RegulationofActivation-associatedMicroRNAAccumulation ... · pathogen-derived molecules as they...

Regulation of Activation-associated MicroRNA AccumulationRates during Monocyte-to-macrophage Differentiation*

Received for publication, July 23, 2014, and in revised form, August 20, 2014 Published, JBC Papers in Press, August 22, 2014, DOI 10.1074/jbc.M114.599316

Renee L. Eigsti‡, Bayan Sudan‡, Mary E. Wilson‡§¶�**, and Joel W. Graff‡§**1

From the ‡Carver College of Medicine and Departments of §Internal Medicine, ¶Microbiology, and �Epidemiology, University ofIowa, Iowa City, Iowa 52242 and the **Veterans Affairs Medical Center, Iowa City, Iowa 52246

Background: A set of miRNAs accumulates in polarized macrophages.Results: The same miRNAs accrued during macrophage differentiation with varied rates depending on exogenous conditionsand were regulated at a preprocessing step.Conclusion: Key monocytic cell miRNAs accumulated in response to activation and differentiation signals.Significance: Accumulation of key miRNAs was tailored to diverse stimuli leading to customized miRNA expression.

Circulating monocytes recruited to tissues can differentiate intomacrophages and adopt unique gene expression programs inresponse to environmental cues. We recently described the regu-lated expression of several microRNAs (miRNAs) in polarizedhuman monocyte-derived macrophages (MDMs). Basal expres-sion of these activation-associated miRNAs was low in monocytesrelative to MDMs. As development occurs in the context of specificcellular environments, we hypothesized that the rate of miRNAaccumulation would be modified in the presence of microbial orcellular products during monocyte-to-macrophage differentia-tion. Indeed, LPS treatment augmented the accumulation of miR-146a and miR-155, whereas IL-4 treatment augmented the accu-mulation of miR-193b and miR-222 during development. Incontrast, some stimuli repressed accumulation of specific miRNAsincluding interferons (IFNs) (miR-27a, miR-125a-5p, and miR-222), IL-4 (miR-125a-5p), and LPS (miR-27a). RT-PCR-basedexpression profiling of monocytes differentiated with distinctmethods showed that activation-associated miRNAs and markersof macrophage polarization were substantially altered in MDMsdifferentiated in the presence of non-monocytic peripheral bloodmononuclear cells due in part to NF-�B and STAT1 pathway acti-vation. Expression of several of these miRNAs was regulated at apreprocessing step because the expression of the primary miRNAs,but not Dicer, correlated with mature miRNA expression. Weconclude that a set of miRNAs is regulated during MDM differen-tiation, and the rate is uniquely modified for each miRNA by envi-ronmental factors. The low basal expression of activation-as-sociated miRNAs in monocytes and their dynamic rates ofaccumulation during MDM differentiation permit mono-cytes to tailor miRNA profiles in peripheral tissues duringdifferentiation to macrophages.

Tissue-resident macrophages established prior to birth fromyolk sac- and fetal liver-derived progenitors replicate to main-tain populations under homeostatic conditions (1–3). Disrup-tion of tissues due to disturbances such as infection, inflamma-tion, and injury can lead to reduced numbers of tissue-residentmacrophages in a phenomenon known as the disappearancereaction (1, 4 – 6). During this time, bone marrow-derivedmonocytes are recruited to the affected tissue and differentiateinto macrophages. In contrast to the resident macrophages, theinfiltrating macrophages often are a transient population. Uponresolution of tissue disturbances, the balance of tissue macro-phage populations is restored through both maintenance ofsome infiltrating macrophages and replication of the remainingoriginal tissue-resident macrophages (2, 3, 5, 7).

Despite their high abundance in most tissues, our under-standing of human tissue-resident macrophages is poor due tolimitations in obtaining these cells (8). Conversely, infiltratingmacrophages can be modeled ex vivo by differentiating periph-eral blood monocytes to MDMs.2 Tissue-infiltrating mono-cytes respond to local environments by dramatically alteringtheir gene expression profiles and consequently their func-tional phenotypes as they differentiate to macrophages. Theaddition of cytokines and other activating stimuli ex vivo tomonocyte cultures can be utilized to represent possible condi-tions encountered by monocytes recruited into tissues. Mono-cytic cell gene expression regulation occurs at many levels.miRNAs have important roles in regulating gene expressionduring innate and adaptive immune responses (9, 10). How-ever, the impact of miRNAs during monocytic cell differentia-tion and activation is relatively understudied. To investigatethis, a better understanding of miRNA expression regulationduring these processes is needed.

Biogenesis of miRNAs is a multistep process (11). In the cellnucleus, a stem loop structure within a primary miRNA (pri-miRNA) transcript is recognized and cleaved by the exonu-clease Drosha. After export to the cytoplasm, cleavage of theterminal loop by a complex containing the exonuclease Dicer

* This work was supported, in whole or in part, by National Institutes of HealthGrants AI080801, AI045540, and AI07511 (to M. E. W.) and T35HL007485 (atraining grant to R. L. E.). This work was also supported by Grants1IK2BX001627 (to J. W. G.), 1I01BX001983 (to M. E. W.), and 5I01BX000536(to M. E. W.) from the Department of Veterans Affairs, Veterans HealthAdministration, Office of Research and Development, Biomedical Labora-tory Research and Development.

1 To whom correspondence should be addressed: Dept. of Internal Medicine,University of Iowa, 400 EMRB, Iowa City, IA 52242. Tel.: 319-335-6807; Fax:319-353-4565; E-mail: [email protected].

2 The abbreviations used are: MDM, monocyte-derived macrophage; Ct, cyclethreshold; miRNA or miR, microRNA; PBMC, peripheral blood mononuclearcell; qPCR, quantitative PCR; pri-miRNA, primary miRNA; M-CSF, macro-phage colony-stimulating factor; TLDA, TaqMan Low Density Array.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 41, pp. 28433–28447, October 10, 2014Published in the U.S.A.

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results in a double-stranded RNA composed of a guide strandand a passenger strand. The guide strand has a long half-life asit is protected within the miRNA-binding cleft of an miRNA-induced silencing complex, whereas the passenger strand isexcluded from the miRNA-induced silencing complex anddegraded rapidly. Watson-Crick base pairing between theguide strand of the miRNA-induced silencing complex and itstargeted mRNAs diminishes gene expression primarily bymRNA destabilization (12). Furthermore, the miRNA-inducedsilencing complex acts to inhibit protein translation at multiplesteps (13).

We recently monitored miRNA expression in MDMs treatedwith a variety of polarizing conditions including IFN� plus LPS(M1-polarized), IL-4 (M2a-polarized), immune complexes plusLPS (M2b-polarized), and TGF� (M2c-polarized). There wereconsistent changes in several miRNA passenger strands, butsurprisingly there were few miRNA guide strands whose abun-dance changed significantly in these conditions (14). With theexception of miR-193b, the amplitude of change in the abun-dance of individual guide strands was less than 2-fold (14). Incontrast to the small changes in miRNA expression describedin our previous study, several studies have described robustinduction of a subset of miRNAs in human monocytes respond-ing to LPS and other inflammatory treatments (15–19). Theseobservations led us to the important discovery that all activa-tion-associated miRNAs tested had higher basal expression lev-els in MDMs than in monocytes (14). Therefore, these miRNAsaccumulated at some point during monocyte-to-macrophagedifferentiation.

In the current study, we characterized the expression regu-lation of the miRNAs that were induced by macrophage-acti-vating conditions (14) during monocyte-to-macrophage differ-entiation under a variety of environmental conditions. Wehypothesized that the low basal expression of these miRNAs inmonocytes would permit monocytes to adopt unique miRNAexpression profiles in response to varied environmental condi-tions during monocyte-to-macrophage differentiation. Sup-porting this hypothesis, the accumulation rate of these miRNAsin monocytic cells was modified by IFN�, IFN�, IL-4, and LPS.We also show that co-culture of monocytes with non-mono-cytic peripheral blood mononuclear cells (PBMCs) during dif-ferentiation, a standard method of generating MDMs ex vivo,profoundly altered the expression of many miRNAs andnumerous mRNA markers of macrophage polarization. Thesefindings suggest that, by maintaining low expression levels ofactivation-associated miRNAs such as miR-155 and miR-193b,monocytes can have appropriately tailored responses to envi-ronmental stimuli such as cytokines, cell-cell interactions, andpathogen-derived molecules as they differentiate to macro-phages in peripheral tissues.

EXPERIMENTAL PROCEDURES

Human Subjects—Human subject protocols were approvedby Institutional Review Boards of the University of Iowa and theIowa City Veterans Affairs Medical Center. Peripheral bloodsamples were obtained anonymously in the form of leukocytereduction system cones from the DeGowin Blood Bank at theUniversity of Iowa.

Cell Culture—Monocytic cells were isolated from blood sam-ples using two standard protocols. Each method started withPBMCs isolated using Ficoll-Paque Plus (GE Healthcare) den-sity gradients.

In the first method, monocytes were isolated on the day ofblood donation directly from PBMCs using CD14 MicroBeads(Miltenyi Biotec). The purified monocytes were then culturedin tissue culture plates at 1 � 106 cells/ml in RP-10 (RPMI 1640medium (Invitrogen) with FBS (10%, v/v; Invitrogen) and L-glu-tamine (2 mM; Invitrogen)) supplemented with M-CSF (50ng/ml). In some experiments, monocytes (1 � 106 cells/ml)were co-cultured with negatively selected, non-monocyticPBMCs from the CD14 MicroBead-mediated sort (5 � 106

cells/ml) in RP-10 supplemented with M-CSF (50 ng/ml).The second method, described in our previous study (14),

selects MDMs from total PBMC cultures based on adherence.Briefly, PBMCs were cultured at 37 °C with 5% CO2 in Petridishes at a density of 5 � 106 cells/ml in RP-10 supplementedwith M-CSF (5 ng/ml; eBioscience). After 4 days of culture,Petri plates were washed extensively with Hanks’ balanced saltsolution lacking divalent cations (Invitrogen) to remove nonad-herent cells. The adherent MDMs were trypsinized and pel-leted. Upon resuspension at 1 � 106 cells/ml in RP-10 withM-CSF (5 ng/ml), the MDMs were incubated at 37 °C in Teflonjars overnight. MDMs were subsequently seeded in tissue cul-ture plates and incubated for 8 h at 37 °C with 5% CO2 prior tostudy.

Monocyte Activation—Activation of purified monocytes wasachieved by supplementing RP-10 with phenol-extracted Esch-erichia coli 055:B5 LPS (Sigma-Aldrich), IL-4 (Peprotech),IFN� (Peprotech), or IFN� (Peprotech) at the indicatedconcentrations.

Reverse Transcription-Quantitative PCR (RT-qPCR)—Totalcellular RNA was purified from monocytes and MDMs usingTRIzol (Invitrogen). For TaqMan MicroRNA Assays, miRNA-specific reverse transcription reactions were performed withMegaScript RT (Applied Biosystems). For TaqMan GeneExpression Assays (Applied Biosystems), random hexamer-primed reverse transcription reactions were performed withSuperScript III RT (Invitrogen). PCR with TaqMan GeneExpression Assays used TaqMan Universal PCR Master Mix(Applied Biosystems). Measurements of �-actin and TATAbox-binding protein were used as endogenous controls for geneexpression assays, whereas RNU48 was used as an endogenouscontrol for miRNA assays. PCR was performed on an 7900HTFast Real-Time PCR System (Applied Biosystems). Cyclethreshold (Ct) values were calculated, and quality control wasperformed using SDS v2.4 (Applied Biosystems). Changes inexpression were calculated using the ��Ct method and con-verted to log2 scale using Excel 2007 (Microsoft).

TaqMan Low Density Array (TLDA) Human miRNA Assays(Applied Biosystems), a gift from Dr. Martha Monick (Univer-sity of Iowa), were used to profile miRNA abundance. Briefly,cDNA was generated using the TaqMan Reverse Transcriptionkit and Megaplex Primer Pool A v2.0. Preamplification was per-formed using TaqMan PreAmp Master Mix and MegaplexPreAmp Primer Pool A v2.0. Finally, TLDA cards were loadedwith preamplified reactions, and PCR was performed on a

MicroRNAs in Human Monocytic Cells

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7900HT Fast Real-Time PCR System. Changes in miRNAexpression levels were calculated (��Ct method) using SDSv2.3 and SDS RQ Manager v1.2 software in tandem. RNU48 wasused as an endogenous control. miRNA expression in MDMsdifferentiated either in the presence or absence of non-mono-cytic PBMCs was compared with that in undifferentiatedmonocytes.

Integrated fluidic circuit-based RT-qPCR was performedusing the Bio-Mark System for Genetic Analysis (Fluidigm).First, RNA was reverse transcribed using random hexamer-primed reactions with SuperScript III RT. Next, preamplifica-tion of the RT product was performed using a pool of 48 Indi-vidual TaqMan Gene Expression Assays with TaqMan PreAmpMasterMix according to recommendations by Fluidigm.Finally, gene expression was monitored by real time PCR on48.48 Dynamic Array integrated fluidic circuits. Ct values werecalculated, and quality control was performed using FluidigmReal-Time PCR Analysis v3.1.3 software (Fluidigm). Measure-ments of ACTB, B2M, and TBP protein were used as endoge-nous controls. Changes in expression were calculated using the��Ct method and converted to log2 scale using Excel 2007.Unsupervised hierarchal clustering of transcripts that had reg-ulated expression (�4-fold in one or more of the samples rela-tive to monocytes harvested immediately after MicroBead-based purification) was performed using Partek Genomic Suitev6.5 (Partek Inc.). Data sets for both the TLDA assays mea-suring miRNA abundance and the integrated fluidic circuit-based RT-PCR experiments are available in the Gene Expres-sion Omnibus as a SuperSeries under accession numberGSE60550.

Immunoblotting—In some experiments, protein lysates werecollected at the indicated times from cultures of purified mono-cytes or from adherent MDMs in SDS-PAGE buffer. In otherexperiments, stocks of control conditioned medium wereobtained on day 5 from cultures of CD14 MicroBead-purifiedmonocytes grown at 1 � 106 cells/ml in RP-10 supplemented withM-CSF (50 ng/ml). Conditioned medium was also collected on day5 from PBMC cultures grown at 5 � 106 cells/ml in RP-10 supple-mented with M-CSF (5 ng/ml). CD14 MicroBead-purified mono-cytes were cultured at 1 � 106 cells/ml in RP-10 with M-CSF (50ng/ml) for 5 days in 24-well plates to obtain MDMs. Cell culturemedium was removed from the MDMs and immediately replacedwith the two conditioned medium types. Lysates were collected inSDS-PAGE buffer at the indicated times.

After separation of protein samples on 10% acrylamide Tris-glycine gels and transfer to nitrocellulose, membranes werestained using Ponceau S to confirm equivalent protein loadingfor each sample. The membranes were then probed with anti-bodies specific for Dicer (Abcam), GAPDH (Abcam), phospho-tyrosine 701 (Tyr(P)-701) STAT1 (Abcam), total STAT1(Abcam), I�B� (Cell Signaling Technology), and �-tubulin(Developmental Studies Hybridoma Bank, University of Iowa)as indicated. HRP-conjugated secondary antibodies weredetected using an LAS-4000 imaging system (Fujifilm) afteradding Pierce ECL Western blotting substrate (ThermoScientific).

Statistical Analyses—Paired, two-tailed t tests or linearmixed model analyses for a randomized block design were used

to compare �Ct values between treatments and controls. Forlinear mixed model analyses, Dunnett’s post hoc tests were per-formed as indicated. p values were calculated using GraphPadPrism 5 (GraphPad Software, Inc.).

RESULTS

LPS and Interferon Treatments Modified the AccumulationRate of Several Activation-associated miRNAs in ex Vivo Mono-cyte Cultures—We reported previously that expression of sev-eral miRNAs is regulated in response to macrophage-activatingconditions. We also observed that these miRNAs wereexpressed at lower abundance in monocytes relative to MDMs(14), leading us to hypothesize that the expression of thesemiRNAs occurred during the process of differentiation in addi-tion to activation. We began our current study of these polar-ization-associated miRNAs by investigating the timing ofexpression level changes during the monocyte-to-macrophagedifferentiation process and whether the accumulation rateswere modified in response to activating conditions. Inuntreated monocytes, the abundance of all seven miRNAstested increased during the first 24 h of ex vivo culture (Fig. 1).The amplitude of miR-125a-5p increase in untreated mono-cytes was large (5.77 on a log2 scale, the equivalent of 55-foldchange), whereas the increase was small for miR-146a, miR-155, and miR-222 during this period.

The data showed that expression of each of the miRNAstested is uniquely modified by three biological agents involvedin induction of inflammatory activation patterns (Fig. 1). Asreported by us and others, LPS treatment augmented the accu-mulation of miR-146a and miR-155 in human monocytic cells(14, 15, 18). In contrast, LPS treatment repressed miR-27a tolevels lower than those observed in monocytes prior to ex vivoculture (Fig. 1). Our prior report showed that IFN� could antag-onize LPS-mediated induction of miR-125a-5p, miR-146a, andmiR-222 but not miR-155 in phorbol 12-myristate 13-acetate-differentiated THP-1 cells (14). In primary human monocytes,Toll-like receptor and IFN signaling pathways also modified theregulation of these miRNAs (Fig. 1). We included the type 1IFN, IFN�, in our analysis to contrast with results seen with thetype 2 IFN, IFN�. Treatment with either IFN significantlyantagonized the accumulation of not only miR-27a but alsomiR-125a-5p and miR-222. Furthermore, each IFN partiallyantagonized LPS-mediated induction of miR-146a; a 19-foldincrease (4.3 on log2 scale) in miR-146a abundance in responseto LPS was diminished to 11-fold (3.5 on log2 scale) in responseto the LPS/IFN combinational treatments.

IFN� and IFN� treatments were not always similar in theirability to modify miRNA expression as exemplified by theireffects on miR-27a and miR-29b. Although each IFN treatmentantagonized miR-27a, the repression in response to IFN� treat-ment was significantly greater than that in response to IFN�treatment. Expression of miR-29b was also significantly lowerin response to IFN� treatment than to IFN� treatment.

IL-4 Treatment Modified the Accumulation Rate of a Subsetof Activation-associated miRNAs in ex Vivo MonocyteCultures—We monitored miRNA expression levels in mono-cytes responding to IL-4 treatment. Accumulation of both miR-193b and miR-222 was augmented in monocytes treated with




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FIGURE 1. The accumulation of some miRNAs during 24 h of monocyte ex vivo culture was modified by LPS, IFN� and IFN�. RT-qPCR was used tocompare miRNA abundance in freshly isolated monocytes versus monocytes maintained in culture for 24 h with or without LPS (10 ng/ml) and/or interferons(each at 20 ng/ml). Graphs represent the mean � S.E. -fold change at 24 h post-treatment (n � 3) relative to monocytes collected immediately after cell sorting.Dotted horizontal lines across each graph mark the expression change in the untreated monocytes at the 24-h time point. Solid horizontal bars indicate statisticalsignificance (p � 0.05) using Tukey’s post-test of mixed model analysis. Error bars represent S.E.




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IL-4 (Fig. 2). It is important to note that miR-222 is one of themost abundant miRNAs in monocytes according to bothmicroarray data (15) and RT-PCR Ct values (data not shown),so the apparently modest increase in this miRNA may have alarger impact on monocyte biology than the small -fold changewould imply.

IL-4 treatment may have reduced the rate at which severalmiRNAs accumulated relative to untreated cells. However,among these miRNAs, only miR-125a-5p reached statisticalsignificance (Fig. 2).

Diverse Rates of Developmentally Induced miRNA Accumu-lation Were Observed during ex Vivo Culture of PurifiedMonocytes—The above results indicated that accumulation ofall seven miRNAs examined began early in the monocyte-to-macrophage differentiation process. The kinetics of accumula-tion was monitored in purified monocytes over the course of 5days of differentiation toward MDMs. Each miRNA accumu-lated in cultured monocytes with a unique rate and amplitude(Fig. 3). Whereas the increase in miR-27a, miR-29b, and miR-125a-5p abundance occurred mostly within the 1st day, theabundance of miR-146a and miR-193b did not reach maximalexpression levels until day 5. The accumulation of miR-155reached statistical significance by day 5 but was small in ampli-tude at each time point. The induction of miR-222 was tran-sient, reaching a 2-fold increase (1.02 on log2 scale) after 1 dayof ex vivo culture but declining thereafter (p � 0.0001 for theincrease on day 1 when data from all six donors described inFigs. 1 and 3 were compiled).

Accumulation of miR-155 and miR-193b Was Augmented inMonocytes Co-cultured with Non-monocytic PBMCs—Not onlydid LPS, interferons, and IL-4 influence the rate of miRNAchange during differentiation, but the presence of other cells inthe environment also exhibited a major impact on miRNAexpression. We compared miRNAs during MDM differentia-tion in two alternate environments. The first method, used inexperiments described up to this point, utilized culture ofmonocytes purified from PBMCs using CD14 MicroBeads onthe day of blood donation. The second method allowed differ-entiation of monocytes into MDMs in cultures of unsortedPBMCs for 4 days followed by an additional day of culture in theabsence of non-monocytic PBMCs. We used the secondmethod to generate MDMs in our previous study to documentlarge differences in expression of some miRNAs betweenmonocytes and MDMs (14). Unexpectedly, the abundance of

miRNAs in MDMs generated by these two methods was sub-stantially different. With the exception of miR-27a, the accu-mulation of the miRNAs under study was equal to or higher inMDMs co-incubated with non-monocytic PBMCs than inMDMs generated from purified monocytes cultured in theabsence of non-monocytic PBMCs (Fig. 3). The difference wasstatistically significant for both miR-155 and miR-193b andnearly significant for miR-29b (p � 0.13), miR-146a (p � 0.12),and miR-222 (p � 0.06).

Dicer Protein and mRNA Expression Levels Were Cons-tant during Monocyte-to-Macrophage Differentiation—Every miRNA that we evaluated in this study had either pro-gressive or transient increased expression levels as purifiedmonocytes differentiated to MDMs. To investigate themechanism, we first considered the possibility that there wasa global enhancement of miRNA processing during MDMdifferentiation. The rationale for this hypothesis was sup-ported by a report that Dicer and other RNAi-associatedproteins accumulate following phorbol 12-myristate 13-ac-etate-induced differentiation of human monocytic cells andprimary monocytes (20). However, we did not detect anyincrease in expression of Dicer protein (Fig. 4A) or mRNA(Fig. 4B) during MDM differentiation.

Pri-miRNA Expression Patterns Correlated with MaturemiRNA Accumulation during MDM Differentiation—Becausethe increase in miRNA abundance was not associated with cor-responding increases in Dicer levels, we hypothesized that thechanges in mature miRNA abundance occurred upstream ofmiRNA processing at the level of pri-miRNA abundance.

To test this hypothesis, we monitored the expression of thefour pri-miRNAs whose mature forms had the largest expres-sion changes during monocyte-to-macrophage differentiationof the seven miRNAs we evaluated. Unfortunately, we wereunable to follow the kinetics of pri-miR-193b expressionbecause its abundance remained too low at all of the time pointsto obtain reliable readings. Among the three pri-miRNAs (pri-miR-125a, pri-miR-146a, and pri-miR-193b) we could measure,each had unique kinetics of expression (Fig. 5). Pri-miR-125atransiently accumulated to �50-fold (5.62 on log2 scale) duringthe 1st day of ex vivo culture before being stably maintained at�20-fold through day 5 of culture. Pri-miR-146a increasedexpression �40-fold above baseline monocyte expression andmaintained this level throughout days 1–5 of culture. Althoughthe kinetics of pri-miR-125a-5p and pri-miR-146a (Fig. 5) were

FIGURE 2. Several miRNAs had altered accumulation rates in human monocytes responding to IL-4 stimulation. RT-qPCR was used to compare miRNAabundance in freshly isolated monocytes versus monocytes maintained in culture for 24 h with or without IL-4 (20 ng/ml). Graphs represent the mean � S.E.-fold change at 24 h post-treatment (n � 3) relative to monocytes collected immediately after cell sorting. Asterisks indicate statistical significance (p � 0.05)between treated and untreated monocytes at the 24-h time point using a paired, two-tailed t test. Error bars represent S.E.




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not identical to the kinetics of the mature forms of these twomiRNAs (Fig. 3), it is important to factor RNA stability into theinterpretation. Mature miRNAs have long half-lives, whereaspri-miRNAs are rapidly cleaved by the microprocessor com-plex (21). Because of the difference in stability, the transient

induction of pri-miR-125a (Fig. 5) was consistent with the pla-teau in mature miR-125a-5p abundance (Fig. 3). The immedi-ate plateau in pri-miR-146a abundance (Fig. 5) was consistentwith the progressive accumulation of a more stable maturemiR-146a (Fig. 3).

FIGURE 3. miRNAs accumulated with varying kinetics during MDM differentiation. RT-qPCR was used to measure miRNA abundance in purifiedmonocytes cultured ex vivo for the indicated number of days. miRNA abundance was also measured in MDMs isolated from PBMC cultures based onadherence. Graphs represent the mean � S.E. -fold change (n � 3) compared with monocyte samples collected immediately after cell sorting. Asterisksindicate statistical significance (p � 0.05) compared with monocyte samples collected immediately after sorting using Dunnett’s post-test of mixedmodel analysis. Horizontal bars indicate statistical significance (p � 0.05) comparing both day 5 samples using a paired, two-tailed t test. Error barsrepresent S.E.




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The profile of pri-miR-155 (Fig. 5) was similar to that ofmature miR-155 (Fig. 3). Specifically, we detected modestincreases in both forms of miR-155 in cultures of purifiedmonocytes during differentiation to MDMs and a markedlygreater increase in MDMs derived from total PBMC culturesthan from purified monocytes. In contrast, differences in MDMdifferentiation conditions minimally altered the expression ofthe primary and mature forms of miR-125a-5p and miR-146a(Figs. 3 and 5). Overall, these observations support a model inwhich the accumulation of developmentally up-regulatedmiRNAs is mechanistically due to changes in expression of thecorresponding pri-miRNA. In the case of miR-155, this up-reg-ulated pri-miRNA abundance seems to occur at least inresponse to signals from non-monocytes (pursued below).

Culturing Monocytes with Non-monocytic PBMCs Increasedthe Expression Levels of miR-155 and miR-193b—Differencesbetween the two MDM preparation methods included expo-sure of purified monocytic cells to CD14 MicroBeads in the firstmethod and co-incubation of monocytes with non-monocyticPBMCs for 4 days in the second method. We hypothesized thatco-culture of monocytes with non-monocytic PBMCs duringMDM differentiation, rather than CD14 MicroBead-mediatedsorting, explained the differences in miR-155 and miR-193baccumulation. To test this, monocytes were positively selectedfrom PBMCs using CD14 MicroBeads and either culturedalone or co-cultured with negatively selected, non-monocyticPBMCs from the CD14 MicroBead-mediated sort. A mono-cyte-to-non-monocyte ratio of 1:5 was chosen for the co-culture condition because monocytes isolated by CD14MicroBeads typically constitute 15–20% of the total PBMCpopulation. After 5 days, the accumulation of both miR-155 andmiR-193b was enhanced in MDMs from the co-culture condi-tions compared with no co-culture, and the level was similar tothe accumulation of these miRNAs in MDMs isolated fromunfractionated PBMC cultures (Fig. 6A).

Expression Profiling of miRNAs Identified Changes That WereEither Dependent on or Independent of Differences in CulturingConditions during Monocyte-to-macrophage Differentiation—The presence of non-monocytic cells had a large impact onmiR-155 and miR-193b expression in MDMs. We profiledmiRNA expression using TaqMan TLDA assays to identifyadditional miRNAs with expression levels that were affected

by differences in culturing conditions during monocyte-to-macrophage differentiation.

We first noted that five of seven miRNAs we evaluated in thisreport (miR-29b, miR-125a-5p, miR-146a, miR-155, and miR-193b) were among the 29 miRNAs up-regulated more than4-fold in the TLDA analysis (Fig. 6B). We then compared ourresults with a previous study that profiled miRNA expression inprimary human CD14 MicroBead-purified monocytes differ-entiated to MDMs in the presence of GM-CSF (19). The mosthighly up-regulated miRNA in both MDM types in both thecurrent study and the MDMs in the previous study (19) wasmiR-511. Additional miRNAs (miR-34a, miR-99b, miR-132,miR-146b-5p, miR-210, miR-212, and miR-449a) were also up-regulated in both the current (Fig. 6B) and the previous studies(19). In the case of miR-132 and miR-212, the regulation wasenhanced by more than 4-fold when non-monocytic PBMCswere present during monocyte-to-macrophage differentiation,leading us to categorize this miRNA as being preferentially up-regulated in the MDMs cultured in the presence of non-mono-cytic PBMCs (Fig. 6B). Because miR-132 and miR-212 areexpressed as a bicistronic pri-miRNA, the similar expressionpattern of these miRNAs suggests that they were regulated at apreprocessing step.

Both Tserel et al. (19) and Wang et al. (22) described miR-150 as an miRNA with repressed expression during monocyte-to-macrophage differentiation. We found that, although thismiRNA was repressed during differentiation of purified mono-cyte cultures, the presence of non-monocytic PBMCs pre-vented the loss of expression for this miRNA in MDMs (Fig.6B). Fig. 6B, right column, describes 24 additional miRNAs thatare differentially regulated in MDMs cultured in the presenceand absence of non-monocytic PBMCs. Considering dataresulting from our work and these publications, there seems tobe a core set of miRNAs that are regulated as a result of mono-cyte-to-macrophage differentiation. The miRNAs that are reg-ulated uniquely in the different MDM culturing conditions mayhighlight a set of miRNAs that are responsive to exogenousstimuli.

Culturing Monocytes with Non-monocytic PBMCs Altered theExpression of Monocyte-to-macrophage Differentiation andMacrophage Polarization Markers—The differences in miRNAexpression between MDMs derived from the two culturing

FIGURE 4. Dicer expression remained constant during monocyte-to-macrophage differentiation. A, immunoblots of Dicer and GAPDH wereperformed using lysates from CD14 MicroBead-purified monocytes and from adherent cells of PBMC cultures after the indicated number of days inculture. B, RT-qPCR was used to measure Dicer transcript abundance in samples collected from monocytes cultured for the indicated number of days andin samples from MDMs isolated after culturing PBMCs for 5 days. Graphs represent the mean � S.E. -fold change (n � 4) compared with monocytesamples collected immediately after cell sorting. No statistically significant (p � 0.05) results were identified using Dunnett’s post-test of mixed modelanalysis. Error bars represent S.E.




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conditions could represent either an alteration in the mono-cyte-to-macrophage differentiation program or an activatedphenotype for the MDMs differentiated in the presence of non-monocytic cells. To distinguish between these possibilities, wemonitored changes in the expression of 48 transcripts as puri-fied monocytes differentiated to MDMs and in MDMs differ-entiated in the presence of non-monocytic PBMCs to charac-terize these MDMs. The panel of transcripts was selected frompublished data sets of a profiling study that characterizedmonocyte-to-macrophage differentiation and macrophage

responses to activating conditions (DataSet Records GDS2429and GDS2430) (23).

The most abundantly changed mRNAs are indicated in theheat map (Fig. 7A). These 25 transcripts had average changes ofmore that 4-fold in one or more of the five sample types relativeto freshly isolated monocytes. Genes reported to be altered dur-ing monocyte-to-macrophage differentiation (23) are indicatedwith color-coded gene names signifying whether the transcriptwas up-regulated or down-regulated in the previous study (Fig.7A). Unsupervised hierarchal clustering analysis showed thatthe gene expression profiles in our panel underwent manychanges by the day 1 time point, continued to change throughday 3, and then stabilized as CD14 MicroBead-purified mono-cytes differentiated to macrophages (Fig. 7A). This is consistentwith microarray results from Martinez et al. (23) showing thatgene expression patterns of purified monocytes change dra-matically during the first 3 days of culture and become relativelystable thereafter.

The gene expression pattern in MDMs from cultures of puri-fied monocytes on day 5 differed drastically from time-matchedsamples from MDMs differentiated in the presence of non-monocytic PBMCs (Fig. 7A). Notably, the expression patternsof MDMs differentiated within cultures of unsorted PBMCswere very similar to the expression pattern of MDMs differen-tiated from purified monocytes co-cultured with non-mono-cytic PBMCs (Fig. 7A). This latter observation indicates that theCD14 MicroBead-mediated monocyte purification proceduredoes not explain the difference in gene expression patternsbetween MDMs derived from purified monocyte cultures andMDMs derived from unsorted PBMC cultures. Because someof the most dramatic differences in transcript expressionbetween MDM types were markers of polarization rather thandifferentiation, we conclude that the MDMs differentiated inthe presence of non-monocytic cells ended up at a differentstate of activation than those differentiated as cultures of puri-fied monocytes. An interaction occurs during monocyte co-in-cubation with non-monocytic PBMCs, drastically altering theabundance of both miRNAs and mRNAs in MDMs.

NF-�B and STAT1 Pathways Were Activated withinMDMs Responding to Secreted Factors in PBMC-conditionedMedium—Several of the mRNAs with altered expression inMDMs due to the presence of non-monocytic cells during dif-ferentiation suggested that pathways associated with inflam-mation such as NF-�B and STAT1 were activated. For example,the IFN-responsive genes ITGB7 and ISG20 were more abun-dant in MDMs cultured in the presence of non-monocyticPBMCs consistent with STAT1 pathway activation. We testedwhether STAT1 and NF-�B pathways were activated in MDMsco-cultured with non-monocytic PBMCs using two experimen-tal approaches.

First, immunoblot analyses were performed to assess activa-tion of STAT1 and NF-�B pathways during the monocyte-to-macrophage differentiation process in the presence or absenceof non-monocytic PBMCs (Fig. 7B). Total STAT1 levelsremained constant and unphosphorylated over time in thepurified monocyte cultures, showing that STAT1 pathwayswere not activated under these conditions. In contrast, weobserved an increased abundance of total STAT1 and a phos-

FIGURE 5. Primary miRNA expression correlated with mature miRNAaccumulation during MDM differentiation. RT-qPCR was used to measuretranscript abundance of the indicated primary miRNAs in samples collectedfrom monocytes cultured for the indicated number of days and in samplesfrom MDMs isolated after culturing PBMCs for 5 days. Graphs represent themean � S.E. -fold change (n � 3– 4) compared with monocyte samples col-lected immediately after cell sorting. Asterisks indicate statistical significance(p � 0.05) compared with monocyte samples collected immediately aftersorting using Dunnett’s post-test of mixed model analysis. Error bars repre-sent S.E.




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phorylated form of STAT1 in MDMs differentiated in the pres-ence of non-monocytic PBMCs, demonstrating that this path-way was activated by this culturing method (Fig. 7B). Moreover,I�B� was present throughout the monocyte-to-macrophagedifferentiation process in cultures of purified monocytes butabsent in MDMs differentiated in the presence of non-mono-cytic cells (Fig. 7B). We hesitate to conclude that the absence of

I�B� indicated that NF-�B was activated under the latter con-dition because the loading control, �-tubulin, was difficult todetect in this sample (Fig. 7B). However, Ponceau S staining(Fig. 7C) suggested that equal amounts of protein were loadedin the samples evaluated in Fig. 7B.

Next, we specifically assessed whether secreted factorspresent in the PBMC cultures were capable of activating

FIGURE 6. The expression levels of many miRNAs differed between MDMs cultured in either the presence or absence of non-monocytic PBMCS. A,RT-qPCR was used to measure miR-155 and miR-193b abundance in MDMs generated using three different methods: culture of purified monocytes, co-cultureof purified monocytes and non-monocytic PBMCs, and unsorted PBMC cultures. In all three methods, MDM purification was based on selecting adherent cells.Graphs represent the mean expression (n � 4) of the indicated miRNA relative to the MDMs differentiated as purified monocyte cultures. Horizontal barsindicate statistical significance (p � 0.05) using a paired, two-tailed t test. B, miRNA expression profiles were determined using TaqMan Low Density Arrayassays in samples from CD14 MicroBead-purified monocytes collected immediately after sorting and from MDMs differentiated in the presence of non-monocytic PBMCs. The heat maps indicate the average miRNA expression level change in the indicated MDM type relative to the expression level in purifiedmonocytes at the time of sorting (n � 3). Expression changes were determined to be increased (), decreased (), or unchanged (0) in each of the two typesof MDMs, resulting in eight expression pattern categories. Asterisks indicate statistical significance (p � 0.05) in MDMs compared with monocyte samplescollected immediately after sorting using a paired, two-tailed t test. Error bars represent S.E.




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MDM STAT1 and NF-�B pathways. Medium in wells ofMDMs derived from culturing CD14 MicroBead-purifiedmonocytes for 5 days was replaced with either PBMC-conditioned medium or purified monocyte-conditionedmedium as a control. Immunoblots showed that STAT1 was

phosphorylated and I�B� was degraded during the first 15min of culturing the MDMs with PBMC-conditionedmedium but not with control medium (Fig. 7D). This sug-gests that both STAT1 and NF-�B pathways were activatedby factors in the PBMC-conditioned medium. The return of

FIGURE 7. Analysis of monocytic cell differentiation and polarization marker abundance revealed a unique signature in MDMs cultured in thepresence of non-monocytic cells. A, a heat map summarizes results from integrated fluidic circuit-based RT-qPCR that measured the abundance of putativemonocyte-to-macrophage differentiation and polarization markers. RNA was collected from CD14 MicroBead-purified monocytes or from MDMs of PBMCcultures after the indicated number of days in culture. Average changes in marker abundance relative to monocyte samples collected immediately after cellsorting are shown (n � 3– 4). Genes and treatments are arranged according to results from unsupervised hierarchal clustering. To facilitate comparison with thestudy by Martinez et al. (23), putative differentiation marker gene names from this report were color-coded (red for up-regulation, blue for down-regulation, andblack for unaltered expression) based on the microarray-based gene expression measurements from purified monocytes after ex vivo culture for 3 days. B,immunoblots were performed to detect the indicated proteins using lysates from CD14 MicroBead-purified monocytes and from adherent cells (MDMs) ofPBMC cultures after the indicated number of days in culture. C, prior to blocking, the nitrocellulose membranes used for immunoblots in B were stained withPonceau S. A representative membrane is shown (L, ladder). D, immunoblots were performed to detect the indicated proteins. Lysates were collected fromMDMs derived from CD14 MicroBead-purified monocytes following the replacement of cell culture medium with conditioned medium either from CD14MicroBead-purified monocytes as a control or from unsorted PBMCs.




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I�B� expression by 1 h further demonstrated that NF-�Bwas activated in response to the PBMC-conditioned mediumbecause I�B� is an NF-�B-induced gene.

Low Basal Expression Levels of miR-155 and MacrophagePolarization Markers in MDMs Derived from Purified Mono-cyte Cultures Poise These Genes for “Large” Induction inResponse to LPS Treatment—A cluster of genes in Fig. 7A(CXCL10, CCL5, TGM2, and CXCL9) had expression profilesthat were similar to miR-155 and miR-193b (Figs. 3 and 6).Specifically, the expression of these genes changed minimallyduring differentiation of purified monocytes, but they wereexpressed at high levels in MDMs differentiated in the presenceof non-monocytic PBMCs. We hypothesized that the differ-ences in basal abundance of these transcripts in the differentMDM populations would inversely correlate with the ampli-tude of expression level changes following treatment with amacrophage-activating factor such as LPS similar to miR-155and miR-193b.

We tested our hypothesis by monitoring transcript inductionin LPS-treated MDMs differentiated in the presence or absenceof non-monocytic PBMCs. In MDMs differentiated withoutnon-monocytic PBMCs, all four transcripts (Fig. 8A) and miR-155 (Fig. 8B) were highly up-regulated following LPS treatmentbut only modestly altered in LPS-treated MDMs co-cultured inthe presence of non-monocytic PBMCs. In contrast to miR-155, another LPS-responsive miRNA, miR-146a, only modestlyaccumulated in MDMs with no difference in amplitude ofresponse when comparing MDMs differentiated in the pres-ence or absence of PBMCs (Fig. 8B). The similar amplitude ofmiR-146a induction in response to LPS treatment was consis-tent with our hypothesis because miR-146a accumulation wassimilar in the MDMs from different culturing methods (Fig. 3).As expected, miR-125a-5p and miR-193b showed neither astrong response to LPS treatment nor consistent differences inexpression between MDMs differentiated in the presence orabsence of non-monocytic PBMCs (Fig. 8B).

Prior to LPS treatment, miR-155 basal levels were lower inMDMs derived from cultures of purified monocytes comparedwith MDMs that were differentiated in the presence of non-monocytic PBMCs (Fig. 9). This confirmed results described inFig. 6 with an independent set of MDMs. After LPS treatment,miR-155 levels were indistinguishable between MDMs regard-less of the culture method used during differentiation (Fig. 9A).Similar trends in �Ct value profiles were observed for CCL5,CXCL9, CXCL10, and TGM2 before and after LPS treatment(Fig. 9B). By interpreting gene expression using �Ct values, weconclude that basal abundance, rather than final abundance,accounts for the difference in amplitude of transcript andmiRNA expression level changes in response to LPS betweenMDMs differentiated in the presence or absence of non-mono-cytic PBMCs. Thus, the surprisingly small changes in miRNAabundance we recently reported for MDMs differentiated inresponse to various activation conditions (14) were a result ofsome activation-associated miRNAs such as miR-155 accumu-lating robustly in response to activation of monocytic cells co-cultured with non-monocytic PBMCs, whereas other activa-tion-associated miRNAs such as miR-146a likely accumulate as

part of a general monocyte-to-macrophage differentiationprogram.

DISCUSSION

Mounting evidence describes many roles for miRNAs in reg-ulating important macrophage functions (9, 10, 24, 25). Moststudies evaluating the role of miRNAs in macrophages havefocused on miRNAs that have regulated expression either dur-ing monocyte-to-macrophage differentiation or in response totreatment with activating stimuli. Our previous report describ-ing miRNA expression regulation in human macrophagesyielded the unexpected observation that the measured -foldchanges in expression of miRNAs in response to strong polar-izing conditions were small in amplitude despite the fact thatthese were biologically relevant changes (14). An importantobservation from the prior study was that polarization-associ-ated miRNAs had low expression in monocytes relative toMDMs. This raised the possibility that the basal levels of theseactivation-associated miRNAs were actually preinduced duringthe process of differentiation from monocyte to macrophageand that we were observing the smaller -fold change in responseto additional stimuli that cause polarization. In the currentstudy, we characterized the accumulation of several miRNAsduring differentiation of human monocytes ex vivo. ThesemiRNAs were selected because their abundance is also regu-lated during macrophage polarization (14). We evaluatedmiRNA expression regulation in monocytic cells responding tovariations in the external environment. First, we determinedthe kinetics of miRNA expression during monocyte-to-macro-phage differentiation in the presence and absence of welldefined activating stimuli. Second, we evaluated miRNA ex-pression in monocytes that were allowed to differentiate toMDMs ex vivo in the presence or absence of non-monocyticPBMCs. We found that the abundance of these miRNAs waslow in monocytes prior to differentiation and that their accu-mulation was dynamic in response to variations in environ-mental stimuli. These modifications of miRNA abundancewere regulated at the level of pri-miRNA expression.

The first set of experiments showed that the rate of miRNAaccumulation during differentiation was modified in responseto cytokines (IFN�, IFN�, and IL-4) and a Toll-like receptoragonist (LPS). We had previously reported that IFN� treatmentcould antagonize the LPS-mediated induction of some miRNAsin phorbol 12-myristate 13-acetate-differentiated THP-1 cells(14). We reassessed the cross-talk in primary human mono-cytes between signaling pathways downstream of LPS and IFN�and expanded the analysis to include the type 1 interferon,IFN�. Two well studied miRNAs, miR-146a and miR-155, wereinduced by LPS in monocytes, but only miR-146a was antago-nized by the addition of either type of interferon. Substantialevidence suggests that miR-146a acts as a negative regulator ofinflammation by targeting TRAF6 and IRAK1 (18, 26, 27).Although sometimes described as anti-inflammatory, miR-155has more commonly been described to act as a positive regula-tor of inflammation by targeting SOCS1 and SHIP1 (for areview, see Ref. 9). In monocytes exposed to inflammatory con-ditions, the simultaneous antagonism of miR-146a and aug-mentation of miR-155 accumulation by interferon treatments




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may function to extend the duration of the inflammatory geneexpression program by these cells.

Treatment of monocytes with IFN� and IFN� regulatedmiRNA expression in a similar fashion with the exceptions ofmiR-27a and miR-29b, which were both expressed at lower lev-els in the IFN�-treated samples. Interestingly, each of thesemiRNAs has been implicated in interferon responses. miR-27aenhances IFN� signaling (28) and is targeted for degradation bya number of viruses (29 –31). In contrast, miR-29b antagonizes

IFN� production in multiple hematopoietic cell types, although aconsensus has not been reached on whether miR-29b acts bydirectly targeting IFN� transcripts or by repressing the expressionof transcription factors involved in IFN� transcription (32, 33).

Our final set of experiments focused on understandingmiRNA and mRNA expression differences in MDMs that werecultured in the presence or absence of non-monocytic PBMCs.We considered the possibility that the MDMs derived from thealternate methods were at different stages of differentiation

FIGURE 8. The amplitude of LPS-induced changes in transcripts or miRNAs in MDMs was inversely related to the abundance of these RNAs prior to LPStreatment. RT-qPCR was used to measure LPS-induced changes in expression of the indicated mRNA (A) or miRNA (B). MDMs were generated using threedifferent methods and treated with LPS (10 ng/ml) for 24 h. Graphs represent the mean � S.E. changes in RNA abundance (n � 4) for the indicated mRNAs andmiRNAs after LPS treatment on day 5 of MDM differentiation. Horizontal bars indicate statistical significance (p � 0.05) using a paired, two-tailed t test. Error barsrepresent S.E.




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after 5 days of ex vivo culture. In agreement with Martinez et al.(23), we observed stabilization in the gene expression profilesfrom cultures of purified monocytes starting at day 3. We inter-pret this observation to indicate that the differentiationprocess may be mostly complete by this time point. The geneexpression profiles obtained from MDMs cultured in thepresence of non-monocytic PBMCs were substantially dif-ferent from those seen in purified monocyte cultures at anytime point. Collectively, this set of observations arguesagainst the non-monocytic PBMCs blocking monocyte-to-macrophage differentiation. Instead, it is possible thatMDMs may each be differentiated toward uniquely polar-ized states. In support of this interpretation, we documentedthat the presence of non-monocytic PBMCs during mono-cyte-to-macrophage differentiation activates STAT1 andNF-�B signaling pathways and that the factors involved inactivation were present in PBMC-conditioned medium.

Our initial experiments showed that treatment with IFNs,presumably through STAT1 activation, did not alter miR-155accumulation, enhanced miR-193b accumulation, repressedmiR-125a-5p accumulation, and partially antagonized LPS-me-diated induction of miR-146a. Thus, the discovery that bothSTAT1 and NF-�B pathways were active in MDMs differenti-ated in the presence of non-monocytic cells correlates well withthe preferential up-regulation of miR-155 and miR-193b, butnot miR-146a and miR-125a-5p, in these MDMs relative toMDMs differentiated in the absence of PBMCs. TLDA-basedRT-PCR detected five additional miRNAs that were preferen-tially up-regulated in MDMs differentiated in the presence ofnon-monocytic PBMCs and independently confirmed this reg-ulation pattern for miR-155 and miR-193b. Notably, inflamma-tory stimuli have been reported to induce miR-146b and miR-147 in murine macrophages as well as the miR-132/miR-212cluster in human macrophages, consistent with our conclusion

FIGURE 9. In MDMs differentiated in the presence or absence of non-monocytic PBMCs, differences in miR-155 and polarization marker expressionexist before, but not after, LPS treatment. RT-PCR was used to monitor miRNA (A) and mRNA (B) expression levels in MDMs. Samples were collected on day5, the day of LPS treatment (“Before LPS”), and on day 6 after treatment with LPS (100 ng/ml) for 24 h (“After LPS”). Error bars represent S.E.




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that MDMs differentiated with non-monocytic PBMCs appearto be activated (34 –36).

Our study highlights many contrasting characteristicsbetween MDMs differentiated in the presence or absence ofnon-monocytic PBMCs. However, it remains unknown whichMDM culturing method best models human MDMs in vivo.Obtaining MDMs from cultures of pure monocytes isolatedusing CD14 MicroBeads is advantageous from a reductionistpoint of view because the culturing conditions are moredefined. Conversely, differentiation of MDMs in the presenceof non-monocytic PBMCs may provide environmental cuesthrough secreted factors, cell-cell interactions, cell density, orsome other intercellular interaction that could more closelymimic in vivo environments.

In this work, we evaluated miRNAs that are regulated bothin response to activating conditions and as part of monocyte-to-macrophage differentiation. Because miRNAs have longhalf-lives, increasing the abundance of activation-associatedmiRNAs may be a more tractable and rapid regulatory mecha-nism than reducing their levels as cells respond to new envi-ronmental cues. We propose that by expressing low basallevels of key miRNAs circulating monocytes are poised toinduce miRNAs in response to diverse stimuli as they differ-entiate to macrophages in peripheral tissues.

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Renee L. Eigsti, Bayan Sudan, Mary E. Wilson and Joel W. GraffMonocyte-to-macrophage Differentiation

Regulation of Activation-associated MicroRNA Accumulation Rates during

doi: 10.1074/jbc.M114.599316 originally published online August 22, 20142014, 289:28433-28447.J. Biol. Chem.

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