miR-155 Knockout Mice Research Articles

The Role of miR-155 in Modulating Gene Expression in CD4+ T Cells: Insights into Alternative Immune Pathways in Autoimmune Encephalomyelitis

CD4+ T cells are considered the main orchestrators of autoimmune diseases. Their disruptive effect on CD4+ T cell differentiation and the imbalance between T helper cell populations can be most accurately determined using experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis (MS). One epigenetic factor known to promote autoimmune inflammation is miRNA-155 (miR-155), which is significantly upregulated in inflammatory T cells. The aim of the present study was to profile the transcriptome of immunized mice and determine their gene expression levels based on mRNA and miRNA sequencing. No statistically significant differences in miRNA profile were observed; however, substantial changes in gene expression between miRNA-155 knockout (KO) mice and WT were noted. In miR-155 KO mice, mRNA expression in CD4+ T cells changed in response to immunization with the myeloid antigen MOG35-55. After restimulation with MOG35-55, increased Ffar1 (free fatty acid receptor 1) and Scg2 (secretogranin-2) expression were noted in the CD4+ T cells of miR-155-deficient mice; this is an example of an alternative response to antigen stimulation.

Regulation of cardiac allograft immune responses by microRNA-155

IntroductionA better understanding of the immune mechanisms involved in allograft rejection after transplantation is urgently needed to improve patient outcomes. As microRNA-155 (miR155) plays a critical role in inflammation, we postulated that a deficiency of miR155 will improve cardiac allograft survival and enhance tolerance induction after heart transplantation. MethodsWe developed an acute rejection mouse model through heterotopic BALB/c cardiac transplantation to C57BL/6 (wild-type) and C57BL/6 miR155 knock-out (miR155KO) mice. Further, we induced tolerance in both groups through a costimulatory blockade with CTLA4-Ig (200 μg; post-transplant day 2) and MRI antibodies (250 μg; post-transplant day 0), targeting CD28/B7 and CD40/CD154 signals, respectively. Finally, we examined the effects of injecting 100 μg of small extracellular vesicles (sEVs) isolated from wild-type mice undergoing rejection into tolerant miR155KO mice. ResultsMean survival time (MST) of the cardiac allografts in wild-type and miR155KO mice was 7 and 15 days, respectively (p < 0.0001). Costimulatory blockade increased MST to 65 days and > 100 days in the wild-type and miR155KO recipients, respectively (p < 0.001). Injection of sEVs isolated from wild-type mice undergoing rejection into tolerant miR155KO mice decreased the allograft survival to 9 days, significantly lower than the tolerant miR155KO mice without injection of sEVs (>100 days; p < 0.0001). ConclusionmiR155KO mice have improved cardiac allograft survival and enhanced induction of tolerance after heterotopic cardiac transplantation. Injection of sEVs from wild-type mice undergoing rejection into the miR155KO mice reversed these benefits.

MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women, is associated with increased obesity, hyperandrogenism, and altered brown adipose tissue (BAT) thermogenesis. MicroRNAs play critical functions in brown adipocyte differentiation and maintenance. We aim to study the role of microRNA-21 (miR-21) in altered energy homeostasis and BAT thermogenesis in a PCOS mouse model of peripubertal androgen exposure.MethodsThree-week-old miR-21 knockout (miR21KO) or wild-type (WT) female mice were treated with dihydrotestosterone (DHT) or vehicle for 90 days. Body composition was determined by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were measured by indirect calorimetry. Androgen receptor (AR), and markers of adipogenesis, de novo lipogenesis, angiogenesis, extracellular matrix remodeling, and thermogenesis were quantified by RT-qPCR and/or Western-blot.ResultsMiR-21 ablation attenuated DHT-mediated increase in body weight while having no effect on fat or BAT mass. MiR-21 ablation attenuated DHT-mediated BAT AR upregulation. MiR-21 ablation did not alter EE; however, miR21KO DHT-treated mice have reduced VO2, VCO2, and RER. MiR-21 ablation reversed DHT-mediated decrease in food intake and increase in sleep time. MiR-21 ablation decreased some adipogenesis (Adipoq, Pparγ, and Cebpβ) and extracellular matrix remodeling (Mmp-9 and Timp-1) markers expression in DHT-treated mice. MiR-21 ablation abolished DHT-mediated increases in thermogenesis markers Cpt1a and Cpt1b, while decreasing CIDE-A expression.ConclusionsOur findings suggest that BAT miR-21 may play a role in regulating DHT-mediated thermogenic dysfunction in PCOS. Modulation of BAT miR-21 levels could be a novel therapeutic approach for the treatment of PCOS-associated metabolic derangements.

The Regulatory Role of microRNA-21 in Coronary Microcirculation in Metabolic Syndrome

Coronary vasculature is essential to supply oxygen and nutrients to the heart and maintain normal cardiac function. Obstruction of large vessels of coronaries (atherosclerosis) is well studied, but coronary microvascular dysfunction (CMD), which is associated with diabetic cardiomyopathy, Takotsubo cardiomyopathy, Ischemia with the non-obstructive coronary artery (INOCA), and HFpEF, is understudied. CMD is characterized by impaired endothelial-dependent vasodilation, but detailed mechanisms have yet to be elucidated. The anti-fibrotic role of microRNA-21 (miR-21) is reported in the HFrEF induced by ischemia/reperfusion or pressure overload, but how miR-21 regulates CMD is not entirely understood. Our recent study shows miR-21 ablation ameliorated impaired vasodilation in isolated coronaries of mice fed on high fat and high sugar (HFHS) diet. In this study, we examined the coronary flow reserve (CFR), the relationship between myocardial blood flow (MBF) and cardiac work (doppler) under hyperemia, cardiac function (echocardiography and exercise exertion test), fibrosis and gene expression of miR-21 knockout and wild-type (WT) mice fed on chow or HFHS diet. Our preliminary data show that MBF and CFR were decreased, cardiac index, stroke volume, and running distance were reduced, and the E/E’ ratio was increased in WT mice fed on the HFHS diet compared to the WT mice fed on the chow diet. The perivascular fibrosis was increased in the WT mice fed on the HFHS diet compared to WT mice on the chow diet. The ablation of miR-21 reversed all these changes in the mice treated with HFHS, suggesting miR-21 deficiency ameliorated CMD in metabolic syndrome. Moreover, we used endothelial-specific miR-21 knockout to study the endothelial miR-21 function. Interestingly, the endothelial mi-R21 KO phenocopied the global knockouts, suggesting the benefit of miR-21 deficiency to coronary microvascular function in metabolic syndrome is from endothelial cells. The study provides the potential therapeutic strategy to target vascular endothelial cells with miR-21 antagomir for CMD in metabolic syndromes. AHA 970663,1R56HL165207,1 R01 HL165207-01A1 to LY and 1 F31 HL156726-01A1 to CJ. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 5689: miR-21: A therapeutic target that delays severe liver disease and hepatocellular carcinoma under high-fat-diet conditions

Abstract Liver disease is a major global health concern, claiming approximately 2 million lives worldwide annually, yet curative treatments remain elusive. In our study, we aimed to investigate the role of microRNA-21-5p (miR-21) in metabolic dysfunction-associated steatotic liver disease (previously NAFLD), metabolic-associated steatohepatitis (previously NASH), and hepatocellular carcinoma (HCC) within the context of a Western high-fat diet (HFD) and offering potential therapeutic insights. We found that reduced miR-21 levels correlated with liver disease progression in WT mice fed on HFD, while miR-21 knockout mice showed exacerbated metabolic dysfunction, including obesity, hepatomegaly, hyperglycemia, insulin resistance, steatosis, fibrosis, and HCC. Our study reveals that miR-21 plays a protective role in metabolic syndrome and in the progression of liver disease to cancer. miR-21 directly targets Transforming growth factor beta-induced (Tgfbi), a gene also known to be significantly upregulated and a potential oncogene in HCC. Further, our study showed that intervention with the administration of a miR-21 mimic in WT livers in HFD conditions effectively improves insulin sensitivity, steatosis, fibrosis, tumor burden, as well as Tgfbi expression. These findings indicate that miR-21 could serve as an effective strategy to delay or prevent liver disease in high-fat-diet environments. Citation Format: Urmila Jagtap, Anan Quan, Yuho Ono, Jonathan Lee, Kylie A. Shen, Sergei Manakov, Gyongyi Szabo, Imad Nasser, Frank J. Slack. miR-21: A therapeutic target that delays severe liver disease and hepatocellular carcinoma under high-fat-diet conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5689.

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

Abstract P2099: Small Proline-rich Protein 1a Is A Key Downstream Effector Of Microrna-150 During Both Maladaptive Cardiac Remodeling In Mice And Human Cardiac Fibroblast Activation

Background &amp; Objectives: MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a ( Sprr1a ) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Methods &amp; Results: Here, using a novel miR-150 knockout (KO); Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased profibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Moreover, primary adult HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A (See Figure). Conclusions: These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

MicroRNA-155 Plays Selective Cell-Intrinsic Roles in Brain-Infiltrating Immune Cell Populations during Neuroinflammation.

The proinflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). Global knockout (KO) of miR-155 in mice confers resistance to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. However, cell-intrinsic roles for miR-155 during EAE have not been formally determined. In this study, we use single-cell RNA sequencing and cell-specific conditional miR-155 KOs to determine the importance of miR-155 expression in distinct immune cell populations. Time-course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells (DCs) in global miR-155 KO mice compared with wild-type controls at day 21 after EAE induction. Deletion of miR-155 in T cells, driven by CD4 Cre, significantly reduced disease severity similar to global miR-155 KOs. CD11c Cre-mediated deletion of miR-155 in DCs also resulted in a modest yet significant reduction in the development of EAE, with both T cell- and DC-specific KOs showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in infiltrating macrophages during EAE, deletion of miR-155 using LysM Cre did not impact disease severity. Taken together, these data show that although miR-155 is highly expressed in most infiltrating immune cells, miR-155 has distinct roles and requirements depending on the cell type, and we have demonstrated this using the gold standard conditional KO approach. This provides insights into which functionally relevant cell types should be targeted by the next generation of miRNA therapeutics.

MicroRNA-223 attenuates hepatocarcinogenesis by blocking hypoxia-driven angiogenesis and immunosuppression

ObjectiveThe current treatment for hepatocellular carcinoma (HCC) to block angiogenesis and immunosuppression provides some benefits only for a subset of patients with HCC, thus optimised therapeutic regimens are unmet needs,...

Deletion of miR-150 Prevents Spontaneous T Cell Proliferation and the Development of Colitis.

To examine the roles of microRNAs in the development of colitis, we conducted the RNA-sequencing studies using RNA derived from normal and colitogenic CD4+ T cells. Colitogenic CD4+ T cells demonstrated the increased expression of miR-150. We focused on the involvement of miR-150 in the colitis. We crossed miR-150 knockout mice and T-cell-specific Rap1KO mice, which is colitis model mice and spontaneously develop the colitis with tubular adenomas in microbiota-dependent manner. MiR-150 silencing completely inhibited the expansion of pathogenic Th17 cells and the development of colitis. MiR-150 is a potential therapeutic target of inflammatory bowel diseases.

Abstract 12156: Small Proline-Rich Repeat Protein 1a is a Significant Functional Target of Microrna-150 in Mouse Hearts and Human Cardiac Fibroblasts

Background and Aims: Cardiac injury induces dynamic changes in the expression of microRNAs (miRs). MiR-150 is conserved between rodents and humans, and its downregulation is markedly associated with heart failure (HF) severity and outcome in humans. Using systemic and cardiomyocyte (CM)-specific knockout (KO) mouse models, we reported that miR-150 plays a cardioprotective role in part by decreasing CM apoptosis, and that pro-apoptotic small proline-rich protein 1a ( Sprr1a ) is a direct and functional target of miR-150 in CMs. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-myocardial infarction (MI), and that Sprr1a is upregulated in mouse cardiac fibroblasts (CFs) isolated from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A in MI and human CF (HCF) activation has never been established. Methods &amp; Results: Here, we demonstrate using a novel miR-150 KO; Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model that Sprr1a knockdown blunts cardiac dysfunction, damage, inflammation, apoptosis and fibrosis post-MI caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased pro-fibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Our primary adult HCF studies further reveal that SPRR1A is upregulated in HCFs subjected to simulated ischemia/reperfusion, while its expression is downregulated in HCFs by a cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. MiR-150 knockdown in HCFs also increases pro-fibrotic SPRR1A . Lastly, protective roles of miR-150 in HCFs are in part mediated by the functional repression of SPRR1A (See Figure). Conclusions: Our findings delineate a pivotal functional interaction between miR-150 and Sprr1a as a novel regulatory mechanism pertinent to CF activation post-MI. This axis could be a therapeutic target for intervention in ischemic HF and cardiac fibrosis.

RF10 | PMON249 MicroRNA-21 Ablation Exacerbates Androgen-Mediated White Adipose Tissue Dysfunction and Impaired Insulin Signaling in a Mouse Model of Polycystic Ovary Syndrome

Abstract Background Polycystic ovary syndrome (PCOS) is characterized by oligo- or anovulation, polycystic ovaries, and hyperandrogenism. PCOS is associated with white adipose tissue (WAT) expansion and dysfunction, which has been linked to impaired insulin signaling in a number of metabolic diseases. MicroRNAs dysregulation in WAT is involved in the development of obesity and its associated complications. MicroRNA-21 (miR-21), the most abundant circulatory microRNA in women with PCOS, regulates androgen receptor (AR) expression in prostate cancer however, its role in WAT dysfunction in PCOS, and the associated systemic insulin resistance, is unknown. Using a well-established PCOS model of hyperandrogenemia we aim to test the hypothesis that miR-21 regulates AR expression to modulate androgen-mediated WAT dysfunction and the associated local and systemic insulin resistance. Methods Three-week-old miR-21 knockout (miR-21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/Silastic tube) or vehicle for 90 days (n=6/group). Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. HOMA-IR index was calculated from fasting serum glucose and insulin levels. Retroperitoneal fat (RPF), a WAT depot that is linked to poor plasma glycemic indices and systemic inflammation, was collected to assess adipocyte size and perform molecular determinations. RPF miR-21 levels, as well as gene and protein expression of AR, and insulin signaling markers (IRS-1/2, insulin receptor-β, PI3K, AKT, Glut4, GSK-α, PTEN) were quantified by RT-qPCR and Western blot. Results In WT mice, DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p&amp;lt;0.05), fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p&amp;lt;0.05), RPF mass (29.68 ± 3.39 vs. 172.64 ± 32.75 g, p&amp;lt;0.05), RPF miR-21 (2.5-fold) and AR expression (2.1-fold). Adipocyte size analysis revealed a hypertrophic response to DHT, which was associated with impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg. min/dL, p&amp;lt;0.05) and downregulation of IRS-1/2, insulin receptor-β, and AKT, indicating impaired insulin signaling. MiR-21 ablation had no effect on DHT-mediated total fat or RPF mass increases, but it exacerbated DHT-mediated AR upregulation, RPF hypertrophy, and insulin resistance as measured by the HOMA-IR index. DHT-mediated reduction in GSK-α, which mediates obesity-induced inflammation, was abolished in miR-21KO mice. Glut4 protein was only downregulated in miR-21KO DHT-treated mice, despite DHT downregulating Glut4 mRNA in both strains. Furthermore, DHT only upregulated PTEN, a negative insulin signaling regulator and known target of miR-21 in miR-21KO mice. Conclusion and significance: These findings imply that WAT miR-21 plays a protective role in PCOS by alleviating androgen-mediated adipose tissue structural and molecular derangements, as well as the associated local and systemic insulin resistance via downregulating AR. Modulation of miR-21 levels in adipose tissue could be a novel therapeutic approach for the treatment of PCOS-related metabolic derangements. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract P3023: The Regulatory Role Of Mir-21 In Heart Failure With Preserved Ejection Fraction (hfpef)

Introduction: Coronary microvascular dysfunction (CMD) is characterized by impaired endothelial-dependent vasodilation. These impairments are seen in diabetic cardiomyopathy, ischemia with no obstructive coronary artery disease, and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated. Moreover, how microRNA (miR-21) regulates CMD in HFpEF is not entirely understood. Methods: miR-21 knockout and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS) for 6 months and blood lipid and glucose were measured. Echocardiography and treadmill exercise exertion tests were performed to assess cardiac function. Myocardial blood flow (MBF) under stress was measured by contrast echocardiography or doppler after the treatment with different dosages of norepinephrine and mean arterial blood pressure was measured simultaneously by femoral pressure catheter. Cardiac fibrosis was detected using trichrome staining and molecular pathways were elucidated via gene and protein analysis. Results: Our preliminary data show decreased MBF during stress in WT mice fed a HFHS diet. While ejection fraction was not changed, cardiac index, stroke volume, and running distance were decreased. Furthermore, E/E’ ratio was increased in WT mice fed HFHS diet compared to the WT mice fed a chow diet, suggesting heart failure and impaired diastolic function with normal systolic function. Moreover, perivascular fibrosis was increased in the WT mice fed a HFHS diet compared to WT mice fed a chow diet. However, the ablation of miR-21 reversed all these changes in the mice fed a HFHS diet. Conclusions: miR-21 regulates CMD and ameliorated HFpEF. Further investigation will elucidate the pathways and mechanisms converging with miR-21 to regulate HFpEF.

MiR-21 upregulation exacerbates pressure overload-induced cardiac hypertrophy in aged hearts.

Young and aging hearts undergo different remodeling post pressure overload, but the regulator that determines responses to pressure overload at different ages remains unknown. With an angiotensin II (Ang II)-induced hypertensive model, miR-21 knockout mice (miR-21−/−) were observed regarding the effects of miR-21 on hypertension-induced cardiac remodeling in young (12 week-old) and old (50 week-old) mice. Although the aged heart represented a more significant hypertrophy and was associated with a higher expression of miR-21, Ang II-induced cardiac hypertrophy was attenuated in miR-21−/− mice. Upon results of cardiac-specific arrays in miR-21-overexpressing cardiomyocytes, we found a significant downregulation of S100a8. In both in vitro and in vivo models, miR-21/S100a8/NF-κB/NFAT pathway was observed to be associated with pressure overload-induced hypertrophic remodeling in aged hearts. To further investigate whether circulating miR-21 could be a biomarker reflecting the aged associated cardiac remodeling, we prospectively collected clinical and echocardiographic information of patients at young (<65 y/o) and old ages (≥65 y/o) with and without hypertension. Among 108 patients, aged subjects presented with a significantly higher expression of circulating miR-21, which was positively correlated with left ventricular wall thickness. Collectively, miR-21 was associated with a prominently hypertrophic response in aged hearts under pressure overload. Further studies should focus on therapeutic potentials of miR-21.

Targeting ETosis by miR-155 inhibition mitigates mixed granulocytic asthmatic lung inflammation.

Asthma is phenotypically heterogeneous with several distinctive pathological mechanistic pathways. Previous studies indicate that neutrophilic asthma has a poor response to standard asthma treatments comprising inhaled corticosteroids. Therefore, it is important to identify critical factors that contribute to increased numbers of neutrophils in asthma patients whose symptoms are poorly controlled by conventional therapy. Leukocytes release chromatin fibers, referred to as extracellular traps (ETs) consisting of double-stranded (ds) DNA, histones, and granule contents. Excessive components of ETs contribute to the pathophysiology of asthma; however, it is unclear how ETs drive asthma phenotypes and whether they could be a potential therapeutic target. We employed a mouse model of severe asthma that recapitulates the intricate immune responses of neutrophilic and eosinophilic airway inflammation identified in patients with severe asthma. We used both a pharmacologic approach using miR-155 inhibitor-laden exosomes and genetic approaches using miR-155 knockout mice. Our data show that ETs are present in the bronchoalveolar lavage fluid of patients with mild asthma subjected to experimental subsegmental bronchoprovocation to an allergen and a severe asthma mouse model, which resembles the complex immune responses identified in severe human asthma. Furthermore, we show that miR-155 contributes to the extracellular release of dsDNA, which exacerbates allergic lung inflammation, and the inhibition of miR-155 results in therapeutic benefit in severe asthma mice. Our findings show that targeting dsDNA release represents an attractive therapeutic target for mitigating neutrophilic asthma phenotype, which is clinically refractory to standard care.

MicroRNA-21-5p promotes mucosal type 2 inflammation via regulating GLP1R/IL-33 signaling in chronic rhinosinusitis with nasal polyps

It has been known that chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammation-dominated disease; however, the reasons causing such type of mucosal inflammation in CRSwNP are not well elucidated. We sought to investigate the role of microRNA-21-5p (miR-21-5p) in regulating mucosal type 2 inflammation in CRSwNP. miR-21-5p expression was detected in nasal mucosa of patients with CRSwNP. Correlations between miR-21-5p and indicators of type 2 inflammation were further analyzed. miR-21 knockout mice were used to explore the role of miR-21-5p in a murine model of eosinophilic (E) CRSwNP. Target gene of miR-21-5p related to type 2 inflammation in CRSwNP was identified. The upregulated miR-21-5p in the nasal mucosa of patients with CRSwNP, compared with control subjects, was expressed higher in patients with ECRSwNP than in patients with nonECRSwNP. miR-21-5p expression was positively correlated with mucosal eosinophil infiltrations and the expression of type 2 inflammatory cytokines. In the CRSwNP mice, miR-21 knockout significantly attenuated type 2 inflammation, as indicated by eosinophil infiltrations and expression of cytokines/chemokines in nasal mucosa and lavage fluid; moreover, genes associated with type 2 inflammation were extensively downregulated at the transcriptome level in miR-21 knockout mice. Glucagon-like peptide-1 receptor, which was negatively correlated with miR-21-5p expression in human nasal mucosa, was identified as the target of miR-21-5p. Overexpression of miR-21-5p induced IL-33 expression, whereas glucagon-like peptide-1 receptor agonist decreased IL-33 production in airway epithelial cells. miR-21-5p aggravates type 2 inflammation in the nasal mucosa of patients with CRSwNP via targeting glucagon-like peptide-1 receptor/IL-33 signaling, which may be a potential therapeutic target for CRSwNP.

Transplantation of bone marrow cells from miR150 knockout mice improves senescence-associated humoral immune dysfunction and arterial stiffness

Background and purposeThe senescence-accelerated mouse P1 (SAMP1) suffers from humoral immune deficiency, arterial stiffness and accelerated aging. In contrast, the microRNA-150 knockout (miR-150-KO) mice show enhanced humoral immune function including increased B cell population and elevated serum immunoglobulin levels and enjoy extended lifespan. The purpose of this study was to investigate whether transplantation of bone marrow cells (BMCs) from miR-150-KO mice affects immune deficiency and arterial stiffening in SAMP1 mice. Methods and resultsPulse wave velocity and blood pressure were increased significantly in SAMP1 mice (10 months), indicating arterial stiffening and hypertension. Interestingly, transplantation of BMCs from miR-150-KO mice significantly attenuated arterial stiffening and hypertension in SAMP1 mice within eight weeks. BMC transplantation from miR-150-KO mice partially rescued the downregulation of B lymphocytes, largely restored serum IgG and IgM levels, decreased inflammatory cytokine and chemokine expression, and attenuated macrophage and T cell infiltration in aortas in SAMP1 mice. BMC transplantation nearly abolished the upregulation of collagen 1, TGFβ1, Scleraxis, MMP-2 and MMP-9 expression and the downregulation of elastin levels in aortas in SAMP1 mice. FISH staining confirmed existence of the transplanted BMCs at end of the experiment. In cultured endothelial cells, IgG-deficient medium invoked upregulation of inflammatory cytokine/chemokine expression which can be rescued by treatment with IgG. ConclusionsAccelerated senescence caused arterial stiffening via impairing the humoral immune function in SAMP1 mice. BMC transplantation from miR-150-KO mice attenuated arterial matrix remodeling and stiffening and hypertension in SAMP1 mice partly via improving the humoral immune function which attenuates vascular inflammation.

Regulatory network identified by pulmonary transcriptome and proteome profiling reveals extensive change of tumor-related genes in microRNA-21 knockout mice.

MicroRNA-21 (miR-21) is a well-known oncomiR and plays key roles in regulating various biological processes related to pulmonary diseases, especially lung carcinoma. The regulatory roles and downstream targets of miR-21 remain far from well understood. We aimed to identify miR-21-gene regulatory network in lung tissue. Transcriptome and proteome analyses were performed on lung tissues from miR-21 knockout (KO) mice and their wildtype (WT) littermates. Differentially expressed genes (DEGs) and proteins (DEPs) between miR-21KO and WT were analyzed, and correlation analysis was performed between transcriptional and translational level. DEPs were used for prediction of miR-21 target genes and construction of co-expression network. Comparing with WT mice, 820 DEGs and 623 DEPs were identified in lung tissues of miR-21KO mice. Upregulated DEGs and DEPs were both significantly enriched in pathways of metabolism of xenobiotics by cytochrome P450, drug metabolism, and chemical carcinogenesis. Of the 31 molecules commonly identified in DEGs and DEPs, 9 upregulated genes were tumor suppressor genes while 8 downregulated genes were oncogenes, and 12 genes showed closely positive correlation between mRNA and protein expression. Real-time PCR validation results were consistent with the omics data. Among the upregulated DEPs in miR-21KO mice, 21 genes were predicted as miR-21 targets. The miR-21 regulatory network was constructed by target genes and their highly co-expressed proteins, which identified the miR-21 target Itih4 as a hub gene. MiR-21-gene regulatory network was constructed in mouse lung tissue. MiR-21KO resulted in extensive upregulation of tumor suppressor genes and downregulation of oncogenes.

Cell-specific roles for miR-155 during neuroinflammation

Abstract The pro-inflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). The whole-body deletion of miR-155 in mice confers resistance to a mouse model of MS, EAE, by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. Here we utilize single-cell RNA sequencing and cell-specific conditional miR-155 knockouts to determine the importance of miR-155 expression in distinct cell populations. Time course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells in whole-body miR-155 knockout mice compared with wild-type controls at day 21. Deletion of miR-155 in T cells, driven by CD4 cre, reduced disease severity similar to whole-body miR-155 knockouts. CD11c cre-driven deletion of miR-155 in dendritic cells also significantly reduced EAE disease score, with both T cell and dendritic cell-specific knockouts showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in macrophages during EAE, deletion miR-155 by LysM cre in did not affect disease severity.

Neutrophil Extracellular Traps Mediate Acute Liver Failure in Regulation of miR-223/Neutrophil Elastase Signaling in Mice.

Background & AimsMarked enhancement of neutrophil infiltration in the liver is a hallmark of acute liver failure (ALF), a severe life-threatening disease with varying etiologies. However, the mechanisms and pathophysiological role corresponding to hepatic neutrophil infiltration during ALF development remain poorly characterized.MethodsExperimental ALF was induced in 10-week-old male microRNA-223 (miR-223) knockout (KO) mice, neutrophil elastase (NE) KO mice, and wild-type controls by intraperitoneal injection of galactosamine hydrochloride and lipopolysaccharide. Age-matched mice were injected with phosphate-buffered saline and served as vehicle controls.ResultsMouse liver with ALF showed evident formation of neutrophil extracellular traps (NETs), which were enhanced markedly in miR-223 KO mice. The blockade of NETs by pharmacologic inhibitor GSK484 significantly attenuated neutrophil infiltration and massive necrosis in mouse liver with ALF. ALF-related hepatocellular damage and mortality in miR-223 KO mice were aggravated significantly and accompanied by potentiated neutrophil infiltration in the liver when compared with wild-type controls. Transcriptomic analyses showed that miR-223 deficiency in bone marrow predominantly caused the enrichment of pathways involved in neutrophil degranulation. Likewise, ALF-induced hepatic NE enrichment was potentiated in miR-223 KO mice. Genetic ablation of NE blunted the formation of NETs in parallel with significant attenuation of ALF in mice. Pharmaceutically, pretreatment with the NE inhibitor sivelestat protected mice against ALF.ConclusionsThe present study showed the miR-223/NE axis as a key modulator of NETs, thereby exacerbating oxidative stress and neutrophilic inflammation to potentiate hepatocellular damage and liver necrosis in ALF development, and offering potential targets against ALF.