Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.

Imaging flow cytometry data analysis using convolutional neural network for quantitative investigation of phagocytosis.

Macrophages play an important role in the adaptive immune system. Their ability to neutralize cellular targets through Fc receptor-mediated phagocytosis has relied upon immunotherapy that has become of particular interest for the treatment of cancer and autoimmune diseases. A detailed investigation of phagocytosis is the key to the improvement of the therapeutic efficiency of existing medications and the creation of new ones. A promising method for studying the process is imaging flow cytometry (IFC) that acquires thousands of cell images per second in up to 12 optical channels and allows multiparametric fluorescent and morphological analysis of samples in the flow. However, conventional IFC data analysis approaches are based on a highly subjective manual choice of masks and other processing parameters that can lead to the loss of valuable information embedded in the original image. Here, we show the application of a Faster region-based convolutional neural network (CNN) for accurate quantitative analysis of phagocytosis using imaging flow cytometry data. Phagocytosis of erythrocytes by peritoneal macrophages was chosen as a model system. CNN performed automatic high-throughput processing of datasets and demonstrated impressive results in the identification and classification of macrophages and erythrocytes, despite the variety of shapes, sizes, intensities, and textures of cells in images. The developed procedure allows determining the number of phagocytosed cells, disregarding cases with a low probability of correct classification. We believe that CNN-based approaches will enable powerful in-depth investigation of a wide range of biological processes and will reveal the intricate nature of heterogeneous objects in images, leading to completely new capabilities in diagnostics and therapy.

PCSK9 Imperceptibly Affects Chemokine Receptor Expression In Vitro and In Vivo.

Proprotein convertase subtilin/kexin type 9 (PCSK9) is a protease secreted mainly by hepatocytes and in lesser quantities by intestines, pancreas, and vascular cells. Over the years, this protease has gained importance in the field of cardiovascular biology due to its regulatory action on the low-density lipoprotein receptor (LDLR). However, recently, it has also been shown that PCSK9 acts independent of LDLR to cause vascular inflammation and increase the severity of several cardiovascular disorders. We hypothesized that PCSK9 affects the expression of chemokine receptors, major mediators of inflammation, to influence cardiovascular health. However, using overexpression of PCSK9 in murine models in vivo and PCSK9 stimulation of myeloid and vascular cells in vitro did not reveal influences of PCSK9 on the expression of certain chemokine receptors that are known to be involved in the development and progression of atherosclerosis and vascular inflammation. Hence, we conclude that the inflammatory effects of PCSK9 are not associated with the here investigated chemokine receptors and additional research is required to elucidate which mechanisms mediate PCSK9 effects independent of LDLR.

Features of the Population of Mouse Peritoneal Macrophages Isolated after Stimulation with Concanavalin A and Thioglycolate.

Murine peritoneal macrophages isolated from the lavage fluid after administration of thioglycolate and concanavalin A are presented by two populations of cells of different diameters. Polarization of macrophages into a proinflammatory (M1) phenotype is accompanied by an increase in number of small cells. Macrophages obtained after administration of thioglycolate demonstrate higher tendency to anti-inflammatory (M2) phenotype, while macrophages isolated after administration of concanavalin A are committed in the proinflammatory direction. Lactate level is increased in M1 macrophages in comparison with M2 cells, which indicates predominance of glycolytic metabolism. Macrophages obtained after administration of concanavalin A have reduced mitochondrial potential, which reflects a tendency to apoptosis. Autophagy activation and inhibition neutralize the differences in pro- and anti-inflammatory properties of polarized macrophages obtained after thioglycolate administration, but have less pronounced effect on macrophages obtained after administration concanavalin A. Autophagy inhibitor increases mitochondrial potential in non-polarized macrophages obtained after administration of concanavalin A. These results demonstrate divergent properties of macrophages obtained after administration of glycolate and concanavalin A due to the difference in the mechanisms of experimental peritonitis.

In vitro and in vivo evaluation of virus-induced innate immunity in mouse.

Innate immunity is the first line of host defense against viral infection. As one of the innate immune cell types, antigen-presenting cells play an important role in the process of antiviral immunity. This protocol describes the analysis of innate immunity induced by vesicular stomatitis virus infection of peritoneal macrophages in vitro and in vivo detection of IFN-ß production and lung injury. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021).

Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum.

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

Scavenger receptor A1 participates in uptake of Leptospira interrogans serovar Autumnalis strain 56606v and inflammation in mouse macrophages.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as a widespread zoonotic disease worldwide. Macrophages mediate the elimination of pathogens through phagocytosis and cytokine production. Scavenger receptor A1 (SR-A1), one of the critical receptors mediating this process, plays a complicated role in innate immunity. However, the role of SR-A1 in the immune response against pathogenic Leptospira invasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages for Leptospira. However, intraperitoneal injection of leptospires into WT mice presented with more apparent jaundice, subcutaneous hemorrhaging, and higher bacteria burdens in blood and tissues than that of SR-A1-/- mice. Exacerbated cytokine and inflammatory mediator levels were also observed in WT mice and higher recruited macrophages in the liver than those of SR-A1-/- mice. Our findings collectively reveal that although beneficial in the uptake of Leptospira by macrophage, SR-A1 might be exploited by Leptospira to modulate inflammatory activation and increase the susceptibility of infection in the host. These results provide our new insights into the innate immune response during early infection by L. interrogans.

Apoptotic Cells induce Proliferation of Peritoneal Macrophages.

The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.

Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells.

Inflammation generally leads to recruitment of monocyte-derived macrophages. What regulates the fate of these cells and to what extent they can assume the identity and function of resident macrophages is unclear. Here, we show that macrophages elicited into the peritoneal cavity during mild inflammation persist long-term but are retained in an immature transitory state of differentiation due to the presence of enduring resident macrophages. By contrast, severe inflammation results in ablation of resident macrophages and a protracted phase wherein the cavity is incapable of sustaining a resident phenotype, yet ultimately elicited cells acquire a mature resident identity. These macrophages also have transcriptionally and functionally divergent features that result from inflammation-driven alterations to the peritoneal cavity micro-environment and, to a lesser extent, effects of origin and time-of-residency. Hence, rather than being predetermined, the fate of inflammation-elicited peritoneal macrophages seems to be regulated by the environment.

Peritoneal macrophages are impaired in cathelicidin-deficient mice systemically challenged with Escherichia coli.

Cathelicidins are small, cationic peptides produced by macrophages with protective effects against infection although their involvement in phagocytosis is not fully understood. This study demonstrates that fewer macrophages were recruited in mice genetically deficient in cathelicidin (Camp-/-) during acute Escherichia coli-induced peritonitis and those macrophages had impaired phagocytosis. These defects seem due to endogenous functions of murine cathelicidin (CRAMP) as phagocytosis was not improved by synthetic human cathelicidin (LL-37) in a murine phagocytic cell line. This knowledge contributes to understanding the function of cathelicidins in the recruitment and function of phagocytic cells and differential roles between endogenous and exogenous cathelicidins.

Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) play a vital role in promoting cholesterol efflux. Although, the dysregulation of these transporters was attributed as one of the mechanisms of atherogenesis, what renders their dysfunction is not well explored. Previously, we have reported that thrombin without having any effect on ABCG1 levels depletes ABCA1 levels affecting cholesterol efflux. In this study, we explored the mechanisms underlying thrombin-induced depletion of ABCA1 levels both in macrophages and smooth muscle cells. Under normal physiological conditions, COP9 signalosome subunit 3 (CSN3) was found to exist in complex with ABCA1 and in the presence of proatherogenic stimulants such as thrombin, ABCA1 was phosphorylated and dissociated from CSN3, leading to its degradation. Forced expression of CSN3 inhibited thrombin-induced ABCA1 ubiquitination and degradation, restored cholesterol efflux and suppressed foam cell formation. In Western diet (WD)-fed ApoE-/- mice, CSN3 was also disassociated from ABCA1 otherwise remained as a complex in Chow diet (CD)-fed ApoE-/- mice. Interestingly, depletion of CSN3 levels in WD-fed ApoE-/- mice significantly lowered ABCA1 levels, inhibited cholesterol efflux and intensified foam cell formation exacerbating the lipid laden atherosclerotic plaque formation. Mechanistic studies have revealed the involvement of Par1-Gα12-Pyk2-Gab1-PKCθ signaling in triggering phosphorylation of ABCA1 and its disassociation from CSN3 curtailing cholesterol efflux and amplifying foam cell formation. In addition, although both CSN3 and ABCA1 were found to be colocalized in human non-lesion coronary arteries, their levels were decreased as well as dissociated from each other in advanced atherosclerotic lesions. Together, these observations reveal for the first time an anti-atherogenic role of CSN3 and hence, designing therapeutic drugs protecting its interactions with ABCA1 could be beneficial against atherosclerosis.

Trypanosoma cruzi infection in Cyclophilin D deficient mice.

Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic in Latin America and around the world through mother to child transmission. The heart is the organ most frequently affected in the chronic stage of the human infection and depends on mitochondria for the required energy for its activity. Cyclophilins are involved in protein folding and the mitochondrial isoform, Cyclophilin D (CyPD), has a crucial role in the opening of the mitochondrial permeability transition pore. In the present study, we infected CyPD deficient mice, with ablation of the Ppif gene, with T. cruzi parasites and the course of the infection was analyzed. Parasite load, quantified by PCR, was significantly lower in skeletal and cardiac tissues of Ppif-/- mice compared to wild type mice. In vitro cultured cardiomyocytes and macrophages from mice lacking CyPD exhibited lower percentage of infected cells and number of intracellular parasites than those observed for wild type mice. Although histopathological analysis of heart and mRNA of heart cytokines showed differences between T. cruzi-infected mice compared to the uninfected animals, no significant differences were found mice due to the ablation of the Ppif gene. Our results suggest that cells deficient for mitochondrial CyPD, inhibited for the mitochondrial membrane potential collapse, reduces the severity of parasite aggression and spread of cellular infection.

Antileishmanial compounds from Connarus suberosus: Metabolomics, isolation and mechanism of action.

Leishmaniasis is a disease impacting public health worldwide due to its high incidence, morbidity and mortality. Available treatments are costly, lengthy and toxic, not to mention the problem of parasite resistance. The development of alternative treatments is warranted and natural products demonstrate promising activity. This study investigated the activity of Connarus suberosus extracts and compounds against Leishmania species. Several C. suberosus extracts were tested against L. amazonensis promastigotes. Active and inactive extracts were analyzed by UHPLC-MS and data evaluated using a metabolomics platform, revealing an unknown neoflavonoid (connarin, 3), isolated together with the pterocarpans: hemileiocarpin (1) and leiocarpin (2). The aforementioned compounds (1-3), together with the benzoquinones: rapanone (4), embelin (5) and suberonone (6) previously isolated by our group from the same species, were tested against: (i) L. amazonensis and L. infantum promastigotes, and (ii) L. amazonensis intracellular amastigotes, with the most active compound (3) also tested against L. infantum amastigotes. Cytotoxicity against murine peritoneal macrophages was also investigated. Compounds 2 and 3 presented an IC50 33.8 µM and 11.4 µM for L. amazonensis promastigotes; and 44.3 µM and 13.3 µM for L. infantum promastigotes, respectively. For L. amazonensis amastigotes, the IC50 of 2 was 20.4 µM with a selectivity index (SI) of 5.7, while the IC50 of 3 was 2.9 µM with an SI of 6.3. For L. infantum amastigotes, the IC50 of 3 was 7.7 µM. Compounds 2 and 3 presented activity comparable with the miltefosine positive control, with compound 3 found to be 2-4 times more active than the positive control, depending on the Leishmania species and form. The extracts and isolated compounds showed moderate toxicity against macrophages. Compounds 2 and 3 altered the mitochondrial membrane potential (ΔΨm) and neutral lipid body accumulation, while 2 also impacted plasma membrane permeabilization, culminating in cellular disorder and parasite death. Transmission electron microscopy of L. amazonensis promastigotes treated with compound 3 confirmed the presence of lipid bodies. Leiocarpin (2) and connarin (3) demonstrated antileishmanial activity. This study provides knowledge of natural products with antileishmanial activity, paving the way for prototype development to fight this neglected tropical disease.

Shen-Hong-Tong-Luo Formula Attenuates Macrophage Inflammation and Lipid Accumulation through the Activation of the PPAR-γ/LXR-α/ABCA1 Pathway.

Atherosclerosis (AS) is the killer of human health and longevity, which is majorly caused by oxidized lipoproteins that attack macrophages in the endarterium. The Shen-Hong-Tong-Luo (SHTL) formula has shown great clinical efficacy and vascular protective effect for over 30 years in China, to attenuate AS progression. However, its pharmacological mechanism needs more investigation. In this study, we first investigated the chemical composition of SHTL by fingerprint analysis using high-performance liquid chromatography. In primary mouse peritoneal macrophages induced by lipopolysaccharide (LPS), we found that SHTL pretreatment suppressed reactive oxygen species accumulation and reversed the increases of the inflammatory factors, TNF-α and IL-6. Moreover, lipid accumulation induced by oxidized low-density lipoprotein (Ox-LDL) in macrophages was inhibited by SHTL. Additionally, network pharmacology was used to predict the potential targets of SHTL as the PPAR-γ/LXR-α/ABCA1 signaling pathway, which was validated in macrophages and ApoE-/- mice by histopathological staining, qPCR, and Western blot analysis. Importantly, the protective effect of SHTL in the LPS- and Ox-LDL-induced macrophages against inflammation and lipid accumulation was attenuated by GW9662, a PPAR-γ antagonist, which confirmed the prediction results of network pharmacology. In summary, these results indicated that SHTL pretreatment reduced inflammation and lipid accumulation of macrophages by activating the PPAR-γ/LXR-α/ABCA1 pathway, which may provide a new insight into the mechanism of SHTL in the suppression of AS progression.

Effects of Monoacyltrehalose Fraction of Rhodococcus Biosurfactant on the Innate and Adaptive Immunity Parameters In Vivo.

The biosurfactant monoacyltrehalose fraction isolated from Rhodococcus ruber IEGM 231 actinobacterium suppresses antibody production, bactericidal potential, and production of IL-1ß by mouse peritoneal cells after intraperitoneal and intramuscular injection and stimulates the production of IL-10 after intraperitoneal injection. The data of in vitro experiments attest to an important role of bacterial glycolipids in the regulation of the functions of splenocytes and peritoneal macrophages.

PEI-modified macrophage cell membrane-coated PLGA nanoparticles encapsulating Dendrobium polysaccharides as a vaccine delivery system for ovalbumin to improve immune responses.

Recently, nanoparticles have been widely used in drug and vaccine adjuvant delivery. Dendrobium devonianum Polygonatum (DP), a main biologically active ingredient isolated from Dendrobium devonianum, has been widely used in the clinic as an immunostimulant to stimulate and improve immune responses, contributing to its excellent biological activity. To increase the immune efficacy of DP, macrophage cell membrane-coated drug nanocrystals featuring homologous immune escape, targeting ability and low toxicity are in high demand. In this study, a new drug and vaccine adjuvant delivery system, PEI-MM-PLGA-DP/OVA, was designed and developed. This study aimed to report the macrophage immunomodulatory activity of PEI-modified macrophage cell membrane-coated PLGA nanoparticles encapsulating Dendrobium devonianum polysaccharides. PEI-MM-PLGA-DP/OVA could promote antigen uptake by macrophage and lymphocyte proliferation, increase the expression levels of MHC II, CD80 and CD86, and upregulate the ratio of CD4+ to CD8+ T cells in immunized mice. PEI-MM-PLGA-DP/OVA induced the highest TNF-α, IFN-γ, IL-4, and IL-6 cytokine secretion levels and the levels of OVA-specific antibodies (IgG) compared with the other groups. The above results indicated that PEI-MM-PLGA-DP/OVA had better adjuvant activity than PLGA-DP/OVA and MM-PLGA-DP/OVA.

Immunosuppressive properties of cytochalasin B-induced membrane vesicles of mesenchymal stem cells: comparing with extracellular vesicles derived from mesenchymal stem cells.

Extracellular vesicles derived from mesenchymal stem cells (MSCs) represent a novel approach for regenerative and immunosuppressive therapy. Recently, cytochalasin B-induced microvesicles (CIMVs) were shown to be effective drug delivery mediators. However, little is known about their immunological properties. We propose that the immunophenotype and molecular composition of these vesicles could contribute to the therapeutic efficacy of CIMVs. To address this issue, CIMVs were generated from murine MSC (CIMVs-MSCs) and their cytokine content and surface marker expression determined. For the first time, we show that CIMVs-MSCs retain parental MSCs phenotype (Sca-1+, CD49e+, CD44+, CD45-). Also, CIMVs-MSCs contained a cytokine repertoire reflective of the parental MSCs, including IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, G-CSF, GM-CSF and TNF-α. Next, we evaluated the immune-modulating properties of CIMVs-MSCs in vivo using standard preclinical tests. MSCs and CIMVs-MSCs reduced serum levels of anti-sheep red blood cell antibody and have limited effects on neutrophil and peritoneal macrophage activity. We compared the immunomodulatory effect of MSCs, CIMVs and EVs. We observed no immunosuppression in mice pretreated with natural EVs, whereas MSCs and CIMVs-MSCs suppressed antibody production in vivo. Additionally, we have investigated the biodistribution of CIMVs-MSCs in vivo and demonstrated that CIMVs-MSCs localized in liver, lung, brain, heart, spleen and kidneys 48 h after intravenous injection and can be detected 14 days after subcutaneous and intramuscular injection. Collectively our data demonstrates immunomodulatory efficacy of CIMVs and supports their further preclinical testing as an effective therapeutic delivery modality.

A Novel Anti-inflammatory Phenotype Transformed by Repetitive Low-dose Lipopolysaccharide in Primary Peritoneal Tissue-resident Macrophages.

BACKGROUND/AIM: Our previous studies suggested that oral administration of lipopolysaccharide (LPS) regulates the progression of various diseases via transformation of tissue-resident macrophages (MΦ). Recently, we characterized microglia transformed by repetitive low-dose LPS treatment (REPELL-microglia) in vitro, and this response was similar to that observed in response to oral administration of LPS in vivo. Here, we examined the characteristics of peritoneal tissue-resident MΦ (pMΦ) transformed by repetitive low-dose LPS treatment (REPELL-pMΦ). MATERIALS AND METHODS: Primary pMΦ were treated with low-dose LPS (1 ng/ml) three times; subsequently, phagocytic activity and gene expression were evaluated. RESULTS: REPELL-pMΦ exhibited high phagocytic activity and elevated expression of Arg1, Gipr, Gdnf, and Fpr2. The gene expression profiles observed in REPELL-pMΦ were distinct from those of REPELL-microglia. CONCLUSION: REPELL-pMΦ have the potential to promote clearance of xenobiotics and to suppress inflammation. The present study also demonstrates the diversity of tissue-resident MΦ transformation that reflect their tissue origin.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.