A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

The effects of microgravity on stem cells and the new insights it brings to tissue engineering and regenerative medicine.

Conventional two-dimensional (2D) cell culture techniques may undergo modifications in the future, as life scientists have widely acknowledged the ability of three-dimensional (3D) in vitro culture systems to accurately simulate in vivo biology. In recent years, researchers have discovered that microgravity devices can address many challenges associated with 3D cell culture. Stem cells, being pluripotent cells, are regarded as a promising resource for regenerative medicine. Recent studies have demonstrated that 3D culture in microgravity devices can effectively guide stem cells towards differentiation and facilitate the formation of functional tissue, thereby exhibiting advantages within the field of tissue engineering and regenerative medicine. Furthermore, We delineate the impact of microgravity on the biological behavior of various types of stem cells, while elucidating the underlying mechanisms governing these alterations. These findings offer exciting prospects for diverse applications.

Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.

Simulated microgravity altered the gene expression profiles and inhibited the proliferation of Kupffer cells in the early phase by downregulating LMO2 and EZH2.

Microgravity is a primary challenge that need to overcome, when human travel to space. Our study provided evidence that Kupffer cells (KCs) are sensitive to simulated microgravity (SMG), and no similar research report has been found in the literature. Using transcriptome sequencing technology, it was showed that 631 genes were upregulated and 801 genes were downregulated in KCs after treatment under SMG for 3 days. The GO analysis indicated that the proliferation of KCs was affected when exposed to SMG for 3 days. CCK-8 assay confirmed that the proliferation of KCs was inhibited in the third day under the environment of SMG. Furthermore, we identified 8 key genes that affect the proliferation of KCs and predicted 2 transcription factors (TFs) that regulate the 8 key genes. Significantly, we found that microgravity could affect the expression of LMO2 and EZH2 to reduce the transcription of Racgap1, Ccna2, Nek2, Aurka, Plk1, Haus4, Cdc20, Bub1b, which resulting in the reduction in KCs proliferation. These finding suggested that the inhibition of KCs proliferation under microgravity may influence the homeostasis of liver, and LMO2 and EZH2 can be the targets in management of KCs' disturbance in the future practice of space medicine.

RichPathR: a gene set enrichment analysis and visualization tool.

Gene set enrichment analysis (GSEA) is an important step for disease and drug discovery. Genomic, transcriptomics, proteomics and epigenetic analysis of tissue or cells generates gene lists that need to be further investigated in the known biological context. The advent of high-throughput technologies generates the vast number of gene lists that are up or down regulated together. One way of getting meaningful insights of the relationship of these genes is utilizing existing knowledge bases linking them with biological functions or phenotypes. Multiple public databases with annotated gene sets are available for GSEA, and enrichR is the most popular web application still requiring custom tools for large-scale mining. richPathR package is a collection of R functions that helps researchers carry out exploratory analysis and visualization of gene set enrichment using EnrichR.

[Differences in Fungal Communities in Different Material Cellar Sediments and Their Correlation with Environmental Factors].

There are rich and diverse fungal communities in rainfall-cellar sediments. Fungi play a key role in the rainfall-cellar ecosystem as a bridge and link for material exchange between the rainfall-cellar ecosystem and the sediments. The changes in fungal community structure are usually closely related to the changes in environmental factors. The 16S rRNA gene Illumina MiSeq high-throughput sequencing technology was used to study the diversity and difference of fungal communities in the cellar sediments under two different catchment environments. The results revealed that the cellar sediments under the concrete catchment environment had higher diversity and richness of fungal communities than those under the loess land catchment environment. The dominant bacteria of the fungal communities under the two catchment environments were the same, namely Ascomycota, Basidiomycota, and Zygomycota, which constituted more than 90% of the abundance of the bacteria; however, the former had better homogeneity and stability. The indicator species based on LEfSe analysis demonstrated that Basidiobolales had the largest contribution to the diversity in the catchment environment of the loess land, and Mycosphaerella had the smallest contribution; Saccharomycetales contributed the most to the diversity in the concrete concentration environment, whereas Periconia contributed the least. The results of the co-occurrence network of the microbial community and environmental factors demonstrated that the positive relationship between fungi and environmental factors was stronger than the negative relationship. The research results have enhanced the understanding of the diversity of fungal communities in the cellar sediments and provided a reference for ensuring the drinking safety of rainwater harvesting cellar water for humans and livestock and improving the quality of cellar water.

Simulated microgravity altered the proliferation, apoptosis, and extracellular matrix formation of L929 fibroblasts and the transforming growth factor-ß1/Smad3 signaling pathway.

Exposure to microgravity can adversely affect the fitness of astronauts. The integrity of the skin plays a crucial role in protecting against mechanical forces and infections, fluid imbalance, and thermal dysregulation. In brief, the skin wound may cause unknown challenges to the implementation of space missions. Wound healing is a physiological process that relies on the synergistic action of inflammatory cells, extracellular matrix (ECM), and various growth factors to maintain the integrity of skin after trauma. Fibroblasts are present almost throughout the entire process of wound repair, especially in the scar formation at the endpoint of wound healing. However, there is limited knowledge about the extent to which fibroblasts are affected by the lack of gravity during wound healing. In this study, we utilized the rotary cell culture system, a ground-based facility that mimics the weightless condition, to study the alterations of L929 fibroblast cells under simulated microgravity (SMG). Our results demonstrated that the SM condition exerted negative influences on the proliferation and ECM formation of the L929 fibroblast. Whereas, the apoptosis of fibroblast was significantly upregulated upon exposure to SMG conditions. Moreover, the transforming growth factor-ß1/Smad3 (TGF-ß1/smad3) signaling pathway of L929 fibroblast related to wound repair was also altered significantly under a weightless environment. Overall, our study provided evidence that fibroblasts are strongly sensitive to SMG and elucidated the potential value of the TGF-ß1/Smad3 signaling pathway modulating wound healing in the future practice of space medicine.

Autophagy in hepatic macrophages can be regulator and potential therapeutic target of liver diseases: A review.

Hepatic macrophages are a complex population of cells that play an important role in the normal functioning of the liver and in liver diseases. Autophagy, as a maintainer of cellular homeostasis, is closely connected to many liver diseases. And its roles are not always beneficial, but manifesting as a double-edged sword. The polarization of macrophages and the activation of inflammasomes are mediated by intracellular and extracellular signals, respectively, and are important ways for macrophages to take part in a variety of liver diseases. More attention should be paid to autophagy of hepatic macrophages in liver diseases. In this review, we focus on the regulatory role of hepatic macrophages' autophagy in a variety of liver diseases; especially on the upstream regulator of polarization and inflammasomes activation of the hepatic macrophages. We believe that the autophagy of hepatic macrophages can become a potential therapeutic target for management of liver diseases.

ITK degradation to block T cell receptor signaling and overcome therapeutic resistance in T cell lymphomas.

Interleukin (IL)-2-inducible T cell kinase (ITK) is essential for T cell receptor (TCR) signaling and plays an integral role in T cell proliferation and differentiation. Unlike the ITK homolog BTK, no inhibitors of ITK are currently US Food and Drug Administration (FDA) approved. In addition, recent studies have identified mutations within BTK that confer resistance to both covalent and non-covalent inhibitors. Here, as an alternative strategy, we report the development of BSJ-05-037, a potent and selective heterobifunctional degrader of ITK. BSJ-05-037 displayed enhanced anti-proliferative effects relative to its parent inhibitor BMS-509744, blocked the activation of NF-kB/GATA-3 signaling, and increased the sensitivity of T cell lymphoma cells to cytotoxic chemotherapy both in vitro and in vivo. In summary, targeted degradation of ITK is a novel approach to modulate TCR signal strength that could have broad application for the investigation and treatment of T cell-mediated diseases.

[Effect of electroacupuncture at "Zusanli" (ST36) and "Tianshu" (ST25) on intestinal function and autonomic nerve balance in rats with irritable bowel syndrome].

OBJECTIVE: To study whether electroacupuncture (EA) of "Zusanli" (ST36) combined with "Tianshu" (ST25) has a synergistic effect in regulating the colonic function and autonomic nerve balance in rats with irritable bowel syndrome (IBS). METHODS: Male Wistar rats were randomly divided into control, model, EA-ST36, and EA-ST36+ST25 groups, with 14 rats in each group. The IBS model was established by using water avoidance stress method. The visceral hypersensitivity was measured using the abdominal wall retraction reflex (AWR). The rectus abdominis electromyogram (EMG), intestinal electrical activity, and electrocardiogram (ECG) were recorded using a PowerLab data acquisition and analysis system. The contents of serum cAMP and cGMP were determined by ELISA, the expression levels of colonic tyrosine hydroxylase (TH) and choline acetyl-transferase (ChAT) proteins were determined by immunofluorescence staining and Western blot, respectively. RESULTS: Compared with the control group, the model group had an evident increase in the levels of AWR, LF, LF/HF, ChAT protein expression, cAMP and cGMP contents and cAMP/cGMP ratio (P<0.001, P<0.05), and a marked decrease in the levels of HF, frequency of slow waves of intestinal EMG, visceral pain threshold (PT), immunoactivity and expression of TH protein (P<0.05, P<0.001). In contrast to the model group, the levels of AWR, LF, LF/HF, ChAT protein expression and immunoactivity, cAMP and cGMP contents and ratio of cAMP/cGMP were significantly reduced (P<0.001, P<0.05, P<0.01), whereas the levels of frequency of slow waves of intestinal EMG, PT, and the immunoactivity and expression of TH were considerably increased (P<0.001, P<0.05) in both EA-ST36 and EA-ST36+ST25 groups. CONCLUSION: EA of both ST36 and ST36+ST25 can relieve visceral pain, and reduce sympathetic activity to improve autonomic nerve balance, but without apparent synergistic effect between EA-ST36 and EA-ST25 in rats with IBS.

A central role for STAT5 in the transcriptional programing of T helper cell metabolism.

Activated lymphocytes adapt their metabolism to meet the energetic and biosynthetic demands imposed by rapid growth and proliferation. Common gamma chain (cγ) family cytokines are central to these processes, but the role of downstream signal transducer and activator of transcription 5 (STAT5) signaling, which is engaged by all cγ members, is poorly understood. Using genome-, transcriptome-, and metabolome-wide analyses, we demonstrate that STAT5 is a master regulator of energy and amino acid metabolism in CD4+ T helper cells. Mechanistically, STAT5 localizes to an array of enhancers and promoters for genes encoding essential enzymes and transporters, where it facilitates p300 recruitment and epigenetic remodeling. We also find that STAT5 licenses the activity of two other key metabolic regulators, the mTOR signaling pathway and the MYC transcription factor. Building on the latter, we present evidence for transcriptome-wide cooperation between STAT5 and MYC in both normal and transformed T cells. Together, our data provide a molecular framework for transcriptional programing of T cell metabolism downstream of cγ cytokines and highlight the JAK-STAT pathway in mediating cellular growth and proliferation.

Proteomic analysis of the effects of simulated microgravity in human gastric mucosal cells.

Microgravity is an ecological factor that affects the environment of the body. In this study, quantitative isobaric labeling (tandem mass tag) method was used to study the changes in human gastric mucosal cells under simulated microgravity for the first time. Comparative proteomic analysis identified 394 (202 upregulated and 192 downregulated) and 542 (286 upregulated and 256 downregulated) proteins differentially regulated by simulated microgravity after 3 and 7 days, respectively. Then the identified proteins were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses for further exploration. The results of the analysis showed that the ribosomes of gastric mucosal cells were significantly impacted after exposure to simulated microgravity for 3 days, and the cells appeared to be in a state of stress and inflammation. Exposure to simulated microgravity for 7 days significantly affected the mitochondria of the cells, oxidative stress became more evident, while inflammation and weakened connections were observed in the cells. The results of this study highlighted the temporal response trend of gastric mucosal cells to the stressor of microgravity at the two time points of 3 and 7 days. These findings will provide insights into the development of methods to protect the gastric mucosa during space flight.

Shared and distinct reward neural mechanisms among patients with schizophrenia, major depressive disorder, and bipolar disorder: an effort-based functional imaging study.

Unwillingness to exert effort for rewards has been found in patients with schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BD), but the underlying shared and distinct reward neural mechanisms remain unclear. This study aimed to compare the neural correlates of such impairments across different diagnoses. The neural responses in an effort-expenditure for reward task (EEfRT) were assessed in 20 SCZ patients, 23 MDD patients, 17 BD patients, and 30 healthy controls (HC). The results found shared activation in the cingulate gyrus, the medial frontal gyrus, and the middle frontal gyrus during the EEfRT administration. Compared to HC, SCZ patients exhibited stronger variations of functional connectivity between the right caudate and the left amygdala, the left hippocampus and the left putamen, with increase in reward magnitude. In MDD patients, an enhanced activation compared to HC in the right superior temporal gyrus was found with the increase of reward magnitude. The variations of functional connectivity between the caudate and the right cingulate gyrus, the left postcentral gyrus and the left inferior parietal lobule with increase in reward magnitude were weaker than that found in HC. In BD patients, the degree of activation in the left precuneus was increased, but that in the left dorsolateral prefrontal cortex was decreased with increase in reward probability compared to HC. These findings demonstrate both shared and distinct reward neural mechanisms associated with EEfRT in patients with SCZ, MDD, and BD, implicating potential intervention targets to alleviate amotivation in these clinical disorders.

Association between depressive symptoms and suicidal risk: Based on self-reported and clinical-interview measurements from a network perspective.

Suicide is commonly found in patients with major depressive disorder (MDD), while the associations among depressive symptoms and their relationships with suicidal risk remain unclear. This study identified the symptoms associated with suicidal risk and the most central symptoms in the MDD networks based on both self-reported and clinical-interview scales. A total of 446 outpatients with MDD were recruited. The Mini International Neuropsychiatric Interview (MINI) was used to assess the suicidal risk. The 13-item Beck Depression Inventory (BDI-13) and 17-item Hamilton Depression Rating Scale (HAMD-17) were used to measure the depressive symptoms. Network analysis was used to estimate the network models. Ten symptoms in the BDI-13 network were related to suicidal risk, among which sadness had the strongest association. Among the six symptoms in the HAMD-17 network that were associated with suicidal risk, guilty feeling was the strongest. Sense of failure was the most central symptom in the BDI-13 network, while depressed mood had the highest centrality in the HAMD-17 network. The depressive symptoms related to suicide risk and the clinical features of MDD showed different characteristics based on different assessment types. Combining self-reported and clinician-rated assessments in future studies and clinical practice might lead to some new findings.

Long non-coding RNA MEG3 regulates autophagy after cerebral ischemia/reperfusion injury.

Severe cerebral ischemia/reperfusion injury has been shown to induce high-level autophagy and neuronal death. Therefore, it is extremely important to search for a target that inhibits autophagy activation. Long non-coding RNA MEG3 participates in autophagy. However, it remains unclear whether it can be targeted to regulate cerebral ischemia/reperfusion injury. Our results revealed that in oxygen and glucose deprivation/reoxygenation-treated HT22 cells, MEG3 expression was obviously upregulated, and autophagy was increased, while knockdown of MEG3 expression greatly reduced autophagy. Furthermore, MEG3 bound miR-181c-5p and inhibited its expression, while miR-181c-5p bound to autophagy-related gene ATG7 and inhibited its expression. Further experiments revealed that mir-181c-5p overexpression reversed the effect of MEG3 on autophagy and ATG7 expression in HT22 cells subjected to oxygen and glucose deprivation/reoxygenation. In vivo experiments revealed that MEG3 knockdown suppressed autophagy, infarct volume and behavioral deficits in cerebral ischemia/reperfusion mice. These findings suggest that MEG3 knockdown inhibited autophagy and alleviated cerebral ischemia/reperfusion injury through the miR-181c-5p/ATG7 signaling pathway. Therefore, MEG3 can be considered as an intervention target for the treatment of cerebral ischemia/reperfusion injury. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China (approval No. XF20190538) on January 4, 2019.

Accurate prediction of acute pancreatitis severity based on genome-wide cell free DNA methylation profiles.

BACKGROUND: Patients with severe acute pancreatitis (SAP) have a high mortality, thus early diagnosis and interventions are critical for improving survival. However, conventional tests are limited in acute pancreatitis (AP) stratification. We aimed to assess AP severity by integrating the informative clinical measurements with cell free DNA (cfDNA) methylation markers. METHODS: One hundred and seventy-five blood samples were collected from 61 AP patients at multiple time points, plus 24 samples from healthy individuals. Genome-wide cfDNA methylation profiles of all samples were characterized with reduced representative bisulfite sequencing. Clinical blood tests covering 93 biomarkers were performed on AP patients within 24 h. SAP predication models were built based on cfDNA methylation and conventional blood biomarkers separately and in combination. RESULTS: We identified 565 and 59 cfDNA methylation markers informative for acute pancreatitis and its severity. These markers were used to develop prediction models for AP and SAP with area under the receiver operating characteristic of 0.92 and 0.81, respectively. Twelve blood biomarkers were systematically screened for a predictor of SAP with a sensitivity of 87.5% for SAP, and a specificity of 100% in mild acute pancreatitis, significantly higher than existing blood tests. An expanded model integrating 12 conventional blood biomarkers with 59 cfDNA methylation markers further improved the SAP prediction sensitivity to 92.2%. CONCLUSIONS: These findings have demonstrated that accurate prediction of SAP by the integration of conventional and novel blood molecular markers, paving the way for early and effective SAP intervention through a non-invasive rapid diagnostic test.

Autophagy in Gastric Mucosa: The Dual Role and Potential Therapeutic Target.

The incidence of stomach diseases is very high, which has a significant impact on human health. Damaged gastric mucosa is more vulnerable to injury, leading to bleeding and perforation, which eventually aggravates the primary disease. Therefore, the protection of gastric mucosa is crucial. However, existing drugs that protect gastric mucosa can cause nonnegligible side effects, such as hepatic inflammation, nephritis, hypoacidity, impotence, osteoporotic bone fracture, and hypergastrinemia. Autophagy, as a major intracellular lysosome-dependent degradation process, plays a key role in maintaining intracellular homeostasis and resisting environmental pressure, which may be a potential therapeutic target for protecting gastric mucosa. Recent studies have demonstrated that autophagy played a dual role when gastric mucosa exposed to biological and chemical factors. More indepth studies are needed on the protective effect of autophagy in gastric mucosa. In this review, we focus on the mechanisms and the dual role of various biological and chemical factors regulating autophagy, such as Helicobacter pylori, virus, and nonsteroidal anti-inflammatory drugs. And we summarize the pathophysiological properties and pharmacological strategies for the protection of gastric mucosa through autophagy.

Near infrared light fluorescence imaging-guided biomimetic nanoparticles of extracellular vesicles deliver indocyanine green and paclitaxel for hyperthermia combined with chemotherapy against glioma.

BACKGROUND: We investigated the therapeutic effect of targeting extracellular vesicles (EVs) loaded with indocyanine green (ICG) and paclitaxel (PTX) on glioma. METHODS: Raw264.7 cells were harvested to extract EVs for the preparation of ICG/PTX@RGE-EV by electroporation and click chemistry. We evaluated the success of modifying Neuropilin-1 targeting peptide (RGE) on the EV membrane of ICG/PTX@RGE-EV using super-resolution fluorescence microscopy and flow cytometry. Spectrophotometry and high performance liquid chromatography (HPLC) were implemented for qualitative and quantitative analysis of the ICG and PTX loaded in EVs. Photothermal properties of the vesicles were evaluated by exposing to 808-nm laser light. Western blot analysis, cell counting kit 8 (CCK-8), Calcein Acetoxymethyl Ester/propidium iodide (Calcein-AM/PI) staining, and flow cytometry were utilized for assessing effects of vesicle treatment on cellular behaviors. A nude mouse model bearing glioma was established to test the targeting ability and anti-tumor action of ICG/PTX@RGE-EV in vivo. RESULTS: Under exposure to 808-nm laser light, ICG/PTX@RGE-EV showed good photothermal properties and promotion of PTX release from EVs. ICG/PTX@RGE-EV effectively targeted U251 cells, with activation of the Caspase-3 pathway and elevated apoptosis in U251 cells through chemotherapy combined with hyperthermia. The anti-tumor function of ICG/PTX@RGE-EV was confirmed in the glioma mice via increased accumulation of PTX in the ICG/PTX@RGE-EV group and an increased median survival of 48 days in the ICG/PTX@RGE-EV group as compared to 25 days in the PBS group. CONCLUSION: ICG/PTX@RGE-EV might actively target glioma to repress tumor growth by accelerating glioma cell apoptosis through combined chemotherapy-hyperthermia.

RNA Externalized by Neutrophil Extracellular Traps Promotes Inflammatory Pathways in Endothelial Cells.

OBJECTIVE: Neutrophil extracellular traps (NETs) are extracellular lattices composed of nucleic material bound to neutrophil granule proteins. NETs may play pathogenic roles in the development and severity of autoimmune diseases such as systemic lupus erythematosus (SLE), at least in part, through induction of type I interferon (IFN) responses via externalization of oxidized immunostimulatory DNA. A distinct subset of SLE proinflammatory neutrophils (low-density granulocytes [LDGs]) displays enhanced ability to form proinflammatory NETs that damage the vasculature. We undertook this study to assess whether NET-bound RNA can contribute to inflammatory responses in endothelial cells (ECs) and the pathways that mediate this effect. METHODS: Expression of newly synthesized and total RNA was quantified in NETs from healthy controls and lupus patients. The ability of ECs to take up NET-bound RNA and downstream induction of type I IFN responses were quantified. RNAs present in NETs were sequenced and specific small RNAs were tested for induction of endothelial type I IFN pathways. RESULTS: NETs extruded RNA that was internalized by ECs, and this was enhanced when NET-bound nucleic acids were oxidized, particularly in lupus LDG-derived NETs. Internalization of NET-bound RNA by ECs was dependent on endosomal Toll-like receptors (TLRs) and the actin cytoskeleton and induced type I IFN-stimulated genes (ISGs). This ISG induction was dependent on NET-associated microRNA let-7b, a small RNA expressed at higher levels in LDG-derived NETs, which acted as a TLR-7 agonist. CONCLUSION: These findings highlight underappreciated roles for small RNAs externalized in NETs in the induction of proinflammatory responses in vascular cells, with implications for lupus vasculopathy.