Examination of Fas-Induced Apoptosis of Murine Thymocytes in Thymic Tissue Slices Reveals That Fas Is Dispensable for Negative Selection.

Chang-Feng Chu,Kuang-Hui Sun,Ivan L Dzhagalov,Hsing-Kai Feng,Chia-Lin Hsu

doi:10.3389/fcell.2020.586807

Abstract

The death receptor Fas can induce cell death through the extrinsic pathway of apoptosis in a variety of cells, including developing thymocytes. Although Fas-induced cell death has been researched and modeled extensively, most of the studies have been done in vitro because of the lethality of Fas triggering in vivo. Thus, little is known about the time line of this type of cell death in vivo, specifically, how does the presence of macrophages and pro-survival cytokines affect apoptosis progression. In addition, although the sequence and timing of events during intrinsic pathway activation in thymocytes in situ have been described, no corresponding data for the extrinsic pathway are available. To address this gap in our knowledge, we established a novel system to study Fas-induced thymocyte cell death using tissue explants. We found that within 1 h of Fas ligation, caspase 3 was activated, within 2 h phosphatidylserine was externalized to serve as an “eat-me” signal, and at the same time, we observed signs of cell loss, likely due to efferocytosis. Both caspase 3 activation and phosphatidylserine exposure were critical for cell loss. Although Fas ligand (FasL) was delivered simultaneously to all cells, we observed significant variation in the entry into the cell death pathway. This model also allowed us to revisit the role of Fas in negative selection, and we ruled out an essential part for it in the deletion of autoreactive thymocytes. Our work provides a timeline for the apoptosis-associated events following Fas triggering in situ and confirms the lack of involvement of Fas in the negative selection of thymocytes.

Highlights

Apoptosis is a fundamental biological process that is important for the immune system (Nagata, 2018)
To ensure that only a small fraction of cells will undergo programmed cell death, avoiding massive apoptosis, we took advantage of lpr mice that have a mutation in Fas and are unresponsive to Fas ligand (FasL) (Watanabe-Fukunaga et al, 1992)
The presence of labeled lpr cells controlled for the efficiency of penetration into the slices and non-specific cell death as they were not affected by the SuperFas ligand (sFasL)

Summary

Introduction

Apoptosis is a fundamental biological process that is important for the immune system (Nagata, 2018). The death signal leads to permeabilization of the outer mitochondrial membrane controlled by members of the Bcl-2 family and the release of molecules that lead to the activation of caspase 3 (Singh et al, 2019). The extrinsic pathway is initiated by ligation of death receptors on cell surface such as Fas (Strasser et al, 2009). Caspase 3 has >1000 substrates (Nagata, 2018), and their destruction interrupts vital cell processes and marks the cell for efferocytosis by macrophages through the production of “eat-me” signals. Caspase 3 inactivates the flippases and activates the scramblase XKR8 (Suzuki et al, 2013) that moves the PS to the plasma membrane’s outer leaflet. While in vitro apoptosis often turns into secondary necrosis, in vivo dying cells are very quickly cleared by macrophages (Nagata, 2018), usually before the appearance of some of the classical features of apoptosis such as nuclear condensation and blebbing (Dzhagalov et al, 2013)

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Results

Conclusion