Early events in Mycobacterium tuberculosis infection in cynomolgus macaques

Abstract

Little is known regarding the early events of infection of humans with Mycobacterium tuberculosis. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose M. tuberculosis; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to M. tuberculosis are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation.

FIG. 1.

M. tuberculosis infection progresses from lymph node to lung granuloma by gross and microscopic histopathology 3, 4, 5, and 6 weeks postinfection in nonhuman primates. Hilar lymph node enlargement and granuloma formation was observed at 3 and 4 weeks postinfection, respectively. Granuloma formation is seen in the lower lung lobes by 5 weeks postinfection. R, right; L, left.

FIG. 2.

Microscopic pathology of granuloma formation in acute M. tuberculosis infection of nonhuman primates in which progression from early unorganized granulomas was observed only in the lymph node. All granulomas identified in the lungs were caseous granulomas. (A) Unorganized aggregates of epithelioid macrophages representing early granuloma formation were observed in the left hilar lymph nodes of a monkey (24702) at 4 weeks postinfection. Magnification, ×10. (B) Caseous granuloma formation observed in the right lower lobe of a monkey (12302) at 4 weeks postinfection. Magnification, ×2. (C) Well-organized caseous granulomas were observed in the hilar lymph nodes of a monkey (21902) by 5 weeks postinfection. Magnification, ×2. (D) Multifocal, coalescing, classic appearing caseous granulomas observed in the right upper lobe of a monkey (21902) at 5 weeks postinfection. Magnification, ×2. (E) Both solid (lower two granulomas) and caseous (upper portion) granulomas are seen within the same monkey lung. Coalescing caseous granulomas in which eosinophilic proteinaceous material are seen centrally with peripherally located epithelioid macrophages and lymphocytes. Magnification, ×2. (F). Higher magnification of the same section seen in panel E demonstrating a classic solid granuloma that is composed predominantly of epithelioid macrophages centrally surrounded by lymphocytes peripherally. Other inflammatory cell types are dispersed throughout the granuloma. No necrosis or caseation is seen (hematoxylin and eosin staining). Magnification, ×10.

FIG. 3.

Phenotypic characterization of T cells from various sites. Cells from PBMCs, BAL fluid, hilar lymph node, and right lower lobe granulomatous lung (RLL gran) were stained for CD3, CD4 (A), and CD8 (B) (top row). Cells were gated on CD4⁺ (A) or CD8⁺ (B) cells and analyzed for expression of activation markers CD69 and CD29 (middle rows of panels A and B) or for chemokine receptors CCR5 and CXCR3 (lower rows of panels A and B). Results for a representative monkey (24302) are shown. Compiled results for all monkeys are presented in Fig. 4.

FIG. 4.

Compartmentalization of the activation markers and chemokine receptors on CD4 and CD8 T cells among PBMCs, hilar lymph node (hilar LN) cells, BAL fluid cells, and lung cells (both granulomatous and nongranulomatous) of eight M. tuberculosis-infected cynomolgus macaques at 3 to 6 weeks postinfection. Cells from monkeys at all time points were analyzed by flow cytometry. Within the lymphocyte gate, cells were gated on CD4 or CD8 cells, then the percentage of single positive cells (CD29, CD69, CXCR3, or CCR5) or double positive cells (CD29⁺ CD69⁺ or CXCR3⁺ CCR5⁺) in the gate were reported. The median of each group is depicted. Patterns of activation markers and chemokine receptors differed in their pattern of expression depending on the location of CD4 and CD8 T cells. There were no differences in the activation marker expression over time. Due to the limited number of lymphocytes obtained in lung samples, lung sections here are from weeks 5 and 6 postinfection.

FIG. 5.

Greater magnitude and broader responses to mycobacterial antigens are seen over time from serial PBMC IFN-γ responses to selected antigens (CFP, 38-1 [CFP-10, Rv3874], 72f [Rv0125 and Rv1196], HTCC-1 [Rv3616c], ESAT6 [Rv3875], and ESAT6 peptides) by ELISPOT assay during acute M. tuberculosis infection. Antigen responses were measured in spot-forming units (SFU) preinfection and every 2 weeks until the time of necropsy (3, 4, 5, and 6 weeks postinfection). y-axis increments are at 0, 25, 50, 1,000, and 2,000 SFU. Responses of at least 25 were considered positive. Five monkeys had positive responses to CFP preinfection, reflecting possible cross-reactivity to other environmental mycobacterial species. In general, responses to only CFP were observed at 3 and 4 weeks. A greater magnitude of responses to a broader spectrum of antigens was seen by 5 and 6 weeks after infection.

FIG. 6.

Broader BAL fluid-, lymph node (LN)-, and lung (with or without granuloma)-specific IFN-γ responses to mycobacterial antigens (CFP, 38-1 [CFP-10, Rv3874], 72f [Rv0125 and Rv1196], HTCC-1 [Rv3616c], ESAT6 [Rv3875], and ESAT6 peptides) seen over time by ELISPOT assay at 3, 4, 5, and 6 weeks after M. tuberculosis infection. Antigen responses were measured in spot-forming units (SFU). y-axis increments are at 0, 25, 50, 1,000, and 2,000 SFU. Responses of at least 25 SFU were considered positive. IFN-γ responses to CFP occurred early in the course of infection. By 5 and 6 weeks after infection, IFN-γ responses were observed with a broader spectrum of antigens. Few responses were observed among lung tissue given the paucity of lymphocytes among tissue samples, with the exception of granulomatous lung tissue that was enriched with lymphocytes.

FIG. 7.

In situ detection of cellular and M. tuberculosis RNAs in a pulmonary granuloma. In situ hybridization was performed on pulmonary tissues from animal 21902, using ³⁵S-UTP-labeled riboprobes specific for CXCL9/Mig (A), IFN-γ (B), mycobacterial 16S rRNA (C), and TNF (D) RNAs. The inset in panel A is a cropped image (same magnification) of ISH with a sense control riboprobe on a subjacent tissue section. Bar, 100 μm.