Macrophages are natural host cells for pathogenic mycobacteria, like Mycobacterium tuberculosis (M.tb). Immune surveillance by T cells and interaction with M.tb infected macrophages is crucial for protection against M.tb reactivation and development of active tuberculosis. Several factors play a role in the control of M.tb infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional in vitro assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional in vitro assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a Mycobacterium bovis BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated in vitro and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. M.tb protein specific CD4+ and CD8+ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4+ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional in vitro assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.

Dilated cardiomyopathy (DCM), an incurable disease of the cardiomyocyte terminating in systolic heart failure (HFrEF), is prevalent, causes hospitalization and is associated with increased mortality. Despite evidence of immune activation in DCM, anti-inflammatory interventions so far did not prove to alter the course of this disease. Here we show that myeloperoxidase (MPO), the principal heme peroxidase expressed by polymorphonuclear neutrophils (PMN) and monocytes, critically contributes to HFrEF in DCM. Muscle LIM protein (MLP) deficient mice, which spontaneously develop DCM, display increased circulating PMN counts and augmented levels of vessel-immobilized MPO. Genetic ablation and pharmacological inhibition of MPO resulted in enhanced nitric oxide (NO) bioavailability of systemic conductance and resistance vessels, and subsequently restoration of systolic left ventricular (LV) function, whereas infusion of MPO worsened systolic LV function. When patients diagnosed for DCM were treated with an orally available MPO inhibitor, systolic LV function increased, natriuretic peptides declined, and functional status improved. Impairment of endothelial NO bioavailability by release of leukocyte-derived MPO evolves as a disease-aggravating mechanism in DCM. MPO inhibition profoundly improved ventricular function by lowering systemic vascular resistance and thus holds promise as a novel and complementary treatment strategy for patients with DCM.