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. 2002 Nov;70(11):5946-54.
doi: 10.1128/IAI.70.11.5946-5954.2002.

Mycobacterium tuberculosis in chemokine receptor 2-deficient mice: influence of dose on disease progression

Holly M Scott  1 , JoAnne L Flynn
Affiliations

Mycobacterium tuberculosis in chemokine receptor 2-deficient mice: influence of dose on disease progression

Holly M Scott et al. Infect Immun. 2002 Nov.

Abstract

Within a Mycobacterium tuberculosis-induced granuloma, lymphocytes and macrophages work together to control bacterial growth and limit the spread of infection. Chemokines and chemokine receptors are involved in cell migration and are logical candidates for a role in granuloma formation. In the present study we addressed the role of CC chemokine receptor 2 (CCR2) in M. tuberculosis infection. In previous studies (W. Peters et al., Proc. Natl. Acad. Sci. USA 98:7958-7963, 2001), CCR2(-/-) mice were found to be highly susceptible to a moderate or high dose of H37Rv administered intravenously (i.v.). We have expanded those studies to demonstrate that the susceptibility of CCR2(-/-) mice is dose dependent. After low-dose aerosol or i.v. infection of CCR2(-/-) mice with M. tuberculosis, there was a substantial delay in cell migration to the lungs and delayed expression of gamma interferon and inducible nitric oxide synthase. The CCR2(-/-) mice had a severe and prolonged deficiency in the number of macrophages in the lungs and an early increase in the number of neutrophils. Despite these deficiencies in cell migration, the CCR2(-/-) mice did not have increased bacterial loads in the lungs compared to wild-type (C57BL/6) mice and successfully formed granulomas. This finding is in contrast to CCR2(-/-) mice infected with a high dose of M. tuberculosis administered i.v. These results indicate that with low-dose infection, a delay in immune response in the lungs does not necessarily have detrimental long-term effects on the progression of the disease. The fact that CCR2(-/-) mice survive with substantially fewer macrophages in the low-dose models implies that the immune response to low-dose M. tuberculosis infection in mice is more robust than necessary to control the infection. Finally, these data demonstrate that, in cases of infectious disease in knockout models, clear phenotypes may not be evident when one is solely evaluating bacterial numbers and survival. Functional assays may be necessary to reveal roles for components of the multifactorial immune system.

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Figures

FIG. 1.
FIG. 1.
Expression of CCR2 ligands, CCL2, CCL7, and CCL12 increases after M. tuberculosis infection. RPA on mRNA isolated from bone marrow-derived macrophages (A) or C57BL/6 mice (B) reveals enhanced expression of CCL2, CCL7, and CCL12 after M. tuberculosis infection. Representative samples from the lung RPA are shown on the left.
FIG. 2.
FIG. 2.
CCR2 deficient mice control low-dose infection with M. tuberculosis. CCR2−/− mice and wild-type C57BL/6 mice were infected via the aerosol route with ∼50 CFU of M. tuberculosis H37Rv (circles) (A), i.v. with 2 × 105 CFU of M. tuberculosis H37Rv (B), or i.v. with 104 CFU of M. tuberculosis Erdman (triangles) or H37Rv (C). CCR2−/− mice (open symbols) controlled growth of M. tuberculosis similar to wild-type (solid symbols) mice in the two lower-dose models (A and C) but had much higher bacterial numbers in the higher-dose model (B). Both lung and spleen CFU results are shown in panel A as indicated; only lung CFU are shown in panels B and C. Each time point represents three to four mice, and the bars represent the standard error. The aerosol route experiment was performed twice with H37Rv and twice with Erdman strain with similar results. The i.v. route experiments were performed twice.
FIG. 3.
FIG. 3.
Macrophages are present in fewer numbers throughout M. tuberculosis infection in the CCR2-deficient mice. The extent of migration of macrophages (A) and neutrophils (B) to the lung after aerosol infection (50 CFU) with M. tuberculosis H37Rv in CCR2−/− mice (□) and CCR2+/+ mice (▴) is charted. Flow cytometric analysis was performed on single-cell suspensions of the lung. Within the macrophage/granulocyte gate (determined by side-scatter versus forward-scatter parameters), macrophages were defined as CD11b+ Gr1− and neutrophils were defined as CD11b+ Gr1+. Each datum point represents three to four mice, and the bars represent the standard error. ✽, P ≤ 0.05; ✽✽, P ≤ 0.005; ✽✽✽, P ≤ 0.001. The experiment was repeated twice with similar results.
FIG. 4.
FIG. 4.
The migration of T lymphocytes to the lung was delayed after low-dose aerosol M. tuberculosis infection. Flow cytometric analysis with anti-CD4 (A) and anti-CD8 (B) antibodies was performed on single-cell suspensions of the lung at various time points postinfection. Each datum point represents three to four CCR2−/− mice (□) or CCR2+/+ mice (▴), and the bars represent the standard error. ✽, P ≤ 0.05; ✽✽, P ≤ 0.005. The experiment was repeated twice with similar results.
FIG. 5.
FIG. 5.
Granulomas form in the lungs with delayed kinetics in CCR2-deficient mice. Hematoxylin and eosin staining was performed on 5- to 6-μm formalin-fixed lung tissue sections at each time point postinfection. Magnifications: ×40, days 13, 21, and 33; ×100, day 48. Infiltration is delayed in CCR2−/− mice, but granulomas do form; however, these granulomas are histologically different from those found in CCR2+/+ mice.
FIG. 6.
FIG. 6.
Expression of IFN-γ and inducible nitric oxide synthase (NOS2) is delayed in the lungs of CCR2−/− mice infected with a low dose of M. tuberculosis H37Rv via aerosol. RPAs were performed on whole-lung RNA preparations as described in the text. Each datum point represents three to four CCR2−/− mice (□) or CCR2+/+ mice (▴), and the bars represent the standard error. ✽, P ≤ 0.05; ✽✽, P ≤ 0.005; ✽✽✽, P ≤ 0.001. The experiment was repeated once with similar results.
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