A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard Laboratory Mice.

The SARS-CoV-2 pandemic has caused extreme human suffering and economic harm. We generated and characterized a new mouse-adapted SARS-CoV-2 virus that captures multiple aspects of severe COVID-19 disease in standard laboratory mice. This SARS-CoV-2 model exhibits the spectrum of morbidity and mortality of COVID-19 disease as well as aspects of host genetics, age, cellular tropisms, elevated Th1 cytokines, and loss of surfactant expression and pulmonary function linked to pathological features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This model can rapidly access existing mouse resources to elucidate the role of host genetics, underlying molecular mechanisms governing SARS-CoV-2 pathogenesis, and the protective or pathogenic immune responses related to disease severity. The model promises to provide a robust platform for studies of ALI and ARDS to evaluate vaccine and antiviral drug performance, including in the most vulnerable populations (i.e., the aged) using standard laboratory mice.

Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis.

Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFß negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Autoimmunity against melanoma differentiation-associated gene 5 induces interstitial lung disease mimicking dermatomyositis in mice.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (DM) is characterized by amyopathic DM with interstitial lung disease (ILD). Patients with anti-MDA5 antibody-associated ILD frequently develop rapidly progression and present high mortality rate in the acute phase. Here, we established a murine model of ILD mediated by autoimmunity against MDA5. Mice immunized with recombinant murine MDA5 whole protein, accompanied with complete Freund's adjuvant once a week for four times, developed MDA5-reactive T cells and anti-MDA5 antibodies. After acute lung injury induced by intranasal administration of polyinosinic-polycytidylic acid [poly (I:C)] mimicking viral infection, the MDA5-immunized mice developed fibrotic ILD representing prolonged respiratory inflammation accompanied by fibrotic changes 2 wk after poly (I:C)-administration, while the control mice had quickly and completely recovered from the respiratory inflammation. Treatment with anti-CD4 depleting antibody, but not anti-CD8 depleting antibody, suppressed the severity of MDA5-induced fibrotic ILD. Upregulation of interleukin (IL)-6 mRNA, which was temporarily observed in poly (I:C)-treated mice, was prolonged in MDA5-immunized mice. Treatment with anti-IL-6 receptor antibody ameliorated the MDA5-induced fibrotic ILD. These results suggested that autoimmunity against MDA5 exacerbates toll-like receptor 3-mediated acute lung injury, and prolongs inflammation resulting in the development of fibrotic ILD. IL-6 may play a key role initiating ILD in this model.

The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.

Coronavirus disease 2019 (COVID-19) is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a pandemic1. The C5a complement factor and its receptor C5aR1 (also known as CD88) have a key role in the initiation and maintenance of several inflammatory responses by recruiting and activating neutrophils and monocytes1. Here we provide a longitudinal analysis of immune responses, including phenotypic analyses of immune cells and assessments of the soluble factors that are present in the blood and bronchoalveolar lavage fluid of patients at various stages of COVID-19 severity, including those who were paucisymptomatic or had pneumonia or acute respiratory distress syndrome. The levels of soluble C5a were increased in proportion to the severity of COVID-19 and high expression levels of C5aR1 receptors were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of acute respiratory distress syndrome. Anti-C5aR1 therapeutic monoclonal antibodies prevented the C5a-mediated recruitment and activation of human myeloid cells, and inhibited acute lung injury in human C5aR1 knock-in mice. These results suggest that blockade of the C5a-C5aR1 axis could be used to limit the infiltration of myeloid cells in damaged organs and prevent the excessive lung inflammation and endothelialitis that are associated with acute respiratory distress syndrome in patients with COVID-19.

Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

GLUT1 Promotes NLRP3 Inflammasome Activation of Airway Epithelium in Lipopolysaccharide-Induced Acute Lung Injury.

Acute lung injury (ALI) is a devastating clinical syndrome caused by different factors, with high morbidity and mortality. Lung injury and inflammation caused by lipopolysaccharide (LPS) can be modulated by NLRP3 inflammasome activation, yet its exact function within the airway epithelium is still unknown. Meanwhile, glucose transporter protein 1 (GLUT1) contributes to a number of inflammatory illnesses, including ALI. The present study aimed to assess GLUT1's function in NLRP3 inflammasome activation of airway epithelium in LPS-induced acute lung injury. BALB/c mice and BEAS-2B cells were exposed to LPS (5 mg/kg and 200 µg/mL, respectively), with or without GLUT1 antagonists (WZB117 or BAY876). LPS up-regulated pulmonary expression of NLRP3 and GLUT1 in mice, which could be blocked by WZB117 or BAY876. Pharmacological inhibition of GLUT1 in vivo significantly attenuated lung tissue damage, neutrophil accumulation, and proinflammatory factors release (TNF-α, IL-6, and IL-1ß) in LPS-exposed mice. Meanwhile, the activation markers of NLRP3 inflammasome (ASC, caspase-1, IL-1ß, and IL-18) induced by LPS were also suppressed. In cultured BEAS-2B cells, LPS induced an increase in GLUT1 expression and triggered activation of the NLRP3 inflammasome, both of which were inhibited by GLUT1 antagonists. These results illustrate that GLUT1 participates in LPS-induced ALI and promotes the activation of the NLRP3 inflammasome in airway epithelial cells.

IL-22 Binding Protein Controls IL-22-Driven Bleomycin-Induced Lung Injury.

The high mortality rates of acute lung injury and acute respiratory distress syndrome challenge the field to identify biomarkers and factors that can be exploited for therapeutic approaches. IL-22 is a cytokine that has antibacterial and reparative properties in the lung. However, it also can exacerbate inflammation and requires tight control by the extracellular inhibitory protein known as IL-22 binding protein (IL-22BP) (Il22ra2). This study showed the necessity of IL-22BP in controlling and preventing acute lung injury using IL-22BP knockout mice (Il22ra2-/-) in the bleomycin model of acute lung injury/acute respiratory distress syndrome. Il22ra2-/- mice had greater sensitivity (weight loss and death) and pulmonary inflammation in the acute phase (first 7 days) of the injury compared with wild-type C57Bl/6 controls. The inflammation was driven by excess IL-22 production, inducing the influx of pathogenic IL-17A+ γδ T cells to the lung. Interestingly, this inflammation was initiated in part by the noncanonical IL-22 signaling to macrophages, which express the IL-22 receptor (Il22ra1) in vivo after bleomycin challenge. This study further showed that IL-22 receptor alpha-1+ macrophages can be stimulated by IL-22 to produce a number of IL-17-inducing cytokines such as IL-1ß, IL-6, and transforming growth factor-ß1. Together, the results suggest that IL-22BP prevents IL-22 signaling to macrophages and reduces bleomycin-mediated lung injury.

Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif-coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI-treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet-neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI-treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

VNS-mediated α7nAChR signaling promotes SPM synthesis via regulation of netrin-1 expression during LPS-induced ALI.

The α7 nicotinic acetylcholine receptor (α7nAChR) plays a crucial role in the cholinergic anti-inflammatory pathway (CAP) during sepsis-associated acute lung injury (ALI). Increasing evidence suggests that specialized pro-resolving mediators (SPMs) are important in resolving α7nAChR-mediated ALI resolution. Our study aims to elucidate the pivotal role of α7nAChR in the CAP during LPS-associated acute lung injury (ALI). By employing vagus nerve stimulation (VNS), we identified α7nAChR as the key CAP subunit in ALI mice, effectively reducing lung permeability and the release of inflammatory cytokines. We further investigated the alterations in SPMs regulated by α7nAChR, revealing a predominant synthesis of lipoxin A4 (LXA4). The significance of α7nAChR-netrin-1 pathway in governing SPM synthesis was confirmed through the use of netrin-1 knockout mice and siRNA-transfected macrophages. Additionally, our evaluation identified a synchronous alteration of LXA4 synthesis in the α7nAChR-netrin-1 pathway accompanied by 5-lipoxygenase (5-LOX), thereby confirming an ameliorative effect of LXA4 on lung injury and macrophage inflammatory response. Concurrently, inhibiting the function of LXA4 annulled the lung-protective effect of VNS. As a result, our findings reveal a novel anti-inflammatory pathway wherein VNS modulates netrin-1 expression via α7nAChR, ultimately leading to LXA4 synthesis and subsequent lung protection.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Characterization of Chemotaxis-Associated Gene Dysregulation in Myeloid Cell Populations in the Lungs during Lipopolysaccharide-Mediated Acute Lung Injury.

During endotoxin-induced acute lung injury (ALI), immune cell recruitment resulting from chemotaxis is mediated by CXC and CC chemokines and their receptors. In this study, we investigated the role of chemokines and their receptors in the regulation of myeloid cell populations in the circulation and the lungs of C57BL/6J mice exhibiting LPS-mediated ALI using single-cell RNA sequencing. During ALI, there was an increase in the myeloid cells, M1 macrophages, monocytes, neutrophils, and other granulocytes, whereas there was a decrease in the residential alveolar macrophages and M2 macrophages. Interestingly, LPS triggered the upregulation of CCL3, CCL4, CXCL2/3, and CXCL10 genes associated with cellular migration of various subsets of macrophages, neutrophils, and granulocytes. Furthermore, there was an increase in the frequency of myeloid cells expressing CCR1, CCR3, CCR5, and CXCR2 receptors during ALI. MicroRNA sequencing studies of vehicle versus LPS groups identified several dysregulated microRNAs targeting the upregulated chemokine genes. This study suggests that chemokine ligand-receptors interactions are responsible for myeloid cell heterogenicity and cellular recruitment to the lungs during ALI. The single-cell transcriptomics allowed for an in-depth assessment and characterization of myeloid cells involved in immune cell trafficking during ALI.

A Synthetic Poly(A) Tail Targeting Extracellular CIRP Inhibits Sepsis.

Sepsis is an infectious inflammatory disease that often results in acute lung injury (ALI). Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA chaperon that binds to mRNA's poly(A) tail. However, CIRP can be released in sepsis, and extracellular CIRP (eCIRP) is a damage-associated molecular pattern, exaggerating inflammation, ALI, and mortality. In this study, we developed an engineered poly(A) mRNA mimic, AAAAAAAAAAAA, named A12, with 2'-O-methyl ribose modification and terminal phosphorothioate linkages to protect it from RNase degradation, exhibiting an increased half-life. A12 selectively and strongly interacted with the RNA-binding motif of eCIRP, thereby preventing eCIRP's binding to its receptor, TLR4. In vitro treatment with A12 significantly decreased eCIRP-induced macrophage MAPK and NF-κB activation and inflammatory transcription factor upregulation. A12 also attenuated proinflammatory cytokine production induced by eCIRP in vitro and in vivo in macrophages and mice, respectively. We revealed that treating cecal ligation and puncture-induced sepsis with A12 significantly reduced serum organ injury markers and cytokine levels and ALI, and it decreased bacterial loads in the blood and peritoneal fluid, ultimately improving their survival. Thus, A12's ability to attenuate the clinical models of sepsis sheds lights on inflammatory disease pathophysiology and prevention of the disease progress.

cGAS Mediates Inflammation by Polarizing Macrophages to M1 Phenotype via the mTORC1 Pathway.

Cyclic GMP-AMP synthase (cGAS), as a cytosolic DNA sensor, plays a crucial role in antiviral immunity, and its overactivation induces excess inflammation and tissue damage. Macrophage polarization is critically involved in inflammation; however, the role of cGAS in macrophage polarization during inflammation remains unclear. In this study, we demonstrated that cGAS was upregulated in the LPS-induced inflammatory response via the TLR4 pathway, and cGAS signaling was activated by mitochondria DNA in macrophages isolated from C57BL/6J mice. We further demonstrated that cGAS mediated inflammation by acting as a macrophage polarization switch, which promoted peritoneal macrophages and the bone marrow-derived macrophages to the inflammatory phenotype (M1) via the mitochondrial DNA-mTORC1 pathway. In vivo studies verified that deletion of Cgas alleviated sepsis-induced acute lung injury by promoting macrophages to shift from the M1 phenotype to the M2 phenotype. In conclusion, our study demonstrated that cGAS mediated inflammation by regulating macrophage polarization through the mTORC1 pathway, and it further provided a potential therapeutic strategy for inflammatory diseases, especially sepsis-induced acute lung injury.

STING mediates LPS-induced acute lung injury by regulating ferroptosis.

The pathogenesis of acute lung injury is not fully understood. Stimulator of interferon genes (STING) and ferroptosis have been implicated in various pathological and physiological processes, including acute lung injury (ALI). However, the relationship between STING and ferroptosis in lipopolysaccharide (LPS)-induced ALI is unclear. We found that LPS stimulation activated STING and ferroptosis. Furthermore, STING knockout and ferroptosis inhibitor alleviated lung inflammation and epithelial cell damage. Also, STING knockout reduced inflammation injury and ferroptosis. Notably, the ferroptosis inducer reversed the alleviation of inflammation caused by STING knockout. These results show that STING participates in the inflammation injury of ALI by regulating ferroptosis. Results also showed that p-STAT3 levels increased after STING knockout, suggesting that STING negatively regulates STAT3 activation. Besides, STAT3 inhibitor aggravated ferroptosis after STING knockout, indicating that STING regulates ferroptosis through STAT3 signaling. In conclusion, STING mediates LPS-induced ALI by regulating ferroptosis, indicating that STING and ferroptosis may be new targets for ALI treatment.

In vivo tracking of mesenchymal stem cell dynamics and therapeutics in LPS-induced acute lung injury models.

Mesenchymal stem cells (MSCs) have been widely used to treat various inflammatory and immune-related diseases in preclinical and clinical settings. Intravital microscopy (IVM) is considered the gold standard for investigating pathophysiological conditions in living animals. However, the potential for real-time monitoring of MSCs in the pulmonary microenvironment remains underexplored. In this study, we first constructed a lung window and captured changes in the lung at the cellular level under both inflammatory and noninflammatory conditions with a microscope. We further investigated the dynamics and effects of MSCs under two different conditions. Meanwhile, we assessed the alterations in the adhesive capacity of vascular endothelial cells in vitro to investigate the underlying mechanisms of MSC retention in an inflammatory environment. This study emphasizes the importance of the "lung window" for live imaging of the cellular behavior of MSCs by vein injection. Moreover, our results revealed that the upregulation of vascular cell adhesion molecule 1 (VCAM1) in endothelial cells post-inflammatory injury could enhance MSC retention in the lung, further ameliorating acute lung injury. In summary, intravital microscopy imaging provides a practical method to investigate the therapeutic effects of MSCs in acute lung injury.