Decrease of miR-146b-5p in monocytes during obesity is associated with loss of the anti-inflammatory but not insulin signaling action of adiponectin

Background

Low adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFκB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFκB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin.

Methods

miR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes.

Results

miR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFκB p65 DNA binding activity and TNFα. As a response IRAK3 and IRS1 protein increased. Mitochondrial and intracellular ROS production did not increase despite more inflammation. In addition, exposure of miR-146b-5p-depleted THP-1 monocytes to high levels of globular adiponectin resulted in an increased production of TNFα and intracellular ROS. Still, they did not lose their potential to increase IRAK3 and IRS1 protein and to decrease mitochondrial ROS.

Conclusion

miR-146b-5p, decreased in monocytes during obesity, is a major mediator of the anti-inflammatory action of globular adiponectin. It appears not to be involved in insulin signaling possibly by protective response of IRAK3 and lack of mitochondrial ROS production.     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.     

miR-21a-5p Promotes Inflammation following Traumatic Spinal Cord Injury through Upregulation of Neurotoxic Reactive Astrocyte (A1) Polarization by Inhibiting the CNTF/STAT3/Nkrf Pathway

Reactive astrocytes are implicated in traumatic spinal cord injury (TSCI). Interestingly, naïve astrocytes can easily transform into neurotoxic reactive astrocytes (A1s) with inflammatory stimulation. Previous studies demonstrated that microRNA(miR)-21a-5p was up-regulated in spinal cord tissue after TSCI; however, it is not clear whether this affected reactive astrocyte polarization. Here, we aim to detect the effects of miR-21a-5p on the induction of A1 formation and the underlying mechanisms. Our study found that the expression of miR-21a-5p was significantly increased while that of Cntfr α was decreased, since naïve astrocytes transformed into A1s 3 days post-TSCI; the binding site between miR-21a-5p and Cntfr α was further confirmed in astrocytes. After treatment with CNTF, the levels of A1 markers decreased while that of A2 increased. The expression of A1 markers significantly decreased with the downregulation of miR-21a-5p, while Cntfr α siRNA treatment caused the opposite both in vitro and in vivo. To summarize, miR-21a-5p/Cntfr α promotes A1 induction and might enhance the inflammatory process of TSCI; furthermore, we identified, for the first time, the effect and potential mechanism by which CNTF inhibits naïve astrocytes transformation into A1s. Collectively, our findings demonstrate that targeting miR-21a-5p represents a prospective therapy for promoting the recovery of TSCI.     

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3′ untranslated region (UTR) but not TRAF6 3′-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.     

Exosomal miR-146a-5p and miR-155-5p promote CXCL12/CXCR7-induced metastasis of colorectal cancer by crosstalk with cancer-associated fibroblasts

C-X-C motif chemokine receptor 7 (CXCR7) is a newly discovered atypical chemokine receptor that binds to C-X-C motif chemokine ligand 12 (CXCL12) with higher affinity than CXCR4 and is associated with the metastasis of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) have been known to promote tumor progression. However, whether CAFs are involved in CXCR7-mediated metastasis of CRC remains elusive. We found a significant positive correlation between CXCR7 expression and CAF activation markers in colonic tissues from clinical specimens and in villin-CXCR7 transgenic mice. RNA sequencing revealed a coordinated increase in the levels of miR-146a-5p and miR-155-5p in CXCR7-overexpressing CRC cells and their exosomes. Importantly, these CRC cell-derived miR-146a-5p and miR-155-5p could be uptaken by CAFs via exosomes and promote the activation of CAFs through JAK2–STAT3/NF-κB signaling by targeting suppressor of cytokine signaling 1 (SOCS1) and zinc finger and BTB domain containing 2 (ZBTB2). Reciprocally, activated CAFs further potently enhanced the invasive capacity of CRC cells. Mechanistically, CAFs transfected with miR-146a-5p and miR-155-5p exhibited a robust increase in the levels of inflammatory cytokines interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and CXCL12, which trigger the epithelial–mesenchymal transition and pro-metastatic switch of CRC cells. More importantly, the activation of CAFs by miR-146a-5p and miR-155-5p facilitated tumor formation and lung metastasis of CRC in vivo using tumor xenograft models. Our work provides novel insights into CXCR7-mediated CRC metastasis from tumor–stroma interaction and serum exosomal miR-146a-5p and miR-155-5p could serve as potential biomarkers and therapeutic targets for inhibiting CRC metastasis.Subject terms: Cancer microenvironment, Colon cancer     

IL4I1 Is a Novel Regulator of M2 Macrophage Polarization That Can Inhibit T Cell Activation via L-Tryptophan and Arginine Depletion and IL-10 Production

Interleukin 4-induced gene-1 (IL4I1) was initially described as an early IL-4-inducible gene in B cells. IL4I1 protein can inhibit T cell proliferation by releasing its enzymatic catabolite, H₂O₂, and this effect is associated with transient down-regulation of T cell CD3 receptor-zeta (TCRζ) expression. Herein, we show that IL4I1 contributes to the regulation of macrophage programming. We found that expression of IL4I1 increased during bone marrow-derived macrophage (BMDM) differentiation, expression of IL4I1 is much higher in primary macrophages than monocytes, and IL4I1 expression in BMDMs could be induced by Th1 and Th2 cytokines in two different patterns. Gene expression analysis revealed that overexpression of IL4I1 drove the expression of M2 markers (Fizz1, Arg1, YM-1, MR) and inhibited the expression of M1-associated cytokines. Conversely, knockdown of IL4I1 by siRNA resulted in opposite effects, and also attenuated STAT-3 and STAT-6 phosphorylation. Furthermore, IL4I1 produced by macrophages catalyzed L-tryptophan degradation, while levo-1-methyl-tryptophan (L-1-MT), but not dextro-1-methyl-tryptophan, partially rescued IL4I1-dependent inhibition of T cell activation. Other inhibitors, such as diphenylene iodonium (DPI), an anti-IL-10Rα blocking antibody, and a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, also had this effect. Overall, our findings indicate that IL4I1 promotes an enhanced M2 functional phenotype, which is most likely associated with the phosphorylation of STAT-6 and STAT-3. Moreover, DPI, L-1-MT, NG-monomethyl-L-arginine, and anti-IL-10Rα blocking antibody were all found to be effective IL4I1 inhibitors in vitro.     

Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology

Background

Despite diverging levels of amyloid-β (Aβ) and TAU pathology, different mouse models, as well as sporadic AD patients show predictable patterns of episodic memory loss. MicroRNA (miRNA) deregulation is well established in AD brain but it is unclear whether Aβ or TAU pathology drives those alterations and whether miRNA changes contribute to cognitive decline.

Methods

miRNAseq was performed on cognitively intact (4 months) and impaired (10 months) male APPtg (APP^swe/PS1^L166P) and TAUtg (THY-Tau22) mice and their wild-type littermates (APPwt and TAUwt). We analyzed the hippocampi of 12 mice per experimental group (n =?96 in total), and employed a 2-way linear model to extract differentially expressed miRNAs. Results were confirmed by qPCR in a separate cohort of 4 M and 10 M APPtg and APPwt mice (n =?7–9 per group) and in human sporadic AD and non-demented control brain. Fluorescent in situ hybridization identified their cellular expression. Functional annotation of predicted targets was performed using GO enrichment. Behavior of wild-type mice was assessed after intracerebroventricular infusion of miRNA mimics.

Results

Six miRNAs (miR-10a-5p, miR-142a-5p, miR-146a-5p, miR-155-5p, miR-211-5p, miR-455-5p) are commonly upregulated between APPtg and TAUtg mice, and four of these (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) are altered in AD patients. All 6 miRNAs are strongly enriched in neurons. Upregulating these miRNAs in wild-type mice is however not causing AD-related cognitive disturbances.

Conclusion

Diverging AD-related neuropathologies induce common disturbances in the expression of neuronal miRNAs. 4 of these miRNAs are also upregulated in AD patients. Therefore these 4 miRNAs (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) appear part of a core pathological process in AD patients and APPtg and TAUtg mice. They are however not causing cognitive disturbances in wild-type mice. As some of these miRNA target AD relevant proteins, they may be, in contrast, part of a protective response in AD.

     

Circular RNA RSF1 promotes inflammatory and fibrotic phenotypes of irradiated hepatic stellate cell by modulating miR-146a-5p

The role of circular RNA (circRNA) in radiation-induced liver disease (RILD) remains largely unknown. In this study, Ras-related C3 botulinum toxin substrate 1 (RAC1) was elevated in irradiated human hepatic stellate cell (HSC) line LX2, the important effector cell mediating RILD. Overexpression of RAC1 promotes cell proliferation, proinflammatory cytokines production, and α-smooth muscle actin expression, which were blocked by microRNA (miR)-146a-5p mimics. CircRNA RSF1 (circRSF1) was upregulated in irradiated LX2 cells and predicted to harbor binding site for miR-146a-5p. Biotinylated-RNA pull down and dual-luciferase reporter detection confirmed the direct interaction of circRSF1 and miR-146a-5p. Enforced expression of circRSF1 increased RAC1 expression by acting as miR-146a-5p sponge to inhibit miR-146a-5p activity, and thus enhanced the cell viability, and promoted inflammatory and fibrotic phenotype of irradiated LX2 cells. These findings indicate a functional regulatory axis composing of circRSF1, miR-146a-5p, and RAC1 in irradiated HSC, which may provide attractive therapeutic targets for RILD.     

METTL3 inhibition ameliorates liver damage in mouse with hepatitis B virus-associated acute-on-chronic liver failure by regulating miR-146a-5p maturation

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) is a clinical syndrome of severe liver damage. HBV infection is affected by N6-methyladenosine (m⁶A) RNA modification. Here, we investigated whether methyltransferase-like 3 (METTL3)-mediated m⁶A methylation can affect ACLF. Human hepatic cells (THLE-2) were treated with lipopolysaccharide (LPS) to induce cell damage. Proliferation, apoptosis and m⁶A modification were measured by MTT assay, flow cytometry and Dot blot assay. Our results showed that HBV infection significantly enhanced the levels of m⁶A modification and elevated the expression of METTL3 and mature-miR-146a-5p in THLE-2 cells, which was repressed by cycloleucine (m⁶A inhibitor). METTL3 overexpression enhanced m⁶A modification and promoted mature-miR-146a-5p expression. METTL3 overexpression promoted HBV replication and apoptosis, enhanced the levels of pro-inflammatory cytokines, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), and repressed cell proliferation in THLE-2 cells, which attributed to repress miR-146a-5p maturation. Moreover, a severe liver failure mouse model was established by HBV infection to verify the impact of METTL3 knockdown on liver damage in vivo. HBV-infection led to a severe liver damage and increase of apoptosis in hepatic tissues of mice, which was abolished by METTL3 knockdown. METTL3 knockdown reduced METTL3 expression and impeded miR-146a-5p maturation in HBV-infected mice. In conclusion, this work demonstrates that METTL3 inhibition ameliorates liver damage in mouse with HBV-associated ACLF, which contributes to repress miR-146a-5p maturation. Thus, this article suggests a novel therapeutic avenue to prevent and treat HBV-associated ACLF.     

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4^?/? mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-β with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.     

miR-146a-5p circuitry uncouples cell proliferation and migration,but not differentiation,in human mesenchymal stem cells

Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton’s jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared with BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed and has high abundance in WJ-MSCs. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration, whereas overexpression of miR-146a-5p in BM-MSCs did not influence their osteogenic and adipogenic potentials. Chemokine (C-X-C motif) ligand 12 (CXCL12), together with SIKE1, which is an I-kappa-B kinase epsilon (IKKε) suppressor, is a direct target of miR-146a-5p in MSCs. Knockdown of miR-146a-5p resulted in the down-regulation of nuclear factor kappa-B (NF-κB) activity, which is highly activated in WJ-MSCs and is known to activate miR-146a-5p promoter. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. These findings suggest that miR-146a-5p is critical to the uncoupling of motility and proliferation of MSCs. Our miRNome data also provide a roadmap for further understanding MSC biology.     

Infection of J774A.1 with different Mycobacterium species induces differential immune and miRNA‐related responses

Macrophages act as a reservoir for Mycobacterium tuberculosis, producing latent infection in approximately 90% of infected people. In this study, J774A.1 mouse macrophage cell line response and microRNA (miRNA) expression during infection with the most relevant mycobacterial strains for humans (M. tuberculosis, M. bovis and M. bovis BCG) was explored. No significant differences in bacillary loads were observed between activate and naive macrophages infected with M. tuberculosis and M. bovis. Nitrite production inhibition and infection control were in accordance with the virulence of the strain. Expression of let‐7e, miR‐21, miR‐155, miR‐210 and miR‐223 was opposite in the two species and miR‐146b* and miR‐1224 expression seemed to be part of the general response to infection.     

MicroRNA-146a and MicroRNA-146b Regulate Human Dendritic Cell Apoptosis and Cytokine Production by Targeting TRAF6 and IRAK1 Proteins

We have previously reported 27 differentially expressed microRNAs (miRNAs) during human monocyte differentiation into immature dendritic cells (imDCs) and mature DCs (mDCs). However, their roles in DC differentiation and function remain largely elusive. Here, we report that microRNA (miR)-146a and miR-146b modulate DC apoptosis and cytokine production. Expression of miR-146a and miR-146b was significantly increased upon monocyte differentiation into imDCs and mDCs. Silencing of miR-146a and/or miR-146b in imDCs and mDCs significantly prevented DC apoptosis, whereas overexpressing miR-146a and/or miR-146b increased DC apoptosis. miR-146a and miR-146b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 expression. Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs enhanced DC apoptosis. By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival. Moreover, silencing of miR-146a and miR-146b expression had little effect on DC maturation but enhanced IL-12p70, IL-6, and TNF-α production as well as IFN-γ production by IL-12p70-mediated activation of natural killer cells, whereas miR-146a and miR-146b overexpression in mDCs reduced cytokine production. Silencing of miR-146a and miR-146b in DCs also down-regulated NF-κB inhibitor IκBα and increased Bcl-2 expression. Our results identify a new negative feedback mechanism involving the miR-146a/b-TRAF6/IRAK1-NF-κB axis in promoting DC apoptosis.     

Down regulation of miR-30a-5p and miR-182–5p in gastric cancer: Clinical impact and survival analysis

Background and aimGastric Cancer (GC) is a leading cause of morbidity and mortality worldwide, particularly in developing nations, only a few suitable gastric cancer serum biomarkers with acceptable sensitivity and specificity exist. This work aims to highlight and uncover miR-30a-5p and miR-182–5p′s diagnostic roles regarding gastric cancer and their roles in predicting prognosis.Methods148 patients participated in this study. Groups I, II, and III had 47 patients with GC, 54 patients with benign gastric lesions, and 47 apparently healthy subjects of coincided age and gender as controls, respectively. All participants were clinically evaluated and subjected to CBC, serum CEA, and CA19-9 by ELISA, and real-time PCR tests of miR-30a-5p and miR-182–5p.ResultsMiR30a-5p and miR-182–5p were down regulated in gastric cancer patients in Group I more than Groups II and III (P < 0.001). ROC curve analysis revealed that miR30a-5p had better AUC, sensitivity, and specificity (0.961%, 93.62%, and 90.74%respectively). When miR-182–5p was gathered with CEA and CA19-9, specificity raised to 98.15% and PPV to 97.6%. Lower miR-30a-5p levels are linked with the presence of distant metastases, advanced TNM stage, and degree of pathological differentiation of tumors in GC patients (p = 0.034, 0.019, 0.049) respectively. According to the multivariate analysis, miR30a-5p expression level could be an independent predictor of GC.ConclusionOur results exhibited that miRNAs, miR-30a-5p and miR182–5p, gene expression have a diagnostic power and can identify patients with GC. MiR-30a-5p displayed the highest diagnostic specificity and sensitivity. Besides other known tumor markers, they could offer simple noninvasive biomarkers that predict gastric cancer.