Mycobacterial trehalose 6,6'-dimycolate induced vascular occlusion is accompanied by subendothelial inflammation

Abstract

Mycobacterium tuberculosis (MTB) is a pathogen that infects and kills millions yearly. The mycobacterium's cell wall glycolipid trehalose 6,6'-dimycolate (TDM) has been used historically to model MTB induced inflammation and granuloma formation. Alterations to the model can significantly influence the induced pathology. One such method incorporates intraperitoneal pre-exposure, after which the intravenous injection of TDM generates pathological damage effectively mimicking the hypercoagulation, thrombus formation, and tissue remodeling apparent in lungs of infected individuals. The purpose of these experiments is to examine the histological inflammation involved in the TDM mouse model that induces development of the hemorrhagic response. TDM induced lungs of C57BL/6 mice to undergo granulomatous inflammation. Further histological examination of the peak response demonstrated tissue remodeling consistent with hypercoagulation. The observed vascular occlusion indicates that obstruction likely occurs due to subendothelial localized activity leading to restriction of blood vessel lumens. Trichrome staining revealed that associated damage in the hypercoagulation model is consistent with intra endothelial cell accumulation of innate cells, bordered by collagen deposition in the underlying parenchyma. Overall, the hypercoagulation model represents a comparative pathological instrument for understanding mechanisms underlying development of hemorrhage and vascular occlusion seen during MTB infection.

Keywords: Cord Factor; Granuloma; Immunopathology; TDM; Trehalose 6,6′-dimycolate; Tuberculosis.

Figure 1.. Comparative gross pulmonary reactivity in models of TDM induced pathology.

The lung weight index (LWI) was calculated post TDM administration, as a measure of gross pulmonary inflammation and cellular recruitment. Lungs from mice injected with TDM-IP (10 μg or 50 μg given IP), TDM-IV Acute (10 μg or 25 μg given IV), or TDM-IPIV Hypercoaculation (10 μg IP plus 10 μg IV) were assessed. Lungs were also isolated for comparison to naïve (non-injected) mice. At day 7 post final TDM administration, lungs were collected and weighed, and compared against weight of the mouse. Individual points represent unique animals. Results represent mean ± standard deviation, with similar data obtained from repeated experiments; 4–6 mice per group per experiment.

Figure 2.. Histological Appearance of Pulmonary Granulomatous Inflammation due to TDM.

TDM was administered as described in the methods, and mice were sacrificed at day 7 post-injection. Histologic examination of lungs revealed acute granulomatous response, increasing with dose of TDM administered, culminating in marked monocytic infiltration and increased cellular aggregation. In contrast, TDM given IP followed 7 days later by IV administration led to marked hemorrhagic response and vascular occlusion. Formalin fixed lung sections were hematoxylin and eosin stained (H&E); representative sections; 20x magnification. Representative sections from repeated experiments; 4–6 mice per group per experiment.

Figure 3.. Inflammation Accompanied with Vascular Occlusion in the Hypercoagulation Model Post TDM Administration.

Acute IV administration reveals accumulation of monocytes and lymphocytes surrounding vascular beds, but limited inflammation in subendothelial regions. In contrast, the hypercoagulation model led to aggressive inflammation throughout the parenchmya, with alterations to subendothelial structure lining vasculature. Formalin fixed lung sections; H&E stain; representative sections, 100x magnification. Representative sections from repeated experiments; 4–6 mice per group per experiment.

Figure 4.. Vascular Remodeling and Collagen Deposition during TDM-Induced Hypercoagulation.

Formalin fixed lung sections were Trichrome stained to assess deposition of collagen material post treatment. Acute inflammation demonstrates no collagen deposition associated with granulomatous response, whereas the hypercoagulation inflammation localized to subendothelial regions shows high matrix formation. Trichrome stain; collagen (blue); representative sections, 100x magnification. Representative sections; 4–6 mice per group per experiment.

Figure 5.. Localization of Inflammation Relative to Vascular Endothelium.

Formalin fixed lung sections were stained with antibody to Vascular Endothelial Cadherin, CD144, (1:200), and visualized using standard HRP techniques. Acute inflammation demonstrates reactivity peripheral to vascular regions, whereas the hypercoagulation inflammation is localized to subendothelial regions, accompanied by relative destruction in continuity of endothelial lined blood vessels. Representative sections, 100x magnification. Representative sections; 4–6 mice per group per experiment.