IMMUNOPATHOLOGY OF DIABETES MELLITUS IN PULMONARY TUBERCULOSIS IS DRIVEN BY NEUTROPHIL HYPER-INFLAMMATORY DYSFUNCTION: DATA FROM A CELLULAR MODEL AND HUMAN COHORT.

Abstract

Intro: Diabetes mellitus (DM) patients with pulmonary tuberculosis (TB) have increased mycobacterial burden and pulmonary cavitation. Neutrophilia, upregulated inflammatory genes and systemic matrix metalloproteinases (MMPs) protein expression are observed, but how neutrophils drive DM-TB immunopathology is unclear. We examined neutrophil function, MMPs, inhibitors of MMPs-tissue inhibitor of matrix metalloproteinases (TIMPs), and chemokine expression in a DM-TB cellular model and patients to identify mechanisms of dysregulation. Methods: Primary neutrophils from type 2 DM patients (HbA1c>8%) and healthy control (HC) were stimulated with Mycobacterium tuberculosis (M.tb). Reactive oxygen species (ROS) was assessed by luminol-amplified chemiluminescence and neutrophil extracellular traps (NETs) by ELISA. Neutrophil lysates were analysed using NF-kβ array. Plasma and sputum from a cohort of 33 TB and DM-TB patients were analysed for proteases and chemokines by luminex bead array. Findings: M.tb-infected DM neutrophils had increased ROS generation and MMP-9 secretion compared to HC (p<0.05). DM-TB patients had suppressed NETs, with lower plasma neutrophil elastase (NE) than non-DM TB patients (p<0.05), while M.tb-infected DM neutrophils resulted in lower NE-DNA release compared to HC (p=0.0736). Tumor necrosis factor receptors TNFR1 and TNFR2 expression were upregulated in M.tb-infected DM neutrophils compared to HC. MMP-8, MMP-9 and IL-8 were upregulated 6.68-, 2.42- and 3.74-fold respectively in DM-TB patients’ sputum compared to non-DM TB patients (all p<0.05). Sputum MMP-8 and -9 strongly associated with acid-fast bacilli burden (both r=0.6, p<0.001), while IL-8 strongly associated with HbA1c% (r=0.62, p<0.01). Upregulated plasma MMP-8 in DM-TB patients is strongly associated with peripheral neutrophil count (r=0.7815, p<0.05). Discussion: The hyper-inflammatory response with increased matrix-degrading phenotype in DM-TB is principally driven by neutrophils, with upregulated MMP-8/-9 in both systemic and respiratory compartments, relative to TB patients without DM. Neutrophil MMPs strongly associated with pulmonary M.tb burden with neutrophil chemokine IL-8 strongly associated with uncontrolled DM. Conclusion: Neutrophil hyper-inflammatory responses drive a worsened DM-TB immunopathology.