Editorial: TRPV1: how thymocytes sense stress and respond with autophagy

Abstract

Discussion on discovery of the roles of TRPV1 in thymocyte autophagy, and its relevance.

Autophagy is an intracellular catabolic process that acts primarily by targeting intracellular components for lysosomal destruction. It is responsible for the turnover of damaged or outdated organelles, aggregated proteins, which in turn, removes sources of cellular cytotoxicity, such as ROS. Autophagy may be used to respond to many cellular stresses, either by eliminating the offending signal(s) or by acting as an alternative form of programmed cell death, termed Type II. Although the signals that induce autophagy are as varied as the cell types in which it occurs (all eukaryotic cells studied thus far appear to have some level of autophagic activity) and the substrates to be catabolized, autophagy remains an indispensable aspect of intracellular homeostasis. At this point in time, it is not fully understood how a cell integrates such a diverse array of input signals to affect the autophagic process. For example, it is not entirely clear how cells integrate signals ranging from ER stress, amino acid or serum starvation, cytokine or TLRs, or antigen receptors to determine the magnitude of the autophagic response. However, new evidence is quickly emerging that is not only filling in the gaps in specific autophagic signaling pathways but is also creating new paradigms and insights into a more general schematic of the machinery needed to induce and sustain this crucial intracellular program.

In this issue, Farfariello and colleagues [1] have proposed and elucidated a stress-sensing pathway for endogenous agonists of the TRPV1, which in turn, activates cytoprotective autophagy (Fig. 1). TRPV1 channels are cation channels expressed in a variety of blood lymphocytes, including naïve and effector CD4⁺ lymphocytes and Jurkat T cells [2]. With the use of capsaicin as a model agonist, exposed murine thymocytes had an influx of Ca²⁺ (a known consequence of TRPV1 activation) and witnessed an increase in activated AMPK. Changes occurring further downstream of TRPV1 activation included a transcriptional up-regulation and oxidation of Atg4c, an important protein in priming LC3 cleavage and lipidation, which in turn, facilitates autophagy induction by recruiting Atg1 [3], elongating the delimiting membrane [4], selecting for specific cargo by binding polyubiquitinated substrates and sequestering them into the nascent autophagosome, and finally, participating in the fusion of autophagic membranes [5]. This autophagy was decidedly prosurvival, as its inhibition led to rapid induction of apoptosis. Apoptosis brought about by the inhibition of autophagy in this system was accompanied by a down-regulation of Bcl-Xl, Atg4C, and Beclin-1, highlighting transcriptional changes associated with autophagy genes in T lineage cells (Fig. 1). Capsaicin treatment made apparent a distinct subset of DP cells, termed DP^dull thymocytes, as they had decreased levels of surface CD4 and CD8 and underwent a vigorous level of autophagy.

Figure 1. Capsaicin induces prosurvival autophagy in CD4⁺CD8⁺ double-positive thymocytes through TRPV1 activation, which is inhibited by chlorpromazine (CPZ).

TRPV1 activation, in turn, results in calcium influx, inhibited by chelation using EDTA, and ROS production, which can be scavenged by N-acetylcysteine (NAC). This leads to AMPK activation, which is inhibited by compound C. Additionally, Beclin-1 and Atg4c are transcriptionally up-regulated, and Atg4c is oxidized. The net result is an activation of microtubule-associated protein 1 LC3 processing and activation of autophagy, which protects the CD4 CD8 DP^dull thymocytes from apoptosis.

Capsaicin is but one of many related lipids, called capsaicinoids, which include dihydrocapsaicin and can activate the TRVP1 channel. Although capsaicin and dihydrocapsaicin make up the major capsaicinoids in chili peppers, the TRVP1 channel can react to several other classes of endovanilloids or eicosanoids, signaling lipids that are important to immune function in general and specifically, to thymocyte development (reviewed in ref. [6]). Leukotrienes and PGs, both derivatives of arachodonic acid, are important mediators of inflammation and have been detected in the thymus, which likely possesses a mechanism for dealing with these stressors. Thymic epithelial cells and thymic phagocytes produce PGs and metabolites from the cyclooxygenase pathway [6]. Although controversial, it has even been demonstrated that thymocytes themselves, when in contact with thymic epithelial cells, can produce PGs, so an autocrine mechanism is even possible [7]. It has even been shown that PGE₂ and other arachodonic acid metabolites can induce apoptosis of primary rat thymocytes and has been suggested that this might play a role in thymic selection of developing T cells [8]. It is tempting to think that as developing thymocytes are committing to apoptosis by the bushel, perhaps in a process releasing arachodonic acid or similar metabolites, bystander thymocytes, which have already undergone the selection process and have up-regulated TRPV1, require cytoprotective mechanisms, such as autophagy, for survival.

Many questions remain unanswered in the model presented here. Most importantly, is the DP^dull thymocyte population a unique DP population that is formed as part of a developmental checkpoint, likely involving autophagy? Put another way, does this population arise from a quality-control mechanism, whereby developing thymocytes are exposed to signaling lipids? Or are Farfariello et al. [1] simply witnessing a population of cells that happens to express TRPV1 in a given space and time and can up-regulate autophagy and down-regulate surface CD4 and CD8 as a consequence of capsaicin treatment? If DP^dull cells do represent a distinct thymocyte population in development, to what other signals are they normally exposed (eicosanoids, hormones, ligands) that would require autophagy as a prosurvival mechanism with which to cope? Are DP^dull cells in any way related to CD8 ISP thymocytes, and if so, does autophagy play a role in the development of CD8 ISP thymocytes? Evidence from genetic ablation of Atg3, Atg5, and Atg7 in mice would suggest that thymocyte autophagy is dispensable for normal T cell development [9, 10], but it remains a possibility that autophagy in developing thymocytes might eliminate a hereto unknown or unappreciated stress that shortens the life of a mature T cell. Perhaps it is merely idle speculation to suggest that an autophagic threshold is set in DP cells to determine how much cellular stress will be tolerated before autophagy is induced as a quality-control mechanism for future encounters with inducers of autophagy. Finally, autophagy in lymphocytes has been shown to be largely cytoprotective [11]. However, calcium influx has also been suggested to be detrimental to thymocyte viability in general [12] and occurs specifically during negative selection [13]. It is of interest to better understand under what circumstances this dichotomy exists. Although it is still not entirely clear what types of stresses thymocytes sense and cope with during their development, Farfariello and colleagues [1] have mapped out a pathway by which endogenous stress-inducing signaling lipids induce autophagy to protect developing DP thymocytes.