Interleukin-13-Overexpressing Mice Represent an Advanced Preclinical Model for Detecting the Distribution of Antimycobacterial Drugs within Centrally Necrotizing Granulomas

Abstract

The Mycobacterium tuberculosis (Mtb)-harboring granuloma with a necrotic center surrounded by a fibrous capsule is the hallmark of tuberculosis (TB). For a successful treatment, antibiotics need to penetrate these complex structures to reach their bacterial targets. Hence, animal models reflecting the pulmonary pathology of TB patients are of particular importance to improve the pre-clinical validation of novel drug candidates. Mtb-infected interleukin-13 overexpressing (IL-13 tg ) mice develop a TB pathology very similar to patients and, in contrast to other mouse models, also share pathogenetic mechanisms. Accordingly, IL-13 tg animals represent an ideal model for analyzing the penetration of novel anti-TB drugs into various compartments of necrotic granulomas by matrix-assisted-laser-desorption/ionization-mass spectrometry imaging (MALDI MS imaging). In the present study, we evaluated the suitability of BALB/c IL-13 tg mice for determining the antibiotic distribution within necrotizing lesions. To this end, we established a workflow based on the inactivation of Mtb by gamma irradiation while preserving lung tissue integrity and drug distribution, which is essential for correlating drug penetration with lesion pathology. MALDI MS imaging analysis of clofazimine, pyrazinamide and rifampicin revealed a drug-specific distribution within different lesion types including cellular granulomas, developing in BALB/c wild-type mice, and necrotic granulomas of BALB/c IL-13 tg animals, emphasizing the necessity of pre-clinical models reflecting human pathology. Most importantly, our study demonstrates that BALB/c IL-13 tg mice recapitulate the penetration of antibiotics into human lesions. Therefore, our workflow in combination with the IL-13 tg mouse model provides an improved and accelerated evaluation of novel anti-TB drugs and new regimens in the pre-clinical stage.

Do Anti-tuberculosis Drugs Reach Their Target?─High-Resolution Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Provides Information on Drug Penetration into Necrotic Granulomas

“…These macrophages are not encapsulated by a collagen rim and could represent a different type of macrophage. The presence of CFZ in the cellular areas of the granuloma is in accordance with our findings of the CFZ distribution in Mtb-infected wildtype mice (BALB/c) in Walter et al 23 Here, a higher intensity was detected in cellular granulomas, which contain a high amount of macrophages, as compared to necrotic granulomas of IL-13 tg mice. Previous studies in mice indicated that CFZ accumulates in macrophages of different tissues.…”

“…These results indicate that in our present mouse study, RIF had not reached steady state. Since we have observed in this and in our other study 23 the same distribution pattern for PZA (detectable inside and outside of necrotic lesions) and CFZ (only detectable outside of necrotic granulomas) as it was observed in TB patients, our results confirm that the IL-13 tg mouse model is suited for preclinical testing of anti-TB drugs. Penetration Analysis.…”

“…Instead of whole organs, we therefore optimized the inactivation of Mtb in lung sections of Mtbinfected IL-13 tg mice and found that Mtb could be inactivated by an energy dose of approx. 6 kGy as described in Walter et al 23 Cryosectioning in the BSL 3 facility was performed without any embedding material to be compatible with the MS imaging experiments. The necrotic tissue makes sectioning even more challenging than it already is for fragile organs such as lung tissue.…”

“…Sections were deposited onto precooled adhesive glass slides (Thermo Superfrost) as we have previously reported 22 and stored at −80 °C. For further processing outside of the BSL 3 facility, Mtb was inactivated by gamma (γ)-irradiation on dry icewe have described the details in Walter et al 23 Following microbiological examination by the National Reference Center for Mycobacteria (NRC, Borstel, Germany) of each sample batch, irradiated lung sections were shipped from the Research Center Borstel to the University of Bayreuth on dry ice and stored at −80 °C. Optical images were taken using a Keyence VHX-5000 digital microscope (Keyence Corporation, Osaka, Japan).…”

“…In the current manuscript, we describe this workflow focusing on MS imaging acquisition and data analysis. Tissue processing (including details of the irradiation protocol) and preclinical interpretation (including comparison of different mouse models) are discussed in a separate publication [Walter et al] . After the development of necrotic granulomas was confirmed, mice were treated with a drug regimen of CFZ, PZA, and RIF.…”