A loss-of-function mutation in Itgal contributes to the high susceptibility of Collaborative Cross strain CC042 to Salmonella infections

Salmonella are intracellular bacteria that are found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. They are one of the leading causes of foodborne infections and a major threat for human populations worldwide. Mouse models have been extensively used to model distinct aspects of the human Salmonella infections in vivo 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 Typhimurium as a model of human systemic invasive infection. In this model, strain CC042 displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weight and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid populations. Uninfected mice also had lower thymus weight with reduced total number of thymocytes and double negative and (CD4+, CD8+) double positive thymocytes. Analysis of bone marrow resident hematopoietic progenitors showed a strong bias against lymphoid primed multipotent progenitors, which are the precursors of T, B and NK cells. An F2 cross between CC042 and C57BL/6N identified two significant QTLs on chromosome 7 (Stsl6 and Stsl7) with WSB-derived susceptible alleles. A private variant in the integrin alpha L (Itgal) gene is carried by CC042 in the Stsl7 QTL region. A quantitative complementation test confirmed the impact of Itgal loss of function in a (C57BL/6JxCC042)F1 background, but not in a C57BL/6J 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. Author summary Salmonella are one of the leading causes of foodborne infections and a major threat for human populations worldwide. Not all humans are equally susceptible to Salmonella infection. Some individuals will develop minor symptoms and recover while others develop severe illness and might die. Mouse models are used to study distinct aspects of human Salmonella infection in vivo. We used a new genetically diverse mouse population to investigate host susceptibility differences to Salmonella infection. We identified one mouse strain with an extreme susceptibility to infection characterized by very high bacterial loads and mortality. Mice of this strain had small thymus and spleen, two organs which are very important for producing a fully mature immune system. We showed that the strain’s immune response is impaired and that its extreme susceptibility to Salmonella infection is due to multiple genes defects. We identified a loss-of-function mutation in the Itgal gene (Integrin Subunit Alpha L) that plays a central role in the immune response to infection. This gene explains part of the susceptibility and other gene(s) involved remain to be identified. Our results emphasize how new genetically diverse animal models can lead to the identification of new host genetic variants controlling susceptibility to pathogens and improve our understanding of human infections.


Introduction
Hematology data for C57BL/6J and CC042 naïve mice aged 14-16 weeks. Values indicate the mean ± SEM.  149 To identify if the reduced splenic size (Fig. 3a) observed in CC042 mice may be due to CD8 + T cells were also significantly reduced in CC042 mice compared to C57BL/6J (Fig. 3e   159 and 3f). Further CD4 + and CD8 + T cells effector function was assessed by measuring 160 intracellular staining of IFNγ and TNFα after activation with anti-CD3 and anti-CD28. We 161 observed diminished IFNγ and TNFα production from CC042 T cells both in naïve mice and 162 after infection (Fig. 3g-3j). Reduced numbers of neutrophils, monocytes, macrophages and B 163 cells were also observed in CC042 prior to infection (Fig. 3k-3n). Overall, CC042 mice present  170 To assess if the reduction in CD8 + T cells and total activated T cells in the spleen of 171 CC042 mice may potentially be due to defective T cell maturation, flow cytometry of the thymus 172 was carried out (gating scheme shown in Supplementary Fig. S1b). In agreement with the 173 previous observation that CC042 mice have reduced thymus size, CC042 mice displayed a two-174 fold reduction in total thymocyte counts (Fig. 4a). While the percentages of DP, SP CD4 + and 175 CD8 + T cells were comparable between CC042 and C57BL/6J mice due to the reduction in 176 thymocyte numbers in CC042 mice, a significant decrease in DN, DP, SP CD4 + and CD8 + T 177 cells cell counts was observed in CC042 thymi ( Fig. 4b and 4c). Further examination of the DN 178 subset revealed significant alterations in CC042 mice ( Fig. 4d and 4e). While no significant 179 difference in the proportion of DN1 cells was observed, a significant reduction in the proportion 180 of DN2 stage thymocytes was present in CC042 thymi compared to C57BL/6J controls.  To characterize the impact of infection on spleen and liver, histopathological 216 examination of CC042 spleens and livers was conducted three days after Salmonella infection.

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The livers of C57BL/6J harbored typical lesions for this strain characterized by multifocal necrotic lesions with aggregation of histiocytes and neutrophils typical of granulomas (Fig. 2b). 219 The necrotic hepatocyte foci were found to be smaller and less abundant in CC042 mice (2-4 foci 220 per 40X field of view) than in C57BL/6J mice (4-5 foci per field of view) (Fig. 2b). CC042 221 spleens were also observed to have reduced neutrophil infiltration during infection compared to 222 C57BL/6J spleens (Fig. 2b). The increased megakaryocyte numbers and erythropoiesis 223 observed in the spleen of uninfected CC042 mice (Fig. 2a) was also present in the spleen of 224 CC042 infected mice (Fig. 2b).   Table S1). Ninety-four F2 mice with the highest or lowest liver bacterial loads were selected for 240 QTL mapping (see raw data in Supplementary Table S2). Since bacterial loads in liver and spleen were strongly correlated in the 94 selected individuals (Fig. 6c), QTL mapping was 242 performed on liver bacterial loads.

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QTL mapping identified only two significant QTLs (at 0.05 genome wide significance 244 level), both on Chromosome 7 ( Fig. 6d and 6e), which were named Salmonella Typhimurium 245 susceptibility locus-6 (Stsl6) and Stsl7. Details for each QTL are given in  (Fig. 6f). The CC042 248 allele at Stsl7 (peak at 123.78Mb) was recessive and homozygotes had 10 times higher liver 249 bacterial loads (Fig. 6g).  resulting in the disruption of the intron 1 splice acceptor site (Fig. 7a). We hypothesized that the  the Itgal KO mutation did not impact liver bacterial load in a B6 inbred background (Fig. 8b).

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The CC is a murine genetic reference population that was developed to reflect the genetic  further upstream as bone marrow hematopoiesis was also defective in CC042 mice.

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Hematopoietic progenitor populations have also been observed to be altered in  The bone marrow containing media was transferred to a 50 mL tube and 2 mL of red blood cell 458 lysis buffer (Sigma Aldrich) was added. After 1 minute of gentle mixing, 15 mL of PBS was 459 added and the tubes were centrifuged for 7 minutes at 1400 RPM and the supernatant discarded.

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The resulting cell pellet was resuspended in 2 mL of PBS.

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The following monoclonal antibodies were used for flow cytometric analysis of spleen,     1-3). A 238 bp PCR product was 866 produced in CC042 mice corresponding to a 100 bp deletion due to exon 2 skipping (lanes 4-6).