Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer.

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1's affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1's in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer's cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1's high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Trained immunity is regulated by T cell-induced CD40-TRAF6 signaling.

Trained immunity is characterized by histone modifications and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to heightened responsiveness to secondary stimuli. Although our understanding of the molecular regulation of trained immunity has increased, the role of adaptive immune cells herein remains largely unknown. Here, we show that T cells modulate trained immunity via cluster of differentiation 40-tissue necrosis factor receptor-associated factor 6 (CD40-TRAF6) signaling. CD40-TRAF6 inhibition modulates functional, transcriptomic, and metabolic reprogramming and modifies histone 3 lysine 4 trimethylation associated with trained immunity. Besides in vitro studies, we reveal that single-nucleotide polymorphisms in the proximity of CD40 are linked to trained immunity responses in vivo and that combining CD40-TRAF6 inhibition with cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)-mediated co-stimulatory blockade induces long-term graft acceptance in a murine heart transplantation model. Combined, our results reveal that trained immunity is modulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells and that this can be leveraged for therapeutic purposes.

The AMPK activator metformin improves recovery from demyelination by shifting oligodendrocyte bioenergetics and accelerating OPC differentiation.

Multiple Sclerosis (MS) is a chronic disease characterized by immune-mediated destruction of myelinating oligodendroglia in the central nervous system. Loss of myelin leads to neurological dysfunction and, if myelin repair fails, neurodegeneration of the denuded axons. Virtually all treatments for MS act by suppressing immune function, but do not alter myelin repair outcomes or long-term disability. Excitingly, the diabetes drug metformin, a potent activator of the cellular "energy sensor" AMPK complex, has recently been reported to enhance recovery from demyelination. In aged mice, metformin can restore responsiveness of oligodendrocyte progenitor cells (OPCs) to pro-differentiation cues, enhancing their ability to differentiate and thus repair myelin. However, metformin's influence on young oligodendroglia remains poorly understood. Here we investigated metformin's effect on the temporal dynamics of differentiation and metabolism in young, healthy oligodendroglia and in oligodendroglia following myelin damage in young adult mice. Our findings reveal that metformin accelerates early stages of myelin repair following cuprizone-induced myelin damage. Metformin treatment of both isolated OPCs and oligodendrocytes altered cellular bioenergetics, but in distinct ways, suppressing oxidative phosphorylation and enhancing glycolysis in OPCs, but enhancing oxidative phosphorylation and glycolysis in both immature and mature oligodendrocytes. In addition, metformin accelerated the differentiation of OPCs to oligodendrocytes in an AMPK-dependent manner that was also dependent on metformin's ability to modulate cell metabolism. In summary, metformin dramatically alters metabolism and accelerates oligodendroglial differentiation both in health and following myelin damage. This finding broadens our knowledge of metformin's potential to promote myelin repair in MS and in other diseases with myelin loss or altered myelination dynamics.