Mitochondrial Transporter SLC25A40 Controls Immune Cell Cytokine Output

Macrophages are frontline immune cells that must carefully balance pro- and anti-inflammatory signals to maintain tissue homeostasis. Mitochondria are central to this balance, generating both ATP and reactive oxygen species (ROS) that influence cytokine production and inflammasome activation. This study asked whether the mitochondrial glutathione (mtGSH) transporter SLC25A40 plays a role in this process — a question that had not previously been investigated in immune cells.

Using murine bone-marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages, the authors first confirmed that SLC25A40 is expressed in both species and is upregulated at the mRNA and protein level following LPS stimulation. This LPS-induced increase suggested a functional role during inflammatory activation. To test this, the team used siRNA to knock down SLC25A40 expression in BMDMs.

SLC25A40 knockdown produced several interconnected defects. First, Western blot analysis using an OXPHOS antibody cocktail revealed destabilization of ISC-rich ETC subunits, most prominently Complex I. Second, both mitochondrial ROS (measured by MitoSOX) and cellular ROS (CellROX) were significantly elevated. Third, the cells mounted a compensatory response by upregulating expression of glutathione biosynthesis genes Gclc and Gclm. Critically, SLC25A40-deficient macrophages showed markedly reduced transcription of Il1b and Il10 following LPS stimulation, and consequently produced less mature IL-1β protein after NLRP3 inflammasome activation by nigericin or ATP. Notably, pyroptosis — measured by LDH release — was unaffected, indicating the effect was specific to cytokine synthesis rather than cell death pathways.

To confirm that these effects were driven specifically by loss of mitochondrial glutathione rather than off-target siRNA effects, the team used mitoCDNB, a mitochondrially-targeted compound that depletes mtGSH. MitoCDNB treatment phenocopied the SLC25A40 knockdown: ETC destabilization, elevated ROS, and reduced IL-1β and IL-10 production. Conversely, supplementing cells with GSH ethyl ester (GSHee), a cell-permeable glutathione precursor, partially restored pro-IL-1β expression and protein levels, strongly supporting a causal role for the SLC25A40–mtGSH axis in cytokine regulation.

These findings establish SLC25A40 as an LPS-inducible, non-redundant regulator of macrophage cytokine production operating through preservation of ETC integrity. The study identifies a new mechanistic link between mitochondrial redox homeostasis and innate immune signaling, with potential implications for inflammatory diseases, neurodegeneration, and conditions where mitochondrial dysfunction intersects with immune dysregulation.