Trehalose 6,6'-dimycolate and lipid in the pathogenesis of caseating granulomas of tuberculosis in mice

Abstract

Trehalose 6,6'-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas.

Figure 1

Caseating granuloma induced by TDM in a sensitized mouse. Animals were injected intraperitoneally with 100 μg of TDM as a 2% oil-in-water emulsion 30 days after immunization with 10⁴ MTB. A: Section of a lesion 21 days after injection of TDM showing dense lymphoid cell infiltration of mesenteric fat containing a smaller area of foamy and degenerating macrophages suggesting early caseous change (arrow) (H&E). B: Section of a lesion 21 days after injection of TDM showing a larger caseating granuloma composed of epithelioid macrophages surrounding a central core of amorphous necrotic material. Many of the holes are remnants of fat cells (H&E). C: Detail of B showing epithelioid cells and fibroblasts. Many lymphocytes were located outside the layer of epithelioid cells (H&E). D: Detail of A at arrow showing fat cells infiltrated and apparently being degraded by foamy macrophages (H&E). E: Section of a lesion 80 days after injection of TDM showing multinucleated macrophages resembling Langhans giant cells (H&E). Original magnifications: ×20 (A, B); ×400 (C, E); ×500 (D).

Figure 2

Granulomas induced by MTB in peritoneal cavities of sensitized mice. The animal was injected intraperitoneally with 10⁹ MTB 30 days after immunization with 10⁴ MTB intraperitoneally and examined on day 28. A: Lesion containing multiple foci of intense infection and infarcted tissue surrounded by a fibrin-fibrous capsule (H&E). B: Detail of blood vessels immediately outside of lesion showing lymphocyte cuffing, medial, and endothelial damage (H&E). Original magnifications: ×20 (A); ×400 (B).

Figure 3

Caseation granuloma in lung after intraperitoneal injection. The animal was injected intraperitoneally with 10⁹ MTB 30 days after immunization with 10⁴ MTB intraperitoneally and sacrificed on day 7. A: Photomicrograph of a caseating granuloma in the mediastinum invading the lung (H&E). B: Acid fast stain of same lesion showing masses of organisms near arrow on A. These were the only AFB found above the diaphragm (AFB). Original magnifications: ×20 (A); ×30 (B).

Figure 4

Caseating granulomas in nonsensitized mice. The animal was injected intraperitoneally with 10⁹ MTB and examined 14 days later. Arrows mark areas shown on B and D. A: Section of a necrotic lesion in adipose tissue adjacent to the pancreas. A separate smaller lesion is present on the top right (H&E). B: Detail of lesion showing epithelioid cells with very few lymphocytes surrounding necrotic material (open arrow on A) (H&E). C: Masses of MTB contained within the lesion (AFB stain). D: Detail showing MTB in fat cells in periphery of lesion (closed arrow on A) (AFB stain). Original magnifications: ×20 (A, C); ×200 (B); ×400 (D).

Figure 5

Persistent caseating granulomas induced by MTB plus TDM. The animal was injected intraperitoneally with 100 μg of TDM in an oil-in-water emulsion and with 10⁶ MTB intravenously 1 day later. The lesions were examined 80 days later. A: Section of an encapsulated lesion consisting of dense connective tissue, fibroblasts, and macrophages surrounding caseation necrosis with focal calcification (H&E stain). B: Connective tissue stain of lesion like A showing a continuous fibrous capsule associated with containment of organisms (reticulum stain). C: Detail of lesion A showing thin layer of foamy macrophages between the capsule and interior necrotic material. Only a few lymphocytes or epithelioid macrophages were present in the outer rim of the lesion (H&E stain). D: Many more MTB were present in this lesion than in any other part of the body. They were evenly distributed in the necrotic tissue and were less acid fast than those observed in earlier lesions (AFB stain). Original magnifications: ×20 (A); ×100 (B); ×400 (C); ×1000 (D).

Figure 6

Histopathology of slowly progressive pulmonary tuberculosis. A: AFB in the lung of a mouse 2 months after infection with 10⁴ MTB i.p. AFB had been cleared from the liver and spleen by this time and small foci of infection had developed in the lung. A single AFB in a foamy alveolar macrophage (inset) was found in sections of the lungs of five mice (arrow). B: Association of AFB with lipid drops in late-stage slowly progressive murine tuberculosis. Section from lung of a mouse 300 days after intravenous infection with MTB strain H37Rv shows the characteristic accumulation of lipid, especially cholesterol crystals, in a focus of infection. Organisms are located within lipid drops in association with cholesterol crystals demonstrating that the association of MTB with lipid in the lung can be physiological (AFB stain). C: Adipose tissue in normal mouse lung. The lungs of mice typically contain small deposits of adipose tissue near the hilus. Caseating granulomas preferentially formed in such tissue (H&E). Original magnifications: ×1000 (B); ×200 (C).

Figure 7

Distribution of caseating granulomas and exudative reactions in lung. Sections of mouse lung 3 days after injection of 10⁹ MTB into the lung. The animals had been infected intraperitoneally with 10⁴ MTB 4 months earlier. A: Photomicrograph showing solitary caseating granuloma (open arrow) in an area of lung with fat cells (closed arrow) near the hilus at 3 days. The remainder of the lung has an exudative reaction (H&E). B: Detail of caseating granuloma (near open arrow on A) showing epithelioid cells surrounding lipid-rich necrotic material (H&E). C: Detail (near closed arrow on A) showing MTB (AFB) in lipid of fat cells at the periphery of the lesion (AFB). D: MTB in the lung at day 3. The large majority of organisms have been cleared from the lung. The remaining AFB are confined to alveolar macrophages (AFB stain). E: MTB in the lung at 1 day. Many AFB are located in alveolar walls, in alveolar macrophages, and associated with dead cells (AFB stain). Original magnifications: ×20 (A); ×400 (B, C, E); ×1000 (D).

Figure 8

Caseating granuloma in a mouse lung. The animal was treated as described in Figure 7 and examined on day 24. A: Photomicrograph of the same lesion showing a caseating granuloma adjacent to the esophagus and invading the lung (H&E). B: Gross photograph of fixed mouse lungs showing solitary white lesion in a central location where adipose tissue is found. C: Detail of edge lesion (near arrow on A) showing necrotic tissue (bottom) surrounded by foamy macrophages, remnants of fat cells, epithelioid macrophages, and finally lymphoid cells (top) (H&E). Original magnifications: ×20 (A); ×200 (C).

Figure 9

Caseating granuloma induced by TDM with MTB in oil. The animal was injected in the lung with 10⁷ MTB plus 100 μg of TDM in a 2% oil-in-water emulsion 30 days after immunization with 10⁴ MTB intraperitoneally and examined on day 14. The section shows a caseating granuloma around lipid droplets of the oil emulsion (H&E). Original magnification, ×40.

Figure 10

Inflammatory reaction to delipidated MTB. Section of mediastinal tissue 6 days after injection of 10⁹ delipidated MTB into the lung. The acute inflammatory reaction in the lung had primarily subsided leaving a resolving acute inflammatory reaction. No caseating or noncaseating granulomas were observed even when the organisms had been injected into adipose tissue (H&E). Original magnification, ×400.