Mitochondrial respiration contributes to the interferon gamma response in antigen-presenting cells
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Abstract
The immunological synapse allows antigen-presenting cells (APCs) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While pathways downstream of toll-like receptors rely on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in murine macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multiscreen approach enabled us to identify novel pathways that preferentially control functionally distinct proteins. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 5 2021, follows.
Summary
In this manuscript, Olive and colleagues used a genetic screen to identify Complex I (CI) of the electron transport chain (ETC) as a regulator of IFNg-mediated gene expression in macrophages. They attribute this role of CI to effects on the activity of the JAK-STAT pathway downstream of the IFNg receptor.
While a potential link between CI activity and the activity of the JAK-STAT pathway would be interesting, the reviewers think that additional analyses are needed to substantiate this claim and rule out alternative interpretations.
Essential Revisions
Lines 204-205: The authors find that sgRNAs targeting other complexes of the ETC, including CIII and CIV, had no effect on the ability of IFNg to stimulate …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 5 2021, follows.
Summary
In this manuscript, Olive and colleagues used a genetic screen to identify Complex I (CI) of the electron transport chain (ETC) as a regulator of IFNg-mediated gene expression in macrophages. They attribute this role of CI to effects on the activity of the JAK-STAT pathway downstream of the IFNg receptor.
While a potential link between CI activity and the activity of the JAK-STAT pathway would be interesting, the reviewers think that additional analyses are needed to substantiate this claim and rule out alternative interpretations.
Essential Revisions
Lines 204-205: The authors find that sgRNAs targeting other complexes of the ETC, including CIII and CIV, had no effect on the ability of IFNg to stimulate expression of cell surface markers. How do the authors interpret these findings, since CI does not work in isolation in the ETC and is rather dependent on CIII and CIV activity?
How does IFNg stimulation affect oxidative metabolism as assessed by Seahorse? In order to corroborate the authors' conclusions regarding activity of individual ETC complexes (point 1 above), Seahorse analysis of individual complexes is also advised.
The authors do some limited analyses in human MDMs to suggest that their findings in the mouse macrophage cell line can be generalized to other macrophage populations. It would be great if the analyses in the human MDMs could be extended to further strengthen the generality of their central findings.
Fig 6D: Not clear whether similar exposures were used in different panels. Would be better to load samples in the same gel so that the same exposure can be used and a direct comparison between conditions can be made.
Fig 6D: Does acute treatment with rotenone (but not inhibitors of other ETC complexes) have similar effects in reducing JAK-STAT signaling as knockdown of CI subunits? If not, then stable, long-term knockdown of CI subunits may have some effect independent of respiration in influencing JAK-STAT signaling (for example, on expression of some component of the JAK-STAT pathway). This interpretation could also explain why knockdown of other components of the ETC do not have similar effects to CI. Rotenone treatment could be tried (and compared with inhibitors of other ETC complexes), and if the data are different from knockdown of CI subunits, then related data in the study could be re-interpreted and conclusions modified.
In Fig. 3H a key control is missing. What about survival of the cells when the import of the only energy substrate is blocked?
The authors could consider placing their findings in the context of the broader literature. (As just one example, Ivashkiv Nat Imm 2015 described a role for mTORC1 and metabolism in IFNg-mediated transcriptional and translational regulation in macrophages.) This would increase the impact of their findings.
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