Genetic and functional characterization of the mouse Trl3 locus in defense against tuberculosis

Abstract

The genetic control of susceptibility to tuberculosis in DBA/2J and C57BL/6J mice is complex and influenced by at least four tuberculosis resistance loci (Trl1-Trl4). To further study the Trl3 and Trl4 loci, we have created congenic mouse lines D2.B6-Chr7 and D2.B6-Chr19, in which resistant B6-derived portions of chromosome 7 (Chr.7) and chromosome 19 (Chr.19) overlapping Trl3 and Trl4, respectively, were independently introgressed onto susceptible D2 background. Transfer of B6-derived Trl3 chromosome 7 segment significantly increased resistance of D2 mice, as measured by reduced pulmonary microbial replication at day 70, and increased host survival following aerosol infection. However, transfer of B6-derived chromosome 19 (Trl4) onto D2 mice did not increase resistance by itself and does not improve on the protective effect of chromosome 7. Further study of the protective effect of Trl3 in D2.B6-Chr7 mice indicates that it does not involve modulation of timing or magnitude of Th1 response in the lung, as investigated by measuring the number of Ag-specific, IFN-gamma-producing CD4(+) and CD8(+) T cells. Rather, Trl3 appears to affect the intrinsic ability of activated macrophages to restrict intracellular mycobacterial replication in an NO synthase 2-independent fashion. Microarray experiments involving parental and congenic mouse lines identified a number of genes in the Trl3 interval on chromosome 7 the level of expression of which before infection or in response to Mycobacterium tuberculosis infection is differentially regulated in a parental haplotype-dependent fashion. This gene list represents a valuable entry point for the identification and prioritization of positional candidate genes for the Trl3 effect on chromosome 7.

Figure 1. Physical position of the Chr.7 (Trl3) and Chr.19 (Trl4) segments from C57BL/6J strain introgressed onto the genetic background of the DBA/2J strain

The locations of the DBA/2 (D2, empty) and C57BL/6J (B6, solid lines) segments introgressed in D2.B6-Chr7 (A) and D2.B6-Chr19 (B) are shown, as delimited by markers D7Mit178-D7Mit193 for Chr.7 and D19Mit69-D19Mit137 for Chr.19. The schematic representation of the recombinant chromosomes are positioned immediately below the LOD score traces defining the Trl3 and Trl4 loci as previously mapped by whole genome scanning in informative (B6 X D2) F₂ mice (see reference (21)). The positions of informative markers, and the length of chromosomes are given in cM, and are drawn to scale.

Figure 2. Replication of M. tuberculosis in the lungs of B6, D2, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines

Resistant B6 and susceptible D2 mice, as well as D2.B6-Chr7 and D2.B6-Chr19 congenic lines were infected via the aerosol route with 10² M. tuberculosis H37Rv, and the number of M. tuberculosis bacilli were enumerated in the lungs (log₁₀ CFU) 30 days and 70 days post-infection. CFU counts for individual mice are shown with means for each group (horizontal line). Asterisks indicate that differences in CFU counts detected between B6 and D2 (*), D2 and D2.B6-Chr7 (**), B6 and D2.B6-Chr7 (***), and B6 and D2.B6-Chr19 (****) are statistically significant (P<0.05, unpaired Student’s t test).

Figure 3. Survival of B6, D2, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines following aerosol infection with M. tuberculosis

B6, D2, D2.B6-Chr7 and D2.B6-Chr19 mice were infected via the aerosol route with 10² M. tuberculosis H37Rv, and survival to infection was monitored during 220 days.

Figure 4. Kinetics of T cell response in the lungs of M. tuberculosis-infected B6, D2, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines

The different control and congenic lines were infected with 10² M. tuberculosis H37Rv by the aerosol route and the number of IFN-γ–producing CD4⁺ (A) and CD8⁺ (B) T cells were enumerated in the lungs at 10, 20, 30 and 50 days post-infection. In parallel, the appearance of antigen-specific, IFN-γ-secreting T cells in the lungs (total cells) was evaluated by Elispot assay (C), using either an ESAT-6 peptide or crude M. tuberculosis sonicate as antigenic stimuli, and RPMI culture medium as unstimulated control. Results are from pooled lung cells obtained from 4 mice per experimental group. Individual experimental points are representative of measurements performed in triplicate.

Figure 5. Lung histopathology of M. tuberculosis-infected mice at day 70 of infection

B6 and D2 control mice together with the D2.B6-Chr7 and the D2.B6-Chr19 congenic lines were infected by the aerosol route with 10² CFUs of M. tuberculosis, and 70 days later lungs were harvested, and processed for immunocytochemistry as described in Materials and Methods. Briefly, tissue sections were stained for acid-fast bacilli (red), while nitric oxide synthase (NOS2) was detected by immunocytochemistry; sections were counterstained with methylene blue. In lung lesions of B6, D2.B6-Chr7, D2.B6-Chr19 and D2 mice, acid fast bacilli were confined to large foamy macrophages that stained positively for NOS2. Macrophages of D2 and D2.B6-Chr19 mice contained many more acid fast bacilli than macrophages of B6 and D2.B6-Chr7 mice. (A) Low power (50X) of B6 lungs; (B) Low power (50X) of D2.B6-Chr7 lungs; (C) Low power (50X) of D2.B6-Chr19 lungs; (D) Low power (50X) of D2 lungs; (E) High power (667X) of B6 lungs; (F) High power (667X) of D2.B6-Chr7 lungs; (G) High power (667X) of D2.B6-Chr19 lungs; (H) High power (667X) of D2 lungs.

Figure 6. Chromosome 7 genes differentially expressed in the lungs of B6/D2.B6-Chr7 mice compared to D2 at day 30 post-infection

The approximate position of annotated genes on Chr.7 (Mouse Ensembl; www.ensembl.org/Mus_musculus/index.html) that show differential expression in the lungs of B6/D2.B6-Chr7 mice vs. D2, 30 days (ANOVA two-by-two interaction) following aerosol infection with 10² CFUs of M. tuberculosis is shown (A). The position of cytogenetically identifiable chromosomal bands is indicated and drawn to scale. The position of the B6 Chr.7 segment (D7Mit178-D7Mit193) introgressed in D2.B6-Chr7 is shown together with the approximate boundaries of the Trl3 locus. (B) Expression levels of the CD22 and Rps9 genes measured either prior to (D0) or 30 days post-infection (D30) in the lungs of B6, D2, and D2.B6-Chr7 lines. Numerical values correspond to the mean signal intensity (Log₂) obtained from three individual samples per experimental group (with standard error shown).