miR-155 In Macrophages Research Articles

M1 macrophage-derived exosomes inhibit cardiomyocyte proliferation through delivering miR-155

BackgroundM1 macrophages are closely associated with cardiac injury after myocardial infarction (MI). Increasing evidence shows that exosomes play a key role in pathophysiological regulation after MI, but the role of M1 macrophage-derived exosomes (M1-Exos) in myocardial regeneration remains unclear. In this study, we explored the impact of M1 macrophage-derived exosomes on cardiomyocytes regeneration in vitro and in vivo.MethodsM0 macrophages were induced to differentiate into M1 macrophages with GM-CSF (50 ng/mL) and IFN-γ (20 ng/mL). Then M1-Exos were isolated and co-incubated with cardiomyocytes. Cardiomyocyte proliferation was detected by pH3 or ki67 staining. Quantitative real-time PCR (qPCR) was used to test the level of miR-155 in macrophages, macrophage-derived exosomes and exosome-treated cardiomyocytes. MI model was constructed and LV-miR-155 was injected around the infarct area, the proliferation of cardiomyocytes was counted by pH3 or ki67 staining. The downstream gene and pathway of miR-155 were predicted and verified by dual-luciferase reporter gene assay, qPCR and immunoblotting analysis. IL-6 (50 ng/mL) was added to cardiomyocytes transfected with miR-155 mimics, and the proliferation of cardiomyocytes was calculated by immunofluorescence. The protein expressions of IL-6R, p-JAK2 and p-STAT3 were detected by Western blot.ResultsThe results showed that M1-Exos suppressed cardiomyocytes proliferation. Meanwhile, miR-155 was highly expressed in M1-Exos and transferred to cardiomyocytes. miR-155 inhibited the proliferation of cardiomyocytes and antagonized the pro-proliferation effect of interleukin 6 (IL-6). Furthermore, miR-155 targeted gene IL-6 receptor (IL-6R) and inhibited the Janus kinase 2(JAK)/Signal transducer and activator of transcription (STAT3) signaling pathway.ConclusionM1-Exos inhibited cardiomyocyte proliferation by delivering miR-155 and inhibiting the IL-6R/JAK/STAT3 signaling pathway. This study provided new insight and potential treatment strategy for the regulation of myocardial regeneration and cardiac repair by macrophages.

Natural Killer Cells Regulate Pulmonary Macrophages Polarization in Host Defense Against Chlamydial Respiratory Infection.

NK cells and pulmonary macrophages both are important components of innate immunity. The interaction between NK cells and pulmonary macrophages during chlamydial infection is poorly understood. In this study, we explored the effect of NK cells on regulation of pulmonary macrophage function during chlamydial respiratory infection. We found that NK depletion led to polarization of pulmonary macrophages from M1 to M2 phenotype, and it is related to reduced miR-155 expression in lung macrophage. Using adoptive transfer approach, we found that the recipients receiving lung macrophages isolated from C. muridarum-infected NK-cell-depleted mice exhibited an increased bacterial load and severe inflammation in the lung upon chlamydial challenge infection when compared with the recipients of lung macrophages from infected isotype control antibody treated mice. Herein, the effects of NK cells on macrophage polarization were examined in vitro. We found that NK cells from chlamydial-infected mice (iNK) significantly induced M1 polarization compared to that from uninfected mice (uNK). Inhibition of miR-155 expression in macrophages reduced M1 polarization induced by iNK, while miR-155 over-expression enhanced it. Furthermore, neutralization of IFN-γ in the coculture system decreased the expression of miR-155 by macrophages, and resulted in weakened M1 polarization. The data indicates that NK cells promote M1 polarization through up-regulation of miR-155 in macrophages by producing IFN-γ during chlamydial infection, and NK-regulated macrophage polarization is functionally relevant to host defense against the infection.

HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines.

Activation of Kupffer cells (KCs) induced that inflammatory cytokine production plays a central role in the pathogenesis of HBV infection. The previous studies from our and other laboratory demonstrated miRNAs can regulate TLR-inducing inflammatory responses to macrophage. However, the involvement of miRNAs in HBV-associated antigen-induced macrophage activation is still not thoroughly understood. Here, we evaluated the effects and mechanisms of miR-155 in HBV-associated antigen-induced macrophage activation. First, co-culture assay of HepG2 or HepG2.2.15 cells and RAW264.7 macrophages showed that HepG2.2.15 cells could significantly promote macrophages to produce inflammatory cytokines. Furthermore, we, respectively, stimulated RAW264.7 macrophages, mouse primary peritoneal macrophages, or healthy human peripheral blood monocytes with HBV-associated antigens, including HBcAg, HBeAg, and HBsAg, and found that only HBeAg could steadily enhance the production of inflammatory cytokines in these cells. Subsequently, miRNAs sequencing presented the up- or down-regulated expression of multiple miRNAs in HBeAg-stimulated RAW264.7 cells. In addition, we verified the expression of miR-155 and its precursors BIC gene with q-PCR in the system of co-culture or HBeAg-stimulated macrophages. Meanwhile, the increased miR-155 expression was positively correlation with serum ALT, AST, and HBeAg levels in AHB patients. Although MAPK, PI3K, and NF-κB signal pathways were all activated during HBeAg treatment, only PI3K and NF-κB pathways were involved in miR-155 expression induced by HBeAg stimulation. Consistently, miR-155 over-expression inhibited production of inflammatory cytokines, which could be reversed by knocking down miR-155. Moreover, we demonstrated that miR-155 regulated HBeAg-induced cytokine production by targeting BCL-6, SHIP-1, and SOCS-1. In conclusion, our data revealed that HBeAg augments the expression of miR-155 in macrophages via PI3K and NF-κB signal pathway and the increased miR-155 promotes HBeAg-induced inflammatory cytokine production by inhibiting the expression of BCL-6, SHIP-1, and SOCS-1.

Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1.

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.

Gold nanoparticle-based miR155 antagonist macrophage delivery restores the cardiac function in ovariectomized diabetic mouse model.

Diabetic cardiomyopathy is a common disease in postmenopausal women, in whom the estrogen deficiency aggravates the pathology. In this study, we have found that estrogen deficiency due to ovariectomy aggravates the inflammation in the hearts of diabetic mice, as depicted by excessive proinflammatory type 1 macrophages (M1) over anti-inflammatory type 2 macrophages (M2). Accordingly, an additional increase of reactive oxygen species, cell apoptosis, cardiac hypertrophy, and fibrosis was observed in the hearts of ovariectomized diabetic mice, in comparison with the diabetes-only group. Significantly, miR155, a potent promoter of M1 polarization, was found to be additionally enhanced in the macrophages and hearts by ovariectomy. Tail vein injection of miR155-AuNP, in which thiol-modified antago-miR155 was covalently conjugated with gold nanoparticle (AuNP), preferentially delivered the nucleic acids into the macrophages via phagocytosis. Together with the increased M2 ratio and reduced inflammation, in vivo delivery of antago-miR155 reduced cell apoptosis and restored the cardiac function. The restoration efficacy of miR155-AuNP was much better than general macrophage depletion by clodrosome. In summary, we revealed that M1/M2 imbalance contributes to the aggravated cardiomyopathy in ovariectomized diabetic mice, and therapeutically reducing miR155 in macrophages by AuNP serves as a promising strategy in improving cardiac function.

Abstract 81: Absence of Macrophage microRNA-155 Affects Cardiac Metabolic and Inflammatory Remodeling Following Diabesotension and Protects Against Development of Heart Failure

The co-existence of diabetes, obesity, hypertension - diabesotension - is a major burden in western societies and often culminates into heart failure (HF). Diabesotension is associated with a chronic status of low-grade inflammation, involving macrophage infiltration into adipose and cardiac tissues, local activation of inflammatory pathways and concomitant insulin-resistance. We showed that the microRNA miR-155 is a powerful modulator of hypertension-induced cardiac inflammation and HF. Here we hypothesize that absence of miR-155 in macrophages protects against adipose tissue and cardiac inflammation during diabesotension and thereby prevents insulin resistance and cardiac dysfunction. Male C57Bl/6J mice received a bone marrow transplantation of miR-155 WT (n=30) or miR-155 KO (n=30) donors with same genetic background. High fat (HFD; n=22/gr) or chow diet (n=8/gr) was given for 20 weeks. An HFD subgroup was subjected to pressure overload by angiotensin II (1.5mg/kg/d) for 4 weeks (HFD+PO; n=12/gr). Glycaemia, glucose tolerance test, cardiac MRI were performed after 16 and/or 20 weeks. Organs were stored for histology, RNA and FACS analysis. After 16 weeks of HFD, body weight (+11% WT; +14% KO; p<0.05) and glycaemia (+16% WT; +18% KO; p<0.05) increased compared to chow diet animals in both genotypes, confirming the status of obesity and pre-diabetes. Absence of macrophage miR-155 protected HFD-mice from glucose intolerance (at T15: WT 25.08; KO 18.15 mmol/L; p<0.05), suggesting that adipose tissue macrophages might contribute to insulin resistance. In addition, HFD+PO caused mild systolic dysfunction in WT but not in miR-155 KO animals compared to control (ejection fraction: -19% WT, p<0.05; -7% KO, n.s.). While cardiac interstitial fibrosis was not affected by absence of macrophage miR-155, cardiac CD45-positive leukocyte infiltration upon HFD+PO was abrogated. HFD+PO-induced cardiac expression of two PPAR-dependent metabolic genes, angiopoietin-like 4 and uncoupling protein 3, is repressed in the absence of macrophage miR-155. Our data suggest that macrophage miR-155 plays a role in the development of insulin resistance and cardiac dysfunction, possibly by affecting local immune cell function and metabolic programming.

TREM-1-accentuated lung injury via miR-155 is inhibited by LP17 nanomedicine.

Triggering receptors expressed on myeloid cell-1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells. Synergy between TREM-1 and Toll-like receptor amplifies the inflammatory response; however, the mechanisms by which TREM-1 accentuates inflammation are not fully understood. In this study, we investigated the role of TREM-1 in a model of LPS-induced lung injury and neutrophilic inflammation. We show that TREM-1 is induced in lungs of mice with LPS-induced acute neutrophilic inflammation. TREM-1 knockout mice showed an improved survival after lethal doses of LPS with an attenuated inflammatory response in the lungs. Deletion of TREM-1 gene resulted in significantly reduced neutrophils and proinflammatory cytokines and chemokines, particularly IL-1β, TNF-α, and IL-6. Physiologically deletion of TREM-1 conferred an immunometabolic advantage with low oxygen consumption rate (OCR) sparing the respiratory capacity of macrophages challenged with LPS. Furthermore, we show that TREM-1 deletion results in significant attenuation of expression of miR-155 in macrophages and lungs of mice treated with LPS. Experiments with antagomir-155 confirmed that TREM-1-mediated changes were indeed dependent on miR-155 and are mediated by downregulation of suppressor of cytokine signaling-1 (SOCS-1) a key miR-155 target. These data for the first time show that TREM-1 accentuates inflammatory response by inducing the expression of miR-155 in macrophages and suggest a novel mechanism by which TREM-1 signaling contributes to lung injury. Inhibition of TREM-1 using a nanomicellar approach resulted in ablation of neutrophilic inflammation suggesting that TREM-1 inhibition is a potential therapeutic target for neutrophilic lung inflammation and acute respiratory distress syndrome (ARDS).

Regulation of Csf1r and Bcl6 in macrophages mediates the stage-specific effects of microRNA-155 on atherosclerosis.

The function of microRNAs is highly context and cell type dependent because of their highly dynamic expression pattern and the regulation of multiple mRNA targets. MicroRNA-155 (miR-155) plays an important role in the innate immune response by regulating macrophage function; however, the effects of miR-155 in macrophages on atherosclerosis are controversial. We hypothesized that the stage-dependent target selection of miR-155 in macrophages determines its effects on atherosclerosis. The expression of miR-155 increased in lesional macrophages of apolipoprotein E-deficient mice between 12 and 24 weeks of a high-cholesterol diet. Mir155 knockout in apolipoprotein E-deficient mice enhanced lesion formation, increased the lesional macrophage content, and promoted macrophage proliferation after 12 weeks of the high-cholesterol diet. In vitro, miR-155 inhibited macrophage proliferation by suppressing colony-stimulating factor-1 receptor, which was upregulated in lesional macrophages of Mir155(-/-) apolipoprotein E-deficient mice. By contrast, Mir155 deficiency reduced necrotic core formation and the deposition of apoptotic cell debris, thereby preventing the progression of atherosclerosis between 12 and 24 weeks of the high-cholesterol diet. miR-155 inhibited efferocytosis in vitro by targeting B-cell leukemia/lymphoma 6 and thus activating RhoA (ras homolog gene family, member A). Accordingly, B-cell leukemia/lymphoma 6 was upregulated in lesional macrophages of Mir155(-/-) apolipoprotein E-deficient mice after 24 weeks, but not after 12 weeks of the high-cholesterol diet. Our findings demonstrate a stage-specific role of miR-155 in lesion formation. miR-155 suppressed macrophage proliferation by targeting colony-stimulating factor-1 receptor in early and impaired efferocytosis by downregulating B-cell leukemia/lymphoma 6 in advanced atherosclerosis. Therefore, targeting the interaction between miR-155 and B-cell leukemia/lymphoma 6 may be a promising approach to inhibit the progression of atherosclerosis.

MicroRNA-155 induction by Mycobacterium bovis BCG enhances ROS production through targeting SHIP1

Macrophages play a critical role in the host immune response against mycobacterial infection. Our previous study has demonstrated that microRNA-155 (miR-155), one of the most important small non-coding RNAs in the immune system, promotes oxygen-independent mycobacterial killing in macrophages. However, little is known regarding the role of miR-155 in modulating oxygen-dependent mycobactericidal response in macrophages, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present study, we demonstrated that miR-155 was increased in macrophages after Mycobacterium bovis bacille Calmette-Guérin (BCG) infection. Moreover, the BCG-induced upregulation of miR-155 in macrophages was dependent on TLR2, NF-κB and JNK signaling pathways. More importantly, our study explored that miR-155 significantly elevated ROS production in macrophages, although miR-155 had no influence on the inducible nitric oxide synthase (iNOS) expression or nitric oxide (NO) production. In addition, our study demonstrated that miR-155 repressed the expression of src homology 2 (SH2) containing inositol 5-phosphatase1 (SHIP1), and knockdown of SHIP1 greatly increased ROS production in BCG-infected macrophages. Collectively, these data indicate that miR-155 modulates ROS but not RNS production by targeting SHIP1, which may provide a better understanding of the host anti-mycobacterial response.

Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis

MicroRNAs (miRNAs) play key roles in inflammatory responses of macrophages. However, the function of miRNAs in macrophage-derived foam cell formation is unclear. Here, we investigated the role of miRNAs in macrophage-derived foam cell formation and atherosclerotic development. Using quantitative reverse transcription-PCR (qRT-PCR), we found that the level of miR-155 expression was increased significantly in both plasma and macrophages from atherosclerosis (ApoE(-/-)) mice. We identified that oxidized low density lipoprotein (oxLDL) induced the expression and release of miR-155 in macrophages, and that miR-155 was required to mediate oxLDL-induced lipid uptake and reactive oxygen species (ROS) production of macrophages. Furthermore, ectopic overexpression and knockdown experiments identified that HMG box-transcription protein1 (HBP1) is a novel target of miR-155. Knockdown of HBP1 enhanced lipid uptake and ROS production in oxLDL-stimulated macrophages, and overexpression of HBP1 repressed these effects. Furthermore, bioinformatics analysis identified three YY1 binding sites in the promoter region of pri-miR-155 and verified YY1 binding directly to its promoter region. Detailed analysis showed that the YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress oxLDL-induced foam cell formation. Importantly, inhibition of miR-155 by a systemically delivered antagomiR-155 decreased clearly lipid-loading in macrophages and reduced atherosclerotic plaques in ApoE(-/-) mice. Moreover, we observed that the level of miR-155 expression was up-regulated in CD14(+) monocytes from patients with coronary heart disease. Our findings reveal a new regulatory pathway of YY1/HDACs/miR-155/HBP1 in macrophage-derived foam cell formation during early atherogenesis and suggest that miR-155 is a potential therapeutic target for atherosclerosis.

MicroRNA-155 Deficiency Results in Decreased Macrophage Inflammation and Attenuated Atherogenesis in Apolipoprotein E–Deficient Mice

microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E-deficient (apoE(-/-)) and miR155(-/-)/apoE(-/-) (double knockout) mice fed a Western diet. Compared with apoE(-/-) mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6C(high) cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE(-/-) mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role. miR155 deficiency attenuates atherogenesis in apoE(-/-) mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.

Abstract 35: Elevated MicroRNA-155 Targets HBP1 to Promote the Formation of Macrophage-Derived Foam Cell and This Can Be Effectively Suppressed by YY1/HDACs Complex

Aims: We aim to investigate the role of microRNAs in the formation of macrophage-derived foam cell. Methods and results: Using quantitative RT-PCR, we found that the level of microRNA-155 (miR-155), a macrophage-associated microRNA, was significantly increased both in plasma of atherosclerotic ApoE-/- mice and in macrophages isolated from this animal model. Indeed, oxLDL effectively induced expression and release of miR-155 in macrophages. We further identified that miR-155 was required to mediate oxLDL-induced lipid uptake and ROS production of macrophages. Importantly, ectopic overexpression and knockdown experiments identified that HMG box-containing protein 1(HBP1) is a novel target of miR-155. Knockdown of HBP1considerably enhanced lipid uptake and ROS production in oxLDL-induced macrophages, and overexpression of HBP1 effectively repressed these effects caused by miR-155 overexpression. Interestingly, bioinformatics analysis identified three YY1 binding sites in promoter region of pri-miR- 155. EMSA and ChIP analyses verified YY1 directly binding to its promoter region. Furthermore, quantitative RT-PCR analysis showed that YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress foam cell formation. Conclusions: In oxLDL-induced macrophages, elevated miR-155 directly targets HBP1 to promote lipid uptake and ROS production, and YY1/HDAC2/4 complex effectively inhibits foam cell formation mediated by miR-155. This study reveals a novel molecular mechanism in the atherosclerosis and suggests miR-155 is a potential therapeutic target.

Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

Inducible microRNA-155 Feedback Promotes Type I IFN Signaling in Antiviral Innate Immunity by Targeting Suppressor of Cytokine Signaling 1

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-κB-dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.