Role of Nod1 in mucosal dendritic cells during Salmonella pathogenicity island 1-independent Salmonella enterica serovar Typhimurium infection

Abstract

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.

FIG. 1.

Nod1 and Nod2 deficiency does not increase susceptibility of mice to WT S. Typhimurium. (A and B) C57BL6 (WT) (n = 15) and Nod1-deficient (n = 13) mice were infected orally with 10⁵ S. Typhimurium organisms. (A) Survival was monitored daily. (B) At 5 days postinfection, the indicated organs (MLN, spleens, and livers) were harvested and homogenized. Serial dilutions of the homogenate were spread on MacConkey agar plates and CFU counted. Means and standard errors of the means are represented. (C) C57BL6 (WT) (n = 10) and Nod2-deficient (n = 10) mice were infected orally with 10⁵ S. Typhimurium organisms. Survival was monitored for a period of 20 days. (D) C57BL6 (WT) (n = 9) and Nod1- Nod2-deficient (n = 12) mice were infected orally with 10⁵ S. Typhimurium organisms. Survival was monitored twice daily.

FIG. 2.

Nod1- but not Nod2-deficient mice are more susceptible to Spi1-deficient S. Typhimurium. (A) C57BL6 (WT) (n = 20) and Nod1-deficient (n = 21) mice were infected orally with 10⁵ WT S. Typhimurium ΔHilA organisms. Survival was monitored for a period of 20 days. (B) At 5 days postinfection, the indicated organs (MLN and spleens) were harvested and homogenized. Serial dilutions of the homogenate were spread on MacConkey agar plates and CFU counted. Bars indicate means and standard errors of the means. *, P < 0.05. (C) C57BL6 (WT) (n = 5) and Nod1-deficient (n = 5) mice were infected orally with 10⁵ WT S. Typhimurium ΔHilA organisms. At 5 days postinfection, the indicated organs (small intestine, cecum, and colon) were harvested and homogenized. Serial dilutions of the homogenate were spread on MacConkey agar plates and CFU counted. Bars indicates means and standard errors of the means. (D) C57BL6 (WT) (n = 12) and Nod2-deficient (n = 15) mice were infected orally with 10⁵ WT S. Typhimurium ΔHilA organisms. Survival was monitored for a period of 20 days. (E) C57BL6 (WT) (n = 19) and Nod1- Nod2-deficient (n = 22) mice were infected orally with 10⁵ WT S. Typhimurium ΔHilA organisms. Survival was monitored for a period of 20 days.

FIG. 3.

Nod1 in the hematopoietic compartment is important for the response to Spi1-deficient bacteria. WT or Nod1^−/− irradiated mice reconstituted with the indicated bone marrow (WT<-WT, n = 8; WT<-Nod1, n = 7; and Nod1<-WT, n = 8) were infected with 10⁵ S. Typhimurium ΔHilA organisms. Survival was monitored twice daily (A), and at day 5 postinfection spleens were harvested and plated for assessment of bacterial numbers (B). Error bars indicate standard errors of the means.

FIG. 4.

Myeloid populations of WT and Nod1^−/− intestinal lamina propria. Small intestines from WT and Nod1^−/− mice were removed and lamina propria cell suspensions prepared. Cells were stained for CD11b and CD11c. Populations of macrophages and DCs were determined by CD11b and CD11c expression in three subsets (R1, CD11b⁺ CD11c⁻; R2, CD11b⁺ CD11c⁺; R3, CD11c⁺). Small intestines of WT and Nod1^−/− mice infected with S. Typhimurium ΔHilA expressing GFP or treated with PBS were harvested and prepared to obtain single-cell suspensions 1 day after infection. (A) Cell populations were analyzed by FACS. Cells were gated and sorted as indicated above. (B to D) The percentage of GFP harboring cells is indicated in the window for CD11b⁺ CD11c⁻ R1 (B), CD11b⁺ CD11c⁺ R2 (C), and CD11b⁻ CD11c⁺ R3 (D) cells. Plots are from one representative out of three independent experiments. Bars represent the means and standard errors of the means (n = 5).

FIG. 5.

Defect of Nod1-deficient DCs in S. Typhimurium ΔHilA infection. BMDCs were infected at a multiplicity of infection of 1 with SL1344 or the ΔHilA mutant. After 30 min, cells were treated with gentamicin. After another 2 h, cells were lysed with sodium deoxycholate and lysates plated for CFU counts (A). Alternatively, cells infected with the ΔHilA mutant were stained for CD11c, CD11b, and intracellular iNOS. iNOS expression in uninfected (dotted line), WT-infected (solid line), and Nod1^−/− mutant-infected cells is plotted (B). After overnight infection, supernatants were collected and the nitric oxide concentration was determined (C).