A Loss-of-Function Mutation in the Integrin Alpha L (Itgal) Gene Contributes to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Collaborative Cross Strain CC042

Abstract

Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8⁺ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4⁺, CD8⁺) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene.

Keywords: QTL mapping; Salmonella; host resistance; immunophenotyping.

FIG 1

CC042 mice display reduced spleen and thymus size relative to body weight. Body (A), spleen (B), and thymus (C) weights are shown for C57BL/6J and CC042 naive mice. Only male mice were used for the calculation of thymus weight, and data were pooled from two experiments. The results in the graphs represent the mean ± SEM. Sidak’s multiple-comparison test (two-way analysis of variance) was used to analyze body (A) and spleen (B) weights, while Welch’s t test was used for thymus weight (C). *, P < 0.05.

FIG 2

Pathological changes in the spleens and livers of infected C57BL/6J and CC042 mice. The images show C57BL/6J and CC042 mouse spleen sections stained with hematoxylin and eosin at day 0 after Salmonella infection (A) and spleen and liver sections stained with hematoxylin and eosin at day 3 after Salmonella infection (B) and are representative of those for 6 C57BL/6J mice and 6 CC042 mice. Foci of necrotic hepatocytes associated with histiocytes and neutrophils were seen in both C57BL/6J and CC042 mice but were smaller and less numerous in the CC042 mouse samples (2 to 4 foci per ×40-magnification field of view in CC042 mice compared to 4 to 5 foci in C57BL/6J mice). Arrows point to inflammatory foci. WP, white pulp; RP, red pulp.

FIG 3

CC042 mice have significantly reduced total splenocyte numbers. Flow cytometry analysis of C57BL/6J and CC042 mouse spleens was performed at day 0 (naive) and day 3 after Salmonella Typhimurium infection. (A and B) Splenic index (A) and total splenocyte count (B) for C57BL/6J and CC042 mice at day 0 and day 3 of infection. (C to F) Total cell counts and total CD69⁺ cells for CD4⁺ T cells (C and D) and CD8⁺ T cells (E and F). (G to J) Percentage of CD4⁺ T cells and CD8⁺ T cells producing IFN-γ (G and H) and TNF-α (I and J) for uninfected splenocytes and Salmonella-infected splenocytes at day 3 postinfection. (K to N) Total cell counts for neutrophils (K), monocytes (L), macrophages (M), and B cells (N). The results in the graphs show the mean ± SEM. Data are representative of those from six experiments in naive mice and three experiments at day 3 of infection. Cell populations were defined as follows: CD4⁺ T cells, TCRβ⁺ CD4⁺; CD8⁺ T cells, TCRβ⁺ CD8α⁺; neutrophils, CD11b⁺ Ly6G⁺ Ly6C^lo; monocytes, CD11b⁺ Ly6G⁻ Ly6C^hi; macrophages, CD11b⁺ Ly6G⁻ Ly6C⁺ F4/80⁺; and B cells, B220⁺ MHC-II⁺. Analysis was conducted using the Benjamini, Krieger, and Yekutieli correction for multiple testing (two-way analysis of variance), where significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

CC042 mice show significantly decreased total thymocyte numbers. (A) Total thymocyte counts for C57BL/6J and CC042 mice. (B to E) Percentage and total numbers of thymocytes by developmental stage analyzed via flow cytometry, where data are for 6 to 7 mice per genotype. The results in the graphs indicate the mean ± SEM for data pooled from two independent experiments. Thymocytes develop through double-negative (DN) stages (stages 1 to 4) to double-positive (DP) stages before splitting to either CD4⁺ (CD4⁺ SP) or CD8⁺ (CD8⁺ SP) T cell populations. Cell populations were gated as follows: DN, CD4⁻ CD8a⁻; DP, CD4⁺ CD8a⁺; CD4⁺, CD4⁺ CD8a⁻; and CD8⁺, CD4⁻ CD8a⁺. DN1, DN2, DN3, and DN4 subpopulations were gated from the DN population as follows: DN1, CD44⁺ CD25⁻; DN2, CD44⁺ CD25⁺; DN3, CD44⁻ CD25⁺; and DN4, CD44⁻ CD25⁻. Multiple t tests using the Holm-Sidak method were used to assess significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

CC042 mice have altered bone marrow-resident hematopoietic progenitor populations. Flow cytometry analysis of hematopoietic progenitors in femoral bone marrow from C57BL/6J and CC042 mice. (A) Schematic diagram of the stages of hematopoietic stem cell differentiation. (B) Total number of bone marrow cells per femur averaged over the total number of cells collected for two femurs. (C) Gating scheme used to analyze Lin⁻ Sca1⁺ cKit⁺ (LSK) cells. (D) Total LSK cells per femur by developmental stage. LSK cells progress through the HSC, MPP1, MPP2, MPP3, and MPP4 subsets. (E) Gating scheme used to analyze Lin⁻ cKit⁺ Sca1⁻ (LKS⁻) and common lymphoid progenitors (CLP). FSC-A, forward scatter area. (F) Total number of LKS⁻ cells per femur grouped by progenitor stage. LKS⁻ cells comprise common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP). (G) Total number of CLPs per femur. The data in the graphs represent the mean ± SEM. Data are representative of those from three independent experiments. The cell populations are defined in Table 2. Significance was determined by Welch’s t test (B and G) and multiple t tests using the Holm-Sidak method (D and F). *, P < 0.05; **, P < 0.01.

FIG 6

Susceptibility of CC042 mice to S. Typhimurium is controlled by two linked loci on chromosome 7. (A and B) Bacterial load in liver (A) and in spleen (B) at day 4 postinfection with S. Typhimurium in C57BL/6NCrl (n = 4), CC042 (n = 5), (C57BL/6NCrl × CC042)F1 (n = 3), and (C57BL/6NCrl × CC042)F2 (n = 196) mice. Bacterial loads in F2 mice spanned the values for the two parental strains. (C) Bacterial loads in the 94 individuals selected for genotyping, showing a strong correlation between the two organs (Pearson’s r = 0.93). (D) Genome-wide QTL mapping of the liver bacterial load identified two statistically significant peaks on chromosome 7. The horizontal dashed lines indicate the 0.05, 0.1, and 0.63 (top to bottom) significance thresholds estimated from 10,000 permutations. (E, H) QTL positions are indicated by vertical lines in liver (E) and in spleen (H). See Table 3 for details on each QTL. (F, G, I, J) The proximal Stsl6 QTL acted semidominantly on liver (F) and on spleen (I) bacterial loads, while the CC042-inherited allele at the distal Stsl7 QTL had a recessive mode of action both for liver (G) and for spleen (J). For both QTLs, the CC042-inherited allele was associated with an increased bacterial load. B, the B6 allele; C, the CC042 allele.

FIG 7

The Itgal mutation in CC042 mice resulted in the skipping of exon 2 and the absence of protein expression. (A) Schematic diagram illustrating the Itgal mutation in CC042 mice. A 15-bp deletion resulted in the loss of the intron 1 splice acceptor site, resulting in the skipping of exon 2 and the formation of a premature stop codon. (B) PCR analysis of Itgal cDNA from C57BL/6J and CC042 mice. Amplification of the region flanking exon 2 produces a 338-bp PCR product in C57BL/6J mice carrying the wild-type Itgal gene (lanes 1 to 3). A 238-bp PCR product was produced in CC042 mice and corresponds to a 100-bp deletion due to the skipping of exon 2 (lanes 4 to 6). (C) Flow cytometry analysis of ITGAL surface expression on CD4⁺ and CD8⁺ T cells. ITGAL gates were constructed using fluorescence-minus-one panels, and significance was calculated using Welch’s t test. ****, P < 0.0001. The results in the graphs represent the mean ± SEM.

FIG 8

The quantitative complementation test confirmed the role of the CC042 Itgal loss-of-function variant in susceptibility to S. Typhimurium. (A) Bacterial load in liver at day 4 postinfection with S. Typhimurium in C57BL/6J (B6/B6) (n = 23), (Itgal^−/− × C57BL/6J)F1 (KO/B6) (n = 17), (C57BL/6J × CC042)F1 (B6/CC042) (n = 31), and (Itgal^−/− × CC042)F1 (KO/CC042) (n = 20) mice determined with data pooled from two independent experiments. On the left are the results for mice which carry one B6 allele and either a B6 or a CC042 allele. On the right are the results for mice which carry one Itgal KO allele and either a B6 allele or a CC042 allele. (Itgal^−/− × CC042)F1 (KO/CC042) mice showed bacterial loads higher by approximately 1 log CFU than the three other groups, indicating the absence of complementation between Itgal KO and CC042 alleles. Differences between groups were assessed by one-way analysis of variance. (B) The interaction between the genetic background (C57BL/6J or CC0042) and Itgal genotype (+/+ or −/−) was assessed by two-way analysis of variance. In an inbred C57BL/6J background, the Itgal genotype had only a mild impact on the susceptibility to S. Typhimurium (x axis, genotype at the Itgal locus; data are for 34, 17, and 23 +/+, +/−, and −/− mice, respectively; P values are from a pairwise Student's t test). NS, not significant.