技术资料

Cytoplasmic stress granules (SG) assemble in response to stress-induced translational arrest and are key signaling hubs orchestrating cell fate and regulating various physiological and pathological processes. However,the role of SG formation in the progression of allergic diseases is incompletely understood. Here,by analyzing the nasal tissues of allergic rhinitis (AR) mouse models and AR patients,we find that SGs assemble specifically in the macrophages within the nasal mucosa and promote AR progression by restraining the efferocytotic ability of macrophages,ultimately resulting in reduced Mres generation and IL-10 production. Mechanistically,intracellular m7G-modified Lrp1 mRNA,encoding for a typical efferocytosis receptor,is transported by the m7G reader QKI7 into stress-induced SGs,where Lrp1 mRNA is sequestered away from the translation machinery,ultimately resulting in reduced macrophage efferocytosis. Therefore,SG assembly impairs macrophage efferocytosis and aggravates AR,and the inhibition of SGs bears considerable potential in the targeted therapy. Cytoplasmic stress granules (SG) regulate cell fate and are involved in several physiological and pathological processes. Here,using mouse models of allergic rhinitis (AR),the authors reveal the formation of SGs within macrophages of the nasal mucosa and implicate SGs in the regulation of Lrp1-mediated efferocytosis and Type 2 cytokine production,aggravating AR symptoms. View Publication

SummarymRNA-based in vivo chimeric antigen receptor (CAR)-T cell engineering offers advantages over ex vivo therapies,including streamlined manufacturing and transient expression. However,current delivery methods require antibody-modified vehicles with manufacturing challenges. In this study,inspired by cardiolipin,we identify cardiolipin-like di-phosphoramide lipids that improve T cell transfection without targeting ligands,both in vitro and in vivo. The T cell-favored tropism is likely due to the lipid’s packing,shape,and rigidity. Encapsulating circular RNA further prolongs mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles,we deliver mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR),alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single-cell sequencing in humans confirms uPAR’s relevance to senescence and inflammation in RA. To facilitate clinical translation,we screen and humanize single-chain variable fragments (scFvs) against uPAR,establishing a PL40 mRNA-encoded humanized uPAR CAR with potential for treating aging-inflamed disorders. Graphical abstract Highlights•Cardiolipin-mimic phosphoramide (CAMP) LNPs transfect T cells without antibody modification•Circular mRNA prolongs mRNA expression•Senolytic in vivo CAR-T treats inflamm-aging disease (liver fibrosis and rheumatoid arthritis)•Develop humanized anti-human uPAR scFv Zhang et al. develop Cardiolipin-mimic phosphoramide (CAMP) lipids,which enable T cell transfection without antibody modification. Using CAMP-based LNPs,they generate senolytic CAR-T cells in vivo to target inflamm-aging diseases. Additionally,they employ circular mRNA to prolong transgene expression. The authors also engineer a humanized anti-human uPAR scFv for clinically relevant applications. View Publication

AbstractBackgroundPatients with head and neck squamous cell carcinoma (HNSCC),particularly the human papillomavirus negative (HPV−) subset,have a dismal prognosis. Furthermore,patients with Fanconi anemia (FA) have a genetic predisposition with a 500-fold to 700-fold higher incidence of HNSCC. Thus,novel and more efficacious therapies are needed. As current immunotherapies often fail due to suppressive elements in the tumor microenvironment (TME),we developed a trispecific killer engager (TriKE) to direct multiple signals to natural killer (NK) cells to overcome the hypoxic TME. This TriKE is comprised of a camelid nanobody that binds to CD16 on NK cells,an interleukin (IL)-15 moiety,and another novel camelid nanobody that binds to the B7-H3 antigen,which is highly and specifically expressed on the tumor cell surface.MethodsThe B7H3 TriKE was generated using a mammalian expression system. Its functionality was evaluated using flow cytometry-based NK cell degranulation,cytokine production,proliferation and live cell imaging cytotoxicity assays. Models of acute and prolonged hypoxia (1% oxygen) were carried out to assess tumor killing. Tumor progression,NK cell persistence,and survival differences between IL-15-treated and TriKE-treated mice were studied using NOD-scidIL2Rgnull (NSG) mice engrafted with human HNSCC.ResultsHigh B7-H3 expression was found in HPV− HNSCC cell lines,even when the FA gene was knocked out,and The Cancer Genome Atlas patient data showed that high B7-H3 expression predicted poor survival in patients with HPV− HNSCC. Similar to the NK cell activity seen with healthy donors,the B7H3 TriKE enhanced activation,expansion and cytotoxicity of NK cells from patients with HPV− HNSCC,a target population for this therapeutic. Additionally,the B7H3 TriKE improved NK cell cytotoxicity in a three-dimensional spheroid model of HNSCC. In both acute and prolonged hypoxia (1% oxygen),the B7H3 TriKE mediated enhanced tumor killing,mitigating impairment of NK cell cytotoxicity in hypoxia. In vivo,the B7H3 TriKE-treated mice demonstrated substantial antitumor activity and prolonged survival.ConclusionsThe B7H3 TriKE is a novel immunotherapeutic approach that can overcome hypoxic suppression of NK cells in the HNSCC TME. These highly translational studies present an innovative therapy for patients with HNSCC and will be developed further for clinical application. View Publication

The maturation process of natural killer (NK) cells,which is regulated by multiple transcription factors,determines their functionality,but few checkpoints specifically targeting this process have been thoroughly studied. Here we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) leads to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibit undermined activation,cytotoxicity and IFN-γ production upon stimulation,as well as weakened responses to IL-15 for NK cell maturation,in vitro. In vivo,NK-specific gp96-deficient mice show increased tumor growth. Mechanistically,we identify Eomes as the downstream transcription factor,with gp96 binding to Trim28 to prevent Trim28-mediated ubiquitination and degradation of Eomes. Our study thus suggests the gp96-Trim28-Eomes axis to be an important regulator for NK cell maturation and cancer surveillance in mice. Natural killer (NK) cell maturation and function are regulated by multiple transcription factors (TF),but detailed molecular insights are scarce. Here the authors show that a TF,Eomes,is important for NK cell responses and cancer surveillance,in which Eomes expression is regulated by gp96 and Trim28 via the ubiquitination and degradation pathways. View Publication

SummaryEnergy metabolism in the context of erythropoiesis and related diseases remains largely unexplored. Here,we developed a primary cell model by differentiating hematopoietic stem progenitor cells toward the erythroid lineage and suppressing the mitochondrial oxidative phosphorylation (OXPHOS) pathway. OXPHOS suppression led to differentiation failure of erythroid progenitors and defects in ribosome biogenesis. Ran GTPase-activating protein 1 (RanGAP1) was identified as a target of mitochondrial OXPHOS for ribosomal defects during erythropoiesis. Overexpression of RanGAP1 largely alleviated erythroid defects resulting from OXPHOS suppression. Coenzyme Q10,an activator of OXPHOS,largely rescued erythroid defects and increased RanGAP1 expression. Patients with Diamond-Blackfan anemia (DBA) exhibited OXPHOS suppression and a concomitant suppression of ribosome biogenesis. RNA-seq analysis implied that the substantial mutation (approximately 10%) in OXPHOS genes accounts for OXPHOS suppression in these patients. Conclusively,OXPHOS disruption and the associated disruptive mitochondrial energy metabolism are linked to the pathogenesis of DBA. Graphical abstract Highlights•Disruptive energy metabolism associates with the pathology of DBA•Suppression of OXPHOS leads to differentiation failure of erythroid progenitors•Energy metabolism disruption decreases overall ribosome levels in erythropoiesis•RanGAP1 is a target of OXPHOS pathway for ribosome biogenesis during erythropoiesis Cellular physiology; Cell biology; Developmental biology View Publication

IntroductionLoss of proteasome function,proteinopathy,and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.MethodsUsing pigs,a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex,we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.ResultsBy Western blotting,proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus,PSMB5 immunoreactivity was diffuse,particulate,and clustered,including perinucleolar decorations. By fluorogenic assay,proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex,proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15–20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay,26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay,MG132-inhibitable proteasome CTL activity was localized to the neuropil,as a mosaic,and to cell bodies,nuclei,and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h,brain proteasome CTL activity was modestly increased.DiscussionThis study shows that the proteasome in the pig brain has relative regional uniformity,prominent nuclear and perinuclear presence with catalytic activity,a mitochondrial association with activity,26S-single cap dominance,and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable. View Publication

AbstractAcute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life‐threatening syndrome with no effective pharmacotherapy. Sepsis‐related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell‐derived EVs (EC‐EVs) play a crucial role in ALI/ARDS development,but the mechanism remains largely unknown. Here,we demonstrated that the number of circulating EC‐EVs was increased in sepsis,exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1),overexpressed on EC‐EVs during sepsis,activated the NF‐κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface,rather than the tissue resident macrophage surface,thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC‐EVs or blocking ITGA4 on monocytes. Furthermore,the number of VCAM1+ EC‐EVs was significantly increased in patients with sepsis‐related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis‐related ALI/ARDS,but also provide potential novel targets and strategies for its precise treatment. During extra‐pulmonary sepsis,more endothelial cell‐derived extracellular vesicles (EC‐EVs) are released,which play a critical role in the development of ALI/ARDS by specifically targeting and reprogramming monocytes. VCAM1,highly expressed on these EVs,activates the NF‐κB pathway by acting on ITGA4,thus promoting the differentiation of monocytes into M1‐type macrophages. View Publication

Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile arthritis,accompanied by cytokine storm and hemophagocytosis. In addition,COVID-19–related hyperinflammation shares clinical features of MAS. Mechanisms that activate macrophages in MAS remain unclear. Here,we identify the role of miRNA in increased phagocytosis and interleukin-12 (IL-12) production by macrophages in a murine model of MAS. MAS significantly increased F4/80+ macrophages and phagocytosis in the mouse liver. Gene expression profile revealed the induction of Fcγ receptor–mediated phagocytosis (FGRP) and IL-12 production in the liver. Phagocytosis pathways such as High-affinity IgE receptor is known as Fc epsilon RI -signaling and pattern recognition receptors involved in the recognition of bacteria and viruses and phagosome formation were also significantly upregulated. In MAS,miR-136-5p and miR-501-3p targeted and caused increased expression of Fcgr3,Fcgr4,and Fcgr1 genes in FGRP pathway and consequent increase in phagocytosis by macrophages,whereas miR-129-1-3p and miR-150-3p targeted and induced Il-12. Transcriptome analysis of patients with MAS revealed the upregulation of FGRP and FCGR gene expression. A target analysis of gene expression data from a patient with MAS discovered that miR-136-5p targets FCGR2A and FCGR3A/3B,the human orthologs of mouse Fcgr3 and Fcgr4,and miR-501-3p targets FCGR1A,the human ortholog of mouse Fcgr1. Together,we demonstrate the novel role of miRNAs during MAS pathogenesis,thereby suggesting miRNA mimic–based therapy to control the hyperactivation of macrophages in patients with MAS as well as use overexpression of FCGR genes as a marker for MAS classification. View Publication

BACKGROUND:Venous thromboembolism is a major health problem. After thrombus formation,its resolution is essential to re-establish blood flow,which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus,we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions.METHODS:Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day,a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed.RESULTS:Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus,and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro,P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly,P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover,blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus,which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin.CONCLUSIONS:Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation,which prevents early thrombus stabilization and facilitates fibrinolysis. View Publication

The immune system has a critical role in orchestrating tissue healing. As a result,regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4–12. However,how neuro–immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils,monocytes and macrophages to inhibit recruitment,accelerate death,enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies,delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro–immune interactions has potential to treat non-healing tissues in which dysregulated neuro–immune interactions impair tissue healing. Experiments in mouse models show that NaV1.8+ nociceptors innervate sites of injury and provide wound repair signals to immune cells by releasing calcitonin gene-related peptide (CGRP). View Publication

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1,two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells,suggesting it has a unique role in mediating the immune system’s response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment,immune checkpoint blockade therapy,which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients. View Publication

Innate antiviral factors are essential for effective defense against viral pathogens. However,the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here,we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN,CIITA,EHMT2,CEACAM3,CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene,we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry,transcription,release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle. Innate immune mechanisms are critical for antiviral defense. Here,the authors developed a CRISPR/Cas9-based HIV-driven approach to identify cellular factors compromising viral transcription,assembly,release or infectivity in human T cells. They identify targets of the Nef protein as antiviral factors. View Publication