Hypoxia and tissue destruction in pulmonary TB

Abstract

Background: It is unknown whether lesions in human TB are hypoxic or whether this influences disease pathology. Human TB is characterised by extensive lung destruction driven by host matrix metalloproteinases (MMPs), particularly collagenases such as matrix metalloproteinase-1 (MMP-1).

Methods: We investigated tissue hypoxia in five patients with PET imaging using the tracer [¹⁸F]-fluoromisonidazole ([¹⁸F]FMISO) and by immunohistochemistry. We studied the regulation of MMP secretion in primary human cell culture model systems in normoxia, hypoxia, chemical hypoxia and by small interfering RNA (siRNA) inhibition.

Results: [¹⁸F]FMISO accumulated in regions of TB consolidation and around pulmonary cavities, demonstrating for the first time severe tissue hypoxia in man. Patlak analysis of dynamic PET data showed heterogeneous levels of hypoxia within and between patients. In Mycobacterium tuberculosis (M.tb)-infected human macrophages, hypoxia (1% pO₂) upregulated MMP-1 gene expression 170-fold, driving secretion and caseinolytic activity. Dimethyloxalyl glycine (DMOG), a small molecule inhibitor which stabilises the transcription factor hypoxia-inducible factor (HIF)-1α, similarly upregulated MMP-1. Hypoxia did not affect mycobacterial replication. Hypoxia increased MMP-1 expression in primary respiratory epithelial cells via intercellular networks regulated by TB. HIF-1α and NF-κB regulated increased MMP-1 activity in hypoxia. Furthermore, M.tb infection drove HIF-1α accumulation even in normoxia. In human TB lung biopsies, epithelioid macrophages and multinucleate giant cells express HIF-1α. HIF-1α blockade, including by targeted siRNA, inhibited TB-driven MMP-1 gene expression and secretion.

Conclusions: Human TB lesions are severely hypoxic and M.tb drives HIF-1α accumulation, synergistically increasing collagenase activity which will lead to lung destruction and cavitation.

Keywords: Tuberculosis.

Figure 1

[¹⁸F] fluoromisonidazole ([¹⁸F]FMISO) PET-CT demonstrates increased tracer uptake within TB lesions. (A) [¹⁸F]FMISO PET-CT images. Transverse, coronal and sagittal slices through the [¹⁸F]FMISO target-to-background (TBR) and CT images of Patient 2. Top row, CT images show consolidation and a pulmonary cavity in the right upper lobe with a smaller, non-cavitating lesion in the left lung. Middle row, [¹⁸F]FMISO TBR map demonstrates intense uptake of tracer in the right upper lobe lesion and the liver, the site of metabolism of [¹⁸F]FMISO. Bottom row, coregistered CT and TBR images. (B) TBR values greater than unity were found for at least one lesion in all patients (each individual bar represents one region of interest, ROI).

Figure 2

Severe hypoxia is present within human pulmonary TB lesions. (A) Patlak K_i images from dynamic PET-CT scanning for Patient 2 demonstrate intense retention of [¹⁸F] fluoromisonidazole ([¹⁸F]FMISO) in the right upper lobe of the lung (middle row). Coregistered PET-CT images (bottom row) confirm localisation of hypoxia to the region of the right upper lobe cavity. (B) Regional K_i values demonstrate heterogeneous trapping of [¹⁸F]FMISO within and between patients. The hypoxia threshold (0.00037/min) was determined from the mean+3 SDs of the Ki values in the normoxic lateral muscle background regions across the patient group. (C) Time–activity measurements of five regions of interest (ROIs) from Patient 3 demonstrate high tissue levels compared with peripheral blood values.

Figure 3

Hypoxia increases Mycobacterium tuberculosis (M.tb)-driven matrix metalloproteinase-1 (MMP-1) expression and secretion by human monocyte-derived macrophages (MDMs). (A) Hypoxia (1% pO₂) increases MMP-1 gene expression in primary human MDMs infected with M.tb at 24 h. (B) Hypoxia increases intracellular MMP-1 accumulation on confocal microscopy at 72 h in MDMs infected with M.tb (MOI=1) compared with normoxia or control uninfected cells. (C) Mtb infection increases MMP-1 secretion by infected MDMs greater than LPS (100 ng/mL) in both normoxia and hypoxia analysed 72 h after infection. (D) MMP-1 activity is increased in M.tb-infected but not control human macrophages analysed by casein zymography, and hypoxia further increases caseinolytic activity. (E) Stabilisation of hypoxia-inducible factor (HIF)-1α by dimethyloxalyl glycine (DMOG) (range 0.05–0.5 mM) significantly increases MMP-1 secretion analysed by ELISA and proteolytic activity measured by zymography in a dose-dependent manner. N=21% O₂; 5% CO₂, H=1% O₂; 5% CO₂. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. LPS, lipopolysaccharide.

Figure 4

Hypoxia upregulates matrix metalloproteinase-1 (MMP-1) in human respiratory epithelial cells. (A) Hypoxia increases MMP-1 secretion from normal human bronchial epithelial (NHBE) cells stimulated with control medium or conditioned media from Mtb-infected monocytes (CoMTb) incubated in 21%, 5% or 1% oxygen. (B) Dimethyloxalyl glycine (DMOG) (range 0.1–0.25 mM) drives dose-dependent MMP-1 secretion from CoMTb-stimulated NHBE cells. (C) In silico analysis of the MMP-1 promoter reveals putative hypoxic response element (HRE)-binding sites as well as consensus NF-κB-binding and AP-1-binding sites. (D) Relative luminescence following transfection of A549 respiratory epithelial cells with either WT-MMP-1 promoter or a series of MMP-1 promoter deletion constructs in normoxia (solid bars) or hypoxia (shaded bars). The effect of hypoxia in increasing promoter activity is absent in constructs −1551, −1194 and −517. (E) IKK-β inhibition in hypoxia with SC-514 in CoMTb-stimulated cells resulted in a dose-dependent decrease in MMP-1 secretion. (F) Site-directed mutagenesis of the NF-κB-binding site at −2878 to −2886 bp decreases MMP-1 promoter activity in response to CoMTb in normoxia (solid bars) or hypoxia (shaded bars). **p<0.01, *p<0.05. AP-1, activated protein-1; IKK-β, inhibitor of nuclear factor kappa-B kinase subunit beta; WT, wild type.

Figure 5

Mycobacterium tuberculosis (M.tb) infection drives hypoxia-inducible factor (HIF)-1α accumulation in normoxia. (A) In A549 cells, conditioned media from Mtb-infected monocytes (CoMTb) stimulation and dimethyloxalyl glycine (DMOG) (0.25 mM) result in early HIF-1α stabilisation. Preincubating A549 cells with DMOG (0.25 mM) and subsequent stimulation with CoMTb markedly increase HIF-1α accumulation, peaking at 6 h. (B) M.tb infection increases HIF-1α accumulation and stabilisation in normoxia in primary human monocyte-derived macrophages (MDMs), peaking at 24 h. (C) Combined infection with M.tb and exposure to hypoxia cause greater HIF-1α accumulation than either stimulus alone, peaking at 4 h and persisting until 24 h. (D) Inhibition of HIF-1α by LW6 (range 50–100 µM) results in decreased matrix metalloproteinase-1 (MMP-1) secretion in M.tb-infected MDMs in hypoxia (hatched bars). **p<0.01.

Figure 6

Hypoxia-inducible factor (HIF)-1α is necessary for matrix metalloproteinase-1 (MMP-1) gene expression and secretion during Mycobacterium tuberculosis (M.tb) infection. (A) HIF-1α western blot at 24 h demonstrates suppression of HIF-1α protein by HIF-1α siRNA in monocyte-derived macrophages (MDMs) in both normoxia and hypoxia, with no knockdown observed with non-targeting (NT) siRNA. (B) HIF-1α siRNA decreases MMP-1 gene expression at 24 h in M.tb-infected MDMs incubated in hypoxia. (C) HIF-1α siRNA causes a significant decrease in MMP-1 secretion at 48 h under conditions of normoxia (solid bars) and hypoxia (hatched bars). No significant change in MMP-1 gene expression or secretion is seen with NT siRNA. (D) Lung biopsies from patients with pulmonary TB (i, ii) are immunoreactive for HIF-1α compared with uninfected control biopsies (iii). HIF-1α staining is pronounced within macrophages and multinucleated giant cells (arrow, ii). Scale bars, 100 µm. ****p<0.0001, *p<0.05. siRNA, small interfering RNA.