Doxycycline host-directed therapy in human pulmonary tuberculosis

Abstract

BACKGROUNDMatrix metalloproteinases (MMPs) are key regulators of tissue destruction in tuberculosis (TB) and may be targets for host-directed therapy. We conducted a phase II double-blind, randomized, controlled trial investigating doxycycline, a licensed broad-spectrum MMP inhibitor, in patients with pulmonary TB.METHODSThirty patients with pulmonary TB were enrolled within 7 days of initiating anti-TB treatment and randomly assigned to receive either 100 mg doxycycline or placebo twice a day for 14 days, in addition to standard care.RESULTSWhole blood RNA-sequencing demonstrated that doxycycline accelerated restoration of dysregulated gene expression in TB towards normality, rapidly down-regulating type I and II interferon and innate immune response genes, and up-regulating B-cell modules relative to placebo. The effects persisted for 6 weeks after doxycycline discontinuation, concurrent with suppressed plasma MMP-1. Doxycycline significantly reduced sputum MMP-1, -8, -9, -12 and -13, suppressed type I collagen and elastin destruction, reduced pulmonary cavity volume without altering sputum mycobacterial loads, and was safe.CONCLUSIONAdjunctive doxycycline with standard anti-TB treatment suppressed pathological MMPs in PTB patients. Larger studies on adjunctive doxycycline to limit TB immunopathology are merited.TRIAL REGISTRATIONClinicalTrials.gov NCT02774993.FUNDINGSingapore National Medical Research Council (NMRC/CNIG/1120/2014, NMRC/Seedfunding/0010/2014, NMRC/CISSP/2015/009a); the Singapore Infectious Diseases Initiative (SIDI/2013/013); National University Health System (PFFR-28 January 14, NUHSRO/2014/039/BSL3-SeedFunding/Jul/01); the Singapore Immunology Network Immunomonitoring platform (BMRC/IAF/311006, H16/99/b0/011, NRF2017_SISFP09); an ExxonMobil Research Fellowship, NUHS Clinician Scientist Program (NMRC/TA/0042/2015, CSAINV17nov014); the UK Medical Research Council (MR/P023754/1, MR/N006631/1); a NUS Postdoctoral Fellowship (NUHSRO/2017/073/PDF/03); The Royal Society Challenge Grant (CHG\R1\170084); the Sir Henry Dale Fellowship, Wellcome Trust (109377/Z/15/Z); and A*STAR.

Keywords: Clinical Trials; Infectious disease; Tuberculosis.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Trial profile.

Safety analysis included all participants who underwent randomization and received an initial dose of placebo or doxycycline. The downstream assessment included all participants who underwent randomization, took at least 24 of the 28 doses of placebo or doxycycline, and returned for follow-up on days 14 and 56 (94).

Figure 2. Doxycycline results in faster normalization of type II interferon and innate immune response genes relative to placebo.

(A and B) PCA analysis of doxycycline-treated (A) and placebo-treated (B) patients at day 0 (orange) and day 14 (blue). First 2 components of PCA are shown and their variances are shown in parenthesis. Doxycycline reduces variation between individuals over the first 2 weeks of treatment. Only patients with day 0 and day 14 samples are plotted. n = 8 placebo, n = 7 doxycycline. (C) Transcript to transcript clustering of 12,977 genes filtered and normalized using TMM identified 27 coexpressed clusters (Pearson r = 0.8; MCL = 1.7; n gene/cluster: 3/10) over the course of doxycycline treatment. The 15 largest clusters (Supplemental Table 3) are shown in different colors. Lines represent the similarity between transcripts, circles represent individual genes. Two major groupings identify genes preferentially expressed in innate immune response (granulocytes, interferon response) and adaptive immune response (T cells). (D–I) Longitudinal analysis of selected genes, IRF1 (D), APOL1 (E), FCGR1A (F), FCGR1B (G), GBP5 (H), and GBP6 (I) from a cluster encoding for type II interferon and innate immune responses. TMM normalized gene expression at days 0, 14, and 56 of patients with TB in placebo (n = 8, green) and doxycycline (Doxy, n = 7, purple) arms, and baseline expression of healthy volunteers (HV, n = 6, blue) are plotted. Box represents 25th and 75th percentile, line is median, with whiskers denoting extremes.

Figure 3. Doxycycline differentially regulates numerous genes in TB pathway relative to placebo.

Comparison of gene expression changes (day 14 vs. day 0) between placebo and doxycycline (Doxy) arms for genes in the KEGG TB pathway (map05152) (–49). The pathway was identified by EGSEA (52) to be significantly regulated by doxycycline treatment (adjusted P = 0.0047). Gene expression changes (day 14 vs. day 0) in the placebo and doxycycline arms are shown on the left and right side, respectively, of each gene box. Red represents upregulation and blue represents downregulation of gene expression.

Figure 4. Doxycycline leads to greater downregulation of type I interferon responses and extracellular matrix genes, and upregulation of B cells markers relative to placebo.

(A) GSEA of DEGs specific to doxycycline treatment ((Doxy_Day14-Doxy_Day0)–(Placebo_Day14-Placebo_Day0)). Blood transcriptional modules (54) were used as gene sets. The top 15 enriched gene sets are shown. Dotted line marks adjusted P = 0.05. Red represents upregulation and blue represents downregulation of gene sets. (B–D) Longitudinal analysis of type I interferon response gene set (M127) (B), extracellular matrix gene set (M2.0) (C), and MMP9 (D) at day 0 and day 14 of placebo (n = 8, green) and doxycycline (n = 7, purple) arms. Median expression levels of genes in the gene sets are plotted, while TMM normalized gene expressions are shown for MMP9. Box represents 25th and 75th percentile, line is median, with whiskers denoting extremes. **Adjusted P < 0.01.

Figure 5. Plasma MMP-1 is suppressed by doxycycline but TIMP-1 and -2 are not affected.

(A–D) Longitudinal analysis of plasma MMP-1 (A), MMP-8 (B), TIMP-1 (C), and TIMP-2 (D) concentrations at days 0, 14, and 56 of placebo (n = 12, green) and doxycycline (Doxy, n = 13, purple) arms. Protein concentrations of each subject were normalized to their day 0 values. Analysis by 2-way ANOVA with Sidak’s multiple comparisons. *Adjusted P < 0.05. Bars represent mean ± SEM.

Figure 6. Sputum MMPs and extracellular matrix degradation activity in patients with TB treated with doxycycline are suppressed with a concurrent decrease in cavity volume.

(A–G) Longitudinal analysis of sputum MMP-1 (A), MMP-8 (B), MMP-9 (C), TIMP-1 (D), TIMP-2 (E), type I collagenase activity (F), and elastase activity (G) at days 0 and 14 of placebo (green) and doxycycline (Doxy, purple) arms. Protein concentrations, as well as functional activity, for each subject were normalized to their day 0 values. n = 13 placebo and n = 13 doxycycline for MMPs; n = 10 placebo and n = 12 doxycycline for TIMPs; n = 12 placebo and n = 9 doxycycline for functional activity. (H) Longitudinal analysis of cavity volume in patients with pulmonary cavities at days 0 and 56. n = 9 placebo (green) and n = 7 doxycycline (Doxy, purple). Analysis by 2-way ANOVA with Sidak’s multiple comparisons. *Adjusted P < 0.05. Bars represent mean ± SEM.