Inhibitory effect of enterohepatic Helicobacter hepaticus on innate immune responses of mouse intestinal epithelial cells

Abstract

Enterohepatic Helicobacter species infect the intestinal tracts and biliary trees of various mammals, including mice and humans, and are associated with chronic inflammatory diseases of the intestine, gallstone formation, and malignant transformation. The recent analysis of the whole genome sequence of the mouse enterohepatic species Helicobacter hepaticus allowed us to perform a functional analysis of bacterial factors that may play a role in these diseases. We tested the hypothesis that H. hepaticus suppresses or evades innate immune responses of mouse intestinal epithelial cells, which allows this pathogen to induce or contribute to chronic inflammatory disease. We demonstrated in the present study that the innate immune responses of intestinal epithelial cells to lipopolysaccharide (LPS) via Toll-like receptor 4 (TLR4) and to flagellin-mediated activation via TLR5 are reduced by H. hepaticus infection through soluble bacterial factors. In particular, H. hepaticus lysate and the soluble component LPS antagonized TLR4- and TLR5-mediated immune responses of intestinal epithelial cells. H. hepaticus lysate and LPS inhibited development of endotoxin tolerance to Escherichia coli LPS. Suppression of innate immune responses by H. hepaticus LPS thus may affect intestinal responses to the resident microbial flora, epithelial homeostasis, and intestinal inflammatory conditions.

FIG. 1.

Adherence of H. hepaticus to intestinal epithelial cells. Murine intestinal cell line m-IC_cl2 was coincubated with H. hepaticus at an MOI of 50 for 4 h, after which nonadherent bacteria were removed. Cell membranes labeled with wheat germ agglutinin coupled to Texas Red are red, and bacteria labeled with H. hepaticus-specific antiserum (see Materials and Methods) are green. A representative confocal plane of the specimen is shown as an overlay of green and red fluorescence.

FIG. 2.

Reactivity of murine cell lines with H. hepaticus and different PRR ligands. (A) Cell line m-IC_cl2 was coincubated for 4 h with live cells of H. hepaticus strains 3B1, 94-2423, 96-284, and 96-1809 (MOI, 50 bacteria per cell), with H. hepaticus bacterial lysates (25 μg/ml), and with E. coli LPS (Ec LPS) (control; 50 ng/ml). (B) Cell lines m-IC_cl2, NCTC 1469, and J774 were coincubated with the following substances: E. coli LPS (10 ng/ml for m-IC_cl2 and J774 and 2 ng/ml for NCTC 1469), Pam3Cys-SK₄ (2 ng/ml for NCTC and 10 ng/ml for J774 and m-IC_cl2), S. enterica serovar Typhimurium FliC flagellin (St flagellin) (100 ng/ml for m-IC_cl2 and 200 ng/ml for NCTC 1469 and J774), 2 μM CpG oligonucleotide 1668, GMDP alone (50 μg/ml for m-IC_cl2 and 10 μg/ml for NCTC 1469 and J774) or combined with E. coli LPS (10 ng/ml for m-IC_cl2 and J774 and 2 ng/ml for NCTC 1469), 20 ng/ml PMA, and the cytokines TNF-α and IL-1β (20 ng/ml for m-IC_cl2 and 10 ng/ml for NCTC 1469 and J774). In panels A and B, MIP-2 release into the cell supernatant (see Materials and Methods) is expressed as the fold induction compared to the induction in the mock-infected control. (C) Transcripts of different PRRs and genes involved in TLR signaling in murine cell lines were detected by semiquantitative RT-PCR (see Materials and Methods). Transcripts of m-IC_cl2 cells were determined in the presence and in the absence of serum as indicated at the top. nc, negative control.

FIG. 3.

Induction of an innate immune response by purified H. hepaticus LPS in different mouse cell lines. m-IC_cl2, NCTC 1469, and J774 cells were coincubated with H. hepaticus strain 3B1 LPS (Hh LPS) (0.6 μg/ml) for 6 h in medium either with or without FBS. 3B1 lysate (25 μg/ml) and E. coli LPS (Ec LPS) (10 ng/ml for m-IC_cl2 and J774 and 2 ng/ml for NCTC 1469) were included as controls. The concentration of MIP-2 in the supernatant is expressed as the fold induction compared to the induction in the mock-infected control.

FIG. 4.

Inhibitory effects of H. hepaticus live bacteria, lysates, and LPS on E. coli LPS-induced stimulation of m-IC_cl2 cells. (A) m-IC_cl2 cells were coincubated for 4 h with different amounts of E. coli LPS and antagonized with 5, 25, or 100 μg/ml 3B1 lysate or with live bacteria at an MOI of 25 or 100 bacteria per cell. (B) m-IC_cl2 cells were stimulated with either 10 ng/ml of E. coli LPS (Ec LPS) alone or 10 ng/ml of E. coli LPS combined with 25 μg/ml H. hepaticus 3B1 lysate (Hh lysate), 0.6 μg/ml H. hepaticus 3B1 LPS, or 0.15 μg/ml H. hepaticus 95-225 LPS for 6 h in medium with or without 2% FBS. The concentration of MIP-2 in the supernatant is expressed as the fold induction compared to the induction in the mock-infected control. An asterisk indicates that the P value is <0.01, and a number sign indicates that the P value is <0.05.

FIG. 5.

Inhibition of the development of endotoxin tolerance in m-IC_cl2 cells by H. hepaticus. m-IC_cl2 cells were coincubated for 6 h with the following stimuli alone or in combination: mock infection, E. coli LPS (Ec LPS) (10 ng/ml), H. hepaticus LPS (Hh LPS) (0.6 μg/ml), and H. hepaticus lysate (Hh lysate) (25 μg/ml). Then the supernatants were harvested (first coincubation), and the cells were washed three times and incubated for 16 h in serum-containing medium. After this, the cells were coincubated for another 6 h with 10 ng/ml E. coli LPS, and the supernatants were collected (second coincubation). The bars indicate the mean MIP-2 concentrations in the supernatants after the first and second coincubations for triplicate experiments, and the error bars indicate the standard deviations. An asterisk indicates coincubation conditions that resulted in a significant change (P < 0.01, as determined by an unpaired, one-sided t test) in MIP-2 induction after the second E. coli LPS stimulation compared to cells that were mock infected during the first coincubation.

FIG. 6.

Amounts of transcripts (as determined by real-time PCR) of marker cytokine genes (MIP-2, TNF-α, IL-10, IL-12p40) after coincubation with E. coli LPS alone or in combination with H. hepaticus lysates or LPS in m-IC_cl2 cells. Real-time PCRs were performed (see Materials and Methods) with cDNA of m-IC_cl2 cells coincubated under different conditions for 30 min or 2, 4, 6, or 24 h. Coincubations were performed with 25 μg/ml 3B1 lysate (Hh lysate), 0.75 μg/ml 3B1 LPS (Hh LPS), 50 ng/ml E. coli LPS (Ec LPS), 50 ng/ml E. coli LPS and 25 μg/ml 3B1 lysate, or 50 ng/ml E. coli LPS and 0.75 μg/ml 3B1 LPS. The amounts of transcripts are expressed as percentages relative to the highest induction value in each panel, which was defined as 100%.

FIG. 7.

Inhibition of flagellin-induced innate immune response by H. hepaticus LPS in human Caco-2 cells. Caco-2 cells were coincubated with either 10 ng/ml S. enterica serovar Typhimurium flagellin or 50 ng/ml PMA alone or combined with 0.6 μg/ml 3B1 LPS (Hh LPS) for 6 h. As controls, the cells were also coincubated with 25 μg/ml 3B1 lysate or 0.6 μg/ml 3B1 LPS alone. The level of IL-8 in the supernatant is expressed as the fold induction compared to the induction in the mock-infected control. The asterisk indicates that the P value is <0.01.