Modulation of Immunopathogenic Matrix Metalloproteinases in Pulmonary Tuberculosis by T helper-17 cytokines & the PI 3-Kinase pathway

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 IL-23/Th17 axis plays a critical role in early Mtb containment. Respiratory stromal cells are important first-line defence and secrete MMPs. Regulation of Matrix metalloproteinases (MMPs), which are substrate-specific proteases causing extracellular matrix (ECM) degradation/ remodelling, was investigated in the context of 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 and intracellular signalling were investigated by luminex, ELISA, zymography, dual-luciferase promoter-reporter, real time RT-PCR and siRNA transfection. MMPs were up-regulated in Human TB BALs (p<0.0001). This positively correlated with cavitation score on CXRs. 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-5 fibroblasts was also increased by CoMTb. AKT inhibition suppressed all MMPs (p<0.01) whereas siRNA and chemical inhibition of the proximal PI3Kp110α 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 regulation was more complex. IL-17 independently and also synergistically with CoMTb, augmented MMP-3 secretion/ gene expression from NHBEs and MRC-5 fibroblasts in a concentration-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). Interleukin-22 augmented MMP-3 from fibroblasts in a TB network, but not from NHBEs. IL-23 did not drive MMPs. MMP-1, MMP-3 and MMP-9 production was also affected by anti-mycobacterial agents in a TB network. In 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 from human airway stromal cells. The PI3Kinase/p110α/p70S6K is a crucial target and its immunomodulation (e.g. with rapamycin) has potential as adjunctive therapy to limit tissue destruction and shorten chemotherapy in TB.