NOS2-deficient mice with hypoxic necrotizing lung lesions predict outcomes of tuberculosis chemotherapy in humans

Abstract

During active TB in humans a spectrum of pulmonary granulomas with central necrosis and hypoxia exists. BALB/c mice, predominantly used in TB drug development, do not reproduce this complex pathology thereby inaccurately predicting clinical outcome. We found that Nos2 −/− mice incapable of NO-production in immune cells as microbial defence uniformly develop hypoxic necrotizing lung lesions, widely observed in human TB. To study the impact of hypoxic necrosis on the efficacy of antimycobacterials and drug candidates, we subjected Nos2 −/− mice with TB to monotherapy before or after establishment of human-like pathology. Isoniazid induced a drug-tolerant persister population only when necrotic lesions were present. Rifapentine was more potent than rifampin prior to development of human-like pathology and equally potent thereafter, in agreement with recent clinical trials. Pretomanid, delamanid and the pre-clinical candidate BTZ043 were bactericidal independent of pulmonary pathology. Linezolid was bacteriostatic in TB-infected Nos2 −/− mice but significantly improved lung pathology. Hypoxic necrotizing lesions rendered moxifloxacin less active. In conclusion, Nos2 −/− mice are a predictive TB drug development tool owing to their consistent development of human-like pathology.Tuberculosis (TB) is a global health threat further worsened by increasing incidences of drug resistance and HIV co-infection 1 . New drugs to combat resistant TB and to shorten the current 6 to 9 month standard-of-care chemotherapy are urgently needed. In most cases, infection starts in the lungs where Mycobacterium tuberculosis induces well-structured granulomas composed of various hematopoietic cells in which the pathogen is contained 2, 3 . During progression to active disease, infected macrophages and neutrophils undergo necrosis at the core of these structure, resulting in the formation of a hypoxic caseum 2 . 18 F-fluorodeoxyglucose positron emission tomography/computed tomography has revealed that pulmonary TB is a highly dynamic disease: coexisting inflammatory hotspots in lungs of TB patients increase in size, shrink or even resolve over time while others are newly formed 4,5 . Recent studies have indicated that pro-and anti-inflammatory signals are spatially segregated within granulomas of humans suggesting that granuloma formation in TB is a direct consequence of the local activity of inflammatory pathways 6 . Among the available animal models, non-human primates mirror the facets of human TB best, but broad application is hampered by prohibitive cost and ethical concerns. To date, mice remain the most widely used

Inhibition of host Lactate dehydrogenase A by a small-molecule limitsMycobacterium tuberculosisgrowth and potentiates bactericidal activity of isoniazid

“…S2B). As previously observed (20), onset of hypoxic and necrotic lesions became apparent at day 56 (at treatment start). Although the number and size of lesions were comparable, further development of necrotic lesions were ceased in the FX11-treated group (Fig.…”

“…TB granulomas in C57BL/6J mice rarely progress into necrosis, whereas Nos2 −/− mice present hypoxic necrotizing lung lesions that are characteristics hallmarks of human TB (18–20). Therefore, in a second experiment, the effect of FX11 (2 mg/kg), either individually or in combination with isoniazid (INH, 25 mg/kg), was evaluated in Nos2 −/− mice (Fig.…”

“…Necrotic lesions in the Nos2 −/− model have been correlated with the evolution of slow/non-growing INH-tolerant subpopulation (20). Accordingly, we interrogated whether FX11-mediated inhibition of progression to necrotic granuloma potentiates INH efficacy possibly by preventing the emergence of the drug-tolerant population.…”

“…*p<0.05. (B) Effect of FX11 (2 mg/kg) as monotherapy or in combination with INH (25 mg/kg) in Nos2 −/− mice with hypoxic necrotizing lung lesions (20). The TNF-α response was neutralized at 2 and 3 weeks of post-infection.…”

“…Six- to eight-week-old C5BL/6J mice were aerosol infected with 100 CFU M. tuberculosis H37Rv. C5BL/6J Nos2 −/− mice were infected as previously reported (2). In brief, six- to eight-week-old female C5BL/6J Nos2 −/− mice were anesthetized (ketamine 65mg/kg, acepromazine 2 mg/kg, xylazine 11 mg/kg) and infected with 1,000 CFU of M. tuberculosis in 20 μl PBS given into the ear dermis.…”