Pharmacologic Inhibition of Host Phosphodiesterase-4 Improves Isoniazid-Mediated Clearance of Mycobacterium tuberculosis

Abstract

The lengthy duration of multidrug therapy needed to cure tuberculosis (TB) poses significant challenges for global control of the disease. Moreover, chronic inflammation associated with TB leads to pulmonary damage that can remain even after successful cure. Thus, there is a great need for the development of effective shorter drug regimens to improve clinical outcome and strengthen TB control. Host-directed therapy (HDT) is emerging as a novel adjunctive strategy to enhance the efficacy and shorten the duration of TB treatment. Previously, we showed that the administration of CC-3052, a phosphodiesterase-4 inhibitor (PDE4i), reduced the host inflammatory response during Mycobacterium tuberculosis (Mtb) infection and improved the antimicrobial efficacy of isoniazid (INH) in both the mouse and rabbit models. In the present study, we evaluated the pharmacokinetics and explored the mechanism underlying the efficacy of a more potent PDE4i, CC-11050, as adjunct to INH treatment in a mouse model of pulmonary Mtb infection. Genome-wide lung transcriptome analysis confirmed the dampening of inflammation and associated network genes that we previously reported with CC-3052. Consistent with the reduction in inflammation, a significant improvement in Mtb control and pathology was observed in the lungs of mice treated with CC-11050 plus INH, compared to INH alone. This important confirmatory study will be used to help design upcoming human clinical trials with CC-11050 as an HDT for TB treatment.

Keywords: Mycobacterium tuberculosis; gene expression; host-directed therapy; immune modulation; mouse model; phosphodiesterase-4 inhibitor; pulmonary tuberculosis.

Figure 1

Expression of host PDE4 genes in Mtb-infected untreated (hatched bars) or CC-11050-treated (solid bars) mouse lungs, relative to the uninfected controls. Experiment was performed in duplicate with four biological replicates (n = 4 per group). *P = 0.0037 for Pde4D and P = 0.015 for Pde4Bi3 between untreated and CC-1150-treated groups.

Figure 2

Genome-wide lung transcriptome of Mtb-infected mice with or without CC-11050 treatment. (A) Principal component analysis (PCA) map showing segregation of uninfected, Mtb-infected untreated, or CC-11050-treated samples (n = 4 per group). The eclipse/oval shape represents 2 × SD from the mean data. (B) Venn diagram showing unique and shared SDEGs between Mtb-infected untreated (red) and CC-11050-treated (blue) mouse lungs after 14 days of treatment. A false discovery rate (FDR) of 5% was applied to select the SDEGs. Microarray data were generated with four biological replicates per each group. Numbers in parenthesis are the total SDEGs.

Figure 3

Intensity plot of TNF-α (A) and lung inflammatory response (B) network gene expression in Mtb-infected mouse lungs with or without CC-11050 treatment. UT, untreated; CC, treated with CC-11050. Only genes expressed in CC are shown in italicized gene symbols. Red color shows upregulation and blue color shows downregulation of gene expression. Scale bar ranges from +3 (red) to −3 (blue).

Figure 4

Gene interaction network and intensity plot of SDEGs associated with Il-17 in Mtb-infected mouse lungs with or without CC-11050 treatment. (A) Interaction of member genes involved in Il-17 expression/regulation. Solid lines represent direct connection, and broken lines indicate indirect link. Red color indicates upregulation, and green color depicts downregulation. (B) Intensity plot of SDEGs associated with Il-17 network. UT, untreated; CC, treated with CC-11050. Red color shows upregulation, and blue color denotes downregulation of gene expression. Scale bar ranges from +3 (red) to −3 (blue).

Figure 5

Interaction and expression pattern of SDEGs involved in canonical interferon signaling pathway in Mtb-infected mouse lungs with or without CC-11050 treatment. (A) Pathway map showing interaction of member genes involved in Ifn signaling pathway. Red color indicates upregulation, and green color indicates downregulation. (B) Intensity plot of SDEGs associated with Ifn signaling. UT, untreated; CC, treated with CC-11050. Red color shows upregulation, and blue color shows downregulation of gene expression. Scale bar ranges from +3 (red) to −3 (blue).

Figure 6

Bacillary load in Mtb-infected mouse lungs with or without CC-11050 and/or INH treatment. (A) Bacterial CFUs at various time points in different treatment groups. Treatment was started at 14 days postinfection. *P = 0.07 between INH (triangle) and INH plus CC-11050-treated groups (diamond) (n = 6 per group per time point). (B) Individual data points at 112 days. One animal out of six from each of INH or INH plus CC-11050-treated groups had unusual CFU pattern that can be an outlier or due to technical error. Excluding these two samples from the analysis led to a significant difference in lung CFUs at 112 days postinfection between the two treatment groups (P = 0.02; n = 5 per group).

Figure 7

Morphometric measurements of lung granulomas in Mtb-infected mice treated with CC-11050 (hatched bars), INH (horizontal bars), both (solid bars), or none (gray bars). *P = 0.0302 between INH and no treatment; P = 0.0259 between INH plus CC-11050 and no treatment at 28 days postinfection. *P = 0.008 and 0.0371, respectively, for INH and INH + CC-11050, compared to no treatment group (n = 4–6 per group per time point).

Figure 8

Histology of Mtb-infected mouse lungs at 112 days postinfection. Representative image of H&E stained mouse lung sections at 10× (A,C,E,G) and 40× (B,D,F,H) magnifications. (A,B) untreated; (C,D) CC-11050-treated; (E,F) INH-treated; and (G,H) INH plus CC-11050-treated. (A,C,E,G) are photographed at 10×, and (B,D,F,H) are photographed at 40× magnification. Note the presence of multiple, coalescent, and larger granulomas in the untreated and CC-11050-treated mouse lungs (A–D); in contrast, the INH-treated mouse had a smaller (E,F), while the INH plus CC-11050-treated animals had the smallest lung lesion (G,H).