[Accepted Manuscript] ESAT-6 Drives MMP-10 Gene Expression and Secretion in Tuberculosis

Tuberculosis (TB) causes disease worldwide and multi-drug resistance is an increasing problem. Matrix metalloproteinases (MMPs), particularly the collagenase MMP-1, cause lung extracellular matrix destruction which drives disease transmission and morbidity. The role in such tissue damage of the stromelysin MMP-10, a key activator of the collagenase MMP-1, was investigated in direct Mycobacterium tuberculosis (Mtb) infected macrophages and in conditioned medium from Mtb infected monocytes (CoMtb)-stimulated cells. Mtb infection increased MMP-10 secretion from primary human macrophages 29-fold, while CoMtb increased secretion by 4.5-fold from pulmonary epithelial cells and 10.5-fold from fibroblasts. Inhibition of MMP-10 activity decreased collagen breakdown. In two independent cohorts of TB patients from different continents, MMP-10 was increased in both induced sputum and bronchoalveolar lavage fluid compared to controls and patients with other respiratory diseases (both p<0.05). Mtb drove 3.5-fold greater MMP-10 secretion from human macrophages than the vaccine strain BCG (p<0.001), whereas both mycobacteria upregulated TNFα secretion equally. Using overlapping short linear peptides covering the sequence of ESAT-6, a virulence factor secreted by Mtb but not BCG, we found that stimulation of human macrophages with a single specific 15 amino acid peptide sequence drove 3-fold greater MMP-10 secretion than any other peptide (p<0.001). Mtb-driven MMP-10 secretion was inhibited in a dose-dependent manner by p38 and ERK MAPK blockade (p<0.001 and p<0.01 respectively), but was not affected by inhibition of NF-ĸB. In summary, Mtb activates inflammatory and stromal cells to secrete MMP-10 and this is partly driven by the virulence factor ESAT-6, implicating it in TB-associated tissue destruction.