Heterogeneous disease progression and treatment response in a C3HeB/FeJ mouse model of tuberculosis

Abstract

Mice are the most commonly used species for non-clinical evaluations of drug efficacy against tuberculosis (TB). Unlike commonly used strains, C3HeB/FeJ mice develop caseous necrosis in the lung, which might alter the representation of drug efficacy in a way that is more like human TB. Because the development of such pathology requires time, we investigated the effect of infection incubation period on the activity of six drugs in C3HeB/FeJ and BALB/c mice. Mice were aerosol infected and held for 6, 10 or 14 weeks before receiving therapy with rifampin (RIF), rifapentine (RPT), pyrazinamide (PZA), linezolid (LZD), sutezolid (PNU) or metronidazole (MTZ) for 4-8 weeks. Outcomes included pathological assessments, pH measurements of liquefied caseum and assessment of colony-forming unit (CFU) counts from lung cultures. Remarkable heterogeneity in the timing and extent of disease progression was observed in C3HeB/FeJ mice, largely independent of incubation period. Likewise, drug efficacy in C3HeB/FeJ mice was not affected by incubation period. However, for PZA, LZD and PNU, dichotomous treatment effects correlating with the presence or absence of large caseous lesions were observed. In the case of PZA, its poor activity in the subset of C3HeB/FeJ mice with large caseous lesions might be explained by the pH of 7.36±0.09 measured in liquefied caseum. This study highlights the potential value of C3HeB/FeJ mice for non-clinical efficacy testing, especially for investigating the interaction of lesion pathology and drug effect. Careful use of this model could enhance the bridging of non-clinical results with clinical outcomes.

Keywords: C3HeB/FeJ; Heterogeneity; Pyrazinamide; Tuberculosis; pH.

Fig. 1.

Aggregate survival of untreated and treated mice from the day of infection. Mice that were sacrificed at predetermined time points were censored. A higher rate of death was observed in C3HeB/FeJ mice compared with BALB/c mice.

Fig. 2.

Lung CFU counts among C3HeB/FeJ mice. The treatment period was 4 weeks except in groups designated by ‘W8’, which received 8 weeks of treatment. Results from each incubation arm are combined. D0, initiation of treatment; RIF, rifampin; RPT, rifapentine; PZA, pyrazinamide; MTZ, metronidazole; LZD, linezolid; PNU, sutezolid.

Fig. 3.

Individual lung CFU distribution by mouse strain. CFU distribution in BALB/c mice is shown on the left (A,C), and in C3HeB/FeJ mice on the right (B,D). Oxazolidinones are presented separately in panels C and D. RIF, rifampicin; RPT, rifapentine; PZA, pyrazinamide; MTZ, metronidazole; LZD, linezolid; PNU, sutezolid; W8, results after 8 weeks of treatment. D0 is the day of treatment initiation.

Fig. 4.

Examples of gross pathology and correlation with lung CFU counts from mice in the 14-week incubation arm following treatment with pyrazinamide or linezolid. PZA, upper panels; LZD, lower panels. Arrows show large, expansile tubercular lesions in the lungs with the highest CFU counts.

Fig. 5.

Histopathology of caseous lesions in untreated C3HeB/FeJ mice at different incubation times. (A) Lung cavity formed in a C3HeB/FeJ mouse after 26 weeks of incubation. (B,C) Cavity wall with H&E (B) and acid-fast bacilli (AFB; C) staining. (D) Sick mouse after 8 weeks of incubation. (E,F) Granuloma wall with monocyte infiltration and foamy macrophages with H&E (E) and AFB (F) staining. Scale bars: 2 mm (A); 200 μm (B); 100 μm (C,E,F); 5 mm (D).

Fig. 6.

Scheme of experiment. Abbreviations: R, rifampin; P, rifapentine; Z, pyrazinamide; L, linezolid; U, sutezolid (PNU-100480); M, metronidazole. Each dot represents a time point for pathology assessment (two untreated mice were included per strain); red dot is the time of infection. Green pentagons represent treatment administered and its duration. Numbers represent the number of mice killed at each time point for each treatment group (above the dot for BALB/c mice and below the dot for C3HeB/FeJ mice). W-14 is 14 weeks prior to initiation of treatment. D0 indicates the day of treatment initiation. W20 is 20 weeks following treatment initiation. This figure represents the scheme as applied to the 14-week incubation cohort. A similar scheme was carried out for the 10-week and 6-week incubation cohorts.