Phosphodiesterase-4 inhibition combined with isoniazid treatment of rabbits with pulmonary tuberculosis reduces macrophage activation and lung pathology

Abstract

Tuberculosis (TB) is responsible for significant morbidity and mortality worldwide. Even after successful microbiological cure of TB, many patients are left with residual pulmonary damage that can lead to chronic respiratory impairment and greater risk of additional TB episodes due to reinfection with Mycobacterium tuberculosis. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α and several other markers of inflammation, together with expression of matrix metalloproteinases, have been associated with increased risk of pulmonary fibrosis, tissue damage, and poor treatment outcomes in TB patients. In this study, we used a rabbit model of pulmonary TB to evaluate the impact of adjunctive immune modulation, using a phosphodiesterase-4 inhibitor that dampens the innate immune response, on the outcome of treatment with the antibiotic isoniazid. Our data show that cotreatment of M. tuberculosis infected rabbits with the phosphodiesterase-4 inhibitor CC-3052 plus isoniazid significantly reduced the extent of immune pathogenesis, compared with antibiotic alone, as determined by histologic analysis of infected tissues and the expression of genes involved in inflammation, fibrosis, and wound healing in the lungs. Combined treatment with an antibiotic and CC-3052 not only lessened disease but also improved bacterial clearance from the lungs. These findings support the potential for adjunctive immune modulation to improve the treatment of pulmonary TB and reduce the risk of chronic respiratory impairment.

Figure 1

Effect of CC-3052 treatment on M. tuberculosis infected rabbit lung pathologic findings. A–D: Gross pathologic findings of rabbit lungs at 12 weeks after infection. A: Untreated. B: CC-3052 treated. C: INH treated. D: INH plus CC-3052 treated. E: Number of subpleural lesions (dorsally) at 4, 8, and 12 weeks after infection. Statistically significant differences in the numbers of lesions were observed between the animals treated with INH plus CC-3052 and INH alone at 8 (asterisk) and 12 (double asterisk) weeks, respectively (P ≤ 0.05).

Figure 2

Morphometric analysis of scanned sections of the lungs of rabbits at 12 weeks after infection, stained with H&E. A: Untreated. B: CC-3052 treated. C: INH treated. D: CC-3052 plus INH treated. Rabbits treated with INH plus CC-3052 had a significantly lower extent of lung infiltration. P ≤ 0.05. Some sections had no granulomas.

Figure 3

Morphometric measurement of the number of lesions (per square centimeters) and the percentage of the lung section occupied by granulomas in the M. tuberculosis infected rabbit lungs. A: Number of lung lesions. B: Extent of lung involvement. Statistically significant difference in the numbers of lesions observed between animals treated with INH plus CC-3052 and INH alone at 8 and 12 weeks after infection (asterisk). P ≤ 0.05.

Figure 4

Histopathology of M. tuberculosis infected rabbit lungs at 12 weeks after infection. A and D: Untreated rabbit. Large confluent granulomas with central necrosis (the area marked by drawing in A and the area under the line in D), surrounded by cuff of lymphocytes (arrows), are observed. B and E: Control untreated rabbit lung with a cavity (the opaque top left area in B and E). Note the layers from the cavity lumen out: an extensive necrotic zone (the area above the marked line in B), with numerous lymphocytes (arrows). The circled area in B is magnified to show the features in E. A fibrotic area adjacent to a highly cellular granulomatous layer is observed below the dotted line in E. C and F: Rabbit treated with INH alone. Large granulomas with central necrosis (the area marked by drawing in C and the area above the marked line in F) and areas of lymphocytes (arrows). Sections were stained with H&E and photographed at ×4 (A–C) or ×40 (D–F) magnification.

Figure 5

Histopathologic analysis of M. tuberculosis infected rabbit lungs at 12 weeks after infection. A and D: Rabbit treated with CC-3052 alone. Big confluent granulomas with central necrosis (the area marked by drawing in A and the area under the line in D) and large numbers of lymphocytes (arrows). B and E: Rabbit treated with the combination of INH plus CC-3052. Few small, well-organized granulomas with a central area of epithelioid macrophages and a peripheral cuff of lymphocytes. No necrosis was observed. C: Control untreated animal. Large number of acid-fast bacilli in the area adjacent to the cavity lumen and in the necrotic zone of the granuloma. F: Rabbit treated with INH plus CC-3052. No acid-fast bacilli can be seen in this section. Arrows indicate lymphocytes. Tissue sections were stained with H&E (A, B, D, and E) or acid-fast stain (C and F) and photographed at ×4 (A and B) or ×40 (C–F) magnification.

Figure 6

Expression of rabbit genes differentially regulated by CC-3052 treatment. Expression of individual genes was quantified by real-time quantitative PCR, normalized against the housekeeping gene GAPDH, and represented as relative fold (log₁₀) compared with the expression levels in uninfected, naive rabbits. Expression level of each gene was measured in triplicates in at least 3 different experiments. *P < 0.05, **P < 0.01 between CC-3052 treated and untreated animals.

Figure 7

Expression of MMP genes in M. tuberculosis infected rabbit lungs with and without CC-3052 treatment (A) or with INH alone or INH plus CC-3052 (B). Expression of individual genes was quantified by real-time quantitative PCR, normalized against the housekeeping gene GAPDH, and represented as relative fold (log₁₀) compared with the expression levels in uninfected, naive rabbit lungs (A) or relative to 8-week untreated rabbits (B). Expression level of each gene was measured in triplicates in at least 3 different experiments. A: *P < 0.05 between 4 weeks and 8 and/or 12 weeks for untreated samples; **P < 0.05 between CC-3052 treated and untreated samples. B: *P < 0.05 between INH alone and INH plus CC-3052 treated samples.

Figure 8

Extent of fibrosis in M. tuberculosis infected rabbit lung tissue from an untreated (A, C, and E) rabbit or an animal treated with CC-3052 (B, D, and F) at 12 weeks after infection. The layered appearance of the fibroblasts below the necrotic center (the area above the marked line) of a granuloma is seen in A (arrows). In contrast, the cells are more loosely aggregated in B and F (the area on the left side of the marked line). A fine network of extracellular matrix components, including collagen, can be clearly visualized by the pink staining in the Van Gieson (arrows in C) and the blue staining in the Gomori's trichrome (arrows in E) stained sections of lungs from untreated animals infected with M. tuberculosis. Less staining is seen in the granulomas of the CC-3052 treated rabbits (D and F). Sections were stained with H&E (A and B) or Van Gieson stain (C and D) or Gomori's trichrome stain (E and F) and photographed at ×40 magnification.