Mitochondrial profiling across macrophage states reveals inhibition of IL-4/IL-13 reprogramming by the integrated stress response

Mitochondria drive cellular reprogramming by integrating metabolism and signaling. In macrophages, mitochondria are central to immunometabolic responses to external cues, but the extent to which they are remodeled and participate in macrophage reprogramming remains unclear. Here, we integrate transcriptomics with whole-cell and purified mitochondrial proteomics to profile LPS/IFNγ- and IL-4/IL-13-stimulated macrophages. We reveal a striking disconnect between mitochondrial transcript and protein levels following either stimulus, and a STAT6-dependent increase in mitochondrial DNA (mtDNA) expression and intramitochondrial translation in IL-4/IL-13 macrophages. We demonstrate that pharmacological inhibition of mitochondrial translation or individual respiratory chain complexes variably impairs reprogramming, whereas ATP synthase inhibition uniquely triggers an HRI-dependent integrated stress response (ISR) through mitochondrial hyperpolarization, thereby preventing IL-4/IL-13 reprogramming. Mechanistically, we show that restoring mitochondrial membrane potential or inhibiting the ISR rescues IL-4/IL-13-mediated reprogramming. Together, we identify mtDNA expression, intramitochondrial translation, and mitochondrial membrane potential as critical, drug-sensitive determinants of the IL-4/IL-13 response.