Limited activity of clofazimine as a single drug in a mouse model of tuberculosis exhibiting caseous necrotic granulomas

Abstract

New drugs and drugs with a novel mechanism of action are desperately needed to shorten the duration of tuberculosis treatment, to prevent the emergence of drug resistance, and to treat multiple-drug-resistant strains of Mycobacterium tuberculosis. Recently, there has been renewed interest in clofazimine (CFZ). In this study, we utilized the C3HeB/FeJ mouse model, possessing highly organized, hypoxic pulmonary granulomas with caseous necrosis, to evaluate CFZ monotherapy in comparison to results with BALB/c mice, which form only multifocal, coalescing cellular aggregates devoid of caseous necrosis. While CFZ treatment was highly effective in BALB/c mice, its activity was attenuated in the lungs of C3HeB/FeJ mice. This lack of efficacy was directly related to the pathological progression of disease in these mice, since administration of CFZ prior to the formation of hypoxic, necrotic granulomas reconstituted bactericidal activity in this mouse strain. These results support the continued use of mouse models of tuberculosis infection which exhibit a granulomatous response in the lungs that more closely resembles the pathology found in human disease.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

CFZ was highly effective at reducing bacterial CFU in BALB/c mice (A) in the lungs (filled symbols) and in the spleen (open symbols). In contrast, CFZ activity was significantly attenuated in the lungs of C3HeB/FeJ mice (B), while comparable activity was observed in the spleen. Data represent mean log10 CFU counts ± SEM; detection limit = 50 CFU. *, P < 0.001.

FIG 2

Pathological progression of disease in BALB/c and C3HeB/FeJ mice. Cellular aggregates in the lungs of BALB/c mice at 3 weeks postinfection (A) (magnification, ×100) were composed predominantly of macrophage cells with distinct regions of lymphocytic perivascular and peribronchiolar cuffing (arrows). C3HeB/FeJ mice at 3 weeks postinfection (B) (×100) exhibited cellular lesions composed predominantly of neutrophilic clusters (arrows) and epithelioid macrophages, with evidence of an early fibrotic response. By 7 weeks (C) (×40), the cellular aggregates in BALB/c mice formed loosely organized inflammatory granulomas lacking a well-defined collagen rim. In contrast, by 7 weeks (D) (×40), highly organized granulomas had formed in the lungs of C3HeB/FeJ mice possessing a hypoxic neutrophilic central caseous necrotic core region (CN) and a layer of foamy macrophages (FM) delineated by a collagen rim (arrows) that encapsulated the granuloma structure. By 7 weeks postinfection, no caseating necrotic granulomas were observed in the spleens of BALB/c (E) (×40) or C3HeB/FeJ (F) (×40) mice.

FIG 3

Attenuation of CFZ activity was related to pulmonary pathology. Administration of CFZ to C3HeB/FeJ mice prior to the formation of well-defined pulmonary granulomas (A) (3 weeks postinfection) reconstituted bactericidal activity in the lungs (filled symbols). Initiation of CFZ treatment after the formation of well-defined pulmonary granulomas with significant caseous necrosis (B) (7 weeks postinfection) resulted in significant attenuation of bactericidal activity in C3HeB/FeJ mice. CFZ exhibited comparable activity in the spleens (open symbols) of C3HeB/FeJ mice, which lack well-defined granulomas and caseous necrosis. Data represent mean log10 CFU counts ± SEM; detection limit = 50 CFU. *, P < 0.001; †, P < 0.05.

FIG 4

CFZ was effective in immunocompromised GKO mice. Gamma interferon gene-disrupted mice were treated for 9 days with 20 mg/kg CFZ beginning on day 13 postinfection. CFZ reduced bacterial loads in the lungs (A) by 3.1 log10 CFU and in the spleens (B) by 3.95 log10 CFU compared to results for untreated controls. Data represent mean log10 CFU counts ± SEM; detection limit = 50 CFU. *, P < 0.001.

FIG 5

CFZ activity decreased as the oxygen concentration decreased. CFZ was highly effective in vitro under high aeration (A). Under low aeration (B), CFZ had reduced but demonstrable activity despite similar bacterial growth. However, CFZ activity was lowest under completely anaerobic conditions achieved in the rapid anaerobic dormancy (RAD) culture model (C). CFZ was added at a final concentration of 50 mg/ml. Data represent mean log10 CFU counts ± SD; detection limit = 50 CFU. *, P < 0.001.