of May 6, 2018.This information is current as

Inflammatory ResponsesTransduction To Potentiate InducibleExpression and MyD88-Mediated Signal Cytomegalovirus Enhances Macrophage TLR

and Lesley E. SmythiesBallestas, Anne Fenton, Satya Dandekar, William J. BrittEvida A. Dennis, Diane Bimczok, Lea Novak, Mary Phillip D. Smith, Masako Shimamura, Lois C. Musgrove,

ol.1302608http://www.jimmunol.org/content/early/2014/10/29/jimmun

published online 29 October 2014J Immunol 

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The Journal of Immunology

Cytomegalovirus Enhances Macrophage TLR Expression andMyD88-Mediated Signal Transduction To PotentiateInducible Inflammatory Responses

Phillip D. Smith,*,† Masako Shimamura,‡ Lois C. Musgrove,* Evida A. Dennis,*,‡

Diane Bimczok,* Lea Novak,x Mary Ballestas,‡ Anne Fenton,{ Satya Dandekar,{

William J. Britt,‡ and Lesley E. Smythies*,x

Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident

macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the

mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macro-

phages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA rep-

lication, significantly enhanced TNF-a, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS)

stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also

potentiated TLR5 ligand–stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated

inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and

TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein

MyD88 and the inducible phosphorylation of IkBa and NF-kB. These findings provide additional information toward elucidating

the mechanism by which HCMV potentiates bacteria-induced NF-kB–mediated macrophage inflammatory responses, thereby

enhancing organ inflammation in HCMV-infected tissues. The Journal of Immunology, 2014, 193: 000–000.

Human CMV (HCMV) is an important cause of diseasein immunocompromised hosts and the most commonlyacquired intrauterine infection in humans (1–3). The

clinical syndromes associated with these disease manifestationscan be correlated with the level of virus replication and respondto treatment with antiviral agents (3). In contrast, some chronicdiseases associated with HCMV infection appear not to have highlevels of virus replication as a correlate of disease (1, 3). Perhapsthe most studied associations with chronic disease are the accel-

erated rate of coronary artery and carotid artery disease in patientswith serologic evidence of HCMV infection (4–10). Similarly, theassociation between HCMV infection and exacerbations of in-flammatory bowel disease is well described (11–17). The mech-anisms responsible for the chronic inflammation and exacerbationof ongoing inflammatory disease in subjects with HCMV infectionare not well described but have not been associated with the lossof immune control of virus replication. Thus, understanding therole of HCMV persistent infection and inflammation could pro-vide insight into mechanisms of disease in chronic conditions suchas inflammatory bowel disease.HCMV infects mononuclear phagocytes at the hematopoietic

stem cell stage of differentiation (18), permitting the release ofHCMV-infected monocytes into the circulation (19, 20). In mye-loid progenitor cells and during the early stages of differentiation,the viral genome can persist without replication, allowing circu-lating CD14+ monocytes to serve as a reservoir for latent HCMV(21). Circulating HCMV-infected monocytes migrate into organtissues, where they differentiate into resident macrophages anddendritic cells (22, 23). Infection with HCMVenhances monocytetransendothelial migration and motility (24, 25), promoting thedissemination of infected monocytes into the tissues. Nonpro-ductive infection is maintained by repression of the immediateearly (IE) genes that drive lytic transcription (26, 27). Repressionof these genes in undifferentiated myeloid cells appears to beachieved through histone suppression of the major IE promoter;however, when the cells differentiate, changes in the histone-modified chromatin structure associated with the IE genes initi-ate gene expression and the lytic transcription program, resultingin release of viral progeny. In compromised immunological orphysiological conditions that impair immune surveillance, thedifferentiation of newly recruited HCMV-infected monocytes intotissue macrophages can activate viral gene expression, leading to

*Division of Gastroenterology, Department of Medicine, University of Alabama atBirmingham, Birmingham, AL 35294; †Veterans Affairs Medical Center, Birmingham,AL 35233; ‡Division of Infectious Diseases, Department of Pediatrics, University ofAlabama at Birmingham, Birmingham, AL 35294; xDepartment of Pathology, Univer-sity of Alabama at Birmingham, Birmingham, AL 35294; and {Department of MedicalMicrobiology and Immunology, University of California, Davis, Davis, CA 95616

Received for publication September 26, 2013. Accepted for publication September29, 2014.

This work was supported by National Institutes of Health Grants DK-64400 (to P.D.S.),AI-59428 and AI-101138 (to M.S.), AI-83539 (to L.E.S.), DK-43183 and AI-432274 (to S.D.), NS-65945 and AI-50189 (to W.J.B.), DK-97144 (to D.B.); andRR-20136 (to P.D.S. and L.E.S.); the Crohn’s and Colitis Foundation of America (toP.D.S.); the Kaul Pediatric Research Initiative of Children’s Hospital of Alabama (toM.S.); the University of Alabama at Birmingham Autoimmunity, Immunology, andTransplantation Steering Committee Pilot Program; the University of Alabama atBirmingham Center for Clinical and Translational Science Pilot Program; and theResearch Service of the Veterans Administration (to L.C.M. and P.D.S.).

Address correspondence and reprint requests to Dr. Phillip D. Smith and Dr. LesleyE. Smythies, University of Alabama at Birmingham, 610 SHEL, 1720 2nd AvenueSouth, Birmingham, AL 35294-2182. E-mail addresses: pdsmith@uab.edu (P.D.S.)and lesmy@uab.edu (L.E.S.)

The online version of this article contains supplemental material.

Abbreviations used in this article: HCMV, human CMV; IE, immediate early; IE1,human CMV immediate early Ag 1; MOI, multiplicity of infection; UC, ultracentri-fuged.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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the local production and release of viral progeny (21, 28–30).Thus, HCMV promotes monocyte dissemination into the tissues,where differentiation-dependent activation of macrophages leadsto virus expression and the release of HCMV throughout the body.HCMV has been identified in all major leukocyte populations in

peripheral blood (31), but the infection of blood monocytes isparticularly relevant to organ inflammatory disease, because bloodmonocytes are the source of macrophages in many tissues (32,33). After recruited monocytes take up residence in tissues such asthe intestinal mucosa and differentiate into macrophages (34), theyare positioned to participate in or orchestrate HCMV-associatedorgan inflammation, especially in response to tissue-invading bac-teria or bacterial products. In this connection, we (35–38) and others(39, 40) have reported that HCMV infection promotes proin-flammatory cytokine and chemokine production by monocytes andmacrophages. Further elucidation of the immunobiology of HCMV-induced inflammatory responses in macrophages could provide newinsight into the mechanism of HCMV-associated organ inflamma-tory disease, a clinical problem of increasing frequency due to theexpanding use of immunosuppressive therapies (41–46).In this study, we show that HCMV infection of monocytes during

their differentiation into macrophages significantly enhanced TLR-induced macrophage inflammatory responses. HCMV infection wasassociated with the maintenance of surface CD14 and TLR4 andTLR5, which declined over time in mock-infected macrophages.HCMV also enhanced macrophage expression of MyD88 and theinducible phosphorylation of both IkBa and NF-kB, enhancinginducible inflammatory cytokine production. These results offera mechanism, at least in part, by which HCMV infection potentiatesmacrophage responses to bacterial components.

Materials and MethodsVirus

HCMV strain TR (a gift of J. Nelson, Oregon Health and Sciences Uni-versity, toW.J. Britt) was originally isolated from the eye of an AIDS patientwith HCMV retinitis (47). The virus was propagated (fewer than threepassages) in human foreskin fibroblasts, harvested at 100% cytopathiceffect, and isolated by centrifugation at 16,000 3 g for 2 h at 4˚C (48).Viral pellets were resuspended in RPMI 1640 plus antibiotics and 10%human AB serum and stored at 280˚C in single-use aliquots. Only viruspassaged fewer than six times was used in the experiments described in thepresent study. Virus titers were determined using our previously describedassay based on the detection of HCMV IE Ag 1 (IE1) (49). Control HCMVincluded 1) UV-inactivated HCMV and 2) HCMV-free fibroblast culturesupernatant. For inactivated HCMV, virus was exposed to UV radiation at150 mJ in a cross-linking chamber (Bio-Rad, Hercules, CA), as describedpreviously (50). For HCMV-free fibroblast culture supernatant, culturesupernatant from HCMV-infected fibroblasts was ultracentrifuged (UC) at30,000 3 g for 1 h, and the absence of infectious virus in the culturesupernatant was confirmed by incubating the supernatant on foreskinfibroblasts. UV-inactivated HCMV and HCMV-free UC fibroblast culturesupernatants were stored at 280˚C.

Monocytes and HCMV infection of monocyte-derivedmacrophages

Mononuclear cells were isolated from blood donated by healthy HCMV-seronegative donors by Ficoll-Hypaque sedimentation, enumerated byautomated cell counter (Beckman Coulter, Fullerton, CA), and plated inserum-free RPMI 1640 in 24-well plates or for immunofluorescenceanalysis onto glass converslips inserted into 24-well plate wells at a con-centration of 23 106 monocytes/well (see “Detection of HCMV infection”below) (51). After 1 h incubation, the nonadherent lymphocytes were re-moved by washing, and the media were replaced with RPMI 1640 con-taining 10% human AB serum, 1% penicillin/streptomycin, and 50 mg/mlgentamicin (complete media) plus HCMV at a multiplicity of infection(MOI) of either 0.5 or 1.0, depending on the experiment. The adherentcells displayed morphological features of macrophages (large size, ec-centric and concave nuclei, phagocytic vacuoles, and cord-like pseudopodsextending from the surface), expressed mRNA transcript and protein for the

monocyte/macrophage markers CD13, HLA-DR, and CD14, and did notdifferentiate into T cells, B cells, NK cells, or dendritic cells (SupplementalFig. 1). After 2 h at 37˚C, the media were removed and replaced withHCMV-free complete media. Parallel cultures of macrophages were mock-infected with either 1) complete media, 2) control fibroblast culture su-pernatant in which the HCMV had been removed by ultracentrifugation, or3) control UV-inactivated HCMV (MOI of 1). The cells then were washedand cultured for up to 6 d in complete media in 24-well plates. At 2, 4, and6 d postinfection, cells were harvested by scraping, enumerated by auto-mated cell counter, and replated in 96-well plates (0.2 ml at 1 3 106 cells/well) in the presence or absence of 1 mg/ml smooth LPS (Salmonellaabortus equi; Alexis Biochemicals/Amgen, Thousand Oaks, CA) and har-vested after either 4 h for cytokine mRNA analysis or 24 h for cytokineprotein determination (see below).

Detection of HCMV infection

Parallel cultures of monocytes (2 3 106) bound to coverslips in 24-wellplates were exposed to HCMV or media, as described above. On day 4,HCMV- and mock-infected macrophages were fixed in 4% paraformal-dehyde and permeabilized using 0.1% Triton X-100. HCMV-infectedmacrophages were detected by immunofluorescence using the Ab p63-27, specific for IE1 Ag (UL123), the major immediate early gene prod-uct, and an FITC-conjugated goat anti-mouse IgG Ab, as we have previ-ously described (50). Cell nuclei were stained with DAPI, and cells wereenumerated by fluorescence microscopy.

HCMV replication also was evaluated using quantitative DNA PCR.Monocytes and primary human foreskin fibroblasts were plated at 2 3 105

cells/well in 96-well plates, inoculated with HCMV (MOI of 1.0), andwashed, after which the cells and supernatants were harvested on days 2,4, and 6. Total DNA was isolated from each sample using the QiagenQiaAMP DNA kit according to the manufacturer’s protocol. QuantitativeDNA PCR was performed by amplification of a fragment of the HCMVUL55 open reading frame and quantified by comparison with a standardcurve generated by amplification of a plasmid encoding a fragment of theHCMV UL55 open reading frame in serial dilution, as we have described(50). Samples were run in triplicate using the described two-step ampli-fication protocol on the Applied Biosystems StepOnePlus real-time PCRcycler. Copy numbers were calculated per cells per well.

Flow cytometric analysis

Monocyte-derived macrophages (2 3 106) were stained with allophyco-cyanin-, PE-, or FITC-conjugated Abs to CD14 (BD Biosciences, San Jose,CA), TLR2, TLR4, and TLR5 (eBioscience, San Diego, CA), and pNF-kBp65 (BD Phosflow, BD Biosciences), respectively, or irrelevant Abs of thesame isotype and fluorochrome, and analyzed by flow cytometry, as pre-viously described (52). Data were evaluated with CellQuest software.

Real-time PCR

HCMV- and mock-infected monocyte-derived macrophages (13 106 cells/ml) incubated for 4 h with LPS or media were harvested, RNAwas isolated(RNeasy kit; Qiagen, Valencia, CA), and cDNA was generated from totalRNA (transcriptor first-strand cDNA synthesis kit; Roche, Indianapolis,IN). Genes were amplified in 25-ml mixtures containing TaqMan UniversalPCR Master Mix and FAM/MGB-labeled primer-probe sets for TNF-a, IL-6, MyD88, and control gene GAPDH (Life Technologies, Carlsbad, CA),as previously described (52). Real-time PCR was run for 40 cycles (15 s at95˚C, 60 s at 60˚C) on a Chromo4 PCR system (Bio-Rad, Hercules, CA)and analyzed with Opticon Monitor software, version 3.1. All PCR reac-tions were performed twice, once with each reference gene, and data arepresented as the geometric mean of both reactions. Relative expressionrates of target genes in stimulated versus unstimulated cells were calcu-lated using the method of Pfaffl (53) and presented as relative RNA ex-pression.

Cytokine protein and gene expression analysis

HCMV- and mock-infected macrophages (1 3 106/ml) were cultured for24 h in the absence or presence of smooth LPS (1 mg/ml) or TLR1–9ligands (InvivoGen, San Diego, CA). TLR ligands included the following:TLR1, Pam3CSK4 (1 mg/ml); TLR2, heat-killed Listeria monocytogenes(HKLM; 108/ml); TLR3, polyinosinic-polycytidylic acid (10 mg/ml);TLR4, LPS (1 mg/ml); TLR5, Salmonella typhimurium flagellin (1 mg/ml);TLR6, Pam2CaDPKH PKSF (FLS1; 1 mg/ml); TLR7, imiquimod (1 mg/ml);TLR8, ssRNA40 (1 mg/ml); and TLR9, ODN2006 (5 mM). Culture super-natants were analyzed for TNF-a and IL-6 protein by immunoassay (R&DSystems, Minneapolis, MN).

2 HCMV POTENTIATES TLR LIGAND–INDUCED RESPONSES

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Total cellular RNA was extracted (RNeasy kit; Qiagen) from bloodmonocytes prior toHCMVinfection, synthesized (SuperScript choice system;Life Technologies) into cDNA utilizing an oligo(dT24) primer, from whichbiotinylated cRNA was generated using a BioArray HighYield RNA tran-scription labeling kit (Enzo Diagnostics) and purified through RNeasynucleic acid columns, using our previously described protocol (52). Afterscanning, fluorescence data were processed by the GeneChip operatingsystem (version 1.1; Affymetrix). Background correction, normalization,generation of expression values, and analysis of differential gene expressionwere performed using dChip analysis software (DNA-Chip analyzer [dChip],version 1.3; Harvard University) in compliance with Minimal Informationabout a Microarray Experiment guidelines (http://www.ncbi.nlm.nih.gov/geo/info/MIAME.html), and data were presented as fluorescence intensity.

NF-kB p65 and IkBa detection

Whole-cell extracts were prepared from 10 3 106 mock-infected orHCMV-infected macrophages cultured in the presence or absence of LPS(1 mg/ml) for 15 min at 37˚C using the RIPA lysis buffer kit (Santa CruzBiotechnology, Santa Cruz, CA). Phosphorylated NF-kB p65 and IkBawere analyzed using the InstantOne ELISA (eBioscience), which detectstotal and phosphorylated NF-kB p65 and IkBa attached to consensusbinding sites in a 96-well plate using the tetramethylbenzidine colorimetricsubstrate and OD at 450 nm (EL 800 ELISA reader, BioTek Instruments,Winooski, VT). Data are presented as phosphorylated NF-kB p65 andIkBa at OD450/10 mg protein.

Electron microscopy

The starting population of monocyte-derived macrophages (prior to HCMVinfection) was prepared and examined using a Zeiss EM 10A electronmicroscope, as previously described (54).

Western blot

The expression of MyD88 protein in HCMV-infected macrophages (10 3106/ml) isolated on day 4 of the infection cycle was determined by im-munoblotting using Abs to MyD88 and actin (Santa Cruz Biotechnology),as previously described in detail (36).

Statistical analysis

Data were analyzed by a t test/Mann–Whitney U test, where appropriate.Data are expressed as means 6 SEM.

ResultsHCMV infects monocytes as they differentiate intomacrophages

The inflammatory lesion in HCMV-infected tissues such as theintestinal mucosa is characterized by the local accumulation ofHCMV-infected macrophages (22, 35, 55). To explore the mechanismby which HCMV infection enhances macrophage inflammatory re-sponses, we first established a reproducible in vitro system to infectmonocyte-derived macrophages with HCMV. Adherent blood mono-cytes, which displayed the features of macrophages (SupplementalFig. 1), from HCMV-seronegative donors were incubated with a clin-ical isolate of HCMV (TR strain; fewer than six passages) at a pre-determined optimal MOI of 0.5 or 1.0 and allowed to differentiate intomacrophages. HCMV was detected in the cells by immunofluores-cence analysis for IE1, and HCMV DNA was quantified by quanti-tative PCR. As shown in Fig. 1A, HCMV IE1 gene product was

FIGURE 1. HCMV infection of monocyte-derived macrophages. Freshly adherent blood monocytes were exposed to media (Mock HCMV), UC culture

supernatant from HCMV-infected human foreskin fibroblasts (UC HCMV, MOI of 0.5), or HCMV also propagated in foreskin fibroblasts (MOI of 0.5),

cultured and analyzed for HCMV infection and DNA replication. (A) Immunofluorescence analysis of mock-infected and HCMV-infected macrophages

shows nuclear localization of IE1 in a representative experiment on day 4 (n = 3; red, IE1; blue, DAPI-stained nuclei) (original magnification, 340). (B)

Percentage of macrophages that contained IE1 on day 4 (n = 9). (C) Quantitative PCR analysis for HCMV DNA in macrophages (Cells) and culture

supernatant (SN) on days (D) 2, 4, and 6 of infection (n = 4).

FIGURE 2. HCMV infection of macrophages enhances LPS-induced cytokine-specific mRNA expression and protein production. (A) Macrophages were

mock- or HCMV-infected, cultured for 4 d, treated with or without LPS (1 mg/ml) for 4 h, harvested, and analyzed for cytokine-specific mRNA by real-time

PCR and expressed as fold increase. Data are the means 6 SEM from two separate cell preparations. (B) Macrophages were mock- or HCMV-infected,

cultured for 2, 4, or 6 d, treated with LPS (1 mg/ml) for 24 h on the indicated day, and the culture supernatants were harvested and analyzed for the indicated

cytokine. Data represent the means 6 SEM from three independent experiments.

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detected in macrophage nuclei as nondiffuse staining, consistent withthat reported by Soderberg-Naucler et al. (29), on day 4 after exposureto HCMV but not in mock-infected cells. Based on the presence ofHCMV IE1 in the macropahges, we routinely achieved an infectionrate of.50% (n = 9) by day 4 of infection (Fig. 1B). Progressive and

substantial increases in the number of copies of HCMV DNA in boththe macrophages and culture supernatant during the 6-d infectioncycle (Fig. 1C) confirmed the replication of viral DNA and its releaseby infected cells. The cells retained a macrophage phenotype withnegligible CD3, CD19, CD69, and CD83 expression in the absence or

FIGURE 3. HCMV infection induces macrophage CD14 and TLR4 expression. Monocyte-derived macrophages were mock- or HCMV-infected, har-

vested on days 2, 4, and 6 of the infection cycle, and then analyzed by flow cytometry for surface CD14 and TLR4 by gating on the CD13+ monocyte-

derived macrophage population. (A) Macrophages from a representative donor were examined before infection on day 0 and on days 2, 4, and 6 after

infection for CD14 and TLR4 by flow cytometry. (B) Mock- and HCMV-infected macrophages from four additional donors were analyzed for CD14 and

TLR4. Inset in (A) and gray histograms in (B) correspond to isotype controls. Horizontal bars in (B) indicate mean values.

FIGURE 4. HCMV infection potentiates macro-

phage TLR expression and TLR ligand–induced cyto-

kine production. (A) Mock- and HCMV-infected (MOI

of 0.5) monocyte-derived macrophages from three

separate donors were cultured for 4 d and analyzed for

the indicated TLR. Data are the mean percentages of

macrophages that expressed the indicated TLR. (B)

Monocyte-derived macrophages from three separate

donors were treated with optimal concentrations of the

indicated TLR ligands on day 4, and 24 h later culture

supernatants were harvested and analyzed for TNF-a

and IL-6. Cytokine levels are the means 6 SEM (pg/ml)

for the three donors. Lower panel inset shows the

level of IFN-a produced by mock- and HCMV-infected

(MOI of 0.5) macrophages after 24 h stimulation with

polyinosinic-polycytidylic acid. HKLM, heat-killed

L. monocytogenes; M-DMs, monocyte-derived mac-

rophages; poly(I:C), polyinosinic-polycytidylic acid;

S. Typhi flagellin, S. typhimurium flagellin.

4 HCMV POTENTIATES TLR LIGAND–INDUCED RESPONSES

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presence of HCMV and/or M-CSF (Supplemental Fig. 1D), and thenumber and viability (.85%) of HCMV-infected macrophages weremaintained during infection (Supplemental Fig. 1B).

HCMV infection of macrophages promotes inducibleinflammatory responses

We next analyzed the effect of HCMV infection on macrophageinflammatory cytokine gene expression and protein production. Onday 4 of infection, macrophages infected with HCMV expressed3-fold and 100-fold more TNF-a and IL-6 mRNA, respectively,than did mock-infected macrophages (Fig. 2A). Predictably, LPSstimulated cytokine-specific mRNA expression by mock-infectedmacrophages, but when the macrophages were preinfected withHCMV, LPS stimulation induced several hundred- to severalthousand-fold more mRNA for both inflammatory cytokinescompared with mock-infected, LPS-stimulated macrophages(Fig. 2A). Consistent with HCMV-enhanced inducible gene ex-pression, HCMV infection significantly increased LPS-stimulatedTNF-a protein production on days 2, 4, and 6 (p , 0.01 to p ,0.001) and IL-6 and IL-8 on days 4 and 6 (p , 0.01 to p , 0.001)of the infection cycle. HCMV infection alone upregulated TNF-aand IL-6 gene transcription (Fig. 2A), but TNF-a and IL-6 proteinproduction by HCMV-infected macrophages did not occur unlessthe cells were subsequently exposed to LPS (Fig. 2B), suggestingthat HCMV-induced cytokine gene transcription required a secondsignal for translation. The ability of HCMV infection to potentiateinducible cytokine production by macrophages was due to theinfection itself, because culture supernatant from HCMV-infectedmacrophages did not enhance cytokine production by noninfectedbystander cells (Supplemental Fig. 2). Inducible cytokine pro-duction also was not significantly affected when the macrophageswere generated in the presence of M-CSF (Supplemental Fig. 3).Macrophages exposed to UC HCMV and UV HCMV, similar tomock-infected macrophages, did not express cytokine-specificmRNA or protein (data not shown). These findings indicate thatHCMV infection primes macrophages for enhanced LPS-induced

production of inflammatory cytokines and that this response isregulated at the level of gene transcription and translation.

HCMV infection promotes maintenance of macrophage CD14and TLR4 expression

To explore the mechanism by which HCMV infection enhancesmacrophage responsiveness to LPS, we first assessed the effect ofHCMV infection on macrophage expression of CD14 and TLR4,the two major components of the LPS receptor complex. As shownin the representative experiment in Fig. 3A, on day 0, 81.2% ofmock-infected monocytes expressed surface CD14 and 54.9%expressed TLR4. During the subsequent 6-d culture period, mock-infected macrophages showed progressive declines in CD14 andTLR4 expression, consistent with previous reports (56, 57).However, HCMV-infected macrophages derived from the samedonor continued to express high levels of both CD14 and TLR4 ateach time point. By day 6, 6.6% of mock-infected cells expressedCD14 and 10.8% expressed TLR4, whereas 45.1% of HCMV-infected cells expressed CD14 and 53.9% expressed TLR4. Theability of HCMV-infected macrophages to maintain expression ofthese components of the LPS receptor was not donor-specific, assignificantly higher proportions of HCMV-infected monocytesfrom four separate donors continued to express both CD14 andTLR4, especially on days 4 and 6 of the infection cycle (Fig. 3B).

HCMV infection promotes TLR ligand–induced cytokineproduction

We next investigated whether the effect of HCMVon macrophageTLR4 gene expression and ligand-induced cytokine productionextended to other TLRs. HCMV infection of monocyte-derivedmacrophages was associated with higher levels of TLR3, TLR5,TLR7, and TLR9 compared with mock-infected macrophages, asshown for cells on day 4 of an infection cycle (Fig. 4A). Addi-tionally, infection with HCMVenhanced TLR2 ligand (heat-killedL. monocytogenes)– and TLR5 ligand (S. typhimurium flagellin)–stimulated production of TNF-a and IL-6 compared with mock-

FIGURE 5. Effect of HCMV infection

on macrophage (A and B) TLR2 and (C

and D) TLR5 expression. HCMV-in-

fected (MOI of 0.5) and mock-infected

monocyte-derived macrophages from (A

and C) a representative donor were cul-

tured for 4 d and from (B) and (D) four

additional donors were cultured for 2, 4,

and 6 d and analyzed for CD14, TLR2

and TLR5 by flow cytometry. Insets in

(A) and (C) show isotype control staining.

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infected, TLR2- and TLR5-stimulated macrophages (Fig. 4B).Thus, HCMV infection potentiated the production of key macro-phage proinflammatory cytokines in response to stimulation bybacterial components. The absence of detectable TLR7 and TLR9ligand–specific responses by mock- and HCMV-infected monocyte-derived macrophages (Fig. 4B) is consistent with the absent tonearly absent TLR7–9-stimulated responses by monocytes and in-testinal macrophages that we previously reported (52).

HCMV infection promotes macrophage TLR2 and TLR5expression

To begin to elucidate the mechanism by which HCMV infectionenhances TLR-mediated inflammatory responses, we evaluatedHCMV-infected macrophages for the expression of TLR2 andTLR5. Similar to CD14 and TLR4 expression (Fig. 3), mock in-fection of macrophages was associated with a progressive declinein TLR2 and TLR5 during a 6-d infection (Fig. 5). However,HCMV infection promoted the upregulation of TLR5, but notTLR2, especially on days 4 and 6 (Fig. 5).

HCMV enhances LPS-stimulated NF-kB signal transductionand nuclear translocation

The inability of HCMV to enhance monocyte-derived macro-phage TLR2 expression (Fig. 4A) despite enhancing TLR2-stimulatedcytokine production (Fig. 4B) suggested that HCMV potentiatedTLR responses through downstream signaling. In the canonicalLPS-induced signal cascade, the binding of LPS to its receptoractivates the recruitment of adaptor proteins, including MyD88,

the master adaptor molecule in the NF-kB signal cascade thatinitiates all TLR, except TLR3, signaling (52, 58–63). In this con-nection, HCMV infection did not enhance TLR3-mediated responses(Fig. 4B, inset). MyD88 binds to the cytoplasmic Toll/IL-1 domain,which triggers the phosphorylation of IL-1R–associated kinase 4with subsequent recruitment and phosphorylation of IL-1R–associ-ated kinase 1, causing the release of TNFR-associated factor 6and propagation of the NF-kB signaling cascade (58–63). Therefore,we examined the effect of HCMV infection on the expression ofMyD88 in monocyte-derived macrophages. HCMV infection in-duced substantial increases in MyD88 mRNA (Fig. 6A, 6B) andprotein (Fig. 6C) in the macrophages. Additionally, infection causeda slight increase in the phosphorylation of IkBa compared withmock-infected macrophages, but infected macrophages stimulatedwith LPS displayed a larger increase in IkBa phosphorylationcompared with mock-infected LPS-stimulated cells on day 4 (Fig.6D, left panel) and day 6 (not shown) in a representative infection.Similarly, HCMV infection alone did not induce significant NF-kBphosphorylation, reflected in nearly the same levels of pNF-kB p65in HCMV- and mock-infected cells during the infection cycle shownin Fig. 6D (right panel), consistent with the inability of HCMVinfection alone to induce inflammatory cytokine release. However,when HCMV-infected macrophages were subsequently stimulatedwith LPS, the level of pNF-kB p65 increased substantially, especiallyon days 4 and 6, compared with mock-infected LPS-stimulatedmacrophages (p , 0.001 and p , 0.03, respectively) (Fig. 6D,right panel). HCMV infection plus LPS stimulation also enhancedthe proportion of macrophages that contained pNF-kB p65 compared

FIGURE 6. HCMV infection enhances LPS-stimulated NF-kB signal protein expression and NF-kB nuclear translocation in macrophages. (A) Mock-

and HCMV-infected (MOI of 0.5) monocyte-derived macrophages were cultured for 4 d and analyzed for MyD88 mRNA by real-time PCR using GAPDH

as a control and expressed as fold change 6 SEM (n = 3). MyD88 protein in HCMV- and mock-infected monocyte-derived macrophages from three

separate donors were harvested on day 4 of an infection cycle and analyzed by (B) Western blot and (C) densitometric comparison of the bands. Mock- and

HCMV-infected (MOI of 0.5) macrophages were treated on the indicated day with media or LPS (1 mg/ml, 15 min), harvested, and cell extracts were

analyzed by ELISA for pIkBa (D, left panel) and pNF-kB p65 (D, right panel). Data are the means 6 SEM values for triplicate cultures from three

experiments. (E) Mock- and HCMV-infected (MOI of 0.5) macrophages (day 4) were analyzed for pNF-kB p65 by flow cytometry.

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with mock-infected LPS-stimulated cells, as detected by flowcytometry (Fig. 6E). Thus, HCMV infection of macrophages inducedMyD88 expression and enhanced inducible phosphorylation of IkBaand NF-kB.

DiscussionMonocytes are an important reservoir for latent HCMV infectionand, after their differentiation into macrophages in the tissues, maycontribute to HCMV-associated inflammatory disease (64, 65).Using a clinical isolate of HCMV to investigate the mechanism ofHCMV-induced macrophage-mediated inflammation, we showedthat macrophages infected with HCMV continued to expressCD14 and TLR4 and TLR5 during a 6-d infection cycle, whereasmock-infected cells displayed a progressive decline in CD14and TLR expression. HCMV infection promoted ligand-inducibleproinflammatory cytokine mRNA expression and TNF-a, IL-6,and IL-8 protein production. Coincident with the enhanced pro-inflammatory response, HCMV infection promoted expressionof the adaptor protein MyD88 and potentiated inducible phos-phorylation of both IkBa and NF-kB, indicating a mechanism forthe increased inducible proinflammatory cytokine production.The ability of HCMV to promote the continued expression of

surface CD14 and TLR4 and TLR5 on monocytes as they dif-ferentiate into macrophages is relevant to mucosal macrophages inparticular. Circulating monocytes, the exclusive source of humanintestinal macrophages (32), lose CD14 as they differentiate intomucosal macrophages (32, 54). Intestinal macrophages also donot activate NF-kB, leading to inflammation anergy (34, 52, 54,66). Thus, our finding that HCMV infection causes monocytes tomaintain CD14 and TLR4 expression and upregulate inducibleIkBa and NF-kB phosphorylation, thereby potentiating inducibleproinflammatory responses by macrophages, could contribute tothe pathogenesis of HCMV-associated mucosal inflammation andis the subject of ongoing investigation.The enhanced proinflammatory response of stimulated macro-

phages may benefit both host and virus. In a novel mouse model,Barton et al. (67) showed that both latent infection with EBVand murine CMV conferred enhanced resistance to the bacterialpathogens L. monocytogenes and Yersinia pestis, suggesting thatlatent herpesvirus infection provides symbiotic protection againstat least some bacterial infections. Consistent with murine CMVinduction of cross-protection against bacteria in mice (67), theability of HCMV to enhance the inflammatory capability of hu-man macrophages suggests that HCMV-enhanced inflammationmay promote containment of bacterial, and possibly fungal,pathogens and, through the induction of inflammatory chemokines(26), recruit HCMV-susceptible target cells to the inflammatorylesion, thereby amplifying host protection against certain patho-gens while perpetuating HCMV infection.The binding of envelope glycoproteins gB (UL55) and gH

(UL75) from high-passaged Towne strain HCMV to embryoniclung fibroblasts in vitro has been shown to activate NF-kB genetranscription within 30–60 min of binding (68, 69). The binding ofHCMV (Towne strain) gB and gH to human monocytes also hasbeen reported to upregulate NF-kB, IkBa, and IL-1 gene tran-scription after 4 h (40). Others have shown that the Towne andAD169 strains inhibit NF-kB signaling in human foreskin fibro-blasts (70). In contrast to these studies, we show in the presentstudy that HCMV infection of human macrophages potentiatedinducible TLR ligand–induced phosphorylation of both IkBaand the transcription factor NF-kB, leading to enhanced gene andprotein expression for proinflammatory cytokines. However, thebinding of gB or gH alone was not responsible for the phenotypeobserved in our studies, as exposure of macrophages to UV-

inactivated virions did not induce the changes caused by incu-bation with infectious virions. Our finding that HCMV infectionenhanced inducible inflammatory responses is important becausethe enhanced responses were potentiated by a low-passaged clin-ical isolate of the virus, persisted throughout the infection cycle,and pertained to primary macrophages, cells that play a funda-mental role in mediating tissue inflammation during HCMV in-fection (23, 35, 55).The ability of HCMV to potentiate inducible macrophage release

of IL-6 is noteworthy for two reasons. First, in the presence ofTGF-b, IL-6 induces the differentiation of Th17 cells (71), whichmediate tissue inflammation through the induction of IL-17. Inmice, for example, IL-17 plays a key role in promoting murineCMV-induced interstitial pneumonia (72). Thus, HCMV-inducedIL-6, as reported in the present study, and the HCMV-inducedTGF-b production that we (37) and others (73, 74) have re-ported could together promote IL-17–mediated tissue inflamma-tion such as that characteristic of Crohn’s disease (75). Second,IL-6 can reactivate HCMV latently infected cells (21), resulting inviral replication and the release of infectious progeny from oth-erwise quiescent macrophages; notably, the virions released inresponse to IL-6–mediated reactivation may be more infectious.Thus, HCMV-induced IL-6 may potently influence both inflam-matory and virological responses. Further elucidation of theimmunobiology of HCMV infection of human macrophagesshould provide new insights into HCMV pathogenesis and helpidentify novel therapeutic strategies for HCMV-associated in-flammatory disease.

DisclosuresThe authors have no financial conflicts of interest.

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