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Michael B. Wallace, Section Editor

olecular Imaging of Murine Intestinal Inflammation With-Deoxy-2-[18F]Fluoro-D-Glucose and Positron Emission Tomography

ARAH BREWER,* MICHAEL MCPHERSON,‡ DAISUKE FUJIWARA,* OLGA TUROVSKAYA,§ DAVID ZIRING,� LING CHEN,*IDETOSHI TAKEDATSU,¶ STEPHAN R. TARGAN,¶ BO WEI,* and JONATHAN BRAUN*,‡

Department of Pathology and Laboratory Medicine, ‡Molecular Biology Institute, and �Department of Pediatrics, University of California Los Angeles, Los Angeles;

La Jolla Institute for Allergy and Immunology, San Diego; and ¶Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, Los Angeles, California

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ackground & Aims: 2-Deoxy-2-[18F]fluoro-D-glucoseFDG) uptake by positron emission tomography (PET),measure of glucose transporter activity, has been used

o detect mucosal inflammation. However, there is lim-ted understanding of the biologic basis of mucosalDG uptake. Methods: A contrast-based computed to-ographic isocontour method was developed to iden-

ify intestinal anatomic regions, and FDG uptake wasntegrated over these regions to achieve reproducibleuantification during longitudinal assessment of indi-idual mice. Intestinal FDG uptake was compared withistologic scores and with glucose transporter 1 levels inucosal immune cells by flow cytometry. Results: In-

estinal FDG uptake quantitatively correlated with dis-ase activity in mild (C3H/HeJ.IL-10�/�) and severe129.G�i2�/�, CD4� CD45RBhigh, and G�i2�/� CD3�

ransfer) murine colitis models at all time points exam-ned (P < .05) and was sufficiently sensitive to detectreclinical inflammation. FDG uptake was correlatedy flow cytometric detection of glucose transporter 1

evels in mucosal CD4� T lymphocyte but not otherntestinal immune cell types. CD4� T-cell transfer inivo confirmed that mucosal FDG uptake was associ-ted with the activated but not quiescent state. Whenntestinal inflammation was increased by treatmentith piroxicam and decreased with anti-TL1A treat-ent, FDG uptake was correspondingly altered.onclusions: This study clarifies the cellular basis ofDG signal in intestinal inflammation and introducesomputed tomographic isocontour analysis of FDG-ET imaging for standardized quantitation of immuneolitis.

nflammatory bowel disease reflects the disruption ofthe homeostasis between intestinal immune cells and

ommensal enteric bacteria.1,2 The localization and mode

ASTROENTEROLOGY 2008;135:744 –755

f inflammation within the gastrointestinal tract fluctu-tes over the course of disease in inflammatory bowelisease and is further complicated by progression toucosal destruction, fibrosis, stricture, or perforation.3

hese different phases and types of inflammation occurecurrently or even concurrently in different intestinalegments, complicating the interpretation of biopsy orndoscopic examination.

Noninvasive methods that biologically categorize anduantitate inflammation can enhance the study of dis-ase pathogenesis and may refine assessment and treat-ent planning in the management of intestinal inflam-ation.3 As a molecular imaging technique, positron

mission tomography (PET) is used to visualize a varietyf in vivo biological processes, including cell interactions,ene expression, and drug metabolism.4 Clinically, 2-eoxy-2-[18F]fluoro-D-glucose (FDG) has emerged as an

mportant molecular biomarker, particularly in PET im-ging of cancer, based on increased glucose transportctivity in the malignant state. Similarly, immune cellctivation requires increased glucose import, primarilyhrough glucose transporter 1 (Glut-1), and a corre-ponding acceleration of glycolysis to meet its new energyequirements. Glut-1 synthesis and translocation to theell surface are mediated though the phosphatidylinosi-ol 3-kinase and Akt pathways,5 including antigen-medi-ted T-cell receptor signaling.6

Accordingly, imaging of FDG uptake by activated lym-hocytes is an appealing strategy to detect immune-ediated inflammation in vivo. Early studies showed

hat concanavalin A or turpentine oil resulted in anncreased FDG uptake by lymphoid and granulation tis-ues.7,8 FDG-PET permitted monitoring of inflammationnd therapeutic intervention in experimental autoim-

Abbreviations used in this paper: CT, computed tomography; FDG,-deoxy-2-[18F]fluoro-D-glucose; Glut-1, glucose transporter 1; ID, in-

ected dose; IL, interleukin; PET, positron emission tomography; ROI,egion of interest; SUV, standard uptake value.

© 2008 by the AGA Institute0016-5085/08/$34.00

doi:10.1053/j.gastro.2008.06.040

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une encephalomyelitis.9 FDG-PET has previously beensed to assess intestinal inflammation in murine anduman subjects. Clinical studies have reported the abilityf FDG-PET to detect intestinal lesions visualized byolonoscopies or histology,10 –13 and a murine study eval-ating free FDG and FDG-tagged white blood cells dis-inguished colitic and healthy mice.14 In the latter study,ntestinal signal from tagged white blood cells but notree FDG correlated with intestinal inflammation. Thus,here is little information defining the biologic correlatesf increased intestinal FDG uptake in inflammatoryowel disease.

In the present study, genetic murine models of inflam-atory bowel disease were used to refine the methodol-

gy of FDG-PET and to clarify the cellular basis of thisoninvasive assessment of immune colitis. To addresshe relationship of FDG uptake to categorical and lon-itudinal changes in disease activity, we used interleukinIL)-10�/� to assess mild colitis and G�i2�/�, CD4�

D45RBhigh, or G�i2�/� CD3� transfer mice to examineevere colitis, respectively. We also used nonsteroidal anti-nflammatory drug (NSAID) treatment to exacerbate co-itis and anti-TL1A to alleviate inflammation to test theensitivity of FDG uptake to short-term changes in in-ammation. Finally, we isolated mucosal immune cellypes and analyzed their Glut-1 expression by flow cy-ometry to evaluate the cellular sources of the intestinalDG signal. Our findings establish a standardized meth-dology for FDG uptake that reflects both disease-asso-iated inflammation and intestinal activity that precedeslinical inflammation. They also show that increased ex-ression of Glut-1 in CD4� T cells is an excellent corre-

ate for the FDG signal in these settings of chronic colitis.

Materials and MethodsMiceC3H/HeOuJ, IL-10�/� (C3Bir.129P2(B6)-Il10tm1Cgn/

t)15 mice were obtained from Jackson Laboratory (Bararbor, ME). G�i2�/� and G�i2�/� (129Sv background)ice16 were bred at the UCLA Department of Laboratory

nd Animal Medicine. Unless otherwise specified, ani-als examined were all female and were age matched to

ontrols within each experiment. All procedures involv-ng animals were performed under approved protocols ofhe UCLA Animal Research Committee.

For G�i2�/� CD3� transfer experiments,17,18 lympho-ytes were harvested from the spleen and mesentericymph nodes and G�i2�/� CD3� T cells were isolatedsing CD90 positive selection (Miltenyi Biotec, Auburn,A). Eight-week-old B6.129 RAG1�/� mice (Jackson Lab-ratory) were injected intravenously with 1 � 106 cellsuspended in 100 �L saline. Anti-TL1A or isotype control

ntibody (500 �g) was administered intraperitoneally on S

he day of T-cell transfer and once weekly for 4 weeksefore PET scans and terminal histology.

For CD4� CD45RBhigh transfer experiments,19,20 CD4�

cells were isolated from Balb/cJ spleen (Charles River,ilmington, MA) using a CD4� T-cell isolation kit

Miltenyi Biotec) and sorted into CD45RBhigh andD45RBhigh�low populations using 2-color fluorescence-ctivated cell sorting (Becton Dickinson FACSVantageE Sorting Flow Cytometer; San Jose, CA). TheD45RBhigh population was �98% pure. C.B-17 scid miceere intravenously injected with 8 � 105 CD4�

D45RBhigh or CD4� CD45RBhigh�low T cells resuspendedn 100 �L saline. Blood samples from mice at 2-weekntervals posttransfer were used to perform blood countserformed by the UCLA Division of Laboratory Animaledicine diagnostic laboratory.For piroxicam experiments,21,22 4-week-old IL-10�/�

ice were dish fed (ad libitum) mouse chow supple-ented with piroxicam (200 ppm, by geometric dilutionith powdered mouse chow) or plain ground chow (neg-tive control) for either 1 or 2 weeks.

Cellular Assessment by Flow Cytometry andHistologic StudiesSmall intestine (including jejunum and ileum)

nd large intestine (including cecum and colon) werearvested from euthanized mice. Superficial lamina pro-ria cells were isolated from these compartments as pre-iously described.23 In some cases, tissue was paraffinmbedded and sectioned, stained with H&E, and scoredy an experienced research pathologist using a histologiccoring system for inflammation and epithelial parame-ers.20

For flow cytometry, lamina propria cells were stainedsing standard 96-well microtiter plate staining tech-ique. For Glut-1 analysis, cells were blocked with 5%oat serum (Sigma Chemical Co, St Louis, MO) for 30inutes, plated at 1 � 106/well (100 �L), incubatedith 0.75 �g of anti–GLUT-1 (ab14683; Abcam, Cam-ridge, MA) for 30 minutes, and then incubated withther surface markers for an additional 30 minutes.ashed cells were incubated with 0.5 �g goat anti-

abbit immunoglobulin (Jackson Immunotech, Westrove, PA) for 30 minutes, washed, and fixed in 200 �L% paraformaldehyde. Anti-mouse monoclonal surfacearker antibodies for CD4, CD8a, CD3, and CD11bere from PharMingen-BD (San Diego, CA) and F4/80ntibody was from eBioscience (San Diego, CA). Iso-ype- and species-matched antibodies were used asegative controls. Data acquisition was performed us-

ng a BD FACSCalibur (BD Biosciences, San Jose, CA)nd analyzed using FCS Express 3 software (DeNovo

oftware, Ontario, Canada).

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StatisticsStatistical analysis used GraphPad Prism software

GraphPad Software, San Diego, CA). Comparison of 2roups was performed by 2-sided Student t test with 95%onfidence interval. Linear regression was used to com-are standard uptake value (SUV) and background sub-racted Glut-1 mean fluorescence intensity values, or per-ent injected dose (ID)/g and histology score, derivedrom individual mice (Prism software; www.graphpad.om). For all statistical tests, significance was defined as� .05.

microPET and microCT Scanning andAnalysisFDG synthesis was performed at the UCLA Cyclo-

ron Facility. Mice were fasted for at least 4 hours beforeracer injection. Age-matched experimental groups werenesthetized with 2% isoflurane gas before 200 �Ci FDGntraperitoneal injection, throughout the 1-hour uptake,nd during scans. For conscious uptake scans, mice werenesthetized 1 hour following injection. For studies re-uiring small intestine analysis, mice were oral gavagedith 100 �L of diatrizoate meglumine and diatrizoate

odium solution (MD-Gastroview; Mallinckrodt Inc, Stouis, MO) 1 hour before PET scanning. For studies

nvolving large intestines, mice were also administered00 �L MD-Gastroview rectally via a 3.5F catheter im-ediately before scan initiation. For all studies, eachouse’s bladder was expressed to minimize bladder sig-

al interference due to normal excretion pathways. Miceere imaged using the microPET FOCUS 220 PET scan-er (Siemens Preclinical Solutions, Knoxville, TN) andicroCAT II CT scanner (Siemens Preclinical Solutions)

n the Crump Institute for Molecular Imaging at UCLA.ach mouse experienced a 10-minute static PET scan

ollowed by an 8-minute computed tomographic (CT)can on 30°C heated scan beds. Upon completion ofoth scans, mice were allowed recovery time before beingeturned to vivarium.

Aligned PET and CT overlays were imported into AMIDEamide.sourceforge.net) for analysis. Using a combinationf whole-body CT cropping and contrast-based isocontouregion of interest (ROI) definitions, small and large intes-ines ROIs were created (Figure 1). Individual decay-cor-ected FDG dosage, cylinder calibration factor, and animaleight were inputted into AMIDE for each mouse. PET

tatistics for ROIs were exported from AMIDE, and theean per voxel SUV and percent ID/g were graphed and

nalyzed. SUV is distinct from percent ID/g by taking intoccount the weight of the animal, which can vary dramat-cally in mice with colitis. For ROIs that were not continu-us, a combined value was obtained through a weighedverage as follows: (Meani � Number of Fraction Voxelsi)/

Number of Fraction Voxelsi). m

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ResultsIdentifying and Quantifying FDG Uptake inthe Small and Large IntestinesThe intestine is a large, serpiginous, and mobile

rgan, and thus it is a challenge to create an objective andeproducible method to define regions of the intestineor microPET imaging. Clinically, intestinal segments arefficiently visualized by ingestion of x-ray contrast mate-ial. Adopting this approach, we found that a combina-ion of gavaged and rectally administered intestinal con-rast agent successfully imaged the intestines by CTFigure 1A).

A second issue was to establish a standardized ROI forptake measurements. Predefined ROIs could not besed, because the intestinal diameter, curvature, position,nd length are dynamic and change even in a singleouse over time. As our first approach, we used a series

f adjacent cylindrical ROIs across the intestines. How-ver, this approach was labor intensive, was subjective,nd underestimated the PET signal mean. Instead, wesed the isocontour feature of an image analysis program

AMIDE) to highlight all areas above a CT thresholdalue. This resulted in an ROI containing the skeletonnd digestive system, from which the skeleton was digi-ally removed (Figure 1B). As a further refinement, thehole intestine ROI was segmented using the cecum as

he boundary to separate the small and large intestine forndividual analysis (Figure 1C).

FDG uptake was represented using 2 values. First,

igure 1. Definition of intestine ROI using AMIDE isocontour functionnd contrast agent. (A) CT imaging of mice with and without contrastgent. (B) Default CT isocontour ROI generated with contrast agent.C) Processed whole intestines ROI separated into large and smallntestines’ ROIs.

ean percent ID/g was determined as a direct represen-

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ation of mean uptake for small and large intestine ROIs.owever, several factors affect intestine segmental ROIs:

etained contrast agent volume, mouse age, inflamma-ion-induced intestinal changes, gavage processing time,nd mass of luminal contents. Therefore, we also calcu-ated mean SUV, which corrects for these factors byepresenting percent ID/g on a per-voxel basis. Together,his set of detection and analytic procedures created aeproducible and reliable methodology to measure FDGignal in various intestinal regions.

Relationship of FDG Uptake andInflammation in G�i2�/� MiceG�i2�/� mice develop spontaneous colitis with

ariable age of onset that originates in the cecum androgresses over time distally into the large intestine.hile a mild form of ileitis is often apparent, the pre-

ominating phenotype (fibrosis, lymphocyte inflamma-ion, crypt abscess formation, mucus loss) as measuredy histology is restricted to the large intestine.16 Weherefore evaluated whether FDG uptake in intestinalOIs could distinguish colitic G�i2�/� mice from age-atched littermate heterozygous controls. In early trials,

t became apparent that G�i2�/� mice had increasedusceptibility to death when anesthetized during the-hour uptake of FDG. Although less ideal due to in-reased muscle uptake of FDG, conscious uptake wassed in these mice (and controls) to decrease mortalityuring scans.As seen in Figure 2A and B, a clear distinction in FDG

ignal was readily observed by unaided comparison ofseudo-colored heat maps of FDG signal. Small and large

ntestine ROIs were quantitated for mean SUV and per-ent ID/g signal of single terminal scans in individual 12-o 24-week-old G�i2�/� and control G�i2�/� mice (Fig-re 2C). The FDG values of control G�i2�/� mice had

ow FDG uptake and clustered tightly regardless ofeight or age. In contrast, FDG uptake was heteroge-eous among individual G�i2�/� mice, with an increasedean FDG uptake in this group that was statistically

ignificant compared with controls. Because the timingnd degree of disease onset in G�i2�/� mice are variable,he heterogeneity of FDG uptake suggested that it mighteflect the authentic variation in disease state. A similarattern of increased FDG uptake was also observed in�i2�/� small intestine. This was unexpected, because

linical or histologic enteritis of the small intestine is notrominent in this model. In young knockouts beforenset of disease, FDG uptake was comparable to age-atched controls. Thus, the FDG signal was inflamma-

ion specific (data not shown).Severe colitis can be induced by transfer of G�i2�/�

D3� T cells into RAG�/� recipients but with the advan-age of more predictable onset of disease.18 TL1A is a

umor necrosis factor–like molecule that promotes lym- o

hocyte activation in the intestinal mucosa. Colitis isssociated with elevated TL1A in inflamed mucosa,24,25 andargeting this molecule reduces the severity of intestinalnflammation.26 We therefore tested the relationship of an-i-TL1A treatment with histologic inflammation and FDGptake.The anti-TL1A–treated mice had significantly lower

DG uptake compared with mice treated with controlntibody by visual inspection (Figure 3A and B) and byuantitation using percent ID/g and SUV (Figure 3C).levated histologic scores in both sets of mice showed theresence of severe colitis. However, both percent ID/gnd histologic score for colitis were decreased in anti-L1A–treated mice (Figure 3D). The correlation of re-uced histologic score and FDG signal with this anti-

nflammatory agent was highly significant (r2 � 0.92, P �003), showing that FDG-PET reflected histologic assess-

ent of response to treatment.

FDG Uptake in Young IL-10�/� Mice WithMild Immune ColitisWhile FDG-PET distinguished colitic mice from

ontrols in a severe model of colitis, we investigatedhether the same divergence would be observable in aodel of milder colitis. Mice bearing the IL-10�/� geno-

ype on a susceptible background predictably developild spontaneous colitis at a young age (�6 weeks).15

hese traits allowed us to follow disease and evaluateDG uptake longitudinally in individual mice. Usingnconscious FDG uptake, tolerated by IL-10�/� mice, wenalyzed 3 independent sets of mice, serially measuredultiple times from early age (5– 6 weeks) to mature

dult age (13–14 weeks).In C3H/OuJ mice, FDG uptake for both small intes-

ine and large intestine was high in very young mice (5– 6eeks), which declined to a lower level in adult mice (�9eeks) (Figure 4C and D). This finding agreed with highbundance and subsequent decline of activated T cellsuring normal intestinal ontogeny.27 In IL-10�/� mice,DG uptake was similarly elevated in 5- to 6-week-oldice before evidence of colitis. However, in contrast to3H/OuJ controls, they did not decline with age. In the

arge intestine, elevated FDG uptake was statistically sig-ificant for IL-10�/� versus control mice by mean SUVnd percent ID/g values at all ages (Figure 4C and D). Anxample of the imaging is shown in Figure 4A and B. Inhe small intestine, FDG uptake was also statisticallylevated for IL-10�/� versus control mice (after the periodf physiologic decline). Notably, enterocolitis in IL-10�/�

ice can affect the duodenum, jejunum, and colon,28

hich may account for FDG uptake in the small intes-ine. Thus, the IL-10�/� condition either prevents thentogenic reduction of intestinal CD4� T-cell activation

r initiates a local process that induces their activation.

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The effect of augmented inflammation in IL-10�/�

ice was assessed with the NSAID piroxicam, whichntensifies colitis by impairing intestinal barrier functionnd altering cytokine profiles.22 After 1 week, FDG SUVnd percent ID/g were increased in treated versus un-reated IL-10�/� mice (Figure 5A and B). The severenflammation in piroxicam-treated mice also was accom-anied with FDG uptake in the mesenteric lymph nodes

Figure 5C–E). These findings indicated that FDG-PETuantitatively delineated inflammation ranging from thereclinical state of young IL-10�/� mice to the exacer-ated inflammation associated with piroxicam treat-ent.The correlation of colonic FDG uptake and inflam-

ation (histology score) was assessed in IL-10�/� and c

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ontrol C3H/OuJ mice (age 5–20 weeks) and in youngL-10�/� mice (age 5 weeks) with piroxicam treatmentFigure 5F). As expected, percent ID/g and histologycore were low and tightly clustered in C3H/OuJ con-rols. However, in young IL-10�/� mice (age 5–14eeks), with or without piroxicam, the 2 measure-ents were not significantly correlated by linear re-

ression (r 2 � 0.15; P � .10). It should be noted thathe acute colitis elicited by piroxicam resulted in 60%–0% animal mortality at 1 week, and those alive at thatoint did not survive anesthesia associated with imag-

ng. Two IL-10�/� mice surviving to later age (�20eeks of age) had both elevated percent ID/g andistology, indicating that histologic change was a late

Figure 2. FDG-PET in G�i2�/� andcontrol mice. (A) Coronal and (B) sag-ittal views of CT-PET overlays andPET images of G�i2�/� and heterozy-gote control mice with large intestinecontrast agent. (C) Quantitation ofmean SUV and mean percent ID/g pervoxel in small and large intestine ROIsfor individual mice. G�i2�/�, squares;G�i2�/� controls, triangles.

orrelate of disease activity.

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Cellular Source of Intestinal FDG UptakeA variety of inflammatory cell types are recruited

o the intestine in colitis associated with IL-10 and G�i2enetic deficiencies and with NSAID treatment.21,29 Weherefore performed a series of measurements to delin-ate the cellular source of the FDG signal. First, wesolated and enumerated small and large intestinal

ononuclear cell types known to uptake FDG (macro-hages8 and T-cell subsets) from individual mice underhese different conditions after scanning and correlatedbsolute numbers with FDG SUV and percent ID/g mea-urements. However, no significant correlations were ob-erved in numbers of immune cell subpopulations andET measurements (data not shown).We therefore considered the idea that FDG uptake

igure 3. FDG-PET in G�i2�/�

D3� cell transfer colitis. (A) Coronalnd (B) sagittal views of PET-CT over-

ays and PET images of mice treatedith anti-TL1A or control antibody.

C) Quantification of mean percentD/g and mean SUV per voxel in thearge intestine for individual mice.D) Values for histology score and per-ent ID/g in the large intestine werelotted and analyzed by linear regres-ion. (C and D) Anti-TL1A and con-rol antibody values are representedy closed and open squares, res-ectively.

eflected increased levels of Glut-1 expression (presum- F

bly due to activation levels) in the intestinal immuneell types. To test this idea, intestinal mononuclearells were isolated from individual mice after FDG-PETnd stained for Glut-1 and subset markers. By flowytometric analysis, macrophages and CD8� T cellshowed no significant elevation in Glut-1 levels inolitic mice over control; in fact, macrophages in alloncolitic mice had higher Glut-1 levels (Figure 6A–C).

n contrast, CD4� T cells displayed significantly ele-ated Glut-1 expression in all colitic mice (G�i2�/�,L-10�/�, and piroxicam-treated IL-10�/� mice). To val-date this observation, FDG signal and Glut-1 expres-ion were compared for each of these intestinal cellypes. IL-10�/� mice with and without piroxicam werenalyzed, because they showed the greatest range of

DG signal. We measured FDG uptake in individual

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ice and then immediately isolated intestinal cellsrom each mouse for flow cytometric analysis of Glut-1xpression. For the correlation analysis, Glut-1 levelsmean fluorescence intensity) were converted to units� mean fluorescence intensity) by subtracting back-

round mean fluorescence intensity; percent ID/g 0

50

ather than SUV was used because it represents abso-ute rather than normalized uptake. In 2 independentxperiments, Glut-1 expression in CD4� T lympho-ytes was distinguished from other intestinal subtypesor their significant correlation with FDG uptake (r �

Figure 4. FDG-PET in IL-10�/� andcontrol mice. (A) Coronal and (B) sag-ittal views of CT and PET overlays ofIL-10�/� and C3H/OuJ control micewith large intestine contrast agent. Av-erage (�SEM) of (C) mean percentID/g and (D) mean SUV for all micescanned in indicated time interval. IL-10�/�, open squares; C3H/OuJ con-trol, closed circles.

.81; P � .05) (Figure 6D–F). These findings suggest

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hat changes in Glut-1 expression in CD4� T cells arehe main driver of FDG signal in these settings.

Relationship of FDG Uptake to QuiescentVersus Active CD4� T Cells: CD45RBhigh ColitisThe foregoing indicated that activated CD4� T

ells, which display elevated Glut-1 levels, were theain cellular source of mucosal FDG uptake. To val-

date this finding in vivo, isolated CD4� T cells wereransferred into lymphocyte-deficient (scid) mice, underonditions where they are known to home to the in-estine but their activation and colitigenicity can be

anipulated.19 Specifically, we compared scid miceransferred with CD4� CD45RBhigh T cells (activationnd colitis), a mixture of CD4� CD45RBhigh�low T cellsminimal activation and no colitis), or control miceithout any cells transferred.CD4� CD45RBhigh mice, but not CD4�

igure 5. Effect of piroxicam-in-uced inflammation on FDG uptakey IL-10�/� mice. (A) Quantitation ofean SUV and (B) percent ID/g in

mall and large intestine ROIs for IL-0�/� mice treated or untreated withiroxicam. (C) Coronal, (D) sagittal,nd (E) transverse views of piroxicam-reated mice. (F) Correlation of histol-gy and mean percent ID/g for individ-al C3H/OuJ (closed circles), IL-10�/�

open squares), and piroxicam-treatedL-10�/� mice (closed squares). ns, notignificant.

D45RBhigh�low mice, displayed elevated FDG uptake g

ompared with scid controls, with significant differ-nces observed at about 3 weeks posttransfer (FigureA and E–G). This finding was associated with colitisy 4 criteria. First, body weight declined in CD4�

D45RBhigh mice (beginning day 27) but not in otherroups (Figure 7B). Second, peripheral blood neutro-hilia occurred in CD4� CD45RBhigh mice, but not inhe other groups, beginning 2 weeks posttransfer (Fig-re 7C). Third, anemia (reduced mean corpuscularemoglobin level) developed exclusively in CD4�

D45RBhigh mice, beginning 3 weeks posttransfer (Fig-re 7D). Fourth, histology scores were elevated inD4� CD45RBhigh mice (6.1 � 3; P � .03 compared with

cid) but were not significantly different in CD4�

D45RBhigh�low mice (0.6 � 0.5) and scid mice (0.6 � 0.3)Figure 7G). By 6 weeks, half of the CD4� CD45RBhigh

ice had died due to complications of colitis, so theolitis scores underestimate the severity of disease in this

roup.

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At 4 and 6 weeks posttransfer, FDG uptake was in-reased in CD4� CD45RBhigh compared with scid mice byercent ID/g and SUV analysis (Figure 7E and F). Inontrast, FDG uptake was low in CD4� CD45RBhigh�low

nd scid mice, and there was no significant differenceetween them. These experiments validated that acti-ated mucosal CD4� T cells, which form in CD45RBhigh

ice due to the absence of Treg control, produced sub-tantial FDG uptake. Conversely, whereas transfer ofD45RBhigh�low CD4� T cells resulted in substantial mu-

osal T-cell accumulation,30,31 they did not induce FDGptake above the levels of lymphocyte-deficient scid mice.hese observations provide further evidence that the el-vated FDG uptake in colitic mice is a manifestation ofctivated CD4� T cells.

DiscussionUsing an improved quantitation methodology for

urine intestinal FDG uptake, this study addresses the e

52

ellular source of intestinal FDG uptake and its relation-hip to different stages of immune colitis. In 2 models ofevere immune colitis (G�i2�/� and CD4� CD45RBhigh

ice), and in anti-TL1A biologic therapy, FDG uptakeas highly correlated with mucosal inflammation aseasured by histology score. Flow cytometry showed thatucosal FDG uptake was specifically correlated withlut-1 (glucose transporter) expression by CD4� T cells.D4� T-cell transfers in vivo confirmed that mucosalDG uptake involved activated but not quiescent cells.hese observations refined the quantitation of FDG-PET

or longitudinal assessment of intestinal inflammationnd identified the major cellular source of FDG uptake inifferent settings of murine colitis.In the mild immune colitis of young IL-10�/� mice,

nd after acute exacerbation with the NSAID piroxi-am, disease activity (clinical symptoms and mortality)orrelated with FDG uptake but occurred without el-

Figure 6. Glut-1 surface expressionby intestinal cell types. Intestinal cellswere isolated, and flow cytometry wasperformed on cells stained for lineagemarkers and Glut-1 in G�i2�/� (blue)and G�i2�/� (red) mice; cells stainedwith negative control for Glut-1 (black).(A) CD3�CD4� T cells, (B) macrophages(CD11b�F4/80�), (C) CD3�CD8� T cells.Glut-1 levels in intraepithelial mononu-clear cell populations. Intraepitheliallymphocyte mononuclear cells wereisolated from the large intestine of in-dividual IL-10�/� mice with or without2 weeks of piroxicam treatment andstained for lineage markers and Glut-1.Cells were gated on (D) CD3�CD4� Tcells, (E) F4/80�CD11b� cells, or(F) CD3�CD8� T cells. Glut-1 levels (�mean fluorescence intensity) were tab-ulated. Glut-1 levels and percent ID/gfor individual mice were graphed, andcorrelation analysis was used to calcu-late P values. n/s, not significant.

vation of histology score. In a variety of mouse colitis

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Imaging and Advanced Technology continued

odels, including piroxicam colitis,21,29 mucosal CD4�

-cell activation is an early event that precedes thexpansion in mucosal T cells through trafficking androliferation.32 Histology score measures the abun-ance of tissue lymphocytes and elements of tissueamage and thus assesses a later stage of tissue inflam-ation. In contrast, FDG uptake specifically detects-cell activation and thus is uniquely useful for detect-

ng this cellular element of disease-associated inflam-atory response. The early-stage colitis in IL-10�/�

ice, and the acuteness of piroxicam exacerbation,ay predominate with activated CD4� T cells before

xtensive T-cell trafficking and is likely to account forhe greater sensitivity of FDG uptake compared with

igure 7. FDG-PET in CD4�

D45RBhigh transfer colitis. (A) Coro-al and sagittal views of CD4�

D45RBhigh, CD4� CD45RBhigh�low,nd nontransferred C.B-17 scid mice.B) Body weight over time (days) forD4� CD45RBhigh (open squares),D4�CD45RBhigh�low (closed trian-les), and C.B-17 scid (closed circles)ice. (C) Peripheral blood neutrophil

umbers over time. (D) Mean corpus-ular hemoglobin levels over time.E) Mean percent ID/g over time.F) Mean SUV over time. (G) Compari-on of histology score and percent ID/gday 42), analyzed by linear regression.or C–F, statistical comparison of eachroup by Student t test to the scidroup is shown by asterisks: *P � .02;*P � .001; ***P � .0001. Comparisonsor CD4� CD45RBhigh�low mice wereonsignificant for all assays and timeoints.

istology score. m

This study confirmed the reported correlation of ele-ated FDG uptake and disease state in mouse colitisodels14 and by extension with human ulcerative colitis

nd Crohn’s disease. Although varying in ROI definitionnd quantification method, 4 independent clinicaltudies involving between 12 and 59 patients positivelyorrelated segmented intestinal FDG uptake to theresence of ileocolonoscopy lesions with sensitivitiesf 73%–95%.10 –13 The methodology of ROI determina-ion where specified used either a normalized or absolute

aximum SUV. Maximum SUV is useful for identifyingocal disease activity and does not require formal defini-ion of ROIs. However, this technique does not permitssessment of surrounding intestinal voxels. Quantitative

easures that integrate the net level of disease activity, or

753

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7

reas of low but clinically significant disease activity, maye a complement or alternative for defining disease ac-ivity.10,11

An interesting issue confronted by these clinical stud-es was the detection of “false positives” (elevated FDGptake without clinically active lesions). For example, Piot al14 and our study showed that FDG uptake in someettings did not directly correlate with disease activity asefined by histology score. The present study suggests anlternate interpretation for such “false positives.” Inoung and piroxicam-treated IL-10�/� mice, elevatedDG uptake was associated with increased Glut-1 expres-ion by intestinal CD4� T cells before elevation of his-ology score but was clearly associated with disease activ-ty (mortality). In chronic, severe colitis (older IL-10�/�

ice as well as CD4� CD45RBhi and G�i2�/� mice), FDGptake, histology score, and other clinical measures ofisease activity were highly correlated. It thus appearshat in early immune colitis, FDG uptake (and, accord-ngly, levels of intestinal activated CD4� T cells) is aensitive correlate of disease activity, even before bulknflux of lymphocytes (detected by histology score). Ac-ordingly, we speculate that in patients such FDG uptakeay also represent sites of elevated CD4� T-cell activa-

ion.Overall, this study contributes to the understanding

nd utility of assessing intestinal inflammation withDG. It establishes the cellular source of the intestinalDG signal (mucosal accumulation of activated CD4� Tells). The ability of FDG-PET to detect both exacerba-ion (piroxicam) and amelioration (anti-TL1A) of colitisupports a basis for its use to monitor disease state andreatment response. It also suggests a plausible explana-ion for false positives observed in clinical FDG studiesssessing inflammatory bowel disease. That is, the pro-ression of FDG uptake to increased histology scorendicates that FDG uptake (levels of activated mucosalD4� T cells) can be more sensitive than total numerical

nflammatory cell accumulation (detected by conven-ional histology) in delineating emerging or early intes-inal inflammation. Accordingly, FDG update may com-lement standard assessment of intestinal inflammationnd therefore may add diagnostic utility in animal oruman settings where disease progression can be attrib-ted to activated CD4� T cells. However, the utility ofhis assessment, and its use in early intervention to avertlinical relapse, will need to be compared with existingiagnostic (CT enteroclysis, magnetic resonance imaging)r prognostic tests (fecal calprotectin) in terms of testccuracy, costs, and risk.

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Received October 18, 2007. Accepted June 5, 2008.Address requests for reprints to: Jonathan Braun, MD, PhD, Depart-ent of Pathology and Laboratory Medicine, UCLA, Mail Code 173216,

os Angeles, California 90095. e-mail: jbraun@mednet.ucla.edu; fax:310) 825-5674.

Supported by National Institutes of Health grants DK46763 (to.R.T., J.B., and O.T.), DK56328 (to S.R.T.), CA86306 (to J.B.),M07185 (to M.M.), AI52031 (to S.B.), and CA016042 (Jonsson Com-rehensive Cancer Center Flow Cytometry).The authors do not have any financial arrangements to disclose.S.B. and M.M. contributed equally to this work.The authors thank CoGenesys Inc (Rockville, MD) for the 19E06A

nti-TL1A hybridoma; El Khansa Kaicer for breeding and experimentalssistance; the staff of the Crump Institute microPET core facility andonsson Comprehensive Cancer Center flow cytometry core facility; Dravid Stout for his suggestions on PET quantitation techniques; Owenitte, Daniel Silverman, and Caius Radu for critical review of theanuscript; Anna Wu for filial and scientific guidance; and members

f the Braun and Gordon laboratories for their advice and support.The authors acknowledge that Sarah Brewer and Michael McPher-

on contributed equally to this work and should be considered co-first

uthors.

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