Fever-Like Hyperthermia Controls T Lymphocyte Persistence by Inducing Degradation of Cellular FLIPshort

Abstract

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIPS) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIPS, the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIPS. Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.

PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability

“…19,24,25 Our previous studies have shown that ubiquitylation-mediated isoform-specific regulation of c-FLIP stability is vital for death receptor responses upon erythroid differentiation and lymphocyte persistence during hyperthermia. 26,27 In this study, we uncovered how c-FLIP is regulated by phosphorylation, as we identified S193 as a novel in vivo phosphorylation site of all c-FLIP proteins and demonstrated that S193 phosphorylation opposes c-FLIP ubiquitylation. Furthermore, although S193A mutation rendered all c-FLIP proteins susceptible for ubiquitylation, S193 phosphorylation selectively increased the stability of c-FLIP S and c-FLIP R .…”

Modeling Reveals That Dynamic Regulation of c-FLIP Levels Determines Cell-to-Cell Distribution of CD95-mediated Apoptosis

“…It has also been found that heat shock sensitizes cells to CD95-mediated apoptosis in Jurkat and HeLa cells, independently of the expression of Hsp70 (9,10). The study demonstrated that a 30-min heat shock treatment is sufficient to decrease the levels of c-FLIP L and c-FLIP S and to sensitize the cells to apoptosis mediated by death receptors.…”

“…To fit experimental data, the model incorporates both the long and short c-FLIP isoforms to account for their different reaction mechanisms. The two short c-FLIP isoforms, c-FLIP S and c-FLIP R , have been shown to affect caspase-8 activation and apoptosis sensitivity in a similar way (8,10,27,33), and they have similar half-lives. As the c-FLIP property that is important for this study is its half-life, and as the half-lives of the short forms of c-FLIP, i.e.…”

“…The study demonstrated that a 30-min heat shock treatment is sufficient to decrease the levels of c-FLIP L and c-FLIP S and to sensitize the cells to apoptosis mediated by death receptors. Hyperthermia also sensitizes activated primary human T lymphocytes to CD95-induced apoptosis, primarily due to induced down-regulation of c-FLIP S (10). It was also demonstrated that the hyperthermia does not affect the synthesis of c-FLIP but rather rapidly enhances the proteasomal degradation of c-FLIP L and c-FLIP S .…”

“…The statistical treatment and Monte Carlo simulations made it possible to compare the outcome of the simulations with results from empirical studies, in which the proportion of apoptotic cells is reported. This approach was also employed to study the effect of down-and up-regulation of c-FLIP under conditions similar to those used in the previously reported heat shock experiments (9,10). To mimic experimental studies of apoptosis in which c-FLIP levels are dynamically regulated and in which this regulation is a key factor for the outcome (6,8,10), the model was here modified to encompass dynamic c-FLIP turnover.…”