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Abstract
Mycobacterium tuberculosis (Mtb) kills 1.7 million people annually. The Th1 paradigm does not explain TB-driven cavitation. Current treatment is lengthy with many adverse effects. The Interleukin-23/Th17 axis plays a critical role in early Mtb containment. Respiratory stromal cells are important first-line defence and secrete Matrix metalloproteinases (MMPs). Role of MMPs, which are substrate-specific proteases causing extracellular matrix degradation/remodelling, was investigated in TB. Human bronchoalveolar lavage (BAL) samples from 35 well-characterised TB and control patients were analysed for MMPs and Th17 cytokines. TB/Control lung biopsies were stained for MMPs/IL-17. Primary normal human bronchial epithelial cells (NHBEs) and MRC-5 fibroblasts were stimulated with IL-17/IL-22/IL-23, alone and in combination with conditioned medium from Mtb-infected monocytes (CoMTb). Secretion, gene expression, gene silencing, intracellular signalling were investigated by luminex, ELISA, zymography, dual-luciferase promoter-reporter, realtime RT-PCR, siRNA transfection. MMPs were up-regulated in Human TB BALs (p<0.0001). This positively correlated with cavitation score on CXRs. TIMPs (tissue inhibitor of metalloproteinase) and IL-17/IL-23 were unaltered but IL-22 was increased in TB BAL. IL-17 and MMP-3 were co-expressed in pneumocytes around granulomas in TB lung biopsies. CoMTb (but not direct infection) up-regulated secretion and gene expression of MMP-1 (collagenase, p<0.0001), MMP-3 (stromelysin, p<0.001) and MMP-9 (gelatinase, p<0.0001) from NHBEs. MMP-3 protein and promoter activity in MRC-5s was also increased (p<0.001). AKT inhibition suppressed all MMPs (p<0.01) whereas siRNA and chemical inhibition of the proximal PI3Kp110a subunit abrogated MMP-3 only (p<0.001). Distally, p70S6K (mTOR) blockade with rapamycin abrogated TB-driven MMP-1 and MMP-3 (p<0.001). MMP-9 production was unaffected by proximal/distal inhibition of PI3K.IL-17 independently and also synergistically with CoMTb augmented MMP-3 secretion/gene expression from NHBEs and MRC-5s in a dose-dependent manner (peak 8ng/ml, p<0.0001). This was p38-dependent, confirmed by p38-specific siRNA. In contrast, IL-17 down-regulated CoMTb-driven MMP-9 to baseline (p<0.01). IL-22 augmented MMP-3 from fibroblasts but not from NHBEs. IL-23 did not drive MMPs.
Summary MMPs are key mediators of tissue damage in human pulmonary TB and are regulated in a cell- and stimulus-specific manner. IL-17 and IL-22 drive MMP-3 but suppress MMP-9 in airway epithelium. The PI3Kinase/p110a/p70S6K pathway is a crucial target and its immuno-modulation (eg, rapamycin) is a potential adjunctive therapy to limit tissue destruction and shorten chemotherapy in TB.
Pulmonary epithelial cells around a TB granuloma expressed IL-17 (A). Strong immunoreactivity for MMP-3 (B) in pulmonary epithelial cells around TB granulomas was also detected. Control lungs (not shown) were negative.