Oral supplementation with selected Lactobacillus acidophilus triggers IL-17-dependent innate defense response, activation of innate lymphoid cells type 3 and improves colitis

Abstract

Live biotherapeutic products constitute an emerging therapeutic approach to prevent or treat inflammatory bowel diseases. Lactobacillus acidophilus is a constituent of the human microbiota with probiotic potential, that is illustrated by improvement of intestinal inflammation and antimicrobial activity against several pathogens. In this study, we evaluated the immunomodulatory properties of the L. acidophilus strain BIO5768 at steady state and upon acute inflammation. Supplementation of naïve mice with BIO5768 heightened the transcript level of some IL-17 target genes encoding for protein with microbicidal activity independently of NOD2 signaling. Of these, the BIO5768-induced expression of Angiogenin-4 was blunted in monocolonized mice that are deficient for the receptor of IL-17 (but not for NOD2). Interestingly, priming of bone marrow derived dendritic cells by BIO5768 enhanced their ability to support the secretion of IL-17 by CD4⁺ T cells. Equally of importance, the production of IL-22 by type 3 innate lymphoid cells is concomitantly heightened in response to BIO5768. When administered alone or in combination with Bifidobacterium animalis spp. lactis BIO5764 and Limosilactobacillus reuteri, BIO5768 was able to alleviate at least partially intestinal inflammation induced by Citrobacter rodentium infection. Furthermore, BIO5768 was also able to improve colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). In conclusion, we identify a new potential probiotic strain for the management of inflammatory bowel diseases, and provide some insights into its IL-17-dependent and independent mode of action.

Figure 1

Capacity of the L. acidophilus BIO5768 to induce the expression of antimicrobial genes downstream of IL-17 and IL-22 and to activate bone marrow derived dendritic cells (BMDC) for supporting production of IL-17- and IL-22. Gene expression by qRT-PCR analysis of Reg3g, Reg3b, Defa4, Defb2, Il22 and Il17a (A) in the small intestine or (B) in the proximal colon of BALB/c mice after 5 days of either fresh (F; n = 6) or lyophilized (L; n = 6) BIO5768 supplementation when compared to controls (n = 6). Results are expressed as relative gene expressions (2^−∆∆ct) values, by comparing the PCR cycle thresholds (Ct) for the gene of interest and for the house keeping gene β-actin (bact), compared with control mice. (C) Percentage of IL-17-producing CD4 T cells and of IL-22 producing NCR- ILC3 within mesenteric lymph nodes of mice that were supplemented with BIO5768 (n = 7) when compared to control animals (n = 6), as determined by flow cytometry. (D) Measurement of IL17 production by ELISA in the supernatant collected after 24 h of coculture between untreated or BIO5768-stimulated BMDCs and naïve CD4 T cells that were sorted by magnetic beads. (E) Comparison of MFI (Mean of Fluorescence Intensity) of activation markers MHCII, CD40 and CD86 in untreated or BIO5768-stimulated BMDCs. Representative results of 6 independent in vitro experiments are shown (bars represent mean ± SEM). * p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

The anti-microbial abilities of L. acidophilus BIO5768 in vivo is independent of NOD2 and IL-17 signaling. GF female WT (n = 9) or deficient for NOD2 (Nod2^−/−; n = 5), RIP2 (Rip2^−/−; n = 4), and IL-17 Receptor A (Il17Ra^−/; n = 4) were mono-colonized with strain BIO5768 by a single administration (5 × 10⁸ CFU/ mice) and compared to untreated GF WT mice (n = 9). (A) After 30 days mono-association, gene expression by qRT-PCR analysis of Reg3g, Reg3b, Ang4, Defa4, Defb2, Il10, Il17a, Il17f and Il22 in the distal colon and caecum of monocolonized and GF mice. Results are expressed as means ± SEM by comparing the PCR cycle thresholds (Ct) for the gene of interest and for the house keeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) of mono-colonized animals compared with GF WT mice. (B) Impact of BIO5768 monocolonization on the percentage of IL-22 producing NCR + and NCR− ILC3 and CD4⁺ T cells within colon and caecum, as determined by flow cytometry. Data represent means values of each group from one gnotobiotic experiment ± SEM. * p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Impact of L. acidophilus BIO5768 supplementation in a mouse model of C. rodentium infection. BIO5768 (5 × 10⁸ CFU) was daily administered to C57BL/6J mice 5 days prior to infection and after the infection until the day of sacrifice. Mice were orally inoculated with 1 × 10⁹ CFU C. rodentium DBS 120 K strain and sacrificed at either 4 or 9 days after infection. (A) Effect of BIO5768 on bacterial load of C. rodentium determined by plating over the course of the experiment, fecal samples dilution on Luria Bertani medium containing 50 µg/ml kanamycin and compared with control infected mice. (B) Effect of BIO5768 supplementation on the colon length. (C) Effect of BIO5768 supplementation on the crypt length. (D) qRT-PCR analysis of the expression of pro-inflammatory genes in the proximal colon of mice after 4 days post infection. (E) qRT-PCR analysis of the expression of pro-inflammatory genes in the distal colon of mice after 10 days post infection. Results are expressed as relative gene expressions (2^−∆∆ct) values, by comparing the PCR cycle thresholds (Ct) for the gene of interest and for the house keeping gene β-actin (bact), compared with control mice. Results of a representative experiment out of 2 are expressed as means ± SEM of uninfected control mice (n = 10) and infected mice that are supplemented (n = 5) or not (n = 10) with BI05768. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Capacity of the mixture to limit Citrobacter rodentium-induced colitis in C57BL/6J mice. (A) Experimental design. Mixture of three bacterial strains (5 × 10⁸ CFU/day/mice, all strains were present in equal amount in the mixture) was daily administered by intragastric preventive gavage for 5 days (n = 10 per group). (B) C. rodentium burden, (C) Colon lengths. (D) qRT-PCR analysis of the expression of pro-inflammatory genes in the proximal colon of mice after 4 days post infection when compared to non-infected animals. (E) qRT-PCR analysis of the expression of pro-inflammatory genes in the distal colon of mice after 10 days post infection when compared to non-infected animals. Results from one experiment are expressed as relative gene expressions (2^−∆∆ct) by comparing the PCR cycle thresholds (Ct) for the gene of interest and for the house keeping gene β-actin (bact), compared with control mice. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 5

Protective effect of L. acidophilus BIO5768 supplementation in a mouse model of TNBS-induced colitis. BIO5768 (5 × 10⁸ CFU of each) was administered to BALB/c mice for 5 consecutive days before and 1 day after TNBS induction (n = 10 mice per group). After 48 h, mice were sacrificed and the impact of BIO5768 was analyzed on (A) Body weight loss, (B) Macroscopic grading of inflammation according to Wallace score, (C) Histological analyses of colon tissue according to Ameho score. (D) Representative histological sections (E) Colon lengths. (F) Gene expression of pro-inflammatory markers as determined by qRT-PCR analysis. Results are expressed as relative gene expressions (2^−∆∆ct) values, by comparing the PCR cycle thresholds (Ct) for the gene of interest and for the house keeping gene β-actin (bact), compared with control mice. Results correspond to means ± SEM of 10 mice per group of a representative experiment from 2 experiments. *p < 0.05.