miR-190b is markedly upregulated in the intestine in response to simian immunodeficiency virus replication and partly regulates myotubularin-related protein-6 expression

of July 15, 2014.This information is current as Protein-6 Expression

Partly Regulates Myotubularin-Related Immunodeficiency Virus Replication andIntestine in Response to Simian miR-190b Is Markedly Upregulated in the

Pyone P. Aye, Xavier Alvarez and Andrew A. LacknerMahesh Mohan, Lawrance C. Chandra, Workineh Torben,

ol.1303479http://www.jimmunol.org/content/early/2014/06/30/jimmun

published online 30 June 2014J Immunol 

MaterialSupplementary

479.DCSupplemental.htmlhttp://www.jimmunol.org/jimmunol/suppl/2014/06/30/jimmunol.1303

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

miR-190b Is Markedly Upregulated in the Intestine inResponse to Simian Immunodeficiency Virus Replication andPartly Regulates Myotubularin-Related Protein-6 Expression

Mahesh Mohan, Lawrance C. Chandra, Workineh Torben, Pyone P. Aye,

Xavier Alvarez, and Andrew A. Lackner

HIV replication and the cellular micro-RNA (miRNA) machinery interconnect at several posttranscriptional levels. To understand

their regulatory role in the intestine, a major site of HIV/SIV replication, dissemination, and CD4+ T cell depletion, we profiled

miRNA expression in colon following SIV infection (10 acute SIV, 5 uninfected). Nine (four up and five down) miRNAs showed

statistically significant differential expression. Most notably, miR-190b expression showed high statistical significance (adjusted

p = 0.0032), the greatest fold change, and was markedly elevated in colon and jejunum throughout SIV infection. In addition,

miR-190b upregulation was detected before peak viral replication and the nadir of CD4+ T cell depletion predominantly in lamina

propria leukocytes. Interestingly non–SIV-infected macaques with diarrhea and colitis failed to upregulate miR-190b, suggesting

that its upregulation was neither inflammation nor immune-activation driven. SIV infection of in vitro–cultured CD4+ T cells and

primary intestinal macrophages conclusively identified miR-190b upregulation to be driven in response to viral replication.

Further miR-190b expression levels in colon and jejunum positively correlated with tissue viral loads. In contrast, mRNA

expression of myotubularin-related protein 6 (MTMR6), a negative regulator of CD4+ T cell activation/proliferation, significantly

decreased in SIV-infected macrophages. Luciferase reporter assays confirmed MTMR6 as a direct miR-190b target. To our

knowledge, this is the first report, which describes dysregulated miRNA expression in the intestine, that identifies a potentially

significant role for miR-190b in HIV/SIV pathogenesis. More importantly, miR-190b–mediated MTMR6 downregulation suggests

an important mechanism that could keep infected cells in an activated state, thereby promoting viral replication. In the future, the

mechanisms driving miR-190b upregulation including other cellular processes it regulates in SIV-infected cells need determi-

nation. The Journal of Immunology, 2014, 193: 000–000.

HIV/SIV infection of the gastrointestinal (GI) immune sys-tem results in massive virus–induced depletion of CD4+/CCR5+ memory T cells, uncontrolled inflammation, and

immune activation that are proximate causes of structural and func-tional damage to the GI tract (1–6). Most of the current knowledgepertaining to the early molecular immunopathological events in theGI tract have been generated predominantly by transcriptome pro-filing studies (7, 8) including our most recent studies done separatelyon the lamina propria leukocyte (LPL) (9) and epithelial cellularcompartments (10) of the intestine in SIV-infected macaques. Thesestudies have revealed significant alterations in the expression of genes

associated with early immune response/inflammation, immune acti-vation, antiviral signaling and T cell, B cell, and macrophage dys-

function in LPLs (7–9). Similarly, genes encoding cell cycle reg-

ulators, Wnt-TCF7L2 signaling, Notch signaling, genes associated

with the formation of adherens junction, hemidesmosomes, desmo-

somes, and so on were found to be significantly dysregulated in the

intestinal epithelium (10) at 21 and 90 d post-SIV infection.Immune/inflammatory responses against viruses are orchestrated

for the most part by signaling pathways activated by IFNs and nu-

merous other proinflammatory cytokines that stimulate transcription

factors including but not limited to, IRFs, STATs, NF-kB, and

CAAAT enhancer binding proteins (C/EBPs) (11, 12). In this context,

we previously demonstrated that GI disease in HIV/SIV infection is

characterized by constitutive activation of proinflammatory tran-

scription factors such as STAT3 and C/EBPb that can generate a self-

perpetuating cycle involving intestinal epithelial damage, inflamma-

tion, immune activation and viral replication (13, 14). Apart from in-

ducing inflammatory gene expression, proinflammatory transcription

factors such as STAT3 (15), C/EBP (16), and NF-kB (17) also acti-

vate the transcription of another novel class of regulatory non-protein

coding RNA called micro-RNAs (miRNAs) and these small RNA

molecules have been shown to be potent regulators of early immune

and inflammatory responses (18) including HIV pathogenesis (19).miRNAs are small (∼21–23 nt) regulatory noncoding RNAs that

are highly conserved and have been shown to suppress the expres-

sion of protein coding genes by targeting mRNAs for translational

repression or degradation (20, 21). They are initially transcribed by

RNA polymerase II as long variable length primary miRNAs, which

are then processed in the nucleus by the microprocessor complex

Division of Comparative Pathology, Tulane National Primate Research Center, Cov-ington, LA 70433

Received for publication December 30, 2013. Accepted for publication May 27,2014.

This work was supported by National Institutes of Health Grants R01DK083929 (toM.M.), AI084793, MH077544, and OD011104 (formerly RR00164).

The TLDA data presented in this article have been deposited to the Gene ExpressionOmnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56624) under ac-cession number GSE56624.

Address correspondence and reprint requests to Dr. Mahesh Mohan, Tulane NationalPrimate Research Center, 18703 Three Rivers Road, Covington, LA 70433. E-mailaddress: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: C/EBP, CAAAT enhancer binding protein; CT, cyclethreshold; DPI, d postinfection; GI, gastrointestinal; IEL, intraepithelial lymphocyte; LPL,lamina propria leukocyte; miRNA, micro-RNA; MTMR6, myotubularin-related protein 6;MUT, mutated; qRT-PCR, quantitative RT-PCR; RT, reverse transcription; TLDA, TaqManlow density arrays; TNPRC, Tulane National Primate Research Center; WT, wild-type.

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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303479

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(Drosha and DGCR8) to precursor miRNAs and subsequently in thecytoplasm by Dicer to generate the mature miRNAs (21). Recentstudies have attributed more significant roles for these small RNAmolecules in regulating the immune response that includes immunecell development, proliferation, activation, and differentiation (18, 22–26). Furthermore, several in vitro studies clearly show that HIV andthe host miRNA machinery criss-cross at several posttranscrip-tional levels (27–31). The restraints exerted by the host miRNAmachinery on HIV replication is clearly evident from the finding thatviral replication kinetics are enhanced in PBMCs following siRNA-mediated knockdown of Drosha and Dicer (32). In addition, severalrecent miRNA profiling studies performed on purified CD4+ T cells,macrophages, and PBMCs of HIV-infected patients provide com-pelling evidence of their dysregulation in HIV infection (33–36).Recent studies also have described dysregulated miRNA expressionin the brain (37) and plasma of SIV-infected rhesus macaques (38)including miRNAs that directly targeted SIV replication in macro-phages (39). Accordingly, we hypothesized that SIV replication inthe GI tract in association with the ensuing immune response in-duces the expression of miRNAs that have the potential to regulateviral replication, including host genes expressed during the immuneresponse. Given the lack of information on the expression of miR-NAs in the GI tract in response to SIV infection, we performedglobal miRNA expression profiling in the intestine of acutely SIV-infected macaques with a focus on the colon. Expression of ninemiRNAs was significantly altered in the colon following acute SIVinfection. miR-190b, in particular, was significantly upregulated inboth colon and jejunum through the course of SIV infection. Fur-thermore, miR-190b upregulation was detected as early as 7 d post-SIV infection even before peak viral replication and the nadir ofCD4+ T cell depletion in both colon and jejunum. Furthermore,using multiple experimental approaches, we show that miR-190b isupregulated specifically in the intestinal lamina propria in responseto SIV infection and targets the expression of myotubularin-relatedprotein 6 (MTMR6), a negative regulator of CD4+ T cell activation/proliferation. Thus, miR-190b could potentially play a significantrole in the pathogenesis of SIV infection in the intestinal mucosa.

Materials and MethodsAnimal ethics statement

All experiments using rhesus macaques were approved by the Tulane In-stitutional Animal Care and Use Committee (protocol number 3574). TheTulane National Primate Research Center (TNPRC) is an Association forAssessment and Accreditation of Laboratory Animal Care Internationalaccredited facility (number 000594). The National Institutes of Health Officeof Laboratory AnimalWelfare assurance number for the TNPRC isA3071-01.All clinical procedures, including administration of anesthesia and anal-gesics, were carried out under the direction of a laboratory animal veter-inarian. Animals were anesthetized with ketamine hydrochloride for bloodcollection procedures. Intestinal resections were performed by laboratoryanimal veterinarians. Animals were preanesthetized with ketamine hydro-chloride, acepromazine, and glycopyrolate; intubated; and maintained ona mixture of isoflurane and oxygen. Buprenorphinewas given intraoperativelyand postoperatively for analgesia. All possiblemeasures are taken tominimizediscomfort of all the animals used in this study. Tulane University complieswith National Institutes of Health policy on animal welfare, the AnimalWelfare Act, and all other applicable federal, state, and local laws.

Animals and tissue collection

Colon and jejunum tissues were collected from 49 Indian-origin rhesusmacaques including 33 animals infected with pathogenic strains of SIV thatuse CCR5 in vivo and 16 animals not infected with SIV. In the SIV-infectedgroup, all animals were inoculated i.v. with 100TCID50 of SIV with theexception of five (CT16, CG32, AT56, H405 and L441) that were inocu-lated intravaginally. For intravaginal inoculation, a viral inoculum dose of1000TCID50 was used. Animal IDs, duration of infection, plasma and in-testinal viral loads in all SIV-infected animals are provided in Table I. Tenacutely SIV-infected animals (Table I) and five uninfected control animals

(Table II) were used for the initial TaqMan low density arrays (TLDA)miRNA profiling. An additional 23 SIV-infected macaques at various stagesof infection (Table I) were used exclusively for characterization of miR-190bexpression through the course of SIV infection. Among the 16 uninfectedanimals, we included eight that were necropsied for chronic diarrhea unre-sponsive to treatment (non–SIV-infected with diarrhea and colitis) (40, 41).These animals were included to assess whether miR-190b upregulation wasin response to viral replication or was simply a byproduct of enterocolitisthat generally accompanies HIV/SIV infection.

Following euthanasia with an i.v. overdose of pentobarbital, all animalsreceived a complete necropsy. Tissue samples (colon and jejunum) from allcontrol, SIV-infected, and non–SIV-infected macaques with diarrhea andcolitis were collected in RNAlater (Life Technologies, Grand Island, NY)over several years (∼6–7 y) and stored at 220˚C for total RNA extraction.In addition, serial resection biopsies (∼6–8 cm long) of jejunum includingcolonic wedge biopsies (2 cm) were collected from eight Indian-originrhesus macaques (HC36, HB31, HF27, HB48, GK31, GA19, HR57 andHV95) at 6 wk before SIV infection and 21 and 90 d after SIV infection forTLDA and miR-190b RT-PCR confirmation studies. For histopathologicalevaluation, colon and jejunum tissues were collected immediately aftereuthanasia and fixed in 10% neutral buffered formalin, embedded in par-affin, sectioned at 6 mM, and stained with H&E for analysis. Sections wereexamined in a blinded fashion, and inflammation was scored semiquanti-tatively on a scale of 0–3 as follows: 0, within normal limits; 1, mild; 2,moderate; and 3, severe. In addition, the presence of crypt dilatation, villusblunting, diverticulosis, and amyloidosis were recorded (Table II).

Global microRNA profiling using TaqMan Low Density Arrays

Total RNAwas extracted from intact colon and jejunum samples using themiRNeasy total RNA isolation kit (Qiagen). RNA integrity was assessed byrunning an aliquot on a denaturing agarose gel, followed by staining withethidium bromide to visualize intact 28S and 18S rRNA bands. For TLDAmiRNA profiling, ∼350 ng total RNA from intact colon tissue was firstreverse transcribed following the ABI microRNA TLDA reverse tran-scription reaction protocol. Briefly, two master mixes were prepared foreach RNA sample representing either TLDA panel (panels A and B) andconsisted of the following reaction components: 0.80 ml MegaPlex reversetranscription (RT) primers (10 times), 0.20 ml 29-deoxynucleoside 59-tri-phosphates with 29-deoxythymidine-59-triphosphate (100 mM), 1.50 mlMultiScribe reverse transcriptase (50 U/ml), 0.80 ml 103 RT buffer, 0.90 mlMgCl2 (25 mM), 0.10 ml RNase inhibitor, and 0.20 ml nuclease-freewater (20 U/ml). Three microliters of total RNA (350 ng) was loaded intoappropriate wells of a 96-well plate containing 4.5 ml of the RT reactionmix and following a brief 5 min incubation on ice was subjected to thefollowing thermal cycling conditions on the ABI 7900 HT Fast PCRsystem: standard or max ramp speed, 16˚C for 2 min, 42˚C for 1 min, 50˚Cfor 1 s (40 cycles), and 85˚C for 5 min (hold).

Approximately 2.5 ml of the resulting cDNA from each sample was mixedwith a total of 22.5 ml preamplification reaction mix consisting of 12.5 mlTaqMan PreAmp Master Mix (2 times), 2.5 ml Megaplex PreAmp Primers(10 times), and 7.5 ml nuclease-free water and preamplified on the ABI 7900HT Fast PCR system, according to the TLDA miRNA preamplification pro-tocol outlined by the manufacturer (Life Technologies). The preamplificationthermal cycling conditions were as follows: hold 95˚C for 10 min; hold 55˚Cfor 2 min; hold 72˚C for 2 min; 12 cycles at 95˚C for 15 s; and 60˚C for 4 min.

The preamplified product was first diluted 4-fold with 75 ml 0.13 TE (pH8) mixed, following which 9 ml of the diluted PreAmp product was mixedwith 450 ml 23 TaqMan Universal PCR Master Mix with no uracil-N-glycosylase (AmpErase) and 441 ml nuclease-free water to bring the finalvolume to 1 ml. After proper mixing and centrifuging, 100 ml of the PCRmix was loaded into each port of the TaqMan Array Human MicroRNAA+B Card Set version 3.0. The TLDA cards were then centrifuged, sealed,and processed on the ABI 7900 HT Sequence Detection System using the384-well TaqMan Low Density Array default thermal-cycling conditions.

Quantitative Real-Time TaqMan Stem loop miRNA and SYBRGreen RT-PCR

Expression of miR-190b was further investigated in both colon and jejunumthrough the course of SIV infection using the TaqMan microRNA prede-signed and preoptimized assays (Life Technologies). Total RNA (500 ng formiR-190b and RNU48 and 100 ng for snoU6 [colon and jejunum], 250 ngfor CD4+ T cells and primary intestinal macrophages] was reverse tran-scribed using the stem loop primers provided in the predesigned kit ina total volume of 15 ml. Similar total RNA concentrations (500–750 ng)have been previously used for miRNA quantitative RT-PCR (qRT-PCR)(42, 43). Approximately 4 ml cDNAwas subjected to 40 cycles of PCR ina total volume of 20 ml on the ABI 7900 HT Fast PCR System (Life

2 miR-190b UPREGULATION IN INTESTINE DURING SIV INFECTION

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Technologies) using the following thermal cycling conditions: 95˚C for10 min, followed by 40 repetitive cycles of 95˚C for 15 s and 60˚C for 1 min.As a normalization control for RNA loading, parallel reactions in duplicatewells to amplify RNU48 or snoU6 or RNU44 in combination with RNU48(CD4+ T cells) were run in the same or different multiwell plate. AlthoughRNU48 worked well for intact intestine, we found snoU6 to be better whenanalyzing miRNA expression in distinct intestinal mucosal compartmentssuch as epithelium and LPLs. RNU44 was used for CD4+ T cells because ithas been previously used for normalization of miRNA expression in HIV-infected CD4+ T cells (44). Comparative real-time PCR was performed induplicate wells including no template controls, and relative change in geneexpression was calculated using the comparative DD cycle threshold (CT)method.

Expression of MTMR6 in in vitro cultured SIV-infected primary in-testinal macrophages was evaluated by SYBR Green qRT-PCR assay.Approximately 1 mg total RNAwas first reverse transcribed in a total vol-ume of 50 ml using the SuperScript III First-Strand Synthesis System forRT-PCR kit (Life Technologies) following the manufacturer’s protocol.Each qRT-PCR (20 ml) contained the following: 23 Power SYBR GreenMaster Mix without uracil-N-glycosylase (12.5 ml), target forward andreverse primer (200 nM), and cDNA (4 ml). Forward and reverse primersequence for MTMR6 and GAPDH is shown in Table III. The PCR am-plification was carried out in the ABI 7900 HT Fast PCR System (LifeTechnologies) using the default thermal cycling conditions for SYBR Greenassays. As a normalization control for RNA loading, parallel reactions in thesame multiwell plate were performed using GAPDH. Relative changes ingene expression were calculated using the DDCT method. PCR efficiencyanalysis was performed using serial 10 fold RNA dilutions (500, 50, 5, and0.5 ng of total RNA) for miR-190b, RNU48, snoU6, or cDNA dilutions (40,4, 0.4, and 0.04 ng) for MTMR6 and GAPDH. The amplification curves forall assays were linear and based on slope values (23.09 to23.24) all assayshad 100–105% efficiency.

In situ hybridization and immunofluorescence for cellularlocalization of SIV and in vitro characterization of primaryintestinal macrophages

In situ hybridization for detecting SIV RNA was performed using SIV-digoxigenin–labeled antisense riboprobes (Lofstrand Laboratories, Gai-thersburg, MD). Briefly, 7-mM thick formalin-fixed, paraffin-embeddedtissue sections were first deparafinized, rehydrated in decreasing concen-trations of ethanol, and pretreated in a microwave with citrate buffer (Agunmasking solution; Vector Laboratories, Burlingame, CA) for 20 min athigh power, according to the manufacturer’s instructions. Thereafter,sections were thoroughly washed, placed in a humidified chamber, andprehybridized at 45˚C with in situ hybridization buffer containing 50%formamide with denatured herring sperm DNA and yeast tRNA at 10 mg/mleach. SIV-digoxigenin–labeled antisense riboprobes (Lofstrand Labora-tories) were used at a concentration of 10 ng/slide in hybridization bufferand hybridized overnight at 45˚C. After hybridization slides were washedwith 43 SSC (SSC buffer), 13 SSC, 0.13 SSC, and blocked with Dakoprotein free blocker (Dako North America, Carpinteria, CA) for 1 h. Fabfragments of an anti-digoxigenin Ab conjugated with alkaline phosphatase(Roche Diagnostics, Penzberg, Germany) were used to detect digoxigenin-labeled probes. Positive signals were detected using permanent red accordingto the manufacturer’s (Dako North America) instructions.

The protocol for detection of SIV RNA in SIV-infected in vitro–culturedprimary intestinal macrophages remained the same as described above.Immunofluorescence for phenotyping SIV-positive cells in tissue and invitro–cultured primary intestinal macrophages was done exactly as describedearlier (13, 14) using unconjugated or Alexa Fluor 647–conjugated CD68(KP1) (Dako North America and Santa Cruz Biotechnology, Dallas, TX),CD163 (Serotec, Raleigh, NC), and CD3 (Dako North America) pri-mary and appropriate Alexa Fluor–conjugated secondary Abs (LifeTechnologies).

Cell isolation from intestinal resection segments, peripheralblood, and in vitro SIV infection of CD4+ T cells and primaryintestinal macrophages

To determine the mucosal compartment contributing to miR-190b upreg-ulation, we separated the intestinal epithelial cells from the underlying LPLsand fibrovascular stroma as described previously (9, 10, 45). Finally, theintraepithelial lymphocytes (IELs) were separated from the epithelial cellsand changes in gene expression were analyzed in the epithelial cells andLPLs separately. To successfully separate all four tissue compartments andensure the availability of sufficient starting material, we obtained intestinalresection segments (6–8 cm long) from the jejunum at 21 and 90 d after

SIV infection. Comparisons in gene expression were made to resectionsegments collected from the same animal 6 wk prior to SIV infection. TheLPLs consist predominantly of lymphocytes (50–60%) but also containsmall numbers of macrophages, neutrophils, plasma, and dendritic cells(Supplemental Fig. 1). The intestinal cell isolation protocol established atthe TNPRC (45) yields epithelial cells with ∼85% purity with minimalcontamination with IELs. Similar purity also has been reported for LPLswith minimal contamination with epithelial cells (45).

To further determine whether miR-190b upregulation was linked to SIVreplication, we isolated peripheral blood CD4+ T cells and primary in-testinal macrophages for in vitro SIV infection. CD4+ T cells from pe-ripheral blood were isolated using the nonhuman primate–specific CD4+

T cell isolation kit (magnetic separation with an LS column) following themanufacturer’s recommended protocol (Miltenyi Biotec, Auburn, CA).Approximately 107 cells were first activated with 1 mg/ml Con A andcultured for 3 d in 10 ml RPMI 1640 medium containing 10% FBS, 2 mML-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Followingactivation, cells were infected with 300 TCID50 SIVmac239 in the pres-ence of 2 ng/ml IL-2 or left uninfected as controls. Syncytia formation wasdetected 48 h postinfection, at which time cells were pelleted and lysed fortotal RNA extraction.

For primary intestinal macrophage isolation, LPLs harvested in theprevious step were transferred to T75 flasks containing RPMI 1640 mediumsupplemented with 10% FBS, 100 mM HEPES, L-glutamine, and 10,000 Upenicillin/streptomycin. Under these culture conditions, lymphocytes willnot attach and become apoptotic by days 3–5 in culture. However, in-flammatory macrophages or monocytes that have recently extravasatedinto the tissues and have not fully differentiated into macrophages willattach and proliferate forming a monolayer after ∼7–10 d in culture (46,47). In vitro–cultured primary jejunal macrophages isolated from SIV-infected (n = 5) and uninfected (n = 6) macaques were pelleted, acti-vated with 20 ng/ml recombinant rhesus macaque GM-CSF (Cell Sciences,Canton, MA), and infected with 100 TCID50 SIVmac251 in a total volumeof 200 ml for ∼2.5 h at 37˚C. Cells were then plated in T25 flasks con-taining RPMI 1640 medium supplemented with 10% FBS, 100 mMHEPES, L-glutamine, and 10,000 U penicillin/streptomycin and culturedfor 4 d post-SIV infection.

Flow cytometry to quantify intestinal CD4+ T cell dynamics

LPLs were isolated as described above and adjusted to a concentration of107/ml. For T cell immunophenotyping, ∼100-ml aliquots were stainedwith appropriately diluted, directly-conjugated mAbs to CD3 (Pacific blue:SP34-2), CD4 (SK3: PerCP-Cy5.5), and CD8 (PE-TR: 3B5) (BD Bio-sciences, San Jose, CA). Samples were stained for 30 min in the dark at 4˚C,fixed in 2% paraformaldehyde, and stored in the dark at 4˚C overnight foracquisition the next day. Samples were acquired on a LSR II flow cytometryequipment (BD Biosciences) and analyzed with Flow Jo software (TreeStar, Ashland, OR). The cells were first gated on singlets, followed bylymphocytes, CD3+ T cells, and then on CD3+CD4+ T cell subsets.

Cloning of 39-untranslated region of MTMR6 mRNA andDual-Glo luciferase reporter gene assay

The 39-untranslated region (UTR) of the rhesus MTMR6 gene containsa single predicted miR-190b binding site (TargetScan 6.2) (48) highlyconserved across several species (Table IV). Accordingly, a short 42–44 ntlong sequence representing the 39-UTR containing the predicted miR-190bsite (59-TCTGTTTATTAAAGTACATATCT-39) was synthesized (IDTDNATechnologies, Coralville, IA) for cloning into the pmirGLO dual luciferasevector (Promega, Madison, WI). A second oligonucleotide with the bindingsite mutated (MUT) (59-TCTGTTTATTAAAGTAGTATAA-39) also wassynthesized to serve as a negative control. Both oligonucleotide sequenceswere synthesized with a PmeI site on the 59 and XbaI site on the 39 end fordirectional cloning. The pmirGLO vector was first cut with PmeI and XbaIrestriction enzymes, gel purified, and ligated with either wild-type (WT)sequence containing the miR-190b binding site (pmirGLO-WT-MTMR6) orMUT sequence (pmirGLO-MUT-MTMR6). HEK293 cells were plated ata density of 5 3 104 cells per well of a 96-well plate. At 50% confluencecells were cotransfected with ∼100 ng pmirGLO-WT-MTMR6 or pmirGLO-MUT-MTMR6 UTR miRNA luciferase reporter vector and 100 nM miR-190b mimic using the Dharmafect Duo transfection reagent (ThermoFisherScientific). In separate wells, cells also were transfected with pmirGLOvector (Promega) as a normalization control. After 48 h, the Dual Glo lu-ciferase assay was performed according to the manufacturer’s recommendedprotocol using the BioTek H4 Synergy plate reader (Bio-Tek, Winooski,VT). The normalized Firefly to Renila ratio was calculated to determine therelative reporter activity. Experiments were performed in six replicates andrepeated thrice.

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Quantitation of plasma and mucosal viral loads

Total RNA samples from plasma, colon, and jejunum tissues of all SIV-infected animals were subjected to quantitative real-time TaqMan two-step RT-PCR analyses to determine viral loads. Briefly, primers andprobes specific to the SIV long terminal repeat sequence were designed andused in the real-time TaqMan PCR assay. Probes were conjugated witha fluorescent reporter dye (FAM) at the 59-end and a nonfluorescent quencherdye at the 39-end. Fluorescence signal was detected with an ABI Prism 7900HT sequence detector (Life Technologies). Data were captured and analyzedwith Sequence Detector Software (Life Technologies). Viral copy numberwas determined by plotting CT values against a standard curve (y =23.2463+39.374) (r2 = 0.998) generated with in vitro–transcribed RNA representingknown viral copy numbers.

Data analysis and availability

TLDA-SDS run files from 10 SIV-infected and 5 uninfected controlmacaques were loaded onto Applied Biosystems Relative QuantificationManager Software version 1.2.2 and analyzed using automatic baselinesettings and a manual threshold of 0.2. The results from the RelativeQuantification manager analysis containing five columns (well, sample,detector, task, and CT values) was saved as a tab-delimited text file, whichwas later imported and analyzed using the DataAssist version 3.01 soft-ware (Life Technologies), a data analysis tool designed to compare sam-ples using the DDCT method for relative quantification of gene expression.miRNA expression data were analyzed using global normalization (49, 50)as this method has been reported to be the most sensitive and accurateapproach for high-throughput miRNA profiling using qRT-PCR comparedwith endogenous controls. In all experiments, the CT upper limit was set to33, meaning that all miRNA detectors with a CT value $ 33 were excluded.Multiple comparisons correction using Benjamini–Hochberg method forfalse-discovery rate was simultaneously applied to all 768 miRNA targetprobes (card A and B combined). TLDA data were deposited with GeneExpression Omnibus (accession number: GSE56624, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56624).

For miR-190b and MTMR6 qRT-PCR studies, one uninfected controlmacaque with the highest (for miR-190b) or lowest (for MTMR6) DCT

value served as the calibrator/reference and assigned a value of 1. Alldifferentially expressed miRNAs or mRNAs in SIV-infected and unin-fected macaques with diarrhea including other macaques in the controlgroup are shown as an n-fold difference relative to this macaque. Thestepwise calculation of fold change using this approach is shown in Sup-plemental Table I. miRNA fold change was also calculated using an av-erage of all control animal DCT values providing very similar results(Supplemental Fig. 2A, 2B). Accordingly, we have used the former ap-proach as it facilitated graphing the control samples so that the variationwithin the control samples can be displayed (Supplemental Fig. 2A). In-testinal CD4+ T cell data and individual miR-190b qRT-PCR data in colon,jejunum, intestinal epithelium, and LPL compartments were analyzed us-ing nonparametric Kruskal–Wallis test and posthoc analysis was doneusing Dunn’s multiple groups comparison employing the GraphPad Prism5 software (La Jolla, CA). p , 0.05 was considered as significant. miR-190b and MTMR6 mRNA qRT-PCR data in SIV-infected macrophageswas analyzed by nonparametric Wilcoxon’s rank-sum test for independentsamples using RealTime STATMINER package, a bioinformatics softwaredeveloped by Integromics on Spotfire DecisionSite. A Spearman’s nonpara-metric one-tailed correlation analysis was performed to determine the degreeof association between tissue viral loads and miR-190b fold expression.Firefly/Renila ratios were statistically analyzed using an unpaired t test.

ResultsMucosal and plasma viral loads, CD4+ T cell dynamics, andintestinal histopathology

The viral loads in plasma, colon and jejunum of all SIV-infectedmacaques are shown in Table I. Intestinal viral loads were sub-stantial and in the colon ranged from 0.06 3 106 to 10,228 3 106

copies/ mg total RNA with a median of 2 3 106 copies/ mg totalRNA. In the jejunum, viral loads ranged from 0.043 106 to 14823106 copies/mg total RNA with a median of 3.5 3 106 copies/mgtotal RNA. Similar to tissue viral loads, plasma viral loads from 7 dpostinfection (DPI) to terminal disease ranged from 0.006 3 106 to500 3 106 copies/ml with a median of 10 3 106 copies/ml. Plasmaviral loads were not available for six animals (IA85, HT44, HV39,HV61, CG32, and CT16).

SIV-infected animals at the 13-14 DPI, 21 DPI and 90 DPI timepoints (Table I) had significant mucosal CD4+ T cell depletion(Fig. 1). Analysis of CD4+ T cell percentages using Kruskal–Wallis (nonparametric method) test revealed statistically signifi-cant differences among groups (p = 0.0005). Post hoc analysisusing Dunn’s multiple comparison test identified all postinfectiontime points with the exception of 7–10 DPI to be significantlydifferent (p , 0.05 to p, 0.01) from the uninfected control group(Fig. 1). Data on CD4+ T cell status in the intestine were notavailable for seven animals. This included two animals at 29 DPI(CG32 and CT16) and five macaques that progressed to AIDS(FT11, HL01, AT56, H405, and L441) (Table I).Histologic evaluation of H&E stained sections of colon and

jejunum from all non–SIV-infected animals with diarrhea revealedthe presence of moderate to severe colitis including other intes-tinal lesions such as crypt dilatation/abscess and diverticulosis(Fig. 2D, 2E, Table II). No bacterial pathogens were detected inseven of the eight non–SIV-infected macaques with diarrhea andcolitis. In contrast, the colonic lamina propria of the SIV-infectedmacaques (7 and 14 DPI) showed minimal to no histological signsof inflammation (Fig. 2A–C) and appeared similar to the unin-fected control macaque (Fig. 2F) (Table II).

Acute SIV infection of the intestinal immune system ischaracterized by marked alterations in miRNA expression

To determine whether SIV infection of the GI tract/immune systemwas associated with alterations in miRNA expression we performedglobal miRNA profiling of colon tissue during acute SIV infectionusing the human microRNATLDA cards. As shown in Table I, oneanimal each was at 7, 8, and 10 DPI, three each at 13 and 21 DPI,and one at 29 DPI. After applying multiple comparisons correc-tion (Benjamini–Hochberg adjusted p values for false-discoveryrate) simultaneously to all miRNA probes (cards A and B com-bined), nine miRNAs (four up and five downregulated) wereidentified as statistically significant (adjusted p , 0.05) and dif-ferentially expressed following analysis using DataAssist softwareversion 3.01 (Table V). Raw CT values shown in Table V provideadditional information on the cellular abundance (high, medium,or low) of each differentially expressed miRNA and the extent ofvariation across samples.Among the four upregulated miRNAs, the expression of one

miRNA, namely, miR-190b exceeded 5-fold (∼6-fold) and in termsof magnitude showed the highest increase in expression follow-ing SIV infection (Table V). The expression levels of the remainingthree miRNAs (miR-222, miR-22*, and miR-223*) ranged from1.5- to 3.0-fold compared with the uninfected control group. Theexpression of five miRNAs (hsa-miR-425*, -199a-5p, -221, -324-5p, and -361-5p) decreased ∼1.5- to 1.7-fold during acute SIVinfection (Table V). These findings suggest that early SIV infec-tion is characterized by significant changes in miRNA expressionin the colon and that the profile is slightly dominated by down-regulated miRNAs.

miR-190b upregulation occurs in both colon and jejunum at allstages of SIV infection and its expression follows a trendobserved with peripheral blood viral loads during SIV infection

miR-190b was selected for further characterization as the mag-nitude of increase was the highest among the four upregulatedmiRNAs (Table V) and ranked second based on p values (adjustedp = 0.0032) making it an ideal target for further characterization.As shown in Fig. 3A and 3B, miR-190b expression significantlyincreased in the colon and jejunum of SIV-infected compared withuninfected controls and non-SIV-infected macaques with diarrheaand colitis (labeled as colitis in Fig. 3A, 3B). With the exception

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of the 21 and 90 DPI time points in both colon and jejunum, miR-190b expression at all other time points showed statistical sig-nificance (p , 0.05) (Fig. 3A, 3B). However, comparison of both(21 and 90 DPI) time points separately with uninfected controlsusing the nonparametric Wilcoxon’s rank-sum test or Mann–Whitney U test revealed statistical significance (p , 0.05) (datanot shown) in colon and jejunum. Furthermore, miR-190b ex-pression in the colon and jejunum of non–SIV-infected macaqueswith diarrhea and colitis did not differ from normal uninfectedcontrols (Fig. 3A, 3B). Although not a longitudinal study, someinteresting trends in miR-190b expression were observed in thecurrent study. From Fig. 3A and 3B, it is clearly evident thatenhanced expression of miR-190b occurred as early as 7 DPI (p,0.05) and peaked between 13 and 14 DPI (coincident with peakviremia) (p , 0.01). The initial elevation in miR-190b at 7 DPIoccurred even before there was a significant loss of mucosalCD4+ T cells (Fig. 1). This indicates that the increase in miR-190bexpression occurs before peak viral loads and the nadir of CD4+

T cell depletion. Subsequent to the peak of viremia between 13and 14 DPI, average miR-190b expression dropped by ∼2-fold incolon and ∼3-fold in jejunum at 21–29 DPI. Interestingly, thisabrupt drop in expression at this time point (21–29DPI) coincideswith the nadir of CD4+ T cell depletion, which may account forthe sudden drop in miR-190b expression. At 90 DPI and in ani-mals with terminal disease, miR-190b expression showed an up-ward trend in both colon and jejunum (Fig. 3A, 3B). Interestingly,miR-190b upregulation was not detected in the colon and jejunumof non–SIV-infected macaques with diarrhea and colitis (Fig. 3A,

3B). Because intestinal tissues from the non–SIV-uninfectedmacaques with diarrhea and colitis were collected over a periodof 6–7 y, there was always the possibility that technical batch

Table I. Animal IDs, duration of infection, SIV inoculum and plasma and intestinal viral loads in SIV-infected macaques

Animal IDDuration of

Infection (DPI) InoculumPlasma Viral LoadsCopies/ml (106)

Viral Load - ColonCopies/mg RNA (106)

Viral Load - JejunumCopies/mg RNA (106)

IA85a 7 SIVmac251 NA 0.1 0.4HI52a 8 SIVmac251 4 900 200AV91a 10 SIVmac251 157 50 100M992a 13 SIVmac251 35 200 9HI63a 13 SIVmac251 24 20 10HI58a 13 SIVmac251 9 20 50HC36a 21 SIVmac251 20 1 2HB31a 21 SIVmac251 10 2 1GA19a 21 SIVmac251 2 10 0.5CT16a 29 SIVmac239 NA 4 40HT44 7 SIVmac251 NA 0.4 3T108 8 SIVmac251 0.06 ND NDHV61 14 SIVmac251 NA 0.8 3HV39 14 SIVmac251 NA 0.2 0.9HF27 21 SIVmac251 9 0.06 5HB48 21 SIVmac251 40 300 6GK31 21 SIVmac251 2 2 0.04HR57 21 SIVmac251 6 0.4 1HV95 21 SIVmac251 2 0.2 0.5CG32 29 SIVmac239 NA ND NDHC36 90 SIVmac251 0.1 0.2 NDHB31 90 SIVmac251 70 7 0.97HF27 90 SIVmac251 10 40 2HB48 90 SIVmac251 20 0.1 100GK31 90 SIVmac251 30 1 0.05GA19 90 SIVmac251 100 5 4HR57 90 SIVmac251 80 2 0.04HV95 90 SIVmac251 7 0.5 0.08FT11 145 SIVmac251 500 2,075 1,176HL01 180 SIVmac251 7 0.8 30L441 170 SIVmac239 1.3 3 21H405 232 SIVmac239 71.4 542 758AT56 1460 SIVmac239 360 10,228 1,482

Colon and jejunum tissues from all 33 animals were used for miR-190b qRT-PCR characterization studies.NA, not available; ND, not detectable.aColon tissue from 10 animals (IA85-CT16) was used for genome-wide miRNA expression profiling using the TLDA platform.

FIGURE 1. Percentage of CD4+ T cells among LPLs isolated at different

time points after SIV infection. The cells were first gated on singlets, fol-

lowed by lymphocytes, CD3 and then on CD3+CD4+ T cell subsets. Data

analysis using Kruskal–Wallis test identified significant differences among

the different groups (p = 0.0004). Post hoc analysis using Dunn’s multiple

groups comparison identified 13–14, 21, and 90 DPI time points to be

significantly different from uninfected controls. The error bars represent SE

of mean CD4+ T cell percentage within each group. *p, 0.05, **p, 0.01.

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effects or RNA degradation could have contributed to the lack ofmiR-190b upregulation in this group. To specifically address this is-sue, we collected colon tissues from a second batch of six non–SIV-infected macaques necropsied within the past 6 mo for chronic di-arrhea nonresponsive to treatment (colitis). Similar to the findingsshown in Fig. 3A, miR-190b expression did not increase (Sup-plemental Fig. 2C) and was no different from the uninfected con-trol group. As shown in Supplemental Table I, average miR-190b CT

values in this group were identical to the uninfected controls (24.2versus 24.3). Furthermore, the colonic lamina propria of the SIV-

infected macaques (7 and 13 DPI) did not show any histologicalsigns of inflammation (Fig. 2A–C) and appeared no different from theuninfected control macaque (Fig. 2F). In contrast, both non–SIV-infected macaques with diarrhea (Fig. 2D, 2E) have moderate tosevere colitis (marked inflammatory cell infiltration of the coloniclamina propria along with other lesions such as crypt abscesses),providing strong evidence that inflammation/immune activation is notdriving miR-190b upregulation.The correlation of miR-190b with increased viral replication in

the intestine is supported by the finding that miR-190b expression

FIGURE 2. H&E stained tissue sections of colon from acutely SIV-infected rhesus macaques (A, 7 DPI), (B and C, 13DPI) and non–SIV-infected

macaques with diarrhea (D and E) and a uninfected normal control macaque (F). In the colon of the non–SIV-infected macaques with diarrhea, moderate to

severe colitis is evident from diffuse cellular infiltrates (arrows), crypt dilations, and crypt abscesses (arrowhead) and a relative paucity of goblet cells (D

and E) compared with acutely SIV-infected (A–C) and normal control (F) macaques. Note that the colons of acutely SIV-infected macaques AV91 (A), HI58

(B), and HI63 (C) show minimal to no histological evidence of inflammation. All figures are original magnification 310.

Table II. List of non–SIV-infected macaques with diarrhea and uninfected control macaques used forTLDA and miR-190b qRT-PCR studies

Intestinal Histopathology

Animal ID Colon Jejunum Bacterial Pathogens

SIV-uninfected with diarrheaDJ15 3 and CD 1 NoneDV87 1 and CD NA NoneDV98 3 and CD NA NoneEB12 3 and CD NA NoneJ053 2 1 NoneEJ54 3 1 NoneEL71 3 and CD, DV 0 NoneEB27 3 and CD, DV 1 Shigella flexneri, Campylobacter coli

Uninfected controlsFF15 0 0 NoneFT23 0 0 NoneHT22 0 0 NoneEL66a 0 0 NoneEH70a 0 0 NoneEH80a 0 0 NoneGI92a 0 0 NoneFK25a 0 0 None

Sections of jejunum and colon were examined and inflammation was scored semiquantitatively on a scale of 0–3 asfollows: 0, within normal limits; 1, mild; 2, moderate; and 3, severe. In addition, the presence of crypt dilatation (CD),diverticulosis (DV), villus blunting, and amyloidosis were recorded. NA, not applicable.

aDenotes uninfected control animals used for TLDA miRNA profiling

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levels in both colon and jejunum of two macaques with unde-tectable tissue viral load (T108 [7–8 DPI], CG32 [21–29 DPI],Table I and arrows in Fig. 3A, 3B) were no different from unin-fected controls. Because miR-190b upregulation in intestine wasonly detected in macaques with detectable tissue viral load, wenext examined the statistical correlation between miR-190b ex-pression levels and intestinal tissue viral load. As shown inFig. 4A and 4B, miR-190b expression levels in colon (r = 0.48,p = 0.0024) and jejunum (r = 0.50, p = 0.0017) were positivelycorrelated with viral loads. Finally, the absence of miR-190b up-regulation in the intestines of non–SIV-infected macaques withdiarrhea and colitis suggests that miR-190b is not a generalmarker of intestinal inflammation or immune activation becausethese animals had moderate to severe colitis.

The intestinal lamina propria and not the epithelialcompartment contributes to miR-190b upregulation inSIV-infected macaques

The failure to detect miR-190b upregulation in the intestines ofnon–SIV-infected macaques with diarrhea and colitis (Fig. 3A,3B) suggested a strong link between miR-190b upregulation andviral replication. The association with viral replication also sug-gested that targets of SIV infection such as CD4+ T cells andmacrophages may be necessary to drive miR-190b upregulation. Ifthis hypothesis is true then miR-190b upregulation should occurpredominantly in the LPL and not the epithelial compartment asboth CD4+ T cells and macrophages reside in the LPL compart-ment. To confirm that the LPL compartment of the intestine wascontributing to the upregulation of miR-190b expression in SIV-infected macaques, we quantified miR-190b expression in isolatedintestinal epithelial cells and LPLs using the individual TaqManstem loop qRT-PCR assay specific for miR-190b. As shown inFig. 5A, miR-190b showed a ∼10- and ∼20-fold increase in theLPL compartment, at 21 and 90 DPI (p , 0.05), respectively. Incontrast, no significant increase in miR-190b expression wasfound in the intestinal epithelial compartment at these same timepoints (Fig. 5B). It is important to note that the presence of a smallpercentage (10–15%) of IELs in the isolated epithelial cells is notlikely to impact miR-190b expression as the vast majority of theIELs are CD8+ and not infected by SIV. Similarly, given the com-plete absence of miR-190b upregulation in the epithelium (Fig.5B), the presence of a small percentage of contaminating epithelialcells (10–15%) in LPLs is also not likely to influence miR-190bexpression because they are not infected by SIV and express basallevels of miR-190b like other uninfected intestinal cells.

miR-190b expression was also quantified in PBMCs isolatedfrom the same four animals shown in Fig. 5A and 5B at the sametime points (preinfection, 21 and 90DPI) as intestinal resectionsegments. Although miR-190b expression was considerably ele-vated in the LPL (p , 0.05 at 90DPI) (Fig. 5A), parallel statis-tically significant upregulation was not observed in PBMCs atthe same time points as shown in Supplemental Fig. 2D, suggest-ing that PBMCs may not contain a sufficient proportion of SIV-infected cells to result in miR-190b upregulation.These findings show that under basal conditions, miR-190b is

expressed in both the intestinal epithelium and LPLs. However,after SIV infection its expression is significantly increased onlyin the LPL compartment. This finding along with the correlationbetween intestinal viral load and miR-190b expression and theabsence of miR-190b upregulation in the intestines of non–SIV-infected macaques with diarrhea and colitis (Fig. 3A, 3B) suggeststhat miR-190b upregulation is linked to viral replication.

miR-190b is expressed in CD4+ T cells and macrophages andits expression is significantly increased in in vitro–culturedCD4+ T cells and primary intestinal macrophages in responseto SIV infection

To further explore the linkage between expression of miR-190band viral replication we examined miR-190b expression inCD4+ T cells and macrophages with and without SIV infection.Accordingly, we isolated peripheral blood CD4+ T cells and pri-mary intestinal macrophages and determined miR-190b expressionseparately in both cell types using stem loop qRT-PCR assayspecific to miR-190b. We preferred this approach to miRNA insitu hybridization because qRT-PCR is highly specific and trulyquantitative. As illustrated in Fig. 6, we were able to obtain high-purity macrophage populations that were consistently positivefor macrophage specific markers CD68 (Fig. 6A) and CD163(Fig. 6B) and negative for T lymphocyte markers such as CD3(Fig. 6C). Furthermore, we successfully infected these cellsin vitro with SIVmac251 as demonstrated by the in situ detectionof viral RNA 4 DPI (Fig 6D, 6E). As shown in SupplementalFig. 3, miR-190b was strongly expressed by both CD4+ T cellsand macrophages. Furthermore, as shown in Fig. 3A and 3B,miR-190b expression increased as early as 7 DPI, peaked at 13–14DPI (peak viral replication), and particularly, its abrupt drop inexpression at 21–29 DPI in both colon and jejunum that coin-cided with the nadir of CD4+ T cell depletion strongly suggeststhat CD4+ T cells are likely to be a major cellular source of ele-vated miR-190b expression during acute infection. Nevertheless, as

Table III. Primer sequences used for real time SYBR Green Two-step qRT-PCR

Gene Name Primer Sequence (forward, reverse)ProductSize (bp)

PrimerConcentration (nM)

MTMR6 59-CAGCAGCCTGGCAGATAATCGTT-3959-TAAGCTGACCACAGCAGGTTCTGA-39

76 200

GAPDH 59-CAACAGCCTCAAGATCGTCAGCAA-3959-GAGTCCTTCCACGATACCAAAGTTGTC-39

100 200

Table IV. Schematic representation of MTMR6 39-UTR depicting predicted binding site for miR-190b

GeneGenBank

Accession No. Site ConservationBinding Siteson 39-UTR Target Site Sequence Prediction Algorithm

MTMR6 NM_004685 Human/chimp/macaque/orangutan/mouse

1239–1245 59...AUUCUGUGUACUAGUACAUAUCU... 39 TargetScan (48),miRanda (54)|||||||

39 UUGGGUUAUAGUUUGUAUAGU...59

Alignment of MTMR6 mRNA sequence with miR-190b: top strand, MTMR6 mRNA; bottom strand, miR-190b.

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previously published (51), and as illustrated in Fig. 6F, SIV in-fection of intestinal macrophages in situ can occur as early as 10DPI.To conclusively verify that miR-190b was upregulated in re-

sponse to SIV replication, we infected peripheral blood CD4+

T cells and primary intestinal macrophages with SIV and found

significant upregulation of miR-190b in CD4+ T cells (Fig. 7A)and macrophages (Fig. 7B) by days 2 and 4 postinfection, re-spectively. miR-190b expression in CD4+ T cells was normalizedto a combination of RNU44 and RNU48 as it yielded better sta-tistical significance (p = 0.0121) compared with RNU44 alone(p = 0.0367). These findings clearly demonstrate that SIV-infected

Table V. Differentially expressed miRNAs in colon during acute SIV infection

miRNA ID

Acute SIV (n = 10) Uninfected Controls (n = 5)Fold

ChangeAdjustedp ValueAV91 HI52 HI58 HI63 M992 CT16 IA85 HC36 HB31 GA19 EL66 EH70 EH80 GI92 FK25

hsa-miR-425* 29.3 28.8 29.3 28.7 29.5 29.1 26.8 28.0 27.7 27.9 28.2 28.6 28.4 29.4 29.0 21.7 0.0024hsa-miR-190b 23.0 23.4 23.0 21.8 22.5 23.1 21.1 22.7 22.7 22.0 25.2 24.7 24.8 26.0 26.4 6.0 0.0032hsa-miR-222 15.6 15.4 15.7 15.1 15.2 15.4 14.3 14.7 14.5 14.7 15.7 15.9 15.5 16.4 16.7 1.5 0.0114hsa-miR-199a-5p 25.3 24.5 25.6 25.4 25.8 24.4 23.8 24.7 24.7 24.4 24.3 24.3 23.8 24.9 24.6 21.7 0.0376hsa-miR-22* 23.4 22.9 23.0 22.5 23.0 23.3 22.2 23.1 22.8 22.6 23.3 23.4 23.4 24.4 24.5 1.5 0.0376hsa-miR-221 21.5 20.9 21.0 21.1 22.0 21.1 20.7 20.7 21.3 21.0 20.4 20.5 20.0 21.3 21.3 21.6 0.0376hsa-miR-223* 26.7 26.1 25.4 25.6 25.0 26.6 24.7 25.0 24.4 24.5 26.5 27.2 26.7 27.0 29.0 2.8 0.0376hsa-miR-324-5p 22.8 22.5 23.0 22.5 23.3 22.3 21.5 22.6 22.7 22.7 22.0 22.2 21.6 22.7 22.7 21.6 0.0376hsa-miR-361-5p 22.0 22.2 22.5 22.0 22.4 22.0 21.6 22.2 22.1 22.1 21.5 21.6 21.1 22.6 22.3 21.5 0.0376

The table shows raw CT and fold change for all differentially expressed (adjusted p , 0.05) miRNAs after applying multiple comparisons correction (Benjamini–Hochbergadjusted p values for false-discovery rate) in the colon of 10 acutely SIV-infected macaques.

FIGURE 3. Elevated miR-190b expression in the

colon (A) and jejunum (B) during SIV infection com-

pared with uninfected normal controls and non–SIV-

infected macaques with diarrhea (indicated as colitis).

Note the absence of miR-190b upregulation in non–

SIV-infected macaques with diarrhea and colitis, sug-

gesting that upregulation of miR-190b is not inflam-

mation driven but occurs in response to viral replication.

Arrows in colon (A) and jejunum (B) point to two animals

(T108 [7 DPI] and CG32 [21–29 DPI]) that had no de-

tectable SIV in intestinal tissue and where the levels of

miR-190b was no different from controls. The error bars

represent SE of mean fold change within each group. Data

analysis using nonparametric Kruskal–Wallis test revealed

differences among groups in both colon (p = 0.0002) and

jejunum (p, 0.0001). Asterisks (*p, 0.05, **p, 0.01)

indicate groups that showed statistical significance com-

pared with uninfected controls following Dunn’s multiple

groups comparison test.

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CD4+ T cells and macrophages directly contribute to miR-190bupregulation. In addition, the data also suggest that although SIV-infected CD4+ T cells are likely to be the primary cellular sourceof miR-190b uregulation during early acute infection (7–10 and13–14 DPI), SIV-infected macrophages could contribute signifi-cantly to miR-190b upregulation at later time points and chronicinfection.

miR-190b can directly regulate the expression of MTMR6 andin doing so potentially play an important role in SIVpathogenesis

To investigate the significance of increased miR-190b expression inresponse to SIV infection, we examined potential targets of miR-190b. TargetScan (version 6.2) (48) lists a total of 186 predictedtargets for mml-miR-190b. Among these is MTMR6, which hasbeen shown to negatively regulate CD4+ T cell activation andproliferation by inhibiting Ca2+-dependent activated potassiumchannel KCa3.1, thereby reducing Ca2+ influx (52). Furthermore,KCa3.1 is also required for macrophage activation (53). Conse-quently, MTMR6 represented a very interesting mR-190b targetfrom the perspective of CD4+ T cell/macrophage activation andHIV replication. Interestingly, MTMR6 mRNA expression de-creased significantly (p = 0.013) in cultured intestinal macro-

phages 4 d post-SIV infection (Fig. 7C). As shown in Fig. 7D,cotransfection of pmirGLO-WT-MTMR6 with miR-190b mimic re-sulted in significant reduction in Firefly/Renila ratios (p = 0.0069).In contrast, cotransfection of pmirGLO-MUT-MTMR6 with miR-190b restored the Firefly/Renila ratios to the level observed withunmanipulated pmirGLO vector (Fig. 7D). These results clearlyshow that miR-190b can physically interact with the 39-UTR andpotentially regulate the expression of MTMR6. The precise effect ofthis regulation on target cell function including its direct or indirectimpact on viral replication in infected cells needs future investigation.

DiscussionHIV/SIV infection is associated with robust viral replication in theintestinal immune system, depletion of mucosal CD4+ T cells andmarked immune activation. The dramatic loss of mucosal CD4+

T cells in the intestine has also been associated with intestinalinflammation, damage and microbial translocation. These changesare associated and in some cases preceded by marked changes ingene expression (7–10). Regulation of gene expression associatedwith the host response to infectious agents is complex and our

FIGURE 4. Correlation of miR-190b in colon (A) and jejunum (B) with

viral loads. A positive statistical (p , 0.05) correlation between miR190b

and SIV viral load was found in both colon and jejunum.

FIGURE 5. The LPL and not the epithelial compartment of the intestine

is the predominant source of miR-190b upregulation in SIV-infected

macaques. Statistically significant increase in miR-190b expression was

detected in the intestinal lamina propria at 90 d post-SIV infection (DPI)

(A) but not in the intestinal epithelium (Epith) (B). Data were analyzed

using nonparametric Kruskal–Wallis test, and post hoc multiple groups

comparison was performed using Dunn’s test. *p , 0.05 compared with

preinfection samples.

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understanding of this process has for the most part been dom-inated by the role played by transcription factors, coacti-vators, corepressors, chromatin modifiers, histone modulators,and so on. The discovery of miRNAs has added another layerof complexity to the standard linear concept of DNA beingtranscribed to mRNA which is then translated to protein.While the regulatory role of miRNAs in immune cell develop-ment including several immunopathological and inflammatoryconditions (18, 22, 23) is receiving a lot of attention, their con-tributions to regulating HIV/SIV replication and host respon-ses, particularly, in the GI tract, an important site of viralreplication, CD4+ T cell depletion and viral persistence remainunknown and unexplored. In the current study, using the rhesusmacaque model of AIDS we investigated genome wide changesin miRNA expression to better understand their regulatory rolein the GI tract following SIV infection. We performed theinitial high throughput miRNA profiling on the colon for nu-merous reasons. First, similar to the jejunum, CD4+ T celldepletion has been well documented to occur in the colon (2,55). Second, from our previously published studies (13, 14) it isclear that although SIV affects the jejunum, the colon is moreseverely impacted. Third, the increased concentration of bac-teria in the colon (1012 bacterial organisms per milliliter ofcontents) (56) makes it an important source of intestinalbacteria/bacterial products that is well documented to translo-cate into the systemic circulation via a disrupted intestinal epi-thelial barrier leading to chronic immune activation and AIDSprogression (6). To our knowledge, the present study for the firsttime describes dysregulated miRNA expression in the GI tract inresponse to SIV infection. We specifically, identified miR-190bto be significantly upregulated in both colon and jejunum ofacutely SIV-infected macaques. Further, we show that miR-190bupregulation is driven by viral replication and not by the immune/inflammatory processes occurring in response to viral replication.Furthermore, our results provide new insights into the role of miR-

190b in regulating host cellular responses, particularly, sustainingthe activated state in SIV-infected cells by inhibiting MTMR6 ex-pression.In recent years, miRNAs have emerged as potent regulators

of immune and inflammatory responses (20–22). Consistent withthese findings and after applying multiple comparisons correctionwe identified a total of 9 miRNAs (Table V) to be markedly al-tered during acute SIV infection. Among these, eNOS suppressingmiR-222 has been linked to regulating vascular inflammation(57) and differentiation of dendritic cells (58). Downregulation ofmiR-199a-5p promoted wound angiogenesis via derepression of theEts-MMP1 pathway (59) suggesting that its reduced expression inthe intestine may be part of the normal host response to repairtissue damage associated with early viral replication. As observedin the current study, miR-221 expression reduced significantly inbronchial epithelial cells in response to respiratory syncytial virusinfection (60). Surprisingly, miR-190b exhibited the highest in-crease in expression following SIV infection and although re-ported to exhibit altered expression in several cancer studies (61,62) has not been previously linked to an infectious disease. How-ever, miR-190/190a, a closely related miRNA originating from adifferent chromosome with distinct primary and precursor sequen-ces has been well characterized (63, 64). Both miR-190 andmiR-190b share identical seed regions and hence have the samepredicted targets. Taken together, acute SIV infection of the GItract results in altered miRNA expression and the markedly ele-vated miR-190b expression as early as 7 d post-SIV infection sug-gests an important role for this miRNA in SIV pathogenesis.As mentioned above, the lack of previously published studies

linking miR-190b to an infectious disease combined with the factthat it showed the highest fold increase among the nine differen-tially expressed miRNAs prompted us to further investigate miR-190b expression at different stages of SIV infection. Surprisingly,miR-190b expression significantly increased in both colon andjejunum as early as 7 DPI. Furthermore, its expression peaked at

FIGURE 6. Characterization of cultured intestinal macrophages. In vitro cultured primary intestinal macrophages express classical macrophage markers

such as CD68 (A) and CD163 (B) but do not express T cell markers such as CD3 (C). All three panels are double labels with CD68 and CD163 in green and

nuclear labeling with Topro3 in blue. Intestinal macrophages were infected with SIVmac251 and in situ hybridization confirmed the presence of viral RNA

(D). Uninfected cells are negative for SIV (E). Both panels involve double labels with viral RNA (red) and Bopro1 (green) for nuclear staining. SIV-infected

macrophages can be detected in the intestine as early as 10 d post-SIV infection (F). Arrows point to SIV-infected (red) macrophages that express CD68

(blue), CD163 (green), or both.

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13–14 DPI (time of peak viral replication), dropped abruptly at21 DPI (coinciding with nadir of CD4+ cell depletion) and thengradually increased as disease progressed (90 DPI and AIDS).These findings are certainly noteworthy and a longitudinal study isdefinitely needed in the future to firmly associate alterations inmiR-190b expression level with key pathogenic events such aspeak viral replication and nadir of CD4+ T cell loss. Subsequently,qRT-PCR quantification of miR-190b expression separately in theintestinal epithelium and LPLs helped us to decisively localize thesource of miR-190b upregulation to the immune cells residing inthe lamina propria. Since viral replication in the intestinal laminaprorpia elicits an immune/inflammatory response there was defi-nitely the possibility that inflammatory cell infiltration in responseto SIV replication may be contributing to miR-190b upregulation.Accordingly, we included a group of macaques with GI disease

(non–SIV-infected with diarrhea and colitis) to determine whetherinflammatory cell infiltration or SIV replication was driving miR-190b expression as this group of animals has moderate to severecolitis, localized immune activation and consequently massivedisruption of the intestinal epithelial barrier. Interestingly, thecomplete absence of miR-190b upregulation in the colon and je-junum of non–SIV-infected macaques with diarrhea and colitisstrongly suggested that SIV replication in the lamina propriatarget cells likely provides the stimulus to drive miR-190b up-regulation. More importantly, the latter finding undoubtedlymeans that shifts in immune cell composition (infiltration by in-flammatory cells) occurring in response to SIV replication doesnot account for the observed increase in miR-190b expression.Finally, infection of in vitro cultured peripheral blood CD4+

T cells and primary intestinal macrophages with SIV conclusively

FIGURE 7. miR-190b expression was markedly elevated in in vitro cultured peripheral blood CD4+ T cells (A) (p = 0.0121) and primary jejunal

macrophages (B) 2 and 4 d post-SIV infection, respectively, suggesting that its upregulation occurs in response to SIV replication. Data were analyzed using

nonparametric Wilcoxon’s rank-sum test for independent samples. The error bars represent SE of mean fold change within each group. *p, 0.05 compared

with uninfected samples. MTMR6 is a direct target of miR-190b (C and D). miR-190b physically associates with the 39-UTR of MTMR6 mRNA. mRNA

expression of MTMR6 is significantly decreased (p = 0.013) in intestinal macrophages 4 d post-SIV infection (C). The error bars represent SE of mean fold

change within each group. Data were analyzed using nonparametric Wilcoxon’s rank-sum test for independent samples. *p , 0.05 compared with un-

infected macrophages. miR-190b physically associates with the 39-UTR of MTMR6 mRNA (D). The MTMR6 39-UTR sequences, WT or MUT, were

inserted into the multiple cloning sites situated in the 39-end of firefly luciferase gene in the pmirGLO vector. HEK293 cells were cotransfected with 100

nM miR-190b mimic and 100 ng luciferase reporter constructs containing WT- or MUT-MTMR6 39-UTR sequences. Firefly and Renila luciferase activities

were detected using the Dual-Glo luciferase assay system 48 h after transfection. The ratio of luciferase activities (Firefly/Renilla) was calculated and

normalized to the wells transfected with only unmanipulated pmirGLO vector. **p , 0.01.

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revealed the SIV-infected cell as the primary cellular source ofmiR-190b upregulation. On the basis of these findings, we canconfidently conclude that miR-190b upregulation occurs primarilyin response to SIV replication and is not simply a nonspecific resultof immune activation and inflammation.To gain further insight into the biological significance of miR-

190b upregulation we next focused on MTMR6, a predictedmRNA target of miR-190b. We focused on MTMR6 as a directmiR-190b target because of its central importance to CD4+ T cell/macrophage activation/proliferation (52, 53). MTMR6 is a phos-phatidylinositol-3 phosphatase that has been well demonstrated tonegatively regulate Ca2+ influx by inhibiting the calcium-dependentactivated potassium channel KCa3.1, thereby, preventing CD4+

T cell activation and proliferation (52). In addition to T cells,KCa3.1 channel activity is also critical for macrophage activation(53). KCa3.1 channel activation requires phosphatidylinositol-3phosphate PI(3)P and MTMR6 downregulates its activity by de-phosphorylating PI(3)P (52). This is evident from studies wheresiRNA mediated silencing of MTMR6 in CD4+ T cells resulted inincreased KCa3.1 channel activity, enhanced calcium influx andfacilitated T cell activation with 10-fold less Ag concentrationcompared with untreated cells (53). In the current study, MTMR6mRNA expression decreased significantly in in vitro cultured pri-mary intestinal macrophages 4 d post-SIV infection. Additionally,the luciferase reporter assay clearly demonstrated the existence ofa physical interaction between miR-190b and the 39-UTR ofMTMR6 and provided strong evidence for a miR-190b–mediatedMTMR6 gene silencing in SIV-infected macrophages. The latterpossibility is strongly supported by the recent study that demon-strated miRNA mediated destabilization of mRNAs as a majormechanism that accounted for .84% of the reduced protein output(65). It has been proposed that MTMR6 functions constitutivelyto tonically inhibit KCa3.1 channel activity and in doing so setsa threshold stimulus for T cell activation (52). Such a mechanismcan be expected to be operational in activated colonic CD4+ T cellsand macrophages of non–SIV-infected macaques with diarrhea andcolitis maintaining basal miR-190b expression levels. Nevertheless,in SIV-infected intestinal CD4+ T cells and macrophages, miR-190blevels were markedly elevated, which in turn provides an additionallayer of regulation to augment KCa3.1 activity by reducing MTMR6levels so that the constitutive Ca2+ influx required for increased cyto-kine production and subsequent immune/inflammatory responses aresustained. Although this represents a protective host response to cur-tail the spread of the virus, paradoxically, miR-190b–mediated silenc-ing of MTMR6 can invariably secure the infected cell in an activatedstate ultimately promoting viral replication. Collectively, these findingssuggest that miR-190b upregulation may be part of the normal cellularstress response to viral replication and its targeting of MTMR6 maybe to promote cellular survival, which eventually benefits the virus.To our knowledge, this is the first report describing genome wide

changes in miRNA expression in the GI tract in response to HIV/SIVinfection. The fact that miR-190b is markedly elevated predominantlyin response to SIV infection suggests important roles for this miRNAin regulating host cell responses to SIV replication. Although wehave identified and confirmedMTMR6 as a direct target of miR-190b,additional studies employing high throughput approaches are neededto validate and identify the roles of the remaining 185 predictedtargets in regulating virus-host cell interactions. It is also intriguingto know whether miR-190b induction is directly triggered by viralproteins or indirectly by host cellular factors responding to viralreplication. To gain detailed insight miR-190b knockdown studies toevaluate its role in viral replication and host cell response are alsoneeded. Finally, although miR-190b expression levels increased inresponse to SIV replication it cannot be concluded that its upregulation

is specific to SIV. Future studies are also needed to determine whethermiR-190b expression levels increase in response to infection withother viruses.

AcknowledgmentsWe thank Ronald S. Veazey, Maurice Duplantis, Vinay Kumar, Yun Te Lin,

Faith R. Schiro, Cecily C. Midkiff, Stephanie Feely, and Robin Rodriguez

for technical assistance in the study.

DisclosuresThe authors have no financial conflicts of interest.

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