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MicroRNAs Distinguish Translational from Transcriptional Silencing during Endotoxin Tolerance*

  • Mohamed El Gazzar
    Correspondence
    To whom correspondence should be addressed: Dept. of Internal Medicine, Section of Molecular Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-8622; Fax: 336-716-1214;
    Affiliations
    From the Department of Internal Medicine, Section of Molecular Medicine, Winston-Salem, North Carolina 27157
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  • Charles E. McCall
    Affiliations
    From the Department of Internal Medicine, Section of Molecular Medicine, Winston-Salem, North Carolina 27157

    Translational Science Institute, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01AI065791 and R01AI079144.
Open AccessPublished:April 30, 2010DOI:https://doi.org/10.1074/jbc.M110.115063
      We reported that gene-selective formation of facultative heterochromatin silences transcription of acute inflammatory genes during endotoxin (LPS) tolerance, according to function. We discovered that reversal of the epigenetically silenced transcription restored mRNA levels but not protein synthesis. Here, we find that translation repression of tumor necrosis factor-α (TNFα) occurs independent of transcription silencing during LPS tolerance. The process required to disrupt protein synthesis followed Toll-like receptor 4 (TLR4)-dependent induction of microRNA (miR)-221, miR-579, and miR-125b, which coupled with RNA-binding proteins TTP, AUF1, and TIAR at the 3′-untranslated region to arrest protein synthesis. TTP and AUF1 proteins linked to miR-221, whereas TIAR coupled with miR-579 and miR-125b. Functional inhibition of miR-221 prevented TNFα mRNA degradation, and blocking miR-579 and miR-125b precluded translation arrest. The functional specificity of the TNFα 3′-untranslated region was demonstrated using luciferase reporter with mutations in the three putative miRNA binding sites. Post-transcriptional silencing was gene-specific, because it did not affect production of the IκBα anti-inflammatory protein. These results suggest that TLR4-dependent reprogramming of inflammatory genes is regulated at two separate and distinct levels. The first level of control is mediated by epigenetic modifications at the promoters that control transcription. The second and previously unrecognized level of control is mediated by TLR4-dependent differential expression of miRNAs that exert post-transcriptional controls. The concept of distinct regulation of transcription and translation was confirmed in murine sepsis. We conclude that transcription- and translation-repressive events combine to tightly regulate pro-inflammatory genes during LPS tolerance, a common feature of severe systemic inflammation.

      Introduction

      Inflammation development and progression are influenced by genetic, epigenetic, and environmental factors (
      • Hao S.
      • Baltimore D.
      ). Sensors such as Toll-like receptors and their cellular communication pathways couple inciters of inflammation to genes, after which epigenetic events reprogram gene expression patterns in a temporal sequence of defense and repair (
      • McCall C.E.
      • Yoza B.K.
      ). In severe systemic inflammation (SSI)
      The abbreviations used are: SSI
      severe systemic inflammation
      TLR
      Toll-like receptor
      IL-1
      interleukin-1
      LPS
      lipopolysaccharide
      RT
      reverse transcription
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      ELISA
      enzyme-linked immunosorbent assay
      ARE
      AU-rich element
      miRNA
      microRNA (also miR)
      miRISC
      miRNA-induced silencing complex
      UTR
      untranslated region
      TNFα
      tumor necrosis factor α
      siRNA
      small interfering RNA
      IP
      immunoprecipitation
      RBP
      RNA-binding protein.
      such as serious infection, a high magnitude response spreads systemically, which often results in death from cellular and organ failure. Such inflammation has distinct features and must be tightly controlled at transcriptional and post-transcriptional levels. Unlike in chronic inflammation, the inciting SSI phase is short-lived (lasts for hours) and progresses to an evolving phase typified by gene-selective reprogramming through histone and DNA methylation reactions that generate facultative heterochromatin (
      • Chan C.
      • Li L.
      • McCall C.E.
      • Yoza B.K.
      ,
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Chen X.
      • Hu J.
      • Hawkins G.A.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Liu T.
      • Yoza B.K.
      • McCall C.E.
      ). This gene-specific paradigm silences transcription of the acute pro-inflammatory genes responsible for the SSI autotoxicity, whereas sustaining transcription of distinctly functional genes like those encoding anti-inflammatory and antibiotic mediators (
      • Chan C.
      • Li L.
      • McCall C.E.
      • Yoza B.K.
      ,
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Chen X.
      • Hu J.
      • Hawkins G.A.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Liu T.
      • Yoza B.K.
      • McCall C.E.
      ,
      • Chen X.
      • Yoza B.K.
      • El Gazzar M.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • Foster S.L.
      • Hargreaves D.C.
      • Medzhitov R.
      ) (see Fig. 1). Depending on the magnitude of the original inciting stimulus, the evolving phase of SSI can dissipate within days, or be sustained for weeks (
      • McCall C.E.
      • Yoza B.K.
      ). Reversal of gene silencing correlates with resolution of SSI and survival. Our studies implicate nuclear factor κB (NF-κB) members p65 and RelB as essential regulators of the inciting, evolving, and restorative phases of SSI (
      • Chan C.
      • Li L.
      • McCall C.E.
      • Yoza B.K.
      ,
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Liu T.
      • Yoza B.K.
      • McCall C.E.
      ,
      • Chen X.
      • Yoza B.K.
      • El Gazzar M.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ).
      Figure thumbnail gr1
      FIGURE 1Gene reprogramming during endotoxin tolerance and SSI. SSI is a germ line and epigenetically controlled gene-specific reprogramming process that evolves in stereotyped stages. This simplified scheme shows three functional and clinically significant gene expression patterns associated with SSI: A, inciting stage: immediate response pro-inflammatory gene set (e.g. TNFα, IL-1β, and others) that are activated by LPS and then become inactive. These mediators initiate SSI. B, evolving stage: feed-forward gene-specific epigenetic switches (e.g. RelB). This establishes transcription silencing. C, resolving stage: reversal of gene reprogramming and survival.
      The NF-κB family is the prototypic master inflammation regulator (
      • Ghosh S.
      • Karin M.
      ). Toll-like receptors (TLRs) usually initiate SSI through NF-κB cytosolic activation with nuclear translocation and promoter binding of p65, which interacts with trans-acting factors. We discovered that temporally distinct de novo NF-κB RelB induction replaces promoter-bound NF-κB p65 after the inciting stage to generate gene set-specific transcription silencing. RelB interacts with and recruits the histone H3K9 methyltransferase, G9a, to dimethylate histone H3K9, leading to recruitment of the DNA methyltransferase Dnmt3a/b and subsequent methylation of promoter CpGs and chromatin condensation (
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Chen X.
      • Hu J.
      • Hawkins G.A.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Liu T.
      • Yoza B.K.
      • McCall C.E.
      ). Gene-selective facultative heterochromatin reverses to euchromatin during the SSI resolution/restorative phase. The evolving phase of SSI with gene-selective silencing is typified by the phenomenon of endotoxin tolerance observed in humans, animals, and cell models (
      • McCall C.E.
      • Yoza B.K.
      ). We found that reversal of condensed heterochromatin to open euchromatin by inhibiting RelB or G9a restored transcription but not translation of pro-inflammatory TNFα and IL-1β (
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Chen X.
      • Hu J.
      • Hawkins G.A.
      • McCall C.E.
      ), suggesting an additional silencing mechanism at the post-transcriptional level.
      The inciting and evolving features of SSI conform to the network motif concept of type 1 incoherent feed-forward loops (
      • Mangan S.
      • Alon U.
      ), in which a transcription activator X (e.g. NF-κB p65) controls a target gene Z (e.g. TNFα) and also sequentially activates a transcription repressor Y (e.g. RelB) of the target gene, thus producing physiologic adaptation, for example tolerance. Recent data support that such gene-encoded adaptive loops may involve microRNAs (miRNAs) (
      • O'Donnell K.A.
      • Wentzel E.A.
      • Zeller K.I.
      • Dang C.V.
      • Mendell J.T.
      ). Feedback repression of inflammatory adhesion genes in endothelial cells by miRNAs recently provides an example (
      • Suárez Y.
      • Wang C.
      • Manes T.D.
      • Pober J.S.
      ). Thus transcription and post-transcription regulatory process may differentially influence the inflammatory phenotype.
      miRNAs are a conserved and abundant class of endogenous non-coding RNAs of ∼22 nucleotides involved in post-transcriptional gene expression by controlling the stability and/or translation of target mRNAs (,
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). miRNAs are processed from long primary transcripts that are transcribed from independent genes or from intronic sequences of protein-coding genes by RNA polymerase II (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). Primary transcripts are processed into ∼60- to 70-nucleotide hairpin precursors, which are exported to the cytoplasm and further processed into ∼22-nucleotide mature miRNAs by the RNase type III enzyme Dicer (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). Mature miRNAs are loaded onto a ribonucleoprotein complex, also known as miRNA-induced silencing complex (miRISC), where they act as guiding molecules to deliver the complex to target mRNA via binding to complementary sequences in the 3′UTR, resulting in mRNA degradation and/or translational repression (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). miRNAs usually base pair to target mRNA with imperfect complementarity, resulting in translational inhibition, whereas perfect base-pairing induces target mRNA degradation (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ,
      • Bushati N.
      • Cohen S.M.
      ). The most stringent requirement for miRNA function is a near perfect base-pairing within the miRNA nucleotides 2–7 (known as the seed region), which nucleates the interaction within the target mRNA 3′UTR (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). Recent studies in mammals implicate miRNAs in a number of biological processes, including regulation of normal immune function, cell differentiation, viral infection, inflammation, signal transduction, and cancer (,
      • Sonkoly E.
      • Ståhle M.
      • Pivarcsi A.
      ,
      • Sheedy F.J.
      • Palsson-McDermott E.
      • Hennessy E.J.
      • Martin C.
      • O'Leary J.
      • Ruan Q.
      • Johnson D.P.
      • Chen Y.
      • O'Neill L.A.
      ).
      Here, we tested whether the SSI-inciting phase might induce an miRNA code that regulates translational events independent of the transcriptional silencing process of selective facultative heterochromatin formation. To do this, we used the THP-1 cell model of SSI as reflected in the generation of endotoxin (lipopolysaccharide (LPS)) tolerance by an inciting TLR4 LPS stimulus. We performed a computational miRNA target prediction algorithm and identified twenty miRNAs with sequences complementary to the TNFα 3′UTR, as a model of post-transcriptional repression of pro-inflammatory genes in LPS tolerance. Expression profiling distinguished three miRNAs (miR-221, miR-579, and miR-125b) whose levels were differentially altered after LPS stimulation in tolerant cells. Importantly, we show that these miRNAs assemble into the miRISC complex and target specific RNA-binding proteins (RBPs) to the TNFα 3′UTR. Functional inhibition revealed that miR-221 directs mRNA degradation, whereas miR-579 and miR-125b inhibit translation of TNFα mRNA. We also found that these miRNAs and RBPs may couple to disrupt TNFα protein synthesis in mice with sepsis. Our studies provide the first evidence linking differential changes in TLR-induced miRNA expression to LPS tolerance, which serves as a cell model of SSI gene reprogramming (
      • McCall C.E.
      • Yoza B.K.
      ,
      • LaRue K.E.
      • McCall C.E.
      ), and suggest that both disruption of transcription and translation independently assure gene-specific silencing during the endotoxin tolerance/evolving phase of SSI.

      DISCUSSION

      Innate immunity employs a multilayered regulatory system to control inflammation through inciting, evolving, and resolving phases (
      • McCall C.E.
      • Yoza B.K.
      ). The distinct and consistent pro- and anti-inflammatory physiologic phases of SSI suggest that gene reprogramming and not TLR specificity ultimately controls host response to infection (
      • Foster S.L.
      • Hargreaves D.C.
      • Medzhitov R.
      ,
      • Cavaillon J.M.
      • Adib-Conquy M.
      ,
      • Foster S.L.
      • Medzhitov R.
      ). Our and other reports on epigenetically controlled chromatin structural modifications during SSI in humans and animals support this concept (
      • Chen X.
      • El Gazzar M.
      • Yoza B.K.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ,
      • El Gazzar M.
      • Yoza B.K.
      • Chen X.
      • Hu J.
      • Hawkins G.A.
      • McCall C.E.
      ,
      • Foster S.L.
      • Hargreaves D.C.
      • Medzhitov R.
      ,
      • Medzhitov R.
      • Horng T.
      ). The formation of facultative heterochromatin at promoters of acute pro-inflammatory genes and not anti-inflammatory genes reversibly silences a set of inflammation inducing genes to switch phenotype during the evolving phase of SSI (
      • McCall C.E.
      • Yoza B.K.
      ). The reversible nature of facultative heterochromatin parallels a return to homeostasis when SSI resolves. Here, we identify an additional, indispensable, and distinct negative regulatory mechanism by which differentially expressed miRNAs disrupt protein synthesis of acute pro-inflammatory TNFα during the evolving anti-inflammatory stage of SSI that is typified by LPS tolerance.
      We identified miRNAs that base pair with sequences in the TNFα 3′UTR. Expression profiling revealed that miR-221, miR-579, and miR-125b were selectively induced in LPS-tolerant cells and assembled with RBPs that target TNFα mRNA for translational repression. We also showed that these miRNAs induced translational repression through a combinatorial effect. miR-221 promoted degradation of endogenous as well as a luciferase reporter mRNA fused to TNFα 3′UTR, whereas miR-579 and miR-125b promoted translation arrest. The selective LPS-mediated induction of miRNAs in LPS-tolerant cells strongly suggests that differential induction of target-specific miRNAs rather than a global change in TLR-mediated pathway is a primary mechanism for translational repression in SSI.
      mRNA instability and translation are mainly regulated by AU-rich elements (AREs) located in the 3′UTR of a variety of short-lived mRNAs, including cytokines (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ,
      • Chen C.Y.
      • Shyu A.B.
      ). Indeed, a recent report indicates that the number of AU sequences correlates with differential expression patterns in the inflammation gene code (
      • Hao S.
      • Baltimore D.
      ). The repressive function of AREs is regulated by protein factors that bind to the 3′UTR. The TNFα 3′UTR contains a ∼70-bp ARE sequence composed of several repeats of AUUUA pentamer (
      • Han J.
      • Beutler B.
      ). The 3′UTR of TNFα influences its synthesis in vivo. Mice expressing TNFα gene lacking the 3′UTR overexpress TNFα protein (
      • Keffer J.
      • Probert L.
      • Cazlaris H.
      • Georgopoulos S.
      • Kaslaris E.
      • Kioussis D.
      • Kollias G.
      ), whereas mice lacking RBPs that target the 3′UTR (e.g. TTP and AUF1) overproduce TNFα protein (
      • Lu J.Y.
      • Sadri N.
      • Schneider R.J.
      ,
      • Carballo E.
      • Lai W.S.
      • Blackshear P.J.
      ). The three miRNA binding sites identified in this study are located within overlapping distances in the TNFα ARE element (see Fig. 13). We found that miR-221 increased mRNA degradation and was co-immunoprecipitated with TTP and AUF1 proteins that are implicated in mRNA instability. In addition, miR-579 and miR-125b induced translation arrest and were co-immunoprecipitated with the translation silencer TIAR. Thus, our results indicate that LPS tolerance is not only associated with differential alteration in miRNAs profile, but also with distinct post-transcriptional mechanisms of mRNA stability and translation. Also, functional analysis showed that TNFα 3′UTR conferred instability and translational arrest on a fused luciferase reporter mRNA in LPS-tolerant cells. Combined mutations in the 3′UTR that disrupted binding by miR-221, miR-579, and miR-125b restored mRNA stability and translation, demonstrating a critical role for the TNFα 3′UTR in its translational repression by miRNAs.
      Figure thumbnail gr13
      FIGURE 13A model of TNFα post-transcriptional repression during SSI. LPS stimulation results in increased expression of miR-221, miR-579, and miR-125b in THP-1-tolerant cells. These miRNAs are then loaded into a ribonucleoprotein complex (miRISC) where they help recruit the complex to the TNFα 3′UTR through binding to complementary sequences (mostly AREs) within this region. RNA-binding proteins TTP, AUF1, and TIAR, which are included in the RISC complex, will bind to the ARE (likely through one or more of these miRNAs) and, jointly with the rest of RISC components, will induce TNFα repression by three possible mechanisms: either mRNA deadenylation (1) and/or decapping (2) resulting in mRNA degradation OR translational repression by inhibiting translation initiation or elongation (3). Note that the constitutive decay element (CDE) has been implicated in mediating mRNA instability, but the one or more proteins involved have not been identified. Our results suggested that this CDE element is targeted by AUF1.
      miRNAs function by recruiting RBPs to target mRNA for translational repression (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ,
      • Peters L.
      • Meister G.
      ). We detected TTP, AUF1, and TIAR proteins bound at the TNFα 3′UTR in tolerant, but not LPS-responsive cells. The binding of these proteins was dependent on miRNAs induction in tolerant cells, because they did not bind in responsive cells despite their normal protein levels (dad not shown). Previous reports indicated that TTP and AUF1 enhance TNFα and IL-1β mRNA degradation in activated macrophages and other cell types (
      • Lu J.Y.
      • Sadri N.
      • Schneider R.J.
      ,
      • Hau H.H.
      • Walsh R.J.
      • Ogilvie R.L.
      • Williams D.A.
      • Reilly C.S.
      • Bohjanen P.R.
      ). TIAR, and the related protein TIA-1, down-regulates TNFα translation (
      • Gueydan C.
      • Droogmans L.
      • Chalon P.
      • Huez G.
      • Caput D.
      • Kruys V.
      ,
      • Piecyk M.
      • Wax S.
      • Beck A.R.
      • Kedersha N.
      • Gupta M.
      • Maritim B.
      • Chen S.
      • Gueydan C.
      • Kruys V.
      • Streuli M.
      • Anderson P.
      ). Our co-immunoprecipitation experiments demonstrated that miR-221 associated with TTP and AUF1 proteins, whereas miR-579 and miR-125b associated with TIAR. These results, together with our finding that functional inhibition of miR-221 stabilized mRNA while miR-579 and miR-125b inhibition enhanced translation of TNFα, suggest that these miRNAs directly interact with and recruit specific RBPs to TNFα 3′UTR, thereby mediating translational repression. In addition, siRNA-mediated knockdown of TTP and AUF1 in tolerant cells stabilized TNFα mRNA, whereas TIAR knockdown increased mRNA translation to almost the same extent seen upon functional inhibition of miRNA expression, indicating that these miRNAs promoted translational repression of TNFα in tolerant cells by recruiting inhibitory RBPs. A recent study reported that a constitutive decay element in the TNFα 3′UTR, downstream of the ARE (see Fig. 13) targeted TNFα mRNA for rapid decay (
      • Stoecklin G.
      • Lu M.
      • Rattenbacher B.
      • Moroni C.
      ). Interestingly, miR-221 binding site is located within this element. Because miR-221 co-immunoprecipitated with AUF1, it is possible that miR221 recruits AUF1 to this decay element. Thus, our results suggest that differentially induced miRNAs couple with specific RBPs to mediate TNFα translational repression in LPS-tolerant phenotype.
      Some RBPs may protect certain mRNAs from miRNA-mediated repression (
      • Dean J.L.
      • Wait R.
      • Mahtani K.R.
      • Sully G.
      • Clark A.R.
      • Saklatvala J.
      ,
      • Ketting R.F.
      ) by facilitating mRNA targeting to polysomes for translation rather than to p-bodies (the sites of mRNA decay and translation arrest) for translational repression (
      • Filipowicz W.
      • Bhattacharyya S.N.
      • Sonenberg N.
      ). For example, HuR protein stabilizes TNFα mRNA in macrophages under normal physiologic conditions (
      • Dean J.L.
      • Wait R.
      • Mahtani K.R.
      • Sully G.
      • Clark A.R.
      • Saklatvala J.
      ), and Dnd1, another RBP, may increase mRNA stability and translation of some tumor suppressor mRNAs by preventing negative regulatory miRNA binding (
      • Ketting R.F.
      ,
      • Kedde M.
      • Strasser M.J.
      • Boldajipour B.
      • Oude Vrielink J.A.
      • Slanchev K.
      • le Sage C.
      • Nagel R.
      • Voorhoeve P.M.
      • van Duijse J.
      • Ørom U.A.
      • Lund A.H.
      • Perrakis A.
      • Raz E.
      • Agami R.
      ). In this study, we observed that HuR and Dnd1 bound to TNFα 3′UTR in responsive but not LPS-tolerant cells (data not shown). This is consistent with these regulators stabilizing TNFα mRNA and translation during the inciting phase of SSI (i.e. in responsive cells). In contrast, during the evolving phase of endotoxin tolerance and gene silencing, HuR and Dnd1 did not bind to TNFα mRNA despite their normal expression in the cytoplasm. Thus, the specific repressive effect conferred by miRNAs on 3′UTR may displace HuR or Dnd1 and override their positive effects.
      Recent studies reported that miR-146a and miR-155 are inflammation modifiers and are induced by LPS in normal monocytes/macrophages (
      • Nahid M.A.
      • Pauley K.M.
      • Satoh M.
      • Chan E.K.
      ,
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      ). miR-146a down-regulates TLR signaling proteins TRAF6 and IRAK1 (
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      ) and thus may reduce TNFα expression and indirectly prevent the generation of endotoxin tolerance (
      • Nahid M.A.
      • Pauley K.M.
      • Satoh M.
      • Chan E.K.
      ). The prevention context of gene silencing should be distinguished from the reversal concept that would be needed to intervene in SSI. miR-155 can attenuate TLR4 signal by targeting IKKϵ kinase (
      • Tili E.
      • Michaille J.J.
      • Cimino A.
      • Costinean S.
      • Dumitru C.D.
      • Adair B.
      • Fabbri M.
      • Alder H.
      • Liu C.G.
      • Calin G.A.
      • Croce C.M.
      ) but can also activate LPS signaling pathways by enhancing TNFα expression (
      • Tili E.
      • Michaille J.J.
      • Cimino A.
      • Costinean S.
      • Dumitru C.D.
      • Adair B.
      • Fabbri M.
      • Alder H.
      • Liu C.G.
      • Calin G.A.
      • Croce C.M.
      ,
      • O'Connell R.M.
      • Taganov K.D.
      • Boldin M.P.
      • Cheng G.
      • Baltimore D.
      ). Although miR-146a and miR-155 can attenuate TLR signaling, they do not appear to directly affect pro-inflammatory gene translational repression in LPS-tolerant cells. Our data indicate that miR-146a and miR-155 expression increases in both responsive and LPS-tolerant cells. Our study does not exclude the possibility that these miRNAs may directly or indirectly influence tolerance.
      Although there are no previous reports to our knowledge implicating miR-221 or miR-579 in regulating TNFα expression, a recent study reported that miR-125b targets TNFα mRNA transcripts for degradation in murine macrophages, which may limit TNFα production under basal normal conditions (
      • Tili E.
      • Michaille J.J.
      • Cimino A.
      • Costinean S.
      • Dumitru C.D.
      • Adair B.
      • Fabbri M.
      • Alder H.
      • Liu C.G.
      • Calin G.A.
      • Croce C.M.
      ). After LPS stimulation, however, the miR-125b level decreases to allow TNFα production in the inciting phase. Our finding of increased miR-125b expression in LPS-tolerant THP-1 cells, which follows the earlier decrease, is consistent with this report in that a reduction of miR-125b during the LPS-inciting phase would support TNFα translation, whereas a subsequent increase in the evolving tolerant phase would feed forward to repress TNFα translation. A recent report further supports that feed-forward repressor loops can limit innate immune responses (
      • Suárez Y.
      • Wang C.
      • Manes T.D.
      • Pober J.S.
      ). TNFα-Induced miRNAs miR-31 and miR-17–3p reduced expression of E-selectin and intercellular adhesion molecule 1 in human endothelial cells to limit endothelial and neutrophil adhesion events after the inciting phase.
      We showed that epigenetic gene reprogramming also occurred in mice with SSI and that this process is reversible by returning silent heterochromatin to euchromatin through G9a inhibition (Fig. 11). The finding, that septic macrophages with reversed epigenetic silencing expressed TNFα mRNA but not protein, suggested a post-transcriptional repression mechanism likely promoted by the induction of negative regulatory miRNAs, similar to that observed in THP-1 cell model of SSI. Recent studies reported selective epigenetic-based gene repression mechanisms using an animal model of inflammation (
      • Foster S.L.
      • Hargreaves D.C.
      • Medzhitov R.
      ,
      • Wen H.
      • Dou Y.
      • Hogaboam C.M.
      • Kunkel S.L.
      ). LPS-tolerant macrophages derived from bone marrow exhibited a differential gene-silencing profile after a second LPS stimulation ex vivo. Such silencing was associated with a loss of H3K4 trimethylation, which usually marks transcriptionally active genes (
      • Foster S.L.
      • Hargreaves D.C.
      • Medzhitov R.
      ,
      • Santos-Rosa H.
      • Schneider R.
      • Bannister A.J.
      • Sherriff J.
      • Bernstein B.E.
      • Emre N.C.
      • Schreiber S.L.
      • Mellor J.
      • Kouzarides T.
      ). A recent study (
      • Wen H.
      • Dou Y.
      • Hogaboam C.M.
      • Kunkel S.L.
      ), using the cecal ligation and puncture model of sepsis, demonstrated that IL-12 gene transcription is associated with a shift from H3K27 trimethylation to monomethylation, which associates with gene silencing (
      • Smale S.T.
      ). Thus, epigenetic silencing seems as a primary process of transcriptional repression in animal systemic and local inflammation. Our studies add on to these findings and, further, reveal an additional miRNA-dependent post-transcriptional silencing mechanism during SSI.
      Based on our results, we propose a model as depicted in Fig. 13, where differentially induced miRNAs bind specifically to TNFα 3′UTR. These miRNAs recruit the RNA-binding proteins TTP, AUF1, and TIAR, which in turn could interact with Ago2 protein, resulting in the assembly of miRISC repressor complexes. Such complexes are then targeted to p-bodies, where mRNA degradation by deadenylation or decapping, and/or translational arrest due to inhibition of translation initiation, occur. This silencing process is gene-specific, allowing for sustained expression of other inflammation modifiers such as IκBα. We found that RBPs and miRNAs were associated with Ago2 at the TNFα 3′UTR in LPS-tolerant but not -responsive cells, and knockdown of Ago2 resulted in dissociation of the complex and relieved translation repression (data not shown). Thus, we conclude that epigenetic modifications generating gene-specific formation of transcription silencing facultative heterochromatin combine with the post-transcriptional repressor events shown herein to direct the physiology of evolving SSI through tightly regulated feed-forward loops. The reversible nature of facultative heterochromatin and the post-transcriptional silencing described in this report and the recent success in using LAN-based antagomirs to silence miRNA expression in mice and primates (
      • Stenvang J.
      • Lindow M.
      • Kauppinen S.
      ), if applied to SSI, might provide a novel approach for therapeutic interventions.

      Acknowledgments

      We thank A. Church, J. Hu, and S. Cousart for technical assistance, T. Liu for Western blotting, and B. Yoza for critical discussion.

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