Matrix Metalloproteinase 12 is Critical for Granuloma Formation in the Murine Model of Granulomatous disease

Abstract

Matrix Metalloproteinase 12 (MMP12) is a protein produced primarily by alveolar macrophages that degrades elastin in the extracellular matrix (ECM) and enables infiltration of immune cells that participate in the inflammatory response. To our knowledge, few studies have been conducted to clarify the role of MMP12 in granulomatous diseases such as sarcoidosis, a chronic inflammatory disease characterized by granuloma formation primarily in the lungs. Previous studies have shown an increase in gene and protein expression of MMP12 in lung tissue and bronchoalveolar lavage (BAL) of patients with sarcoidosis as well as a correlation between MMP12 elevation and disease severity. Our murine model uses multiwall carbon nanotubes (MWCNT) to mimic the characteristics (gene, protein expression, and granuloma formation) observed in sarcoidosis patients. Based on these observations, we hypothesized that MMP12 plays a role in the acute and late inflammatory response in pulmonary sarcoidosis. MMP12KO mice were used to address this hypothesis. Analysis of gene expression of BAL cells in C57BL/6 (wildtype) mice shows a significant elevation in MMP12 after oropharyngeal instillation of MWCNT at all time points (3, 10, 20, 60, 90 days). We observed similar trends in proinflammatory genes chemokine (C-C motif) ligand 2 (CCL2), matrix metalloproteinase 14 (MMP14), and interferon-gamma (IFNÎđ) at all time points and osteopontin (OPN) at 20, 60, and 90 days. MMP12 protein levels increased in BAL cells at all time points. Evaluation of BAL cells from MMP12KO mice shows a similarity in the expression of all proinflammatory genes explored with wildtype at 10 days. CCL2 and MMP14, identified through gene expression profiling of the wildtype to be directly regulated by MMP12, is significantly reduced at 60 days in MMP12KO MWCNT instilled mice compared to wildtype. Histological analyses at 3, 10, 20, and 60 days shows increasing exacerbation in wildtype and continuous attenuation of granuloma formation in MMP12KO mice after exposure to MWCNT. A proposed mechanism for the reduction of granulomas at 60 days in MMP12KO, lead to an investigation into the relationship between MMP12, a pro-inflammatory mediator and PPAR[gamma], an anti-inflammatory modulator. Gene analysis showed a significant increase in MMP12 in PPAR[gamma]KO mice compared to wildtype and a substantial rise in PPARÎđ in MMP12KO mice compared to wildtype. Interestingly, MMP12 significantly increased, and PPAR[gamma] decreased dramatically in African American sarcoid patient's vs. controls when adjusted for race. MMP12 is seemingly instrumental in driving granuloma pathogenesis during inflammation. Evaluation of genes in MMP12KO mice suggests that the macrophage-secreted cytokines and matrix genes explored are necessary for granuloma formation. The significant increase in PPAR[gamma] intrinsically and after instillation with MWCNT in MMP12KO and its' decrease in wildtype mice after MWCNT instillation at 60 days suggests an inverse relationship between MMP12 and PPAR[gamma]. A reduction in granuloma formation in MMP12KO mice compared to wildtype supports a critical role for MMP12 in granuloma formation.