Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis (original) (raw)
Fig 5
AMPK regulates neutrophil MMP-8 secretion in TB in vitro.
(A) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from n = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (B) CoMTB stimulation phosphorylates AMPKα1/2 analyzed by western blotting and gel densitometry. Neutrophils were stimulated for 30 minutes. Bars represent mean ± s.e.m from n = 3 donors. (C) Neutrophils were infected with M.tb MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. M.tb caused maximal phosphorylation at 120 mins. (D) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (E) Compound C (Comp C) was pre-incubated for 30 minutes before stimulation with CoMCont or CoMTB for 24 hours with MMP-8 gene expression analyzed by real-time PCR normalized to GAPDH. Bars represent mean ± s.d. of an experiment performed in biological triplicates on at least 2 occasions. *P<0.05, ***P<0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.