Fettrelet, T. | Search bioRxiv

bioRxiv2025

Mitochondrial Dysfunction Rewires Macrophage Metabolism, Driving Pro-inflammatory Priming and Immune System Remodeling

Macrophage activation is tightly coupled to cellular metabolism: classically activated pro-inflammatory (M1) macrophages rely on glycolysis and a disrupted tricarboxylic acid cycle, whereas alternatively activated (M2) macrophages depend on oxidative phosphorylation (OXPHOS) and fatty acid oxidation. Although mitochondria are central to this metabolic plasticity, it remains unclear whether mitochondrial dysfunction itself can dictate macrophage polarization. Using macrophage-specific OPA1 knockout mice, we investigated how mitochondrial dysfunction influences macrophage metabolism and immune homeostasis. Loss of OPA1 caused severe impairment of OXPHOS, reduced mitochondrial membrane potential, and a compensatory glycolytic shift, driving M0 and M2 macrophages toward an M1-like bioenergetic state. Integrative metabolomic and transcriptomic analyses revealed strong priming of OPA1-deficient macrophages towards classical activation, including accumulation of M1-associated metabolites (lactate, succinate, itaconate) and upregulation of NF-{kappa}B-driven and other inflammatory gene programs, resulting in increased secretion of IL-6 and TNF even in the absence of stimulation. Functionally, this metabolic shift primed non-activated and M2 macrophages toward partial M1 polarization with enhanced bactericidal capacity, while simultaneously suppressing M2-associated processes such as proliferation, efferocytosis, and expression of Arg1, CD206, and RELM. In vivo, OPA1{Delta}M mice displayed reduced peritoneal macrophage abundance, impaired self-renewal after IL-4 complex stimulation, and compensatory monocyte recruitment. The remaining macrophages exhibited increased MHCII and reduced RELM expression, consistent with partial M1 skewing and loss of alternative activation. These local alterations were mirrored systemically: blood profiling revealed enhanced T-cell activation and sex-specific remodeling of immune composition during inflammation and aging. Collectively, these findings demonstrate that mitochondrial dysfunction serves as a cell-intrinsic cue that primes macrophages toward a glycolytic, pro-inflammatory phenotype while constraining their M2 properties and proliferative capacities. This dual metabolic and functional rewiring highlights mitochondrial integrity as a pivotal determinant of macrophage immunometabolic identity and reveals how its disruption can reshape both local and systemic immune homeostasis.

immunology↗

bioRxiv2021

The Card19 locus of murine chromosome 13 regulates terminal cell lysis downstream of caspase activation and Gasdermin-D cleavage

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1{beta} release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection. Author SummaryProgrammed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

immunology↗