Functionally Overlapping Variants Control Tuberculosis Susceptibility in Collaborative Cross Mice

Abstract

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (αL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The Itgal^CC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross.IMPORTANCE The variable outcome of Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.

Keywords: Collaborative Cross mice; Mycobacterium tuberculosis; adaptive immunity; host genetics; host response; host-pathogen interactions.

FIG 1

CC042 mice are susceptible to low-dose aerosol M. tuberculosis infection. The numbers of lung CFU (A), the numbers of spleen CFU (B), body weight (C, D), and total IFN-γ levels in lung (E) or spleen (F) homogenates at 14, 21, 28, and 33 days after infection by low-dose aerosol (∼50 to 100 CFU) of M. tuberculosis strain H37Rv are shown. All mice were infected in one batch, and 3 males and 3 females of each strain were used for analysis at each time point. The data in the graphs represent the mean ± SD. One-way analysis of variance with Sidak’s multiple-comparison test was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 2

Changes in lung pathology during M. tuberculosis infection. Lung lobes obtained from B6 and CC042 mice at days 21, 28, and 33 postinfection were stained with hematoxylin and eosin. Images are representative of those from 6 mice per strain per time point. (Insets) Magnified images from day 33 show lymphocytes (yellow arrow), macrophages (red arrow), and neutrophils (green arrow).

FIG 3

TB disease traits in a (CC001 × CC042)F₂ intercross population. At 28 to 31 days postinfection, the following traits were quantified in the parental strains and the F₁ and F₂ offspring: the numbers of spleen CFU (A), IFN-γ levels from lung homogenate (B), and the numbers of lung CFU (C). (D) The Pearson correlation between measured traits. The distribution of each measured phenotype (numbers of lung CFU, numbers of spleen CFU, and IFN-γ levels) is shown on the diagonal. Scatter plots depicting the correlation for each pair of phenotypes are shown below the diagonal. Above the diagonal, the correlation coefficient and significance are shown. ***, P < 0.001. The data shown in all panels are for the following population sizes: for the F₂ population, n = 201 total mice, n = 101 females, and n = 100 males; for the F₁ population, n = 65 total mice, n = 32 females, and n = 33 males; for CC001 parent mice, n = 33 total mice, n = 15 females, and n = 18 males; and for CC042 parent mice, n = 37 total mice, n = 18 females, and n = 19 males. The mice were infected in 4 batches, and values were adjusted for batch differences using coefficients from multiple regressions.

FIG 4

QTL mapping identifies four loci underlying TB susceptibility. (A, B) QTL scans of the numbers of lung CFU, the numbers of spleen CFU, and IFN-γ abundance in the lung identify four tuberculosis immunophenotype (Tip) loci on chromosomes 7, 15, and 16. The dashed lines in panel B represent a 5% false discovery rate for each trait based on permutation analysis. (C to E) The Tip loci on each chromosome are indicated at the marker with the peak LOD. (F to J) Allele effect plots for the indicated Tip loci. One hundred seventy F₂ mice (86 female and 84 male mice) were successfully genotyped and used for QTL mapping.

FIG 5

The susceptibility of CC042 mice correlates with altered numbers of lung leukocytes. The total numbers of the following cells were enumerated in the lungs of B6 and CC042 mice: T cells (lymphocytes > single cells > CD3⁺ CD19⁻ cells) (A), B cells (lymphocytes > single cells > CD3⁻ CD19⁺ cells) (B), neutrophils (single cells > Gr1⁺ CD11b⁺ cells) (C), and monocytes/macrophages (single cells > Gr1⁻ CD11b⁺ cells) (D). All mice were infected in one batch, and 3 males and 3 females of each strain were used for analysis at each time point. The data in the graphs represent the mean ± SD. One-way analysis of variance with Sidak’s multiple-comparison test was used to determine significance. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 6

CC042 mice have a defect in T cell recruitment to the lung and lack CD11a expression. (A, B) Intracellular cytokine staining (ICS) of CD4 (A) and CD8 (B) T cells reveals a defect in the number of cytokine-producing T cells in the lungs of CC042 mice. (C to E) Total number of CD4 and CD8 T cells in the lung (C), mediastinal lymph node (D), and spleen (E). (F to H) Total number of ESAT-6 (CD4) or TB10.4 (CD8) tetramer-positive cells in the lung (F), mediastinal lymph node (G), and spleen (H). Bar plots show the mean + SD. Welch’s t test was used to determine significance. *, P < 0.05; **, P < 0.01; ns, not significant. (I) Histograms of CD4 (left) and CD8 (right) T cells stained for activation and migration markers CD44, CD62L, CD69, and CD11a for the isotype control (top, light gray trace), CC042 mice (middle, teal trace), and B6 mice (bottom, gray trace).

FIG 7

A private mutation in Itgal in CC042 mice explains the Tip2-driven susceptibility. (A) Histograms depicting CD11a staining in CD4 (left) and CD8 (right) T cells from B6 (gray shading), WSB (purple shading), CC011 (light blue shading), and CC042 (teal shading) mice. The isotype control antibody staining is shown on each plot as a dotted gray trace. (B) PCR primers flanking the putative private Itgal mutation were used to amplify cDNA from B6, WSB, and CC042 mouse RNA. PCR products were separated by gel electrophoresis on a 3% agarose gel. The second and third lanes show for B6 band WSB (the parental allele for CC042), respectively, the 197-bp amplicons. The 100-bp size decrease in the CC042 mouse-derived product is consistent with the loss of exon 2 (fourth lane and schematic). Sanger sequencing traces from the CC042 and WSB mouse amplicons are shown. WT, wild type; SA, splice acceptor; SV, splice variant; PTC, premature termination codon. (C to E) TB immunophenotypes were reevaluated in F₂ progeny of the CC001 × CC042 mice that were homozygous for each parental allele at the Itgal locus (probe UNC13811649): numbers of lung CFU (C), numbers of spleen CFU (D), and lung IFN-γ levels (E). Welch’s t test was used to determine significance. *, P < 0.05; ****, P < 0.0001. Box-and-whiskers plots indicate the median and minimum-maximum values.