Rotenone Treatment Reveals a Role for Electron Transport Complex I in the Subcellular Localization of Key Transcriptional Regulators During T Helper Cell Differentiation

Abstract

Recent advances in our understanding of tumor cell mitochondrial metabolism suggest it may be an attractive therapeutic target. Mitochondria are central hubs of metabolism that provide energy during the differentiation and maintenance of immune cell phenotypes. Mitochondrial membranes harbor several enzyme complexes that are involved in the process of oxidative phosphorylation, which takes place during energy production. Data suggest that, among these enzyme complexes, deficiencies in electron transport complex I may differentially affect immune responses and may contribute to the pathophysiology of several immunological conditions. Once activated by T cell receptor signaling, along with co-stimulation through CD28, CD4 T cells utilize mitochondrial energy to differentiate into distinct T helper (Th) subsets. T cell signaling activates Notch1, which is cleaved from the plasma membrane to generate its intracellular form (N1ICD). In the presence of specific cytokines, Notch1 regulates gene transcription related to cell fate to modulate CD4 Th type 1, Th2, Th17, and induced regulatory T cell (iTreg) differentiation. The process of differentiating into any of these subsets requires metabolic energy, provided by the mitochondria. We hypothesized that the requirement for mitochondrial metabolism varies between different Th subsets and may intersect with Notch1 signaling. We used the organic pesticide rotenone, a well-described complex I inhibitor, to assess how compromised mitochondrial integrity impacts CD4 T cell differentiation into Th1, Th2, Th17, and iTreg cells. We also investigated how Notch1 localization and downstream transcriptional capabilities regulation may be altered in each subset following rotenone treatment. Our data suggest that mitochondrial integrity impacts each of these Th subsets differently, through its influence on Notch1 subcellular localization. Our work further supports the notion that altered immune responses can result from complex I inhibition. Therefore, understanding how mitochondrial inhibitors affect immune responses may help to inform therapeutic approaches to cancer treatment.

Regulatory T cell differentiation is controlled by αKG-induced alterations in mitochondrial metabolism and lipid homeostasis

“…(legend continued on next page) Cell Reports 37, 109911, November 2, 2021 9 Article ll OPEN ACCESS However, in Th17-polarizing conditions, oligomycin has recently been reported to enhance Treg differentiation (Shin et al, 2020). Our results show that agents which completely blocked OXPHOS--oligomycin, rotenone, and antimycin A (Figure S5E)--significantly attenuated FoxP3 expression under Treg-polarizing conditions (Figure S5F), as previously reported (Ozay et al, 2018;Raud et al, 2018). Notably though, in contrast with these agents, 3-NP--rescuing Treg differentiation in the presence of aKG --did not block basal respiration but rather attenuated the cell's SRC (Figure S5E).…”

The role of the electron transport chain in immunity

“…25,26 In T H 17-polarising conditions, rotenone abrogated Notch1 and RAR-related orphan receptor gamma 2 (RORγt) colocalization in the nucleus and reduced T H 17 differentiation. 27 In terms of function, again, complex I was required for IL-17 production in T H 17 cells, but dispensable for IFN-α production in T H 1 or IL-4 in T H 2 cells. 23 However, conflicting results were shown by Ozay and colleagues, where rotenone treatment attenuated IFN-γ and IL-2 production.…”

“…23 However, conflicting results were shown by Ozay and colleagues, where rotenone treatment attenuated IFN-γ and IL-2 production. 27 In effector and memory CD8 + T cells, rotenone ablated T cell cytotoxicity as measured by reduced IFN-γ and TNF-α production and degranulation. 24 Together, it is suggested that complex I plays an integral role in lymphocyte differentiation, although its exact role remains to be elucidated.…”

“…In terms of function, again, complex I was required for IL‐17 production in T H 17 cells, but dispensable for IFN‐α production in T H 1 or IL‐4 in T H 2 cells 23 . However, conflicting results were shown by Ozay and colleagues, where rotenone treatment attenuated IFN‐γ and IL‐2 production 27 . In effector and memory CD8 + T cells, rotenone ablated T cell cytotoxicity as measured by reduced IFN‐γ and TNF‐α production and degranulation 24 .…”

“…29 Rotenone treatment also selectively reduced Foxp3 expression and cytokine production during inducedregulatory T cell (iT reg ) differentiation but did not affect Foxp3 expression in fully differentiated iT reg cells, suggesting a critical role of complex I in iT reg differentiation. 27,30 Rotenone treatment or complex I mutation (ND6 P25L ) also altered T reg suppressive functions, as illustrated by a lower capacity to limit T eff -cell proliferation. 31,32 The five complexes in the respiratory chain, except complex II, can dynamically assemble as larger molecular supercomplexes (SCs) in the mitochondrial inner membrane, 33 with complex I forming SCs with either complex III and complex IV, or with complex III alone.…”