The Journal of ImmunologyMicroRNA-127 Inhibits Lung Inflammation by Targeting IgGFcgReceptor ITing Xie,*,†Jiurong Liang,* Ningshan Liu,* Qingguo Wang,†Yuhang Li,†Paul W. Noble,*and Dianhua Jiang*The molecular mechanisms of acute lung injury are incompletely understood. MicroRNAs (miRNAs) are crucial biological reg-ulators that act by suppressing their target genes and are involved in a variety of pathophysiologic processes. miR-127 appears to bedownregulated during lung injury. We set out to investigate the role of miR-127 in lung injury and inflammation. Expression of miR-127 significantly reduced cytokine release by macrophages. Looking into the mechanisms of regulation of inflammation by miR-127,we found that IgG FcgRI (CD64) was a target of miR-127, as evidenced by reduced CD64 protein expression in macrophagesoverexpressing miR-127. Furthermore, miR-127 significantly reduced the luciferase activity with a reporter construct containingthe native 39untranslated region of CD64. Importantly, we demonstrated that miR-127 attenuated lung inflammation in an IgGimmune complex model in vivo. Collectively, these data show that miR-127 targets macrophage CD64 expression and promotesthe reduction of lung inflammation. Understanding how miRNAs regulate lung inflammation may represent an attractive way tocontrol inflammation induced by infectious or noninfectious lung injury. The Journal of Immunology, 2012, 188: 2437–2444.Acute lung injury (ALI) is characterized by hypoxemia,pulmonary edema, reduced lung compliance, and im-paired gas exchange (1). Severe lung injury leads to acuterespiratory distress syndrome (ARDS), characterized by severelung inflammation and profound hypoxemia, and frequently resultsin multiple organ failure (2). Both ALI and ARDS are major causesof morbidity and mortality. The molecular and immunologicalmechanisms of ALI remain incompletely understood.When the lung encounters an exogenous insult, epithelial cellsand macrophages are the primary lines of defense. The injured cellstrigger a cascade of events including acute inflammatory response,recruitment of immune cells such as monocytes/macrophages, andrelease of cytokines (IL-1, IL-6, and TNF-a), chemokines, growthfactors, and PGs (3). Through innate immunity, the structures ofinvading microorganisms, including lipids, carbohydrates, pep-tides, and nucleic acids, are first recognized by pattern-recognitionreceptors (4). Pattern-recognition receptors include the TLR fam-ily, including TLR4 (4). TLR4 is essential for responses to bac-terial LPS as well as to various endogenous ligands, such ashyaluronan (HA) fragments (5, 6). Engagement of the TLR4 re-ceptor triggers the activation of an intracellular signaling pathway,resulting in subsequent cytokine/chemokine production and re-lease (6). Through the adaptive immune system, Fc receptorsrecognize the Fc domain of Ig and thereby link the Ab-mediatedimmune response to cellular effector functions including phago-cytosis, release of inflammatory mediators, and clearance of im-mune complexes (IC). FcgRs belong to the Ig superfamily and arethe most important Fc receptors for phagocytosis of opsonizedmicrobes, mainly including FcgRI, FcgRII, FcgRIII, and FcgRIV(7). Whereas FcgRI (CD64) is constitutively present on onlymonocytes and macrophages, FcgRIII is expressed in many tis-sues, but absent in lymphocytes. FcgRII is present on almost allhematopoietic cells. FcgRI, FcgRIII, and FcgRIV function asactivating receptors, in which FcgRII acts as a negative regulator.Alveolar macrophages express FcgRI, FcgRII, and FcgRIII (8).FcgRI-deficient mice showed impaired cytokine release, phago-cytosis, and cellular cytotoxicity in IC-induced inflammation,suggesting a critical role for FcgRI in IgG2a-IC–dependent im-mune functions (9).Numerous human diseases are thought to result from the failureto regulate the production and clearance of IC. Circulating IC werefound in patients with systemic lupus erythematosus (10), rheu-matoid arthritis (11), Goodpasture syndrome (12), and nephritis(13). In the respiratory system, basement membrane destructionby IC is found in patients with ARDS (14), idiopathic interstitialpneumonias (15), and hypersensitivity pneumonitis/alveolitis (16).Studies suggest that the anti–IL-8 autoantibody–IL-8 IC werefound in lung fluids from patients with ALI/ARDS and correlatedboth with the development and outcome of ARDS (14, 17). Anti-keratinocyte chemoattractant (KC)–KC complexes induced lunginflammation in mice and were associated with the developmentof severe pulmonary inflammation (18). A recent study suggestedthat anti-chemokine autoantibody–chemokine IC may contributeto the pathogenesis of lung inflammation by inducing activation ofendothelial cells through engagement of the IgG receptor FcgRIIa(14). The molecular mechanisms by which the IC regulates in-flammatory responses are largely unclear.*Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medi-cine, Duke University School of Medicine, Durham, NC 27710; and†School of BasicMedical Science, Beijing University of Chinese Medicine, Beijing 100029, ChinaReceived for publication April 13, 2011. Accepted for publication December 29,2011.This work was supported by the National Institutes of Health Grants R01-HL 060539and R01-HL 077291 (to P.W.N). T.X. was supported in part by a Ph.D. studentfellowship from the China Scholarship Council.The complete microarray data set presented in this article has been submitted to theNational Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE27642.Address correspondence and reprint requests to Dr. Dianhua Jiang, Division of Pul-monary, Allergy, and Critical Care Medicine, Duke University School of Medicine,106 Research Drive, 2089 Medical Science Research Building II, Durham, NC27710. E-mail address: dianhua.jiang@duke.eduThe online version of this article contains supplemental material.Abbreviations used in this article: ALI, acute lung injury; ARDS, adult respiratorydistress syndrome; BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid;HA, hyaluronan; IC, immune complex; KC, keratinocyte chemoattractant; LNA,locked nucleic acid; miRNA, microRNA; 29-OME, 29-O-methyl; PGN, peptidogly-can; UTR, untranslated region.Copyright Ó2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101070 on February 22, 2012www.jimmunol.orgDownloaded from MicroRNAs (miRNAs) are critical regulators of gene expression.Mature miRNAs bind target mRNAs at complementary sites in 39untranslated regions (UTRs) or coding sequences and therebytrigger downregulation and suppression of target gene expression(19). Emerging evidence also shows that miRNAs play an im-portant role in both adaptive and innate immunity (20). miRNAsare involved in innate immunity through the regulation of TLRsignaling and cytokine responses. For example, reports showedthat miR-146, miR-155, and miR-132 are strongly upregulatedafter LPS treatment in human monocyte THP1 (21–23). miR-146may target IL-1R–associated kinase-1 and TNFR-associated fac-tor 6, two essential components of the TLR signaling pathways,thereby acting as a negative regulator of the inflammatory re-sponse (21). miR-147 is induced by TLR stimulation and canregulate the macrophage inflammatory response by decreasingLPS-induced TNF-aand IL-6 production (24). miRNAs alsoregulate adaptive immune responses. For example, silencing ofmiR-126 by antagomir impairs Th2 responses in the lung (25).miR-181a appears to act as a negative regulator of TCR signalingby targeting Bcl-2 and CD69, mediating positive selection (26,27). Furthermore, miR-155 has a role in regulating Th cell dif-ferentiation and the germinal center reaction to produce an opti-mal T cell-dependent Ab response (28).We have recently analyzed miRNA expression after bleomycin-induced lung injury and repair (29) and identified that miR-127expression was decreased 1 d after bleomycin treatment. In thisreport, we examined miR-127 expression in both noninfectiousand infectious lung injury. To examine the role of miR-127 inmacrophages, we established a miR-127 stable overexpression ofMH-S cells. We demonstrated that miR-127 directly down-regulates CD64 and attenuated lung inflammation in vivo. Takentogether, these data suggest that miR-127 plays a previously un-recognized role in preventing exaggerated inflammatory responsesto complement injury by negatively regulating FcgRI.Materials and MethodsReagentsPeptidoglycan (PGN) from Bacillus subtilis and LPS from Escherichia coli0111:B4 were purchased from Sigma-Aldrich. HA fragments (∼200,000kDa in molecular mass, LPS-free) were from ICN. IC were preparedaccording to previous reports (30). Briefly, IgG-BSA IC were produced byslowly adding four volumes IgG anti-BSA (5 mg/ml; MP Biomedicals) toone volume BSA (10 mg/ml; Sigma-Aldrich) and incubating at 37˚C for 30min. The complexes were recovered by centrifugation and resuspended inRPMI 1640 medium. Macrophage cell lines MH-S and RAW246.7, as wellas human monocyte cell line U937, were stimulated with IgG-BSA IC(100 mg/ml) or RPMI 1640 medium as control.CellsThe mouse macrophage cell line RAW264.7, alveolar macrophage cell lineMH-S and human monocyte cell line U937 were purchased from theAmerican Type Culture Collection. Cells were cultured in RPMI 1640 plus10% FBS at 37˚C and 5% CO2. U937 were differentiated and activatedwith PMA (100 nM; Sigma-Aldrich) before other treatment.miRNA oligonucleotides29-O-methyl (29-OME) modified miRNAs were chemically synthesized byIDT. All bases were 29-OME modified, and a cholesterol moiety waslinked to the 39terminus. miR-127: 59-mUmCmG mGmAmU mCmCmGmUmCmU mGmAmG mCmUmU mGmGmC mU/3CholTEG/-39, in which\"m” denotes 29-OME modification. CEMR67: 59-mUmCmA mCmAmAmCmCmUm CmCmUm AmGmAm AmAmG mAmG/3CholTEG/-39.Custom locked nucleic acid (LNA) oligonucleotides were synthesized byExiqon: LNA–miR-127, 59-+T+CGG+ATC+CGT+CTG+AGC+TTG+G+C-39; LNA-anti–miR-127, 59-+G+CCA+AGC+TCA+GAC+GGA+TCC+G+A-39; and control probe (CEMR67), 59-+T+CACA+ACCT+CCT+AGA+AAG+A+G-39, in which the plus (+) sign denotes the LNA modi-fication.miR-127 precursor, inhibitor, and control probes were purchased fromAmbion.Expression constructs and stable transductionmiR-127 lentiviral construct was generated as follows: a region of 101nucleotides containing premiR-127 was amplified from mouse genomicDNA by using the following primers: 59MIR127, 59-GAT CAC TGT TAACCA GCC TGC T-39; and 39MIR127, 59-TTA ACT CGA GGG AGGCAG ATG A-39. They were then cloned into the HpaI/XhoI sites of a pSicovector (31) named pSico-miR-127. Viral particles were produced by cal-cium phosphate-mediated transfection into 293T cells as described (32).Lentiviral supernatants were collected 48 h after transfection, passedthrough a 0.22-mm filter, and used directly to infect MH-S cells. GFP-positive cells were sorted 3 to 4 d postinfection and resulted in MH-S-SicoGFP and MH-S–miR-127GFP cells. Recombinant adenoviral stocksexpressing Cre recombinase were purchased from the Gene TransferVector Core facility of the University of Iowa College of Medicine (IowaCity, IA). Infections of GFP-positive cells were performed by using 100PFU virus per cell. Four days after adenovirus infections, the GPF-negativecells were sorted from GFPposinfected with adeno-Cre as MH-S-Sico andMH-S–miR-127 cells. Cre recombination was discriminated by PCR am-plification of genomic DNA with the following primers: pSico-Cre-UP: 59-GGG ACA GCA GAG ATC CAG TT-39and pSico-Cre-DN: 59-ACC GGAACC CTT AAA CAT GA-39. The unrecombined viral DNA results in a1921-bp band, and the recombination viral DNA results in a 495-bp band.Animals, IgG IC-induced lung injury, bleomycin-induced lunginjury, and bronchoalveolar lavage analysisAll experiments were carried out using 8–12-wk-old C57BL/6 female mice(The Jackson Laboratory). All studies were conducted in accordance withNational Institutes of Health guidelines for the care and use of animals andwith approval from the Duke University Animal Care and Use Committee.Rabbit anti-BSA IgG (catalog number 55275; MP Biomedicals; 10 mgin20 ml PBS) was administered intranasally, and 5 mg/kg BSA (A8412; Sigma-Aldrich) in 200 ml PBS was injected i.v. immediately thereafter. Control micereceived BSA i.v. in the absence of an intranasal dose of anti-BSA. To assesspermeability, 0.25% Evans blue (catalog number A16774; Alfa Aesar) wasadministrated with BSA i.v. Because neither CEMR67 nor PBS had anyeffect, either CEMR67 or PBS was used as control in certain experiments.When miR-127 and control probe CEMR67 were used, 100 mgin40mlPBSwasinstilledintranasally2hbeforeanti-BSAIgGadministration.Animals were sacrificed 4 h after IgG IC-induced alveolitis according toreports (33). The trachea was catheterized, and lungs were lavaged with 0.8ml ice-cold PBS three times. The first 0.8 ml bronchoalveolar lavage fluid(BALF) was collected for ELISA, and the total and differential cells inBALF were counted by use of a hemocytometer. Cell differentials wereperformed on cytospin preparations stained with the protocol Hema3 stainset (Fisher Healthcare). Evans blue concentration in BALF was determinedby an ELISA reader under 405 nm for lung permeability analysis. Totalprotein level was determined by bicinchoninic acid protein assay.Under anesthesia, 2.5 U/kg bleomycin (Blenoxane; Mayne Pharma,Paramus, NJ) dissolved in sterile PBS was administered via trachea aspreviously described (6, 34, 35). Lung tissues were harvested 1 d post-bleomycin challenges or from untreated mice.RNA analysisRNA was isolated from cells with TRIzol reagent (Invitrogen) or themirVana miRNA Isolation Kit (Ambion). The expression of miR-127 wasnormalized to sno202 and analyzed using TaqMan miRNA assays (AppliedBiosystems).Luciferase assayConstruct generation. The full-length FcgRI 39-UTR was amplified byPCR from mouse genomic DNA with the following primers: FcgRI-Forward, 59-GGT GAC TAG TGG ACC CGA GCA GCC-39;andFcgRI-Reverse, 59-AAG TGA GCT CAG GTC CTC TGG AAG-39. Theamplified 1404-bp product was inserted into the SpeI/SacI sites of theluciferase expression vector pMIR-REPORT (Applied Biosystems) im-mediately downstream of the luciferase gene and termed pMIR-REPORT[FcgRI–39-UTR]. A mutant construct was generated with two primers:FcgRI-3m-Forward, 59-GAC TGG CCG CGG CTG AGA CAA GCT GGGTAA TCA GAC-39, and FcgRI-3m-Reverse, 59-GCC GCG GCC AGTCTG TAT ATT TGC TTT ATT TAA GAG TTG CAT GCC-39cloned intopMIR-REPORT, resulting in pMIR-REPORT [mFcgRI–39-UTR].Transfection. RAW264.7 cells were cotransfected with 0.4 mg firefly lu-ciferase report vector and 0.08 mg control vector containing Renilla lu-2438 miR-127 INHIBITS LUNG INFLAMMATION on February 22, 2012www.jimmunol.orgDownloaded from ciferase pRL-TK (Promega) in 24-well plates by using Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol. For each well,10 nM miR-127 precursor and miR-127 inhibitor (Ambion) were used. Forluciferase assay, Firefly and Renilla luciferase activities were measuredconsecutively by using dual luciferase assays (Promega) 48 h after trans-fection according to the manufacturer’s suggestions.Cytokine and complement productionTNF-a, IL-6, IL-1b, MIP-1b, MIP-2, MCP-1, KC, and complement 5fragment a (C5a) protein levels in supernatant collected from cell cultureor in BALF of injured mice were measured immunologically using com-mercial ELISA kits (R D Systems). Complement C5a in BAL of injuredmice were determined with ELISA with Abs from BD Pharmingen (cap-ture Ab: purified rat anti-mouse C5a, catalog number 558027; standard:purified recombinant mouse C5a, catalog number 622597; detect Ab: bi-otin rat anti-mouse C5a, catalog number 558028).Protein analysisProtein extracts were isolated from cells or lung tissues. CD64, STAT3, andphospho-Stat3 (Tyr705) levels were determined using goat polyclonal anti-CD64 (R D Systems), mouse monoclonal anti-Stat3 (Cell Signaling Tech-nology), or rabbit monoclonal anti-phospho-Stat3 (Tyr705) (Cell SignalingTechnology) and normalized to GAPDH (Cell Signaling Technology).Microarray analysisMicroarray analysis was performed using the Affymetrix Mouse Genome430 2.0 Array (Affymetrix). Gene chips were scanned by the GenePix4000B scanner (Molecular Devices, Sunnyvale, CA). Data and images wereobtained using GenePix Pro 6.1 (Molecular Devices, Sunnyvale, CA)according to the manufacturer’s instructions. RNAs extracted from MH-S-Sico cells were hybridized on Cy3, and RNAs extracted from MH-S–miR-127 cells were hybridized on Cy5. Data were background subtracted andnormalized within the array using the LOESS normalization by GenespringGX 11 software. The complete microarray data set is available at theNational Center for Biotechnology Information Gene Expression Omnibus(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE27642.Flow cytometryRAW264.7 and MH-S cells were stained with PE-conjugated anti-mouseCD64 Ab (catalog 558455; BD Pharmingen), and flow cytometry wasused to determine the CD64 staining-positive cells in MH-S-Sico and MH-S–miR-127 cells, to determine the CD64 staining-positive cells in miR-127precursor, inhibitor, and control probe-transfected Raw 264.7 cells, and toselect the top 1% of highest CD64-expressing cells as CD64hiand thebottom 1% of lowest CD64-expressing cells as CD64lo.Synthetic 2’-OME–, cholesterol-modified, and Fam (59)-labeled miR-127 oligonucleotide or control oligonucleotide without Fam labeling (100mgin40ml PBS) was intranasally administered to C57BL/6J mice. BALcells were isolated 2 h later and stained with CD64-PE (catalog number558592; BD Biosciences) and F4/80-allophycocyanin (catalog numberMCA497APCT; AbD Serotec) Abs. Macrophages uptaking Fam-labeledmiR-127 oligonucleotide and CD64 expression on BAL cells isolated frommiR-127 and control oligonucleotide-treated mice were analyzed with flowcytometry.HistologyTo morphologically assess lung injury 4 h after IgG IC deposition, thelungswere inflated with 10% formalin via a tracheal cannula and removed fromthe thoracic cavity. Tissue was fixed overnight, embedded in paraffin, andsectioned for staining with H E.StatisticsData are expressed as the mean 6SEM where applicable. We assesseddifferences in measured variables using the unpaired two-sided Student ttest or Wilcoxon rank-sum test. Differences between multiple groups werecalculated using one-way ANOVA with Tukey-Kramer posttest or two-wayANOVA with Bonferroni multiple comparisons. Statistical difference wasaccepted at p,0.05. Prism 5.0 software (GraphPad) was used to performstatistical analysis.ResultsMature miR-127 is regulated during inflammationThere are limited reports suggesting that miR-127 may play a rolein development (36) and tumor progression (37). However, there isno report on miR-127 targets or on its role in inflammation. Werecently analyzed miRNA expression after bleomycin-inducedlung injury with an miRNA array study (29) and identified thatmiR-127 expression was regulated after injury. Bleomycin treat-ment led to downregulation of miR-127 at day 1 after bleomycin(Fig. 1A). We also found that miR-127 was downregulated afterIgG IC-induced lung injury (Fig. 1B). We reasoned that miR-127may play a role in regulating lung injury and inflammatory re-sponse. When we measured the expression of miR-127 in murineRAW264.7 macrophages and MH-S alveolar macrophages uponLPS stimulation, we found that overnight treatment with LPSshowed a marginal downregulation of miR-127 expression in bothMH-S (Supplemental Fig. 1A) and RAW264.7 macrophages(Supplemental Fig. 1B).Overexpression of miR-127 decreased cytokine expressionWe next determined the ability of miR-127 to modulate the in-flammatory response in macrophages in vitro. To accomplish this,we transduced MH-S cells with a lentivirus encoding miR-127(pSico–miR-127) or an empty lentivirus (pSico) as control (31).Pre–miR-127 gene was cloned into a pSico vector in which theCMV-GFP cassette is located downstream of the U6 promoter andis flanked by loxP sites such that Cre-mediated recombination isexpected to result in the loss of GFP expression and activationof miRNA expression (Supplemental Fig. 2A). PCR confirmationof Cre-mediated recombination was performed to amplify therecombined and unrecombined viral DNA (Supplemental Fig.2B). High-efficiency transduction by the vector was achieved asindicated by uniform GFP expression in infected cells (Supple-mental Fig. 2C). After superinfection with a Cre-expressing re-combinant adenovirus, near complete recombination with con-comitant loss of GFP fluorescence was observed (SupplementalFig. 2C). One week after Cre expression, .2-fold higher level ofmiR-127 expression was detected in cells infected with pSico-miR-127 versus control cells (Supplemental Fig. 2D). This sug-gested we had successfully established the miR-127 overexpressedMH-S cell line.To test the hypothesis that the gain of miR-127 function plays arole in the release of cytokines during inflammation, MH-S–miR-127 and control cells were treated with LPS. Overexpression ofmiR-127 in MH-S cells significantly decreased production of pro-inflammatory cytokines induced by LPS, such as TNF-a, IL-1b,and IL-6 (Fig. 2A–C). These data suggest that miR-127 functionsas a negative regulator of inflammatory response. Given the abilityof miR-127 to downregulate TLR4-induced inflammatory re-sponses, we next investigated if miR-127 also modulates activa-tion of PGN, hyaluronan fragments, and IgG IC-stimulated cells.As shown in Fig. 2, miR-127 significantly attenuated TNF-aandIL-1bproduction induced by PGN and hyaluronan fragments (Fig.2A, 2B), as well as IL-6 production induced by PGN and IgG IC(Fig. 2C).FIGURE 1. Lung injury regulated miR-127 expression. (A) Total RNAwas isolated from normal lungs and lungs 1 d after intratracheal bleomycininstillation (2.5 U/kg). miR-127 expression was determined with TaqManmicroRNA assay. (B) miR-127 was decreased after IgG IC-induced lunginjury. *p,0.05.The Journal of Immunology 2439 on February 22, 2012www.jimmunol.orgDownloaded from To further address this question, RAW264.7 cells, MH-S cells,and PMA-treated human monocyte U937 cells were transientlytransfected with a specific miR-127 precursor, miR-127 inhibitor,or control probes. miR-127 precursor markedly inhibited IgG IC-induced IL-6 by RAW264.7 cells (Fig. 2D), TNF-aby MH-S cells(Fig. 2E), and human TNF-aby U937 cells (Fig. 2F). However,miR-127 precursor, miR-127 inhibitor, or the control probes alonedid not alter cytokine production by RAW264.7, MH-S cells, orU937 cells (Fig. 2D–F). These results demonstrated that miR-127possesses the ability to suppress cytokine releases in various in-flammation models in vitro.CD64 is a target of miR-127We next focused our study on miR-127 regulation in IgG IC-induced inflammation because putative mRNA targets predictedin silico (MicroCosm Targets Version 5 [http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/]) were members of path-ways implicated in FcgR-mediated immune response in macro-phages, including FcgRI, P13K category class IA, VAV-1, PIP5KI,and myosin II. Notably, CD64 was predicted to have a strong miR-127 binding site (Fig. 3A) and showed a minimum free energyof 226.24 kcal/M.We then performed cDNA microarray analysis to determine ifCD64 expression is different between MH-S–miR-127 and controlMH-S–Sico cells. The microarray analysis of RNA isolated fromMH-S–miR-127 and MH-S–Sico cells revealed a downregulationof FcgRI transcripts in MH-S–miR-127 cells compared with MH-S–Sico cells (Supplemental Fig. 3). These data display an inverserelationship between CD64 expression and miR-127 expression,indicating that CD64 is a putative target of miR-127.Next, Western blot analysis of CD64 was performed in MH-S–miR-127 and control cells with and without LPS treatment. CD64protein expression was induced by LPS in control cells, but pro-tein expression of CD64 in MH-S–miR-127 cells was significantlyinhibited in response to LPS stimulation (Fig. 3B). Because CD64is an integral membrane glycoprotein, we performed FACS (9) toinvestigate if membrane CD64 is regulated by miR-127. As ex-pected, flow cytometry analysis showed a decrease in CD64-positive staining on MH-S–miR-127 cells when compared withcontrol cells (Fig. 3C). We also found decreased CD64-positivestaining in miR-127 precursor-transfected RAW 264.7 cells com-pared with the transfection with miR-127 inhibitor or controlprobe (Fig. 3D). Together, these data demonstrate that CD64 isa potential target of miR-127 and is downregulated by miR-127overexpression.To further obtain direct evidence that CD64 is a target of miR-127 and determine whether miR-127 targets and represses CD64directly through a 39-UTR interaction, we constructed a luciferasereporter vector containing the full-length CD64 39-UTR (pMIR-REPORT [FcgRI-39-UTR]) and analyzed the effect of miR-127on luciferase expression. As anticipated, cotransfection of miR-127 attenuated expression of luciferase activity from the pMIR-REPORT [FcgRI-39-UTR] reporter (Fig. 3E), whereas the inhi-bition was not observed when either control miRNA or miR-127inhibitor were used (Fig. 3F). Correspondingly, we also generateda mutant reporter (pMIR-REPORT [mFcgRI-39-UTR]) in whichthe seed binding sequence in 39-UTR of CD64 was mutated. Theluciferase activity in pMIR-REPORT [mFcgRI-39-UTR] and miR-127–cotransfected cells showed no differences when comparedwith pMIR-REPORT [mFcgRI-39-UTR] and control miRNA ormiR-127 inhibitor cotransfection (Fig. 3G). These suggest thatmiR-127 reduced the luciferase activity by binding to CD64–39-UTR, but had no effect on CD64 mutant 39-UTR. Therefore, weconcluded that CD64 is a bona fide target of miR-127.Expression of CD64 is positively correlated with IgGIC-induced inflammatory responseTo test whether CD64 functionally regulates IgG IC-induced cy-tokine expression, RAW264.7 and MH-S cells were stained withanti-CD64 Ab, and flow cytometry analysis was used to sort out thetop 1% of the highest CD64-expressing cells as CD64hiand thebottom 1% of the lowest CD64-expressing cells as CD64lo. Next,the highest and lowest CD64-expressed RAW264.7 and MH-Scells were treated with IgG IC, and cytokine expression in the con-ditioned media was measured. RAW264.7-CD64locells showeda decrease in TNF-a, IL-1b, and IL-6 release after IgG IC stim-ulation as compared with RAW264.7-CD64hicells (Fig. 4A).Similarly, MH-S–CD64locells showed a decrease in proinflam-matory cytokines such as IL-6, after IgG IC stimulation, as com-pared with MH-S–CD64hicells (Fig. 4B). RAW264.7-CD64locellsalso showed a decrease in IL-1brelease after LPS stimulation ascompared with RAW264.7-CD64hicells (Fig. 4C). These datademonstrate that expression of CD64 is closely associated with theinflammatory response of macrophages activated by IgG IC.miR-127 attenuates IgG IC-induced lung injury in vivoWe have shown that the IgG IC-induced inflammation is inhib-ited by miR-127 in vitro. We next investigated whether miR-127FIGURE 2. Stable expression of miR-127 in MH-S cells attenuatedinflammatory response. (A–C) MH-S–miR-127 and control cells weretreated with LPS, HA, PGN, and IgG IC for 4 h. The levels of TNF-a(A),IL-1b(B), and IL-6 (C) in the supernatants were determined by ELISA. (Dand E) miR-127 precursor attenuated IgG IC-induced inflammatory re-sponse in RAW264.7 cells and MH-S cells. RAW264.7 cells (D) and MH-Scells (E) were transiently transfected with 10 nM control probes, miR-127precursor, and miR-127 inhibitor. At 48 h after transfection, the cells werecultured with or without IgG IC for 4 h. The levels of IL-6 in the super-natants were then determined. (F) Human monocyte U937 cells were ac-tivated with PMA at 100 nM for 24 h, and the cells were washed and thencultured in regular medium for 24 h before transfection. The cells weretransiently transfected with 10 nM control probes, miR-127 precursor, andmiR-127 inhibitor. At 48 h after transfection, the cells were culturedwithout or with IgG IC for 4 h. The levels of IL-6 in the supernatants werethen determined. miR-127 precursor attenuated IgG IC-induced TNF-aproduction of human U937 cells. Values are presented as mean 6SEM. n=3 to 4. The experiments were repeated three times. *p,0.05, **p,0.01,***p,0.001, ****p,0.0001 compared with control group.2440 miR-127 INHIBITS LUNG INFLAMMATION on February 22, 2012www.jimmunol.orgDownloaded from mediates regulation of inflammatory response in complement-mediated ALI in vivo. We administered miR-127 probes or con-trol (CEMR67 or PBS) intranasally 2 h before onset of the IgG ICand then analyzed the extent of lung inflammation 4 h after onset ofthe IgG IC model. Neither CEMR67 nor PBS had any effect. Flowcytometry revealed that more than half of macrophages hadtaken up Fam-labeled miR-127 (Supplemental Fig. 4A). RT-PCRrevealed an accumulation of miR-127 in the lungs of the miR-127treatment group (Supplemental Fig. 4B). miR-127–treated mac-rophages expressed less cell-surface CD64 when compared withcontrol oligonucleotide CEMR67 (or PBS)-treated macrophages(Supplemental Fig. 4C). IgG IC induced a typical lung pathol-ogy (30, 33) characterized by lung hemorrhage, edema, fibrindeposition, and accumulation of inflammatory cells. However,treatment with miR-127 displayed a significant reduction inpathological changes (Fig. 5A). We collected bronchoalveolarlavage fluid (BALF) from mice of control, IgG IC model, andmiR-127 treatment groups. Examination of the inflammatory re-sponse to IgG IC revealed a significant attenuation in total proteinlevel (Fig. 5B) and Evans blue concentration (Fig. 5C). Further-more, miR-127 also markedly reduced inflammatory cell infiltra-tion (Fig. 5D, 5E) as well as neutrophil influx (Fig. 5F). Thesedata suggest that miR-127 significantly attenuated IgG IC-inducedlung injury.miR-127 inhibits IgG IC-induced lung inflammation in vivoNext we examined the role of miR-127 in the effect on cytokinerelease. Consistent with our in vitro observation, miR-127 sig-nificantly reduced cytokine release into the alveolar space. Allmajor cytokines and chemokines (IL-6, TNF-a, MIP-1b, MIP-2,MCP-1, and KC) were reduced by miR-127 in IgG IC-inducedlung injury (Fig. 6A).Complement activation is an important component of IC-mediated tissue injury (38). C5a has been demonstrated to berequired for the full development of injury and neutrophil accu-mulation in an IgG IC model of lung injury (33, 39). Anti-C5a Abreduced lung vascular permeability and lung myeloperoxidase andsuppressed upregulation of ICAM-1 and TNF-ain the IgG ICmodel of lung injury (33). Anti-C5a Ab attenuated ARDS andsome of the systemic manifestations of sepsis in nonhuman pri-FIGURE 3. FcgRI (CD64) is a target of miR-127. (A) Predicted binding site for miR-127 in 39-UTR of CD64. miR-127 seed sequence was in blue. (B)Western blot analysis was performed to determine the levels of CD64 protein in MH-S–miR-127 and control cells without or with LPS (100 ng/ml)treatment for 4 h. (C) Flow cytometry was performed to determine the percent of CD64 staining positive cells in MH-S–miR-127 and control cells. (D)RAW264.7 cells were transfected with 10 nM control probes, LNA–miR-127, and LNA-anti–miR-127. Forty-eight hours after transfection, the cells werestained with CD64 Ab. The percent of CD64 staining positive cells were then determined. Luciferase activity of miR-127 precursor (E), miR-127 inhibitor(F), or control probe and pMIR-REPORT [FcgRI-39-UTR] cotransfection. (G) Luciferase activity of miR-127 precursor, miR-127 inhibitor, or control probeand pMIR-REPORT [mFcgRI-39-UTR] (containing a mutated 39-UTR of FcgRI) cotransfection (n= 3 to 4). The experiments were repeated three times.*p,0.05, **p,0.01, ****p,0.0001.FIGURE 4. Expression of CD64 correlated with IgG IC-induced in-flammatory response. (A) RAW264.7 cells were sorted for the highest orlowest 1% cells expressing CD64. CD64hiand CD64locells were treatedwithout or with IgG IC for 4 h. The levels of IL-1b, IL-6, and TNF-ain thesupernatants were then determined by ELISA. (B) MH-S cells were sortedfor CD64 highest or lowest 1% expressing cells. The levels of IL-6 in IgGIC-treated supernatant of CD64hiand CD64loMH-S cells were then de-termined. (C) CD64hiand CD64loRAW264.7 cells were treated with orwithout LPS for 4 h. The levels of IL-1bin the supernatants were thendetermined by ELISA. Values are presented as mean 6SEM. n= 3–6. Theexperiments were repeated three times. ****p,0.0001 compared withcontrol group.The Journal of Immunology 2441 on February 22, 2012www.jimmunol.orgDownloaded from mates (40). Therefore, we wanted to determine whether comple-ment C5a was affected. IgG IC markedly induced C5a in mouseBAL. miR-127 treatment significantly reduced C5a protein levelsin the BAL (Fig. 6B).STAT3 is a transcription factor transducing signals in response tocytokines and growth factors. STAT3 is activated in the lung duringIgG IC-induced ALI (30). Therefore, we wanted to determineif miR-127 treatment influences STAT3 activation. STAT3 andp-STAT3 protein levels were examined in total lung proteins.p-STAT3 protein levels were elevated in the IgG IC onset lungs,but diminished by miR-127 treatment (Fig. 6C). Thus, miR-127appears to function in attenuating IgG IC-induced inflammatoryresponse in vivo through the regulation of CD64.DiscussionIn this study, we found that miR-127 expression was regulatedduring inflammation both in vitro and in vivo. Functionally, miR-127 regulated cytokine release by macrophages in vitro and at-tenuated lung injury in an IgG IC-induced lung injury modelin vivo. Mechanistically, miR-127 directly targeted the 39-UTR ofCD64, resulting in the downregulation of CD64.Several studies have demonstrated that miRNAs regulate in-flammation and the immune response. For example, a recent studyshowed that miR-21 was upregulated in the lungs of mice withbleomycin-induced injury and also in the lungs of patients withidiopathic pulmonary fibrosis and that miR-21 may regulate lunginjury and fibrosis through targeting Smad7 (24). The downreg-ulation of let-7d was found in the patients with idiopathic pul-monary fibrosis and let-7d targets HMGA2 in epithelial cells (41).Let-7g was reported to be downregulated in LPS-induced acuteinflammation in circulating leukocytes (42). miR-126 promotesTh2 responses in the lung (25). miR-146a has been shown to playa role in innate immunity (43). However, the role of miR-127 inimmune response and inflammation has not been investigated, andthe targets of miR-127 have not been identified.There are limited reports suggesting that miR-127 may playa role in development (36) and tumor progression (37, 44, 45). Werecently analyzed miRNA expression after bleomycin-inducedlung injury with a miRNA array study (29) and identified thatmiR-127 expression was decreased at 1 d after bleomycin treat-ment. In this study, we showed that miR-127 expression is mar-ginally downregulated in IgG IC-induced lung injury in vivo anddecreased in macrophages when stimulated with inflammatorystimuli in vitro, suggesting miR-127 may play a role during in-flammatory response. We focused our study on miR-127 regu-lation in IgG IC-induced inflammation because putative mRNAtargets predicted in silico were members of pathways impli-catedinFcgR-mediated immune response in macrophages, in-cluding FcgRI, P13K cat class IA, VAV-1, PIP5KI, and myo-sin II.We provide several lines of evidence suggesting that FcgR1 isa genuine target of miR-127. First, using bioinformatics tools,targets of miR-127 were identified, and CD64 was selected forexperimental validation, given its known role in the inflamma-tory response (8). cDNA array analysis identified that CD64 wasdownregulated in miR-127–overexpressing macrophages. We fur-ther showed that overexpression of miR-127 resulted in a decreasein mRNA, protein, and cell-surface expression of CD64. Lastly,reduced luciferase activity was observed in a reporter systemcontaining the full length of 39-UTR of CD64, demonstrating thatmiR-127 directly targets CD64. However, we did not detect anyeffect of miR-127 inhibitor on IgG IC-induced cytokine release,cell-surface CD64 expression, or CD64-luciferase activity inmacrophages. It is possible that the endogenous miR-127 ex-pression is low in lung tissue (36) as well as in macrophages,whereas CD64 expression is abundant on macrophages. Intro-duction of anti–miR-127 into macrophages may not be able tofurther reduce endogenous miR-127 levels, so that anti–miR-127may not upregulate CD64 expression in the cells. On the contrary,introduction of miR-127 mimics into cells can readily increaseFIGURE 5. miR-127 attenuated IgG IC-induced ALI in mice. Mice received either PBS or miR-127 intranasally 2 h before onset of IgG IC model (10mg/mouse anti-BSA in 40 ml PBS intranasal instillation and 5 mg/kg BSA together with 0.25% Evans blue in 200 ml PBS intravascular injection). At 4 hafter IgG IC onset, mice were sacrificed, and BALF was collected. Lung tissues were fixed, embedded in paraffin, and sectioned for staining with H E. (A)miR-127 decreases lung inflammation and alveolar hemorrhage on lung sections stained by H E. Scale bar, 100 mm. (B) Total protein levels in BALF wereperformed by bicinchoninic acid assay. (C) Evans blue concentrations in BALF were measured. (D) Total cell counts in BALF were performed to determineinflammatory cell infiltration. (E) Representative cytospin preparations stained with Protocol Hema3 stain set. Original magnification 3400. (F) Differentialcell counts were performed on cytospin preparations of BAL. Values are presented as mean 6SEM. n= 3–6 in each group. The experiments were repeatedthree times. *p,0.05, **p,0.01 compared with IgG IC group. LYM, lymphocytes; MAC, macrophages; PMN, neutrophils.2442 miR-127 INHIBITS LUNG INFLAMMATION on February 22, 2012www.jimmunol.orgDownloaded from miR-127 levels in the cells and lead to significantly downregulatedCD64 expression.By establishing stable or transient miR-127 overexpression inmacrophage cell lines,we observed decreased TNF-a,IL-1b, or IL-6release correlated with overexpression of miR-127 when stimu-lated with LPS, HA, PGN, or IgG IC. We also present a functionalrole for the miR127–CD64 axis in the signaling pathways inducedin response to IgG IC. IgG IC or LPS treatment of sorted highestand lowest CD64 expression macrophages revealed a correlation ofdecreased cytokine releases and the loss of CD64.Next we determined the role of miR-127 in an IgG IC-inducedALI model in vivo (8). miR-127 attenuated the inflammatory re-sponse and lung permeability in IgG IC-induced ALI, as indi-cated by a change in lung morphology, decreased inflammatorycell infiltration, cytokine releases, total protein level, Evans blueconcentration, and complement activating protein C5a. C5a hasbeen demonstrated to be required for the full development of in-jury and neutrophil accumulation in an IgG IC model of lunginjury (39). It has shown that C5a directly activated chemokineproduction by neutrophils and macrophages. In the presenceof IgG IC, C5a and C5b–C9 membrane attack complex causea synergistic intrapulmonary generation of MIP-1b, MIP-2, andMCP-1 (46). Also, we performed Western blot to analyze the totalSTAT3 and p-STAT3 levels in whole lung tissue. As previousreports indicate, STAT3 is activated in the lung during IgG IC-induced ALI, and its activity is chiefly regulated at the post-translational level (30). We found that miR-127 could prevent IgGIC-induced phosphorylation of STAT3.In our study, we found that overexpression of miR-127 leadsto downregulation of CD64 expression. CD64 is one member ofthe IgG receptor family. Binding of IgG to FcgRs triggers awide variety of cellular functions including phagocytosis, Ab-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as clearance of IC and regulation of Ab pro-duction (7). FcgRs have been indicated to be important immune-regulatory receptors involved in various disease states of allergy,autoimmunity, and inflammation, especially IgG-mediated humandisease (7). By using a variety of FcgR2/2mice, Ioan-Facsinayand colleagues (9) were able to show that in the absence of FcgRI,IgG2a-IC–induced cellular processes of phagocytosis, cytokinerelease, cellular cytotoxicity, and Ag presentation are impaired.Furthermore, FcgRI contributes significantly to enhanced PMNinfiltration in IC-induced peritonitis (47). Mice lacking FcgRIshowed reduced inflammatory responses in an IC-induced reversepassive Arthus model (48). Given the importance of FcgRI in IgGIC-mediated inflammatory responses, our findings determined thatmiR-127 acts as an anti-inflammatory factor by targeting FcgRIboth in vitro and in vivo. Interestingly, the microarray analysis ofdifferent gene expression revealed an increase of FcgRII subunitsin MH-S–miR-127 cells when compared with control cells. Asindicated before, FcgRII is a negative regulator of IC signaling.It is reported that FcgRII prevents inappropriate activation ofeffector responses. Mice with FcgRII deficiency were hyperre-sponsive to subthreshold levels of cytotoxic Abs (49) and IC (50).This suggests to us that miR-127 may result in increased ex-pression of FcRI and decreased expression FcRII, which collab-oratively inhibit the inflammatory response. Hence, our data showthe potential of miR-127 as a target for the treatment of inflam-matory diseases such as ALI and ARDS.As we focused on the role of miR-127 in IC-induced lung in-flammation, our data suggest CD64 is the target of miR-127. Wehave also showed that miR-127 may have a role in the inflammatoryresponse induced by LPS, PGN, and HA fragments. It is possiblethat the effect of miR-127 on cytokine production stimulated byTLR ligands is through CD64, because it has been suggested thatCD64 contributes to protection from bacterial infection (9), andthere are crosstalk pathways between TLRs and FcgRs (51).However, it is more plausible that miR-127 targets a set of genes,of which CD64 plays a key role in IC-induced inflammation. Inaddition to CD64, there are at least 24 other genes putativelytargeted by miR-127 involved in immune response based on insilico analysis. By GeneGo pathway analysis (http://www.genego.com), these genes are involved in phosphatidylinositol-(3,4,5)-triphosphate signaling, NFAT signaling, CD28 signaling, and trig-gering receptor expressed on myeloid cells 1 signaling. Bytargeting a set of genes including CD64, miR-127 may thereforeplay a role in regulation of the inflammatory response to a varietyof IgG receptor as well as TLR stimulations.In summary, we demonstrate that an miRNAwas involved in IgGIC-induced lung injury. Specifically, we identify that miR-127targeted CD64. miR-127 administration inhibited the inflamma-tory response of IgG IC-induced lung injury. We believe that thesefindings have important implications in the regulation of the adap-tive immune response in ALI. Further investigation into the miR-127–CD64 axis would pave the way to developing a novel ther-apeutic approach to the treatment of infectious and noninfectiouslung inflammation.FIGURE 6. miR-127 inhibited cytokine production and complementcomponent in IgG IC-induced ALI in vivo. Mice received either PBS ormiR-127 probes intranasally 2 h before onset of IgG IC model. At 4 h afterIgG IC onset, mice were sacrificed, and BALF or lung tissue was collected.(A) Protein levels of IL-6, TNF-a, IL-1b, MIP-1b, MIP-2, MCP-1, and KCin BALF were measured with ELISA. (B) Complement C5a protein inBALF was measured with ELISA. Values are presented as mean 6SEM.n= 3–6 in each group. The experiments were repeated three times. (C)Western blot was performed to determine STAT3 and p-STAT3 proteinlevels in lung tissues. *p,0.05, **p,0.01, ***p,0.001 compared withIgG IC group.The Journal of Immunology 2443 on February 22, 2012www.jimmunol.orgDownloaded from DisclosuresThe authors have no financial conflicts of interest.References1. Matthay, M. A., and R. L. Zemans. 2011. The acute respiratory distress syn-drome: pathogenesis and treatment. Annu. Rev. Pathol. 6: 147–163.2. Wheeler, A. P., and G. R. Bernard. 2007. Acute lung injury and the acute re-spiratory distress syndrome: a clinical review. Lancet 369: 1553–1564.3. Crosby, L. M., and C. M. Waters. 2010. 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Moreover, numerous miRNAs are involved in the progression of pneumonia; miR-302e reduced inflammation during infantile pneumonia via the NF-κB signaling pathway (11), miR-146a-5p regulated lipopolysaccharide (LPS)-induced cell apoptosis and inflammation via CC motif chemokine ligand 5 during acute pneumonia (12), and miR-370-3p modulated LPS-induced cell apoptosis and inflammation via toll like receptor 4 during acute pneumonia (13). It has also been reported that miR-127 may reduce lung inflammation by targeting immunoglobulin (Ig) G Fcγ receptor I (14). Additionally, miR-127-5p expression, which is reduced in bronchoalveolar lavage fluid, may serve as a biomarker for the diagnosis of the disease (15). ...... miR-127 serves a role in numerous lung diseases. For example, miR-127 inhibits lung inflammation by targeting IgG Fcγ receptor I (14). Additionally, miR-127-5p, which is downregulated in bronchoalveolar lavage fluid, serves as a novel biomarker for the diagnosis of severe pneumonia (15). ...MicroRNA‑127‑5p attenuates severe pneumonia via tumor necrosis factor receptor‑associated factor 1ArticleJul 2020Exp Ther MedCunrong ChenSen LinLili ZhouMingzhi ChenPneumonia is a persistent and pervasive disease, the effects of which can be severe. MicroRNA (miR)-127-5p has been utilized as a novel biomarker for the diagnosis of severe pneumonia. The present study aimed to investigate the function of miR-127-5p during severe pneumonia. An in vitro model of severe pneumonia in Ana-1 murine macrophages was established using lipopolysaccharide (LPS). Subsequently, reverse transcription-quantitative PCR and ELISA were performed to detect the mRNA and protein expression levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. Western blotting was also performed to measure the activity of AKT and NF-κB. The results indicated that compared with the control group, LPS treatment increased TNF receptor-associated factor 1 (TRAF1) expression levels and reduced miR-127-5p expression levels. Furthermore, the results revealed that the 3 -untranslated region of TRAF1 was targeted by miR-127-5p. miR-127-5p mimic reduced LPS-induced increases in IL-1β, IL-6 and TNF-α expression by targeting TRAF1, which was potentially mediated by inactivation of the AKT and NF-κB signaling pathways. Collectively, the results demonstrated that miR-127-5p may attenuate severe pneumonia by reducing LPS-induced inflammatory cytokine production, and inactivating the AKT and NF-κB signaling pathways by targeting TRAF1.ViewShow abstract... Table S3). Strikingly, all of these significant DE miRNAs have previously been linked to the regulation of inflammation at both cellular and systems levels (Xia et al., 2018;Xie et al., 2012;Akehurst et al., 2015;Chu et al., 2019;Garcia-Lacarte et al., 2019). To identify the biological processes in which the four miRNAs are involved, we first searched for the potential target genes using multiple target prediction databases and then performed a GO enrichment analysis on the ranked targets (Fig. 1D). ...... We identified four miRNAs, miR-127-5p, miR-206, miR-337-3p and miR-1185-1-3p, which displayed lower expression in SCZ astroglia expressions relative to CTRL astroglia. All four of these miRNAs are involved in the regulation of inflammation in various systemic and tissue microenvironment-specific contexts (Xia et al., 2018;Xie et al., 2012;Akehurst et al., 2015;Chu et al., 2019;Garcia-Lacarte et al., 2019). We then ran gene enrichment analyses to gain insight into the biological processes the four miRNAs may regulate. ...Transcriptome analysis reveals disparate expression of inflammation-related miRNAs and their gene targets in iPSC-astrocytes from people with schizophreniaArticleFull-text availableFeb 2021BRAIN BEHAV IMMUNIbrahim A. AkkouhTimothy HughesVidar M. Steen Attila SzaboDespite the high heritability of schizophrenia (SCZ), details of its pathophysiology and etiology are still unknown. Recent findings suggest that aberrant inflammatory regulation and microRNAs (miRNAs) are involved. Here we performed a comparative analysis of the global miRNome of human induced pluripotent stem cell (iPSC)-astrocytes, derived from SCZ patients and healthy controls (CTRLs), at baseline and following inflammatory modulation using IL-1β. We identified four differentially expressed miRNAs (miR-337-3p, miR-127-5p, miR-206, miR-1185-1-3p) in SCZ astrocytes that exhibited significantly lower baseline expression relative to CTRLs. Group-specific differential expression (DE) analyses exploring possible distinctions in the modulatory capacity of IL-1β on miRNA expression in SCZ versus CTRL astroglia revealed trends toward altered miRNA expressions. In addition, we analyzed peripheral blood samples from a large cohort of SCZ patients (n=484) and CTRLs (n=496) screening for the expression of specific gene targets of the four DE miRNAs that were identified in our baseline astrocyte setup. Three of these genes, LAMTOR4, IL23R, and ERBB3, had a significantly lower expression in the blood of SCZ patients compared to CTRLs after multiple testing correction. We also found nominally significant differences for ERBB2 and IRAK1, which similarly displayed lower expressions in SCZ versus CTLR. Furthermore, we found matching patterns between the expressions of identified miRNAs and their target genes when comparing our in vitro and in vivo results. The current results further our understanding of the pathobiological basis of SCZ.ViewShow abstract... 12 Accumulating evidence suggests that microRNAs are involved with lung diseases, among which miR-146a, miR-24, miR-155, and miR-127 have been proved to alleviate pulmonary inflammation. [13][14][15][16] As a member of microRNAs family, miR-130a is involved in many diseases, such as cancers, 17 spinal cord injury, 18 and kidney diseases. 19 The miR-130a-3p has been shown to mediate inflammatory responses in LPSinduced BV2 cells via IGF-1/IGF-1R pathway. ...Chicoric acid alleviates LPS-induced inflammatory response through miR-130a-3p/IGF-1pathway in human lung A549 epithelial cellsArticleFull-text availableAug 2021To investigate the effects and potential mechanisms of chicoric acid (CA) on LPS-induced inflammatory response in A549 cells. 0–800 μM CA was added to A549 cells to determine the toxicity of CA using MTT assay. Then, 2 μg/mL LPS and 50 μM CA were simultaneously added to A549 cells to investigate the effects of CA. In order to investigate the effects of miR-130a-3p and IGF-1 on LPS-induced A549 cells, cells were infected with inhibitor of miR-130a-3p and si RNA IGF-1. The levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α were measured by real-time RT-PCR and enzyme-linked immunosorbent (ELISA) assay. The IGF-1 pathway and NF-κB expression were measured using immunoblot assay. Moreover, a luciferase activity assay was used to indicate the binding site of miR-130a-3p on the 3′UTR of IGF-1. 0–50 μM CA had no toxicity on A549 cells. Thus, we chose 50 μM CA for the following study. CA attenuated the inflammatory response by LPS through decreasing IL-1β, IL-6, and TNF-α levels and increasing IGF-1/IGF-1R expression. Inhibition of miR-130a-3p reduced the inflammatory response and restored IGF-1/IGF-1R pathway induced by LPS. Furthermore, luciferase activity results indicated that miR-130a-3p directly targeted IGF-1 to regulate inflammatory response. CA alleviates LPS-induced inflammatory response through miR-130a-3p/IGF-1pathway in A549 cells.ViewShow abstract... miR-1246 loss reduced cellular apoptosis in mice with ALI, IL-1β release as well as infiltration of neutrophils through suppressing its downstream target ACE2 [41]. Some studies have suggested that miR-127 expression significantly decreased in lung damage in vivo; however, miR-127 probe could alleviate lung inflammation by regulating CD46 in macrophages [42]. In another animal research, upregulation of miR-146a inhibited IL-6, IL-1β, and TNF-α secretion in ALI model induced by LPS via suppressing TRAF-6 and IRAK-1 [43]. ...miR-128-3p reduced acute lung injury induced by sepsis via targeting PEL12ArticleFull-text availableAug 2021Shinan LiuShuai GaoZhaoyu Yang Peng ZhangObjectiveAcute lung injury (ALI) caused by sepsis is clinically a syndrome, which is featured by damage to the alveolar epithelium and endothelium. In this study, we employed mice models of cecal ligation and puncture (CLP) and primary mice pulmonary microvascular endothelial cells (MPVECs) in vitro to investigate the effect of miR-128-3p in ALI caused by sepsis.MethodsmiR-128-3p agomir or randomized control were injected into adult male C57BL/6 mice 1 week before the CLP surgery. We used miR-128-3p agomir or scrambled control to transfect MPVECs and then employed lipopolysaccharide (LPS) stimulation on the cells. Pellino homolog 2 (PELI2) was predicted to be a direct target of miR-128-3p via luciferase reporter assay. MPVECs were cotransfected with lentiviral vector that expressed PELI2 (or empty vector) as well as miR-128-3p-mimics 1 day before LPS stimulation in rescue experiment. Transcriptional activity of caspase-3, cell apoptosis rate, and the expression levels of miR-128-3p, interleukin-1β (IL-1β), interleukin-6 (IL-6), and PELI2 were analyzed.ResultsCompared with the sham group, the lung of mice in the CLP group showed pulmonary morphological abnormalities, and the expression of IL-6 and IL-1β, caspase-3 activity, and apoptosis rate were significantly upregulated in the CLP group. Inflammatory factor levels and apoptosis rate were also significantly induced by LPS stimulation on MPVECs. Upregulation of miR-128-3p effectively inhibited sepsis-induced ALI, apoptosis as well as inflammation. miR-128-3p also played a role in antiapoptosis and anti-inflammation in MPVECs with LPS treatment. PEL12 upregulation in MPVECs alleviated miR-128-3p-induced caspase-3 activity inhibition and pro-inflammatory factor production.ConclusionsmiR-128-3p enabled to alleviate sepsis-induced ALI by inhibiting PEL12 expression, indicating a novel treatment strategy of miR-128-3p for sepsis-induced ALI.ViewShow abstract... In our miRNA-sequencing results, the expression of hsa-miR-103a-3p was also decreased in EAT from CAD patients. In addition, we also found that pro-inflammatory miRNAs (miR-146a-5p [26], miR-155-3p [26], miR-206 [27] and miR-223-5p [28]) were up-regulated and anti-inflammatory miRNA Let-7i [29] and miR-127-5p [30] were down-regulated. These data suggested that AS was an inflammatory process and the EAT pro-inflammatory environment could promote the progress of AS in the coronary artery. ...MicroRNA-200b-3p promotes endothelial cell apoptosis by targeting HDAC4 in atherosclerosisArticleFull-text availableApr 2021BMC Cardiovasc DisordFan ZhangNaixuan ChengJie Du Congcong ZhangBackgroundEpicardial adipose tissue (EAT) shares the same microcirculation with coronary arteries through coronary arteries branches, and contributes to the development of atherosclerosis. MicroRNAs (miRNAs) are involved in the formation of atherosclerosis. However, the alteration of miRNA profile in EAT during atherosclerosis is still uncovered.MethodsThe miRNA expression profiles of EAT from non-coronary atherosclerosis disease (CON, n = 3) and coronary atherosclerosis disease (CAD, n = 5) patients was performed to detect the differentially expressed miRNA. Then the expression levels of miRNA in other CON (n = 5) and CAD (n = 16) samples were confirmed by realtime-PCR. miR-200b-3p mimic was used to overexpress the miRNA in HUVECs. The apoptosis of HUVECs cells was induced by H 2 O 2 and ox-LDL, and detected by Annexin V/PI Staining, Caspase 3/7 activity and the expression of BCL-2 and BAX.Results250 miRNAs were differentially expressed in EAT from CAD patients, which were associated with metabolism, extracellular matrix and inflammation process. Among the top 20 up-regulated miRNAs, the expression levels of miR-200 family members (hsa-miR-200b/c-3p, miR-141-3p and miR-429), which were rich in endothelial cells, were increased in EAT from CAD patients significantly. Upregulation of miR-200 family members was dependent on the oxidative stress. The overexpression of miR-200b-3p could promote endothelial cells apoptosis under oxidative stress by targeting HDAC4 inhibition.ConclusionsOur study suggests that EAT derived miR-200b-3p promoted oxidative stress induced endothelial cells damage by targeting HDAC4, which may provide a new and promising therapeutic target for AS.ViewShow abstract... Several previous studies have suggested the importance of endogenous anti-inflammatory pathways for the control of organ inflammation. These pathways include the hypoxiainducible factor signaling pathway [61,64,65], purinergic signaling pathway [66][67][68][69][70][71][72][73][74][75], microRNAs [19,20,[76][77][78], resolvins [79][80][81], and many others [82][83][84][85][86][87][88][89][90]. Along these lines, those pathways not only facilitate tissue protection during pulmonary injuries but also contribute to other inflammatory organ conditions such as myocardial infarction [91][92][93][94][95][96][97][98], kidney injury [99][100][101], and inflammatory bowel diseases [102][103][104][105][106][107][108]. ...Characterization of a Murine Model System to Study MicroRNA-147 During Inflammatory Organ InjuryArticleFull-text availableAug 2021Inflammation Boyun KimVictor GuareguaXuebo Chen Xiaoyi YuanInflammatory organ injury and sepsis have profound impacts on the morbidity and mortality of surgical and critical care patients. MicroRNAs are small RNAs composed of 20–25 nucleotides that have a significant contribution to gene regulation. MicroRNA-147 (miR-147), in particular, has been shown to have an emerging role in different physiological functions such as cell cycle regulation and inflammatory responses. However, animal model systems to study tissue-specific functions of miR-147 during inflammatory conditions in vivo are lacking. In the present study, we characterize miR-147 expression in different organs and cell types. Next, we generated a transgenic mouse line with a floxed miR-147 gene. Subsequently, we used this mouse line to generate mice with whole-body deletion of miR-147 (miR-147 −/−) by crossing \"floxed” miR-147 mice with transgenic mice expressing Cre recombinase in all tissues (CMVcre mice). Systematic analysis of miR-147 −/− mice demonstrates normal growth, development, and off-spring. In addition, deletion of the target gene in different organs was successful at baseline or during inflammation, including the heart, intestine, stomach, liver, spleen, bone marrow, lungs, kidneys, or stomach. Moreover, miR-147 −/− mice have identical baseline inflammatory gene expression compared to C57BL/6 mice, except elevated IL-6 expression in the spleen (7.5 fold, p 0.05). Taken together, our data show the successful development of a transgenic animal model for tissue and cell-specific deletion of miR-147 that can be used to study the functional roles of miR-147 during inflammatory organ injury.ViewShow abstract... MiR-127 can be significantly induced in an inflammation-related lung injury. 31,32 Increased expression of miR-127 in macrophages augments proinflammatory cytokine production. 33 Similarly, miR- (1) 155 is a major regulator of TLR signaling, and plays an important role in regulating immune cell development, function, and disease. ...Kinetics of changes in gene and microRNA expression related with muscle inflammation and protein degradation following LPS-challenge in weaned pigletsArticleFull-text availableNov 2020INNATE IMMUNPing KangXingfa HuangZhicheng Wan Yulan LiuTo test the dynamic changes of the expression of genes and microRNA in the gastrocnemius muscle after LPS challenge, 36 piglets were assigned to a control group (slaughtered 0 h after saline injection) and LPS groups (slaughtered at 1 h, 2 h, 4 h, 8 h, and 12 h after LPS treatment, respectively). After LPS treatment, the mRNA expression of IL-1β, IL-6, and TNF-α reached maximal levels at 1 h, 2 h, and 1 h, respectively ( P 0.05), and mRNA expression of TLR4, NODs, muscle-specific ring finger 1, and muscle atrophy F-box peaked at 12 h ( P 0.05). Moreover, the expression of miR-122, miR-135a, and miR-370 reduced at 1 h, 1 h, and 2 h, respectively ( P 0.05), and miR-34a, miR-224, miR-132, and miR-145 reached maximum expression levels at 1 h, 1 h, 2 h, and 4 h, respectively ( P 0.05). These results suggested that mRNA expression of pro-inflammatory cytokines was elevated in the early stage, mRNA expression of genes related to TLR4 and NODs signaling pathways and protein degradation increased in the later phase, and the expression of microRNA related to muscle inflammation and protein degradation changed in the early stage after LPS injection.ViewShow abstract... Therefore, we explored the specific molecular mechanism of NF-κB on LPS -induced ALI. Acting as important biological regulators, miRNAs are involved in many pathophysiological processes by inhibiting the expression of target genes [19]. Moreover, several studies have demonstrated that miRNAs regulate inflammation and the relevant immune response. ...Suppressed nuclear factor-kappa B alleviates lipopolysaccharide-induced acute lung injury through downregulation of CXCR4 mediated by microRNA-194ArticleFull-text availableJun 2020Respir ResRuidong ChenFei XieJie ZhaoBin YueAcute lung injury (ALI) is a highly lethal pulmonary disease that causes edema, hypoxemia and respiratory failure. Recent evidence indicates that nuclear factor-kappa B (NF-κB) plays a crucial role in ALI development. However, the regulatory mechanism of NF-κB on ALI remains enigmatic. In this study, we investigated potential molecular mechanism of NF-κB on ALI induced by lipopolysaccharide (LPS). BALB/c mice were subjected to intratracheal spraying of LPS to generate an ALI mode, with the activity of NF-κB in mice tissues being detected by enzyme linked immunosorbent assay (ELISA), and the number of inflammatory cells in bronchoalveolar lavage fluid being counted. Then, the macrophage cell line RAW264.7 exposed to LPS were treated with ammonium pyrrolidinedithiocarbamate (PDTC) (inhibitor of NF-κB), miR-194 mimic, or oe-chemokine receptor type 4 (CXCR4) separately or in combination. After that, ELISA and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression level of IL-1β, IL-6, TNF-α, miR-194 and CXCR4, respectively. In addition, the targeting relationship between miR-194 and CXCR4 was verified by dual-luciferase reporter gene assay. The dry/wet ratio of lung and the MPO activity were also measured to assess the inflammatory response in mice. Activation of NF-κB down-regulated the miR-194 expression in LPS-induced ALI. Overexpression of miR-194 alleviated LPS-induced ALI and reduced the expression of inflammatory factors IL-1β, IL-6 and TNF-α via targeting CXCR4. In LPS-induced ALI, NF-κB mediates the CXCR4 expression by inhibiting the expression of miR-194, thus promoting the inflammatory injury of lung.ViewShow abstractMacrophages in Lung Injury, Repair, and FibrosisArticleFull-text availableFeb 2021Peiyong ChengShuangyan LiHuaiyong ChenFibrosis progression in the lung commonly results in impaired functional gas exchange, respiratory failure, or even death. In addition to the aberrant activation and differentiation of lung fibroblasts, persistent alveolar injury and incomplete repair are the driving factors of lung fibrotic response. Macrophages are activated and polarized in response to lipopolysaccharide- or bleomycin-induced lung injury. The classically activated macrophage (M1) and alternatively activated macrophage (M2) have been extensively investigated in lung injury, repair, and fibrosis. In the present review, we summarized the current data on monocyte-derived macrophages that are recruited to the lung, as well as alveolar resident macrophages and their polarization, pyroptosis, and phagocytosis in acute lung injury (ALI). Additionally, we described how macrophages interact with lung epithelial cells during lung repair. Finally, we emphasized the role of macrophage polarization in the pulmonary fibrotic response, and elucidated the potential benefits of targeting macrophage in alleviating pulmonary fibrosis.ViewShow abstractThe Role of MicroRNAs in Acute Respiratory Distress Syndrome and Sepsis, From Targets to Therapies: A Narrative ReviewArticleSep 2020Anesth AnalgLisa K. Lee Lejla Medzikovic Mansoureh EghbaliXiaoyi YuanAcute respiratory distress syndrome (ARDS) is a significant cause of morbidity and mortality in the intensive care unit (ICU) and is characterized by lung epithelial and endothelial cell injury, with increased permeability of the alveolar-capillary membrane, leading to pulmonary edema, severe hypoxia, and difficulty with ventilation. The most common cause of ARDS is sepsis, and currently, treatment of ARDS and sepsis has consisted mostly of supportive care because targeted therapies have largely been unsuccessful. The molecular mechanisms behind ARDS remain elusive. Recently, a number of microRNAs (miRNAs) identified through high-throughput screening studies in ARDS patients and preclinical animal models have suggested a role for miRNA in the pathophysiology of ARDS. miRNAs are small noncoding RNAs ranging from 18 to 24 nucleotides that regulate gene expression via inhibition of the target mRNA translation or by targeting complementary mRNA for early degradation. Unsurprisingly, some miRNAs that are differentially expressed in ARDS overlap with those important in sepsis. In addition, circulatory miRNA may be useful as biomarkers or as targets for pharmacologic therapy. This can be revolutionary in a syndrome that has neither a measurable indicator of the disease nor a targeted therapy. While there are currently no miRNA-based therapies targeted for ARDS, therapies targeting miRNA have reached phase II clinical trials for the treatment of a wide range of diseases. Further studies may yield a unique miRNA profile pattern that serves as a biomarker or as targets for miRNA-based pharmacologic therapy. In this review, we discuss miRNAs that have been found to play a role in ARDS and sepsis, the potential mechanism of how particular miRNAs may contribute to the pathophysiology of ARDS, and strategies for pharmacologically targeting miRNA as therapy.ViewShow abstractShow moreComprehensive microRNA analysis in bleomycin-induced pulmonary fibrosis identifies multiple sites of molecular regulationArticleFull-text availableMay 2011Physiol Genom Ting Xie Carol Jiurong LiangRishu Guo Dianhua JiangThe molecular mechanisms of lung injury and fibrosis are incompletely understood. MicroRNAs (miRNAs) are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiological processes. To gain insight into miRNAs in the regulation of lung fibrosis, total RNA was isolated from mouse lungs harvested at different days after bleomycin treatment, and miRNA array with 1,810 miRNA probes was performed thereafter. MiRNAs expressed in lungs with bleomycin treatment at different time points were compared with miRNAs expressed in lungs without bleomycin treatment, resulting in 161 miRNAs differentially expressed. Furthermore, miRNA expression patterns regulated in initial and late periods after bleomycin were identified. Target genes were predicted in silico for differentially expressed miRNAs, including let-7f, let-7g, miR-196b, miR-16, miR-195, miR-25, miR-144, miR-351, miR-153, miR-468, miR-449b, miR-361, miR-700, miR-704, miR-717, miR-10a, miR-211, miR-34a, miR-367, and miR-21. Target genes were then cross-referenced to the molecular pathways, suggesting that the differentially expressed miRNAs regulate apoptosis, Wnt, Toll-like receptor, and TGF-β signaling. Our study demonstrated a relative abundance of miRNA levels in bleomycin-induced lung fibrosis. The miRNAs and their potential target genes identified may contribute to the understanding of the complex transcriptional program of lung fibrosis.ViewShow abstractMechanistic Role of MicroRNA-146a in Endotoxin-Induced Differential Cross-Regulation of TLR SignalingArticleFull-text availableFeb 2011J ImmunolMd A NahidMinoru Satoh Edward K.L. ChanHuman TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.ViewShow abstractResveratrol decreases the levels of miR-155 by upregulating miR-663, a MicroRNA targeting JunB and JunDArticleFull-text availableSep 2010Carcinogenesis Esmerina TiliJean-Jacques MichailleBrett AdairCarlo M CroceAn inflammatory component is present in the microenvironment of most neoplastic tissues, including those not causally related to an obvious inflammatory process. Several microRNAs, and especially miR-155, play an essential role in both the innate and adaptative immune response. Resveratrol (trans-3,4 ,5-trihydroxystilbene) is a natural antioxidant with anti-inflammatory properties that is currently at the stage of preclinical studies for human cancer prevention. Here, we establish that, in human THP-1 monocytic cells as well as in human blood monocytes, resveratrol upregulates miR-663, a microRNA potentially targeting multiple genes implicated in the immune response. In THP-1 cells, miR-663 decreases endogenous activator protein-1 (AP-1) activity and impairs its upregulation by lipopolysaccharides (LPS), at least in part by directly targeting JunB and JunD transcripts. We further establish that the downregulation of AP-1 activity by resveratrol is miR-663 dependent and that the effects of resveratrol on both AP-1 activity and JunB levels are dose dependent. Finally, we show that resveratrol impairs the upregulation of miR-155 by LPS in a miR-663-dependent manner. Given the role of miR-155 in the innate immune response and the fact that it is upregulated in many cancers, our results suggest that manipulating miR-663 levels may help to optimize the use of resveratrol as both an anti-inflammatory and anticancer agent against malignancies associated with high levels of miR-155.ViewShow abstractInhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4ArticleFull-text availableJun 2010J Clin Investig Dianhua Jiang Carol Jiurong LiangGabriele S V Campanella Paul W NoblePulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.ViewShow abstractInhibition and Role of let-7d in Idiopathic Pulmonary FibrosisArticleFull-text availableJul 2010Kusum V. PanditDavid L. Corcoran Hanadie Yousef Naftali KaminskiIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.To determine changes in expression and role of microRNAs in IPF.RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. Measurements and MainEighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).ViewShow abstractEpithelial repair mechanisms in the lungArticleFull-text availableApr 2010Am J Physiol Lung Cell Mol Physiol Lynn CrosbyChristopher M WatersThe recovery of an intact epithelium following lung injury is critical for restoration of lung homeostasis. The initial processes following injury include an acute inflammatory response, recruitment of immune cells, and epithelial cell spreading and migration upon an autologously secreted provisional matrix. Injury causes the release of factors that contribute to repair mechanisms including members of the epidermal growth factor and fibroblast growth factor families (TGF-alpha, KGF, HGF), chemokines (MCP-1), interleukins (IL-1beta, IL-2, IL-4, IL-13), and prostaglandins (PGE(2)), for example. These factors coordinate processes involving integrins, matrix materials (fibronectin, collagen, laminin), matrix metalloproteinases (MMP-1, MMP-7, MMP-9), focal adhesions, and cytoskeletal structures to promote cell spreading and migration. Several key signaling pathways are important in regulating these processes, including sonic hedgehog, Rho GTPases, MAP kinase pathways, STAT3, and Wnt. Changes in mechanical forces may also affect these pathways. Both localized and distal progenitor stem cells are recruited into the injured area, and proliferation and phenotypic differentiation of these cells leads to recovery of epithelial function. Persistent injury may contribute to the pathology of diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. For example, dysregulated repair processes involving TGF-beta and epithelial-mesenchymal transition may lead to fibrosis. This review focuses on the processes of epithelial restitution, the localization and role of epithelial progenitor stem cells, the initiating factors involved in repair, and the signaling pathways involved in these processes.ViewShow abstractNF-{kappa}B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responsesArticleAug 2006Konstantin D TaganovMark P. BoldinKuang-Jung ChangDavid BaltimoreActivation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-{kappa}B-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3 UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.ViewShow abstractThe Acute Respiratory Distress Syndrome: Pathogenesis and TreatmentArticleNov 2010Annu Rev PatholMichael A MatthayRachel L ZemansThe acute respiratory distress syndrome (ARDS) causes 40% mortality in approximately 200,000 critically ill patients annually in the United States. ARDS is caused by protein-rich pulmonary edema that causes severe hypoxemia and impaired carbon dioxide excretion. The clinical disorders associated with the development of ARDS include sepsis, pneumonia, aspiration of gastric contents, and major trauma. The lung injury is caused primarily by neutrophil-dependent and platelet-dependent damage to the endothelial and epithelial barriers of the lung. Resolution is delayed because of injury to the lung epithelial barrier, which prevents removal of alveolar edema fluid and deprives the lung of adequate quantities of surfactant. Lymphocytes may play a role in resolution of lung injury. Mortality has been markedly reduced with a lung-protective ventilatory strategy. However, there is no effective pharmacologic therapy, although cell-based therapy and other therapies currently being tested in clinical trials may provide novel treatments for ARDS.ViewShow abstractRegulation of Non-Infectious Lung Injury, Inflammation, and Repair by the Extracellular Matrix Glycosaminoglycan HyaluronanArticleJun 2010 Dianhua Jiang Carol Jiurong Liang Paul W NobleAn important hallmark of tissue remodeling is the dynamic turnover of extracellular matrix (ECM). ECM performs a variety of functions in tissue repair including scaffold formation, modulation of fluid dynamics, and regulating cell behavior. During non-infectious tissue injury ECM degradation products are generated that acquire signaling functions not attributable to the native precursor molecules. Hyaluronan (HA) is a non-sulfated glycosaminoglycan which is produced in great abundance following tissue injury. It exists both in a soluble form and as side chains on proteoglycans. HA has critical roles in development as well as a variety of biological processes including wound healing, tumor growth and metastasis, and inflammation. HA fragments share structural similarities with pathogens and following tissue injury can be recognized by innate immune receptors. Elucidating the protean roles of HA in tissue injury, inflammation, and repair will generate new insights into mechanisms of diseases characterized by chronic inflammation and tissue remodeling.ViewShow abstractInnate Immune Recognition in Infectious and Noninfectious Diseases of the LungArticleFeb 2010 Bastian OpitzV van LaakJulia Eitel Norbert SuttorpDiseases of the respiratory tract are among the leading causes of death in the world population. Increasing evidence points to a key role of the innate immune system with its pattern recognition receptors (PRRs) in both infectious and noninfectious lung diseases, which include pneumonia, chronic obstructive pulmonary disease, acute lung injury, pneumoconioses, and asthma. PRRs are capable of sensing different microbes as well as endogenous molecules that are released after cell damage. This PRR engagement is the prerequisite for the initiation of immune responses to infections and tissue injuries which can be beneficial or detrimental to the host. PRRs include the Toll-like receptors, NOD-like receptors, RIG-I-like receptors, and cytosolic DNA sensors. The PRRs and their signaling pathways represent promising targets for prophylactic and therapeutic interventions in various lung diseases.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectStem cells in lung fibrosis Dianhua JiangPlease see recent paper: Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice.Liang J, Zhang Y, Xie T, Liu N, Ch en H, Geng Y, Kurkciyan A, Mena JM, Stripp BR, Jiang D, Noble PW.Nat Med. 2016 Oct 3. doi: 10.1038/nm.4192.PMID: 27694932.https://www.ncbi.nlm.nih.gov/pubmed/27694932. ... [more]View projectArticleDelay of LPS-induced acute lung injury resolution by soluble immune complexes is neutrophil dependen...September 2009 · Shock (Augusta, Ga.) Chieh-Liang WuLiang-Yi LinHsueh-Mei Yeh[...] Chi-Mei HsuehThe pathophysiological role of soluble immune complexes (SICXs) and its relationship with neutrophils were investigated in LPS-induced acute lung injury (ALI) animal model (Sprague-Dawley rat) and through the in vitro studies. Results showed that LPS-induced SICX was timely related to changes of tumor necrosis factor alpha and macrophage inflammatory protein 2 (inflammatory cytokines) in ... [Show full abstract] bronchoalveolar lavage fluid. In vitro study showed that SICX can bind to Fc gammaR (CD64 and CD32 or CD16) to prevent the apoptosis of neutrophils. The SICX-mediated apoptosis inhibition was extracellular signal-regulated kinase (ERK) or phosphoinositide 3 kinase dependent and was interrupted by PD98059 and LY294002. In vivo, additional amount of SICX exacerbated the lung injury caused by LPS. LPS-induced lung injury and macrophage inflammatory protein 2 release, however, were prevented by CD64 and CD32 blockers (decoy antibodies). In conclusion, excessive amount of SICX in lung can act through Fc gammaRs to protect bronchoalveolar lavage fluid neutrophils from apoptosis that eventually lead to delayed resolution of ALI caused by LPS. Blockade of SICX engagement of CD32 and CD64 (with decoy antibodies) could interrupt SICX-mediated protection of neutrophils and protect lung from LPS-induced injury. The decoy antibodies may therefore have therapeutic utility in ALI.Read moreArticleFull-text availableInterleukin-3 plays a vital role in hyperoxic acute lung injury in mice via mediating inflammationOctober 2018 · BMC Pulmonary MedicineZhijian HuangWei ZhangJian Yang[...]Hongwei ZhouBackground: Interleukin (IL)-3 amplifies inflammation. However, the effect of IL-3 in acute lung injury (ALI), an acute inflammatory disease, is unclear. The aim of this study was to test the hypothesis that IL-3 plays an important role in hyperoxia-induced ALI.Methods: Hyperoxic ALI was induced in wild-type (WT) and IL-3 gene disrupted (IL-3-/-) mice by exposure to 100% O2 for 72 ... [Show full abstract] h.Results: Hyperoxia increased IL-3 levels in plasma and lung tissues in WT mice. Pulmonary inflammation and edema were detected by histological assay in WT mice exposed to 100% O2 for 72 h. However, the hyperoxia-induced lung histological changes were improved in IL-3-/- mice. The hyperoxia-induced elevation of neutrophils in bronchoalveolar lavage fluids and circulation were reduced in IL-3-/- mice. Meanwhile, the levels of tumor necrosis factor-α and IL-6 were suppressed in IL-3-/- mice compared with WT mice. Moreover, the hyperoxia-induced the activation of IκBα kinase (IKK) β, IκBα phosphorylation, and nuclear factor-κB translocation were inhibited in IL-3-/- mice compared with WT mice.Conclusions: Our results suggest IL-3 is a potential therapeutic target for hyperoxia-induced ALI.View full-textArticleThe value of the lipopolysaccharide-induce acute lung injury model in respiratory medicineDecember 2010 · Expert Review of Respiratory Medicine Hong Chen Chunxue BaiXiangdong WangAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a syndrome characterized by pulmonary edema and acute inflammation. Lipopolysaccharide (LPS), a major component in Gram-negative bacteria, has been used to induce ALI/ARDS. LPS-induced animal models highlight ways to explore mechanisms of multiple diseases and provide useful information on the discovery of novel biomarkers and ... [Show full abstract] drug targets. However, each model has its own merits and drawbacks. The goal of this article is to summarize and evaluate the results of experimental findings in LPS-induced ALI/ARDS, and the possible mechanisms and treatments elucidated. Advantages and disadvantages of such models in pulmonary research and new directions for future investigations are also discussed.Read moreArticleRole of Oxygen Metabolites in Immune Complex Injury of LungJuly 1981 · The Journal of Immunology Katee Jean JohnsonP A WardAcute lung injury in the rat has been produced by the deposition of immune complexes, by using the model developed earlier in this laboratory. The airway instillation of antibody (rabbit IgG) to bovine serum albumin (BSA) and the i.v. injection of BSA results in acute alveolar injury. As measured quantitatively by the leakage of 125I-rat IgG into the lung parenchyma, acute injury occurring over a ... [Show full abstract] 4-hr period can be markedly suppressed, in a dose-dependent manner, by the simultaneous airway instillation of antibody and catalase but not by chemically inactivated catalase, by superoxide dismutase (SOD), or by antiproteases (soybean trypsin inhibitor, α1-antitrypsin inhibitor from human serum) that are known to be potent inhibitors of the neutral proteases derived from rat lysosomal granules. Instillation of radiolabeled catalase, SOD, α1-antitrypsin, and soybean trypsin inhibitor into lungs of normal rats reveal average lung retention rates at 3 hr of 67, 54, 84, and 83%, respectively, suggesting that the inability of antiproteases to protect from acute injury is not related to their rapid disappearance from the lung. By immunofluorescence, the catalase-suppressed lung reactions contain deposits of immune complexes. Histologically, the suppressed lung reactions contain infiltrates of neutrophils in an interstitial and intralveolar location, but hemorrhage and intra-alveolar edema are absent. These studies suggest that a major mechanism for damage occurring in immune complex-triggered, complement and neutrophil-dependent acute lung injury may be related to the production of H2O2 and/or its metabolic products.Read moreArticleSynthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammat...June 2016 · The Journal of Immunology Alexander BocharovTinghuai Wu Irina Baranova[...] Konstantin G BirukovSynthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier ... [Show full abstract] dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.Read moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! 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