Infection Susceptibility in Gastric Intrinsic Factor (Vitamin B12)-Defective Mice Is Subject to Maternal Influences

Abstract

Mice harboring a mutation in the gene encoding gastric intrinsic factor (Gif), a protein essential for the absorption of vitamin B12/cobalamin (Cbl), have potential as a model to explore the role of vitamins in infection. The levels of Cbl in the blood of Gif(tm1a/tm1a) mutant mice were influenced by the maternal genotype, with offspring born to heterozygous (high Cbl, F1) mothers exhibiting a significantly higher serum Cbl level than those born to homozygous (low Cbl, F2) equivalents. Low Cbl levels correlated with susceptibility to an infectious challenge with Salmonella enterica serovar Typhimurium or Citrobacter rodentium, and this susceptibility phenotype was moderated by Cbl administration. Transcriptional and metabolic profiling revealed that Cbl deficient mice exhibited a bioenergetic shift similar to a metabolic phenomenon commonly found in cancerous cells under hypoxic conditions known as the Warburg effect, with this metabolic effect being exacerbated further by infection. Our findings demonstrate a role for Cbl in bacterial infection, with potential general relevance to dietary deficiency and infection susceptibility.

Importance: Malnutrition continues to be a major public health problem in countries with weak infrastructures. In communities with a high prevalence of poor diet, malnourishment and infectious disease can impact vulnerable individuals such as pregnant women and children. Here, we describe a highly flexible murine model for monitoring maternal and environmental influences of vitamin B12 metabolism. We also demonstrate the potential importance of vitamin B12 in controlling susceptibility to bacterial pathogens such as C. rodentium and S Typhimurium. We postulate that this model, along with similarly vitamin deficient mice, could be used to further explore the mechanisms associated with micronutrients and susceptibility to diseases, thereby increasing our understanding of disease in the malnourished.

FIG 1

Gif^tm1a/tm1a mice exhibit signatures of Cbl deficiency. (a) Immunofluorescence analysis of the glandular stomach regions of wild-type (i) and Gif^tm1a/tm1a (ii) mice stained (red) for the presence of Gif with specific antiserum (×400 magnification). (b) Representative photograph of 8-week-old Gif^tm1a/tm1a and wild-type pups from Gif^tm1a/tm1a (left), Gif^tm1a/+(middle), or wild-type (right) dams. (c) Mean body weights of F₁ Gif^tm1a/tm1a (red triangles), F₂ Gif^tm1a/tm1a (purple triangles), and wild-type (blue circles) mice between 3 and 16 weeks of age. (d) Blood plasma Cbl concentrations Gif^tm1a/tm1a and wild-type mice. Dashed lines show the detection limits of the analyzer. Black bars represent geometric mean values. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (ANOVA with Dunn’s multiple-comparison post hoc test).

FIG 2

F₂ Gif^tm1a/tm1a mice are susceptible to C. rodentium and S. Typhimurium pathogen challenges. (a, part i) Kaplan-Meier curve showing percent survival following infection of F₂ Gif^tm1a/tm1a and wild-type mice with C. rodentium (n = 8). (a, part ii) Enumeration of C. rodentium bacteria in cecal, colon, spleen, and liver tissues of surviving F₂ Gif^tm1a/tm1a and wild-type mice at day 14 p.i. (b, part i) Kaplan-Meier curve showing percent survival following infection of F₂ Gif^tm1a/tm1a and wild-type mice with S. Typhimurium (n = 8). (b, part ii) Enumeration of S. Typhimurium bacteria in the spleens and livers of F₂ Gif^tm1a/tm1a and wild-type mice at day 14 p.i. Gray circles represent wild-type mice. Black triangles represent F₂ Gif^tm1a/tm1a mice. Black bars represent geometric mean values. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (ANOVA with Dunn’s multiple-comparison post hoc test).

FIG 3

Representative hematoxylin- and eosin-stained sections from naive and infected F₂ Gif^tm1a/tm1a and wild-type mice. (a) Liver sections from naive wild-type (i) and F₂ Gif^tm1a/tm1a (ii) mice. A single asterisk indicates an abnormal mitotic figure, and double asterisks indicate enlarged cellular nuclei in an F₂ Gif^tm1a/tm1a mouse (×400 magnification). (b) Colon sections obtained on day 14 after a C. rodentium challenge of wild-type (i) and F₂ Gif^tm1a/tm1a (ii) mice. The asterisk indicates a crypt abscess in an F₂ Gif^tm1a/tm1a mouse (×100 magnification). (c) Liver sections obtained on day 8 after an S. Typhimurium challenge of wild-type (i) and F₂ Gif^tm1a/tm1a (ii) mice. Both images show inflammatory cellular infiltration and granuloma formation. Asterisks indicate the large necrotic regions seen in F₂ Gif^tm1a/tm1a mice (× 200 magnification).

FIG 4

Immune cell profiling of naive and S. Typhimurium-challenged F₂ Gif^tm1a/tm1a and wild-type mice. (a) Analysis of peripheral blood leukocytes from 16-week-old naive mice showing percentages of CD3⁺ (i) and CD4⁺ (ii) T cells. (b) Analysis of spleen cells from S. Typhimurium-infected mice on day 14 p.i. for total CD3⁺ (i) and total CD4⁺ (ii) T cells. Samples were analyzed on a BD LSR Fortessa or BD LSR II apparatus. Interpretation of the results was performed with FlowJo v9. Black bars represent geometric mean values. *, P < 0.05 (one-way ANOVA followed by Dunn’s multiple-comparison post hoc test).

FIG 5

Metabotyping of blood serum from F₂ Gif^tm1a/tm1a and wild-type mice reveals metabolic abnormalities. (a) PCA score plot separating naive F₂ Gif^tm1a/tm1a mice (green circles) from naive wild-type mice (blue circles) with 92% of the variables explained (R²_cum) by using the first four components with 72% predictability minus Q2. (b) Multivariate data analysis results comparing the blood serum of uninfected F₂ Gif^tm1a/tm1a (ui_gif; red circles) and wild-type (ui_wt; yellow circles) mice and that of S. Typhimurium-infected F₂ Gif^tm1a/tm1a (si_gif; green circles) and wild-type (si_wt; blue circles) mice. Principal component 1 discriminates on the basis of the presence or absence of infection, while principal component 2 separates different mouse genotypes, regardless of infection status. Ninety percent of the variables (R²_cum) were explained by using the first five components with 73% predictability.

FIG 6

Cbl supplementation of F₂ Gif^tm1a/tm1a mice alters their susceptibility to an S. Typhimurium challenge. Shown is a Kaplan-Meier curve of percent survival of F₂ Gif^tm1a/tm1a mice left untreated or treated with cyanocobalamin and then infected with S. Typhimurium (n = 8).