In the crosshairs: Visualizing lytic granules by high resolution microscopy and electrophysiology

Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules, at the CTL-target cell interface (the immunological synapse; IS). The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes. In this review we highlight and discuss three high-resolution techniques that have proven invaluable in the effort to decipher key features of the mechanism of CTL effector function and in particular lytic granule maturation and fusion. Correlative light and electron microscopy allows the correlation between organelle morphology and localization of particular proteins, while total internal reflection fluorescence microscopy (TIRFM) enables the study of lytic granule dynamics at the IS in real time. The combination of TIRFM with patch-clamp membrane capacitance measurements finally provides a tool to quantify the size of fusing lytic granules at the IS

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair. ?? 2019 Elsevier Inc.TNF-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. Kusnadi et al. find that TNF stimulation of macrophages results in late-phase activation of cholesterol regulator SREBP2. SREBP2 binds to inflammatory and interferon response target genes and promotes inflammation. Inhibition of SREBP activity promotes M2-like polarization and improves wound healing