Nod1 and Nod2 regulation of inflammation in the Salmonella colitis model

Abstract

The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS.

FIG. 1.

DKO mice have reduced inflammation following SL1344 infection. Streptomycin-treated WT or DKO mice were either left uninfected or infected with 5 × 10⁷ CFU of SL1344 for 24 to 72 h prior to sacrifice, and then their ceca were examined for histological changes. A pathological score was assigned to each sample, and the average score for each analyzed feature (edema, neutrophil recruitment, goblet cell depletion, and epithelial erosion) for all mice from each group was calculated (A) as well as the average total sum of the pathological scores (B). The line graphs show the average numbers of neutrophils (C) and goblet cells (D) observed per microscopic field from infected samples over the period of infection. Error bars represent 1 standard error of the mean. Significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 2.

Reduced cytokine production in cecal tissue from DKO mice. ELISAs were used to measure the level of KC and IL-1β in samples from streptomycin-treated WT and DKO mice infected with 5 × 10⁷ CFU of SL1344 for 24 to 72 h. Line graphs depict the average serum levels of KC (A) and the average cecal tissue levels of KC (B) and IL-1β (C) in infected samples over time. Error bars represent 1 standard error of the mean. Significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 3.

Increased colonization of cecal tissue from DKO mice at 72 h postinfection. Streptomycin-treated WT and DKO mice were infected with 5 × 10⁷ CFU of SL1344 for 24 to 72 h, and then CFU counts were performed on spleen (A), pellet (B), and cecal tissue (C) samples. In addition, the colonization of the cecum of streptomycin-treated Rip2-deficient mice infected with SL1344 for 72 h was determined (D). Hematoxylin- and eosin-stained cecum samples from 72-h-infected WT (top panels) and DKO (bottom panels) mice were also stained with Alcian Blue to visualize mucus (E). Arrowheads indicate mucinous material in the cecal lumen that is stained with Alcian Blue. The left panels show 2.5× magnification, while the right panels show 20× magnifications of the inset black rectangles. All graphs show the median CFU levels, and error bars depict the interquartile ranges. Significance: *, P < 0.05; **, P < 0.01.

FIG. 4.

Nod1/2 from both hematopoietic and nonhematopoietic compartments contribute to Salmonella colitis. Lethally irradiated WT and DKO mice were reconstituted with WT or DKO bone marrow, thus generating chimeric mice with DKO nonhematopoietic cells with WT hematopoietic cells (WT→DKO), mice with WT nonhematopoietic cells with DKO hematopoietic cells (DKO→WT), or control mice with both compartments derived from either WT (WT→WT) or DKO (DKO→DKO) mice. These chimeric mice were streptomycin treated, infected with 5 × 10⁷ CFU of SL1344 for 72 h, and then sacrificed, and their ceca were analyzed for pathological changes and cytokine production. A pathological score was assigned to each sample, and the average score for each analyzed feature (edema, neutrophil recruitment, goblet cell depletion, and epithelial erosion) for all mice from each group was calculated (A) as well as the average total sum of the pathological scores (B). ELISAs were also used to measure and calculate the average levels of KC (C) and IL-1β (D) in the cecum samples. Error bars represent 1 standard error of the mean. Significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 5.

Infection using SL1344 grown under SPI-1- or SPI-2-inducing growth conditions influences the role of Rip2 in Salmonella colitis. Streptomycin-treated Rip2-deficient mice or their WT littermates were infected with 5 × 10⁷ CFU of SL1344 grown under either SPI-1- or SPI-2-inducing conditions. After 72 h of infection, the mice were sacrificed and their ceca were analyzed for pathological changes and cytokine production. A pathological score was assigned to each sample, and the average score for each analyzed feature (edema, neutrophil recruitment, goblet cell depletion, and epithelial erosion) for all mice from each group was calculated (A) as well as the average total sum of the pathological scores (B) (error bars represent 1 standard error of the mean). ELISAs were used to measure KC (C) and IL-1β (D) in the cecum samples; the circles in the scatter plots show the data from individual mice, and the horizontal bars represent the means. Significance: *, P < 0.05; **, P < 0.01.