Introduction The interactions between macrophages and inflamed white adipose tissue (WAT) have been implicated to play a role in several diseases including diabetes mellitus type 2, obesity, and breast cancer. Macrophages degrade dead adipocytes through a process known as “exophagy”, which is extracellular lysosomal hydrolysis. They form crown-like structures (CLSs), encircling dead or dying adipocytes. The macrophages create a tight seal on the adipocytes and their lysosomal contents are secreted into these seals or compartments which are known as lysosomal synapses. This project was investigating the way that macrophages digest dead adipocytes, specifically if the autophagosome protein marker LC3 is recruited to the apoptotic cell phagosome in CLS macrophages. Autophagy and phagocytosis have traditionally been thought of as completely separate processes, but recent studies have shown that there are some autophagy proteins which are involved in the phagocytosis of apoptotic cells (Florey et al, 2012). These autophagy proteins can lipidate microtubule-associated protein light chain 3 (LC3) onto phagosomes in a way that is independent of the autophagosome. This recruitment of autophagy proteins LC3 and Beclin1 to phagosomes has been called LC3-associated phagocytosis, or LAP, and ends in the acidification and lysosome fusion with phagosomes. Even though LAP was originally only thought to be used for the phagocytosis of pathogens, autophagy is also being shown to play a role in apoptotic cell engulfment, with some research showing that autophagy proteins play a role within phagocytes (such as macrophages) for regulating the degradation of apoptotic cells. Methods The adipocytes used in this model system were differentiated 3T3 L1 cells. In order to induce apoptosis in the adipocytes, they were incubated with 25 nM TNF-a for 24 hours, or on one occasion exposed to 365 nm UV radiation for 1 hour. The macrophages that were used in this project were from the J774 cell line and also the stable cell line J774 GFP LC3. In order to replicate the in vivo setting of macrophages surrounding a dead adipocyte, the in vitro CLS cell culture model was carried out based on preliminary work that had been already established in the Maxfield lab. All fluorescence microscopy experiments were carried out using the confocal microscope Zeiss LSM 880 equipped with the high resolution detector, Airyscan. After the adipocytes were incubated with TNF-a for 24 hours to induce apoptosis, they were labeled with NHS-Alexa633, and the macrophages were trypsanized in order to add them to the adipocytes. Several different time points were used for this experiment, starting every 30 min from 30 min post addition to 210 minutes. Macrophages were added to each dish containing adipocytes and the dishes were incubated for the specified times. At the end of the incubation time, the macrophages and adipocytes were fixed by adding PFA for 10 min, and then the PFA was washed off and blocking IF solution was added for 1 hour. Primary antibody diluted in BSA was added after the blocking step. The dishes were then left in the cold room overnight. The following day the secondary antibody was added, and left for 60 min at room temperature. This was then washed off and the dishes were left in PBS until imaging with the confocal microscope Zeiss LSM 880 equipped with the high resolution detector, Airyscan. In order to have a control for the experiment, previous researchers had used zymosan, since it is known to induce LAP, and that LC3 is translocated to the zymosan containing phagosome. We therefore had dishes with macrophages and zymosan instead of the apoptotic adipocytes. There were also dishes solely with macrophages imaged however these were also simply a control and not leading to any results. Results The results obtained from the confocal imaging of the macrophages with apoptotic adipocytes were consistent in showing no recruitment of LC3 to the macrophage-adipocyte contact sites. The adipocytes were labeled in blue with the NHS-Alexa633, the macrophages and LC3 with the Alexa546, and the plasma membrane of the macrophages were labeled in green with the Alexa488. Discussion The results of the study showed that the hypothesis was rejected, in that LC3 is not recruited to the phagosome in the digestion of dead adipocytes by macrophages, and therefore the process is not a form of LC3- associated phagocytosis. Although past research has shown that the ideal time to image LC3 being recruited would be at 120-150 minutes, images taken both before and after those times yielded no results in this case. Because this experiment had never been done before by the lab or by any other researchers, there was no reason to either specifically refute or support the fact that LC3 would be involved and the process would be LAP for CLS macrophages with dead adipocytes. In the future, the search for regulatory molecules that act in this process may lead to improved strategies to prevent or treat type 2 diabetes, metabolic disorders and certain types of cancers.


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