The complex direct and indirect interplay between adipocytes and various adipose tissue (AT)-resident immune cells plays an important role in maintaining local and whole-body insulin sensitivity. Adipocytes can directly interact with and activate AT-resident invariant natural killer T (iNKT) cells through CD1d-dependent presentation of lipid antigens, which is associated with anti-inflammatory cytokine production in lean AT (IL-4, IL-10). Whether alterations in the microenvironment, i.e., increased free fatty acids concentrations or altered cytokine/adipokine profiles as observed in obesity, directly affect adipocyte-iNKT cell communication and subsequent cytokine output is currently unknown. Here we show that the cytokine output of adipocyte-iNKT cell interplay is skewed by a lipid-rich microenvironment. Incubation of mature 3T3-L1 adipocytes with a mixture of saturated and unsaturated fatty acids specifically reduced insulin sensitivity and increased lipolysis. Reduced activation of the CD1d-invariant T-Cell Receptor (TCR) signaling axis was observed in Jurkat reporter cells expressing the invariant NKT TCR, while co-culture assays with a iNKT hybridoma cell line (DN32.D3) skewed the cytokine output toward reduced IL-4 secretion and increased IFNγ secretion. Importantly, co-culture assays of mature 3T3-L1 adipocytes with primary iNKT cells isolated from visceral AT showed a similar shift in cytokine output. Collectively, these data indicate that iNKT cells display considerable plasticity with respect to their cytokine output, which can be skewed toward a more pro-inflammatory profile in vitro by microenvironmental factors like fatty acids.

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Invariant Natural Killer T (iNKT) cells act at the interface between lipid metabolism and immunity, due to their restriction to lipid antigens presented on CD1d by antigen presenting cells (APC). How foreign lipid antigens are delivered to APC remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), Isoglobotrihexosylceramide (iGb3) and OCH, a sphingosine-truncated analogue of αGalCer, with very-lowdensity (VLDL) and/or low-density (LDL) lipoproteins in vitro and in vivo. We demonstrate LDL receptor (LDLR)-mediated uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a novel mechanism of lipid antigen delivery to APCs, and provides further insight in the immunological capacities of circulating lipoproteins.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.M.C.P. is supported by a Netherlands Cardiovascular Research Initiative (CVON) grant (REMAIN 2014B27) and J.P.G.S is supported by Horizon2020 ERC-2016-COG-EVICARE [725229] and BRAVE (grant number 874827. Purpose Restoring perfusion, either by coronary angioplasty or bypass anastomosis, is performed around 2 million times per year throughout Europe to treat patients after myocardial infarction. Although this treatment is highly efficient to resolve myocardial ischemia, reperfusion is accompanied by damage through the release of reactive oxygen species (ROS). This ischemia/reperfusion (I/R) injury is manifested by cardiomyocyte death, caused to a certain extent by necroptosis. As a potential means of cardioprotection, we applied the small molecule compound 547 inhibiting RIP1-kinase, a central mediator of the necroptosis pathway, in an in vitro model of human cardiac I/R injury. Methods Human foetal cardiomyocyte progenitor cells (hfCPCs) were cultured and characterized for cell type defining markers before being subjected to simulated I/R damage using hydrogen peroxide (H2O2). Cardioprotective effects of 547 were assessed 24 hours after induction of damage and application of the compound. Cell viability and necroptosis pathway activation were assessed by flow cytometry, immunocytochemistry, and western blotting. Furthermore, mitochondrial damage was determined by JC-1 staining. Results hfCPCs expressed indicative markers SCA1, GATA4, PECAM-1, VEGF and C-KIT. Treatment with compound 547 significantly protected hfCPCs from H2O2 induced damage (viable cells: 84,3 ± 4,3% vs 1,6 ± 0,31%; P<0,0001). 547 decreased the necrotic cell population (DAPI+ AnnV-) (3,0 ± 1,1% vs 10,1 ± 1,5%, P<0,0001) and reduced phosphorylation of necrosome components RIP1 (1,3 ±  0,35 fold vs 3,7 ±  0,78 fold; P< 0,01), RIP3 (1,0 ± 0,46 fold vs 4,3 ± 1,5 fold; P< 0,05), and MLKL (1,1 ± 0,15 fold vs 5,7 ± 1,7 fold; P< 0,01) without decreasing unphosphorylated protein or mRNA levels. Interestingly, 547 treatment prevented oxidative stress induced loss of nuclear RIP1 and RIP3 as measured by immunocytochemistry and western blotting of cell fractions (nuclear/cytoplasmic ratio RIP1: 0,80 ±  0,0006 [Control] vs 1,5 ±  0,17 fold [547]; P< 0,01; nuclear/cytoplasmic ratio RIP3: 0,76 ±  0,10 [Control] vs 1,6 ± 0,18 fold [547]; P< 0,01). Remarkably, compound 547 also decreased H2O2–induced mitochondrial depolarization (JC-1 staining/membrane depolarization index: 4,5 ± 0,85% vs 1,2 ±  0,38%; P< 0,05)) and expression and activation of CAMKII (0,46 ± 0,13 fold; P<0,01), a protein mediating mitochondria-dependent cell death. Conclusions Administration of compound 547 led to inhibition of necroptosis, increased cell viability and reduced mitochondrial damage in an in vitro human cardiac I/R injury model, emphasizing the potential of necroptosis inhibition for cardioprotection. Assessing clinical applicability will require further studies in relevant in vivo models.