Aug 2026· Journal of Biological Chemistry· pp.
113452
· 0 citations· 49 references
Medicine
TL;DR
Translational control is redefined as a central checkpoint in macrophage activation, revealing how GCN2 mitigates ribosomal stress to prevent inflammatory hyperactivation, with potential therapeutic implications for TNFα-driven inflammatory diseases.
Abstract
Macrophages orchestrate inflammation through rapid and extensive proteome remodeling, yet the translational programs governing macrophage activation remain poorly defined. Here, we show that classically activated macrophages (LPS+IFNγ-treated) and alternatively activated macrophages (IL-4-treated) engage fundamentally distinct translational trajectories. Whereas alternatively activated macrophages sustain elevated protein synthesis, classically activated macrophages undergo a rapid but transient increase in translation that is subsequently restrained by the integrated stress response (ISR) kinase General Control Nonderepressible 2 (GCN2). Using puromycin incorporation, polysome profiling, and quantitative proteomics, we demonstrate that GCN2-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) limits global translation and constrains the pro-inflammatory response. Genetic loss of GCN2 results in excessive translation and hyperinflammation driven by the ribosome-associated stress sensor ZAKα (MAP3K20). Importantly, pharmacological inhibition of ZAKα in GCN2-deficient macrophages selectively normalizes tumor necrosis factor α (TNFα) secretion, establishing a functional regulatory axis in which GCN2 suppresses ZAKα-dependent inflammatory signaling. Together, these findings redefine translational control as a central checkpoint in macrophage activation, revealing how GCN2 mitigates ribosomal stress to prevent inflammatory hyperactivation, with potential therapeutic implications for TNFα-driven inflammatory diseases.
Glucose metabolism is pivotal in regulating innate immune responses in primary human monocyte-derived macrophages (MDMs). Lipopolysaccharide (LPS) stimulation induces both inflammatory and antiviral programs; however, despite the established importance of glucose metabolism in these responses, its precise role in coordinating them remains poorly defined. Here, we identify the STAT1/NF-κB/IRF5 signaling axis as a key mediator linking glucose metabolism to inflammatory responses through the upregulation of the rate-limiting glycolytic enzyme PFKFB3. We found that LPS triggered delayed expression and activation of NF-κB p65, accompanied by increased expression of inflammatory target genes, including CD38 and CD40. Using complementary pharmacological and genetic approaches, we demonstrate that glycolysis and PFKFB3 activity are required for NF-κB p65 expression and activation. Strikingly, inhibition of PFKFB3 also suppressed LPS-induced STAT1 activation and nuclear translocation, revealing a glucose-dependent amplification loop that potentiates STAT1-mediated antiviral and NF-κB p65-mediated inflammatory responses. Collectively, these findings establish a mechanistic link between glycolytic metabolism and STAT1/IRF5- and NF-κB-dependent transcriptional programs in human MDMs responding to LPS, highlighting potential therapeutic targets for modulating innate immune responses in inflammatory disease.
Adebola A. Owolabi, Yetunde I Kayode, Deanna C. Clemmer et al.· bioRxiv· 0 citations
Sepsis-associated liver dysfunction is a life-threatening condition with a high mortality rate and no mechanism-based therapy. In this study, we identify the cross-linking enzyme transglutaminase 2 (TG2) as a driver of liver inflammation by activating macrophages in a mouse model of sepsis. Pharmacological inhibition of TG2 improves survival and reduces multiorgan inflammation, with the liver as a primary therapeutic target. Mechanistically, TG2 activity was up-regulated in macrophages, where it cross-linked vimentin to promote oligomerization and intermediate filament remodeling. Genetic ablation of TG2 or vimentin suppressed macrophage cytokine production and attenuated lipopolysaccharide-induced inflammation. Notably, vimentin-deficient macrophages exhibited enhanced proteasome recruitment to detergent-insoluble protein aggregates, accelerating the degradation of proinflammatory mediators such as Traf6, thereby dampening nuclear factor κB signaling. Proteomic profiling revealed a previously unrecognized Rab27a-positive vesicle trafficking pathway for inflammatory aggregate clearance. Together, these findings define a TG2–vimentin axis that controls macrophage activation through proteostasis regulation, linking cytoskeletal remodeling to inflammatory signaling.
Ya-Li Xu, Ting Su, Hricha Mishra et al.· Science Advances· 0 citations
The cGAS-STING pathway senses cytosolic DNA derived from both pathogens and host cells and plays a central role in innate immune responses. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes alkylation-induced DNA lesions and modulates macrophage inflammatory responses. Here, we investigated the role of MGMT in macrophage responses to STING activation. Bone marrow-derived macrophages (BMMs) from Lyz2ΔMgmt mice produced higher levels of IL6, TNFα, and IFNβ following stimulation with the STING agonist DMXAA, accompanied by increased phosphorylation of TBK1 and IRF3. Lyz2ΔMgmt BMMs also exhibited increased expression of CD86, CD40, and CD120a (TNFRI), but reduced MHC class II expression. Metabolic flux analysis revealed enhanced mitochondrial oxidative respiration, increased ATP production, and greater maximal respiratory capacity, whereas glycolytic capacity remained unchanged. In addition, DMXAA-stimulated Lyz2ΔMgmt BMMs displayed increased γH2AX levels and reduced activation of the energy sensor AMPK and autophagy. Transcriptomic analysis further identified enrichment of pathways associated with cellular respiration. Collectively, these findings indicate that MGMT deficiency is associated with enhanced STING-induced inflammatory responses, altered cellular metabolism, and increased DNA damage in macrophages.
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 lipopolysaccharide (LPS)/interferon-γ (IFN-γ)– and interleukin-4 (IL-4)/IL-13–stimulated macrophages. We reveal a notable disconnect between mitochondrial transcript and protein levels following either stimulus and a signal transducer and activator of transcription 6 (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 a heme-regulated inhibitor (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.
Joan Blanco-Fernández, Miriam Lisci, Mads M. Foged et al.· Science Advances· 0 citations
Recognition of the bacterial product lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) initiates inflammatory responses. The unfolded protein response (UPR) elicited by endoplasmic reticulum (ER) stress can strengthen TLR4-dependent inflammatory responses. Here, we report that the ER-localized E3 ubiquitin ligase TRIM13 restrained LPS-induced inflammatory responses in macrophages and in mice by protecting the cells from ER stress. TRIM13 mediated Lys33-linked polyubiquitylation of the ER-localized Ca2+ sensor STIM1, promoting its degradation. TRIM13 deficiency caused STIM1 accumulation and activated store-operated Ca2+ entry (SOCE) and the inositol-requiring enzyme 1 α (IRE1α) branch of the UPR. Suppressing SOCE, chelating extracellular Ca2+, relieving ER stress, or blocking IRE1α activation inhibited the amplification of inflammatory responses caused by the loss of TRIM13 in macrophages. Pharmacological inhibition of IRE1α ameliorated chemically induced colitis in TRIM13-deficient mice. Our study suggests that TRIM13 restrains inflammation by limiting LPS-induced activation of SOCE and the IRE1α branch of the UPR.
N6-methyladenosine (m6A) RNA modification regulates pulmonary inflammation, yet the precise function of the m6A reader IGF2BP2 in acute lung injury (ALI) remains elusive. Here, we reveal that IGF2BP2 and global m6A levels are significantly upregulated in lipopolysaccharide (LPS)-induced ALI. Prophylactic genetic silencing or pharmacological inhibition of IGF2BP2 robustly mitigated pulmonary hyperinflammation, epithelial apoptosis, and pathological tissue damage. Mechanistically, transcriptome profiling identified MARCKSL1 as a direct downstream target. We demonstrate that IGF2BP2 binds and post-transcriptionally stabilizes MARCKSL1 mRNA in a partially METTL3/m6A-dependent manner. Elevated MARCKSL1 subsequently drives NF-κB p65 nuclear translocation and activation, fueling the inflammatory cascade. Strikingly, we trace this IGF2BP2 dysregulation upstream to inflammatory metabolic reprogramming: elevated alveolar glycolysis drives lactate accumulation, which triggers EP300-mediated histone lactylation (H3K18la) at the IGF2BP2 promoter to activate its transcription. Pharmacological manipulation of glycolysis confirmed this metabolic-epigenetic coupling. Collectively, our findings decipher a novel H3K18la/IGF2BP2/MARCKSL1 signaling cascade that integrates metabolic reprogramming with m6A epitranscriptomics in ALI, highlighting IGF2BP2 as a promising target for preventive intervention in airway epithelial inflammation.
Yangdi Peng, Junyao Zhang, Yang Yang et al.· Molecular Immunology· 0 citations
