in addition to heat-inactivated Salmonella Typhimurium and their subsequent protective capacity against S. Typhimurium challenge. Intact probiotic bacteria were orally administered for comparison. We find that previous oral feeding with probiotics or their sub-cellular fraction reduce bacterial burden in spleen and liver after Salmonella challenge. Antibody responses after pathogen challenge were negligible, characterized by not major changes in the antibody-mediated phagocytic activity, and in the levels of total and Salmonella-specific intestinal sIgA and serum IgG, respectively. Conversely, the beneficial effect of probiotic-derived CW after S. Typhimurium challenge were ascribed to a Th1-type cell-mediated immunity which was characterized by augmentation of the delayed-type hypersensitivity response. The cell-mediated immunity associated with the oral feeding with probiotic-derived CW was accompanied with a Th1-cell polarizing cytokines, distinguished by increase IFN-γ/IL-4 ratio. Similar results were observed with the intact probiotics. Our study identified molecular events associated with the oral administration of sub-cellular structures derived from probiotics and their adjuvant capacity to exert immune modulatory function.

Keywords: Salmonella Typhimurium; Th1-type response; cell wall; immunomodulation; mucosal adjuvant; probiotic.

Figure 1

Study design. BALB/c mice (6 weeks-old) were given probiotic bacteria, Lc431 or Lp1518, in the drinking water for 7 and 5 consecutive days, respectively. CW431 or CW1518, were administered by gavage for 7 consecutive days. Mice were immunized with heat-inactivated S. Typhimurium via oral route on days 1, 3, and 5 of either probiotics or CW administration. After 21 days, animals were challenged with one dose of virulent S. Typhimurium via oral route and a group of mice received PBS (oral) and served as control (uninfected); mice were euthanized 72h.

Figure 2

Probiotic and their CW did not enhance antibody-mediated phagocytosis against S. Typhimurium. Percentage of phagocytosis of S. Typhimurium opsonized with serum obtained from uninfected or infected animals. Non-immunized animals (littermate mice) were used as cut-off (dashed line). Data are shown as mean ± SEM from three independent experiments with 3 mice per group. P values were calculated with one-way ANOVA with Šidak correction for multiple comparisons. *P < 0.05.

Figure 3

Probiotic and their CW modulate mucosal antibody response against S. Typhimurium. (A, B) Total and Salmonella-specific sIgA levels in intestinal fluid of uninfected and S. Typhimurium infected mice. (C) Salmonella-specific IgG titer in serum of uninfected and S. Typhimurium infected mice. (D, E) Salmonella-specific IgG subclasses determined in serum of uninfected (D) and S. Typhimurium infected (E) mice. Data are shown as mean ± SEM. Data are pooled from two independent experiments (A, B) or representative of three independent experiments (C–E) with 3-6 mice per group. P values were calculated with one-way ANOVA with Šidak correction for multiple comparisons (A–C) and non-parametric Mann-Whitney test (D, E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Probiotic and their CW enhance Th1 response against S. Typhimurium. (A) Intradermal injection with heat-inactivated S. Typhimurium were performed in one footpad at day 21 of probiotic or CW administration and oral immunization with heat-inactivated S. Typhimurium as was described in Figure 1 . PBS was injected in the contralateral footpad as control. After 24h, animals were challenged with one dose of virulent S. Typhimurium via oral route and a group of mice received PBS (oral) and served as control (uninfected); mice were euthanized 48h from intradermal injection. (B) Footpad swelling measurement of uninfected and S. Typhimurium infected mice. Non-immunized animals (littermate mice) were used as cut-off (dashed line). Data are shown as mean ± SEM from three independent experiments with 3 mice per group. P values were calculated with one-way ANOVA with Šidak correction for multiple comparisons. ***P < 0.001; ****P < 0.0001.

Figure 5

Probiotic and their CW modulate cytokine production. (A–F) Cytokines measured in the supernatant of splenocytes of uninfected and S. Typhimurium infected. Data are shown as mean ± SEM from three independent experiments with 3-4 mice per group. P values were calculated with one-way ANOVA with Šidak correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Cytokine ratios were calculated dividing IFN-γ levels by IL-4 (G), and IL-10 by TNF-α (H) and IL-12p70 (I). P values were calculated by Student’s t test. *P < 0.05.

Figure 6

Probiotic and their CW reveal similar modulatory effect on the immune system during S. Typhimurium infection. Principal component analysis (PCA) plots of uninfected (A) and S. Typhimurium infected (B) mice. Variables analyzed: phagocytosis, sIgA, Salmonella-specific sIgA, IgG, IgG1, IgG2a, footpad swelling, IFN-γ, TNF-α, IL-12p70, IL-6, and IL-10.