Salmonella enterica causes more severe inflammatory disease in C57/BL6 Nramp1G169 mice than Sv129S6 mice

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) causes systemic inflammatory disease in mice by colonizing cells of the mononuclear leukocyte lineage. Mouse strains resistant to S. Typhimurium, including Sv129S6, have an intact Nramp1 (Slc11a1) allele and survive acute infection, whereas C57/BL6 mice, homozygous for a mutant Nramp1 allele, Nramp1(G169D) , develop lethal infections. Restoration of Nramp1 (C57/BL6 Nramp1(G169) ) reestablishes resistance to S. Typhimurium; mice survive at least 3 to 4 weeks postinfection. Since many transgenic mouse strains are on a C57/BL6 genetic background, C57/BL6 Nramp1(G169) mice provide a model to examine host genetic determinants of resistance to infection. To further evaluate host immune response to S. Typhimurium, we performed comparative analyses of Sv129S6 and C57/BL6 Nramp1(G169) mice 3 weeks following oral S. Typhimurium infection. C57/BL6 Nramp1(G169) mice developed more severe inflammatory disease with splenic bacterial counts 1000-fold higher than Sv129S6 mice and relatively greater splenomegaly and blood neutrophil and monocyte counts. Infected C57/BL6 Nramp1(G169) mice developed higher proinflammatory serum cytokine and chemokine responses (interferon-γ, tumor necrosis factor-α, interleukin [IL]-1β, and IL-2 and monocyte chemotactic protein-1 and chemokine [C-X-C motif] ligand 1, respectively) and marked decreases in anti-inflammatory serum cytokine concentrations (IL-10, IL-4) compared with Sv129S6 mice postinfection. Splenic dendritic cells and macrophages in infected compared with control mice increased to a greater extent in C57/BL6 Nramp1(G169) mice than in Sv129S6 mice. Overall, data show that despite the Nramp1 gene present in both strains, C57/BL6 Nramp1(G169) mice develop more severe, Th1-skewed, acute inflammatory responses to S. Typhimurium infection compared with Sv129S6 mice. Both strains are suitable model systems for studying inflammation in the context of adaptive immunity.

Keywords: Nramp1; Salmonella enterica; anemia; dendritic cells; inflammation; macrophages; mouse; typhoid fever.

Figure 1

Comparison of spleens from C57/BL6 Nramp1^G169 and Sv129S6 mice at 3 weeks postinfection with Salmonella enterica serovar Typhimurium (S. Typhimurium). (a) C57/BL6 Nramp1^G169 mice have greater splenic bacterial loads (colony-forming units, CFU) of S. Typhimurium per gram of spleen compared with Sv129S6 mice. No bacteria were recovered from control spleens. (b) Infected C57/BL6 Nramp1^G169 mice have more pronounced splenomegaly, based on spleen weight as a percentage of body weight, and (c) decreased total non–heme iron per gram of spleen occurs in both mouse strains post-infection. Data shown are from 5 controls (both strains), 10 S. Typhimurium–infected Sv129S6 (Sv129) mice (white bars), and 9 C57/BL6 Nramp1^G169 (B6-N1R) (black bars). C, control; Inf, infected.

Figure 2

C57/BL6 Nramp1^G169 mice develop greater peripheral blood neutrophilia, monocytosis, and lymphopenia 3 weeks following infection with Salmonella enterica serovar Typhimurium (S. Typhimurium) and more severe myeloid hyperplasia compared with Sv129S6 mice. Mean myeloid to erythroid (M:E) ratios for Sv129S6 and C57/BL6 Nramp1^G169 are 2.8:1 and 7:1, respectively, compared with control mice at 1.6:1. Anemia is more severe in C57/BL6 Nramp1^G169 mice 3 weeks postinfection. Data shown are from 5 controls (both strains), 10 S. Typhimurium–infected Sv129S6 (Sv129) mice (white bars), and 9 C57/BL6 Nramp1^G169 (B6-N1R) (black bars). C, control; Inf, infected; MCV, mean cell volume.

Figure 3

Erythrocyte microcytosis and poikilocytosis, including schistocytes, keratocytes (fragmentation; arrows) and echinocytes (arrowheads), are present in peripheral blood of anemic C57/BL6 Nramp1^G169 mice. (a) Control mouse and (b) infected anemic C57/BL6 Nramp1^G169 mouse 3 weeks following infection with Salmonella enterica serovar Typhimurium. Wright-Giemsa.

Figure 4–7

Hematopathology 3 weeks after oral Salmonella enterica serovar Typhimurium (S. Typhimurium) infection in C57/BL6 Nramp1^G169 and Sv129S6 mice. Figures 4 and 5. Bone marrow myeloid hyperplasia is more marked as indicated by increased myeloid to erythroid (M:E) ratios in C57/BL6 Nramp1^G169 mice compared with Sv129S6 mice. Figure 4. Bone marrow cytology of (a) Sv129S6 control, M:E ratio 1.5:1; (b) Sv129S6 infected, M:E ratio 3.0:1; (c) C57/BL6 Nramp1^G169 control, M:E ratio 1.8:1; and (d) C57/BL6 Nramp1^G169 infected, M:E ratio 7:1; white arrows indicate erythroid precursor cells. Wright-Giemsa. Figure 5. Bone marrow histology of representative mid-femoral sections demonstrating increased bone marrow cellularity (reduced adipose) and myeloid hyperplasia (arrows) in infected mice (b, d) compared with controls (a, c). Hematoxylin and eosin (HE). Figure 6. Disruption of normal splenic architecture with loss of splenic lymphoid follicles and increased extramedullary hematopoiesis (EMH) (arrows) following infection (b, d) compared to controls (a, c). Megakaryocytes and erythroid precursors are shown (insets) within areas of expanded EMH in infected mice (b, d). HE. Figure 7. Markedly decreased iron staining (blue) of splenic reticuloendothelial cells in Salmonella-infected Sv129S6 and C57/BL6 Nramp1^G169 mice (b, d) compared to controls (a, c). Prussian blue. For all rows: (a) Sv129S6 control, (b) Sv129S6 infected, (c) C57/BL6 Nramp1^G169 control, and (d) C57/BL6 Nramp1^G169 infected.

Figure 8

Density plots demonstrating a flow cytometric gating procedure for individual animal splenic leukocyte populations. Data shown are from a representative Salmonella enterica serovar Typhimurium–infected C57BL6 Nramp1^G169 mouse, including splenic neutrophils (CD68−, Gr1^high), inflammatory monocytes (CD68+, Gr1^int), macrophages (CD68+, CD11c−, Gr1^low/−), dendritic cells (CD11c+), and plasmacytoid dendritic cells (CD11c+, Gr1+). Isotype controls were rat IgG2a-RPE (CD68), Armenian Hamster IgG (CD11c), and rat IgG2b-K-PE-Cy7 (Gr1).

Figure 9

Splenic macrophage and dendritic cell populations are significantly greater in C57BL6 Nramp1^G169 than in Sv129S6 mice at 3 weeks postinfection with Salmonella enterica serovar Typhimurium. Mean and SEM of splenic leukocyte cell numbers from control (C; n = 3 for each strain) and infected (Inf) mice. Sv129S6 (Sv129) mice (white bars; n =4 infected mice). C57BL6 Nramp1^G169 (B6-N1R) mice (black bars; n =5 infected mice).

Figure 10

C57/BL6 Nramp1^G169 mice have significantly greater increases in proinflammatory serum cytokine concentrations and decreases in anti-inflammatory serum cytokine concentrations than Sv129S6 mice 3 weeks following Salmonella enterica serovar Typhimurium infection. Concentration of serum cytokines as indicated above each graph. Data shown are from 5 controls (both strains), 10 S. Typhimurium–infected Sv129S6 (Sv129) mice (white bars), and 9 C57/BL6 Nramp1^G169 (B6-N1R) (black bars). C, control; CXCL-1, chemokine (C-X-C motif) ligand 1; IFN-γ, interferon-γ; IL, interleukin; Inf, infected; MCP-1, monocyte chemotactic protein–1; TNF-α, tumor necrosis factor–α.