Modulation of the immune response by probiotic strains in a mouse model of gluten sensitivity

Probiotic strains play an important role in modulating activities in the gut-associated lymphoid tissue. Elucidation of the mechanisms that mediate probiotic-driven immunomodulation may facilitate their therapeutic application for specific immune-mediated diseases or for prophylaxis. In this study, we explored the effect of different Lactobacillus spp. and Bifidobacterium lactis in transgenic mice expressing the human DQ8 heterodimer, a HLA molecule linked to Celiac Disease (CD). In vitro analysis on immature bone marrow-derived dendritic cells (iBMDCs) showed that all strains up-regulated surface B7-2 (CD86), indicative of DC maturation, however, with different intensity. No strain induced appreciable levels of 1110 or IL-12 in iBMDCs, whereas TNF-alpha expression was essentially elicited by Lactobacillus paracasei and Lactobacillus fermentum. Interestingly, these strains were found also to increase the antigen-specific TNF-alpha secretion in vivo, following co-administration of probiotic bacteria in mice mucosally immunized with the gluten component gliadin. Together these findings highlighted the ability of probiotics to exert strain-specific inductive rather than suppressive effects both on the innate and adaptive immunity in a mouse model of food antigen sensitivity. (C) 2009 Elsevier Ltd. All rights reserved

PPARγ depletion from intestinal epithelium augments the immune response to <i>S.</i> Typhimurium infection.

<p>(A and B) Nuclear extracts from colonic scrapings of mock (Con)- or <i>S.</i> Typhimurium (Sal)-infected PPARγVillinCre+ (Cre+) or littermate control PPARγVillinCre− (Cre−) mice were assessed for NFκB (A) and AP-1 (B) activities by electromobility shift assay (6–8 mice per group). The expression levels of TNF-α (C), IL-6 (D), IL-17 (E), and IL-22 (F) in the colons of mock- or <i>S.</i> Typhimurium-infected PPARγVillinCre+ or PPARγVillinCre− mice were measured by real-time PCR. Error bars = ± standard error of the mean. *p<0.001, **p<0.01.</p

Mechanism of <i>S.</i> Typhimurium-induced intestinal damage during colitis.

<p>(A) Secretion of MMP-9 and MMP-2 in the colons of mock (Con)- or <i>S.</i> Typhimurium (SaI)-infected Lcn2^−/− mice was analyzed by gelatin zymography using gelatin-agarose-purified PBS extracts (6–8 mice per group). (B) Quantitation of gelatinolytic activities from the zymogram in panel A and from another representative zymogram from independent experiments. (C) MMP-9 protein levels in the colons of mice from the respective groups were assessed by immunoblotting purified PBS extracts under non-reducing conditions. Flow-through from the purification was used as a loading control. (D) Quantitation of changes in protein levels from the immunoblot in panel C and from another representative blot from independent experiments. (E) MMP-9 expression levels in the colons of mock- or <i>S.</i> Typhimurium-infected Lcn2^+/+ and Lcn2^−/− mice were measured by real-time PCR. Error bars = ± standard error of the mean. *p<0.0001 vs. Lcn2^−/− control. NS, not significant. (F) <i>S.</i> Typhimurium down-regulates PPARγ in intestinal epithelial cells. The subsequent decrease in PPARγ activity leads to the activation of NFκB and AP-1 and the release of TNF-α and IL-6. Through activation of the acute-phase immune response, TNF-α and IL-6 induce IL-17 and IL-22. NFκB and AP-1 activation either directly or in conjunction with IL-17 and IL-22 induce Lcn2 expression in epithelial cells and its consequent secretion into the intestinal milieu. Extracellular binding of Lcn2 to secreted MMP-9 increases MMP-9 stabilization and activity, resulting in extensive tissue damage during infectious colitis.</p

Colitis severity is more pronounced in PPARγVillinCre⁺ mice than in wild-type mice.

<p>Sections of colon from mock- or S. Typhimurium-infected wild-type (C57BL/6) and PPARγVillinCre⁺ mice were stained with hematoxylin and eosin (6 mice per group). (A and B) Wild-type mice mock-infected. (C and D) Wild-type mice infected with S. Typhimurium. PPARγVillinCre− mice mock-infected (E and F), or infected with S. Typhimurium (G and H). PPARγVillinCre⁺ mice mock-infected (I and J), or infected with S. Typhimurium (K and L). All scale bars are 100 µm. Pathology scoring was performed for neutrophil infiltration (M). (N) Myeloperoxidase (MPO) activity in colonic extracts of mice, measured per mg of total protein. In panels M and N, mock (Con)- or S. Typhimurium (Sal)-infected PPARγVillinCre⁺ (Cre⁺) or littermate control PPARγVillinCre− (Cre−) mice were examined. Error bars  = ±standard error of the mean. **p<0.01.</p

Elevated levels of Lcn2 in the colonic milieu of <i>S.</i> Typhimurium-infected mice stabilize proMMP-9.

<p>(A) The activities of MMP-9 and MMP-2 in gelatin-agarose-purified PBS (secreted) extracts of mock (Con)- or <i>S.</i> Typhimurium (SaI)-infected PPARγVillinCre+ (Cre+) or PPARγVillinCre− (Cre−) mouse colonic tissues were analyzed by gelatin zymography (6–8 mice per group). (B) Quantitation of gelatinolytic activities from the zymogram in panel A and from two other representative zymograms from independent experiments. Expression levels of MMP-9 (C), MMP-2 (D), and TIMP-1 (E) in the colons of the respective groups were measured by real-time PCR. (F) Protein levels of MMP-9 and Lcn2 in the colons of the respective mouse groups were assessed by immunoblotting using purified PBS extracts under non-reducing conditions. Flow-through from gelatin-agarose purification was probed for β-actin as a loading control. (G) Quantitation of changes in protein levels from the immunoblot in panel F and from another representative blot from independent experiments. Error bars = ± standard error of the mean. *p<0.001 vs. PPARγVillinCre- mice infected with <i>S</i>. Typhimurium. NS, not significant.</p

<i>S.</i> Typhimurium down-regulates PPARγ while inducing colitis in C57BL/6 mice.

<p>(A, B, and C) Groups of 8–10-week-old streptomycin-pretreated C57BL/6 mice were mock- (Con) or <i>S.</i> Typhimurium-infected (Sal) and sacrificed after 24 h (10 mice per group). PPARγ expression in colonic scrapings was analyzed by real-time PCR (A) and by immunoblotting (B). (C) Electromobility shift assay of PPARγ activity in the nuclear extracts of colonic scrapings. (D and E) Age-matched, streptomycin-pretreated TLR4^−/− mice were mock- or <i>S.</i> Typhimurium-infected and sacrificed after 24 h (5 mice per group). Colonic expression of PPARγ was analyzed by real-time PCR (D) or immunoblotting (E). (F) Metronidazole-pretreated C57BL/6 mice were mock- or <i>Citrobacter rodentium</i>-infected, sacrificed 6 days after infection, and PPARγ expression in the colon was analyzed by real-time PCR. (G) HT-29 cells were mock- or <i>S.</i> Typhimurium-infected for 6 h, incubated for another 18 h without the pathogen, and PPARγ expression was analyzed by real-time PCR. (H) Macroscopic image of whole cecum after mock or <i>S.</i> Typhimurium infection in C57BL/6 (WT), PPARγVillinCre− (Cre−), or PPARγVillinCre+ (Cre+) mice. (I) Quantitation of colon lengths in the respective mouse groups. Recovery of <i>S.</i> Typhimurium from cecum tissue (J) and spleen (K) 24 h after infection. Error bars depict ± standard error of the mean. *p<0.001, **p<0.05. CFU, colony-forming units; NS, not significant.</p

Lcn2^−/− mice are markedly protected from <i>S.</i> Typhimurium-induced colitis.

<p>(A and B) Groups of 8–10-week-old, streptomycin-pretreated Lcn2^−/− mice were mock (Con)- or <i>S.</i> Typhimurium (Sal)-infected and sacrificed after 24 h (10 mice per group). (A) Macroscopic image of whole cecum 24 h after infection. (B) Quantitation of colon lengths in mock- or <i>S.</i> Typhimurium-infected Lcn2^−/− mice. (C–F) Sections of colon from mock- or <i>S.</i> Typhimurium-infected Lcn2^−/− mice were stained with hematoxylin and eosin (scale bars, 100 µm). Lcn2^−/− mice mock-infected (C and D), or infected with <i>S.</i> Typhimurium (E and F). Pathology scoring was performed for neutrophil infiltration (G). (H) Myeloperoxidase (MPO) activity was determined in the colonic extracts of mice, as measured per mg of total protein. Recovery of <i>S.</i> Typhimurium from cecum tissue (I) and spleen (J), 24 h after infection. Error bars = ± standard error of the mean. **p<0.05. NS, not significant.</p

Colonic Lcn2 expression in PPARγVillinCre+ mice increases after <i>S.</i> Typhimurium challenge.

<p>(A and B) Expression levels of Lcn2 in the colons of mock (Con)- or <i>S.</i> Typhimurium (SaI)-infected PPARγVillinCre+ (Cre+) or PPARγVillinCre− (Cre−) mice (6–8 mice per group) were measured by real-time PCR (A) and by immunoblotting (B). (C) Quantitation of changes in protein expression from the immunoblot in panel B and from another representative blot from independent experiments. (D) Expression levels of Reg3γ in the colons of mock- or <i>S.</i> Typhimurium-infected mice were measured by real-time PCR. (E and F) HT-29 cells were mock- or <i>S.</i> Typhimurium-infected for 6 h, incubated for another 18 h without the pathogen, and Lcn2 expression (E) and secretion (F) were analyzed by real-time PCR and immunoblotting, respectively. (G–I) HT-29 cells were treated with siRNA directed against PPARγ, and the expression of PPARγ (G) and Lcn2 (H) in uninfected cells was analyzed by real-time PCR. The secretion of Lcn2 was analyzed by immunoblotting using concentrated culture supernatant (I). Error bars = ± standard error of the mean. *p<0.005, **p<0.01.</p

Absence of intestinal PPARγ aggravates acute infectious colitis in mice through a lipocalin-2-dependent pathway.

To be able to colonize its host, invading Salmonella enterica serovar Typhimurium must disrupt and severely affect host-microbiome homeostasis. Here we report that S. Typhimurium induces acute infectious colitis by inhibiting peroxisome proliferator-activated receptor gamma (PPARγ) expression in intestinal epithelial cells. Interestingly, this PPARγ down-regulation by S. Typhimurium is independent of TLR-4 signaling but triggers a marked elevation of host innate immune response genes, including that encoding the antimicrobial peptide lipocalin-2 (Lcn2). Accumulation of Lcn2 stabilizes the metalloproteinase MMP-9 via extracellular binding, which further aggravates the colitis. Remarkably, when exposed to S. Typhimurium, Lcn2-null mice exhibited a drastic reduction of the colitis and remained protected even at later stages of infection. Our data suggest a mechanism in which S. Typhimurium hijacks the control of host immune response genes such as those encoding PPARγ and Lcn2 to acquire residence in a host, which by evolution has established a symbiotic relation with its microbiome community to prevent pathogen invasion