of February 15, 2018.This information is current as

in Response to TLR7/8 StimulationPhenotype at Steady State and Produce IL-23Dendritic Cells Display an Activated

+Human Intestinal Lamina Propria CD1c

and Cara C. WilsonLydia A. Hostetler, Jonathan Buhrman, Martin D. McCarter Stephanie M. Dillon, Lisa M. Rogers, Rawleigh Howe,

http://www.jimmunol.org/content/184/12/6612doi: 10.4049/jimmunol.10000412010;

2010; 184:6612-6621; Prepublished online 7 MayJ Immunol 

Referenceshttp://www.jimmunol.org/content/184/12/6612.full#ref-list-1

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The Journal of Immunology

Human Intestinal Lamina Propria CD1c+ Dendritic CellsDisplay an Activated Phenotype at Steady State and ProduceIL-23 in Response to TLR7/8 Stimulation

Stephanie M. Dillon,* Lisa M. Rogers,* Rawleigh Howe,* Lydia A. Hostetler,*

Jonathan Buhrman,* Martin D. McCarter,† and Cara C. Wilson*

Intestinal dendritic cells (DCs) play key roles in mediating tolerance to commensal flora and inflammatory responses against

mucosal pathogens. The mechanisms by which intestinal “conditioning” influences human DC responses to microbial stimuli

remain poorly understood. Infections with viruses, such as HIV-1, that target mucosal tissue result in intestinal epithelial barrier

breakdown and increased translocation of commensal bacteria into the lamina propria (LP). It is unclear whether innate LP DC

responses to concurrent viral and bacterial stimuli influence mucosal HIV-1 pathogenesis. In this study, direct ex vivo phenotype

and in vitro constitutive cytokine production of CD1c+ DCs in human intestinal LP were compared with those in peripheral blood

(PB). To evaluate innate responses to viral and bacterial stimuli, intracellular cytokine production by LP and PB DCs following

stimulation with ligands for TLRs 2, 4, 5, and 7/8 was evaluated. At steady state, LP CD1c+ DCs expressed higher levels of

activation markers (CD40, CD83, CD86, HLA-DR, and CCR7) than did PB CD1c+ DCs, and higher frequencies of LP CD1c+ DCs

constitutively produced IL-6 and -10 and TNF-a. LP DCs had blunted cytokine responses to TLR4 ligand and TLR5 ligand

stimulation relative to PB DCs, yet similarly produced IL-10 in response to TLR2 ligand. Only synthetic TLR7/8 ligand, a mimic

of viral ssRNA, induced IL-23 production by LP CD1c+ DCs, and this proinflammatory cytokine response was synergistically

enhanced following combined TLR7/8 and TLR4 stimulation. These findings highlight a potential mechanism by which viruses

like HIV-1 may subvert homeostatic mechanisms and induce inflammation in the intestinal mucosa. The Journal of Immunology,

2010, 184: 6612–6621.

The intestinal immune system maintains tolerance to com-mensalmicrobes and foodAgs, yet itmustmount protectiveresponses against invading bacterial and viral pathogens.

Dendritic cells (DCs) are APCs that bridge innate and adaptiveimmunity and serve as sentinels of the intestinal immune system (1,2). An intricate maturation and activation process allows them tosense the presence of invading pathogens, migrate to local lymphnodes, and stimulate the activation and expansion of naive and Ag-specific T cells (3, 4). In the intestinal mucosa, DCs in the laminapropria (LP) were shown to play a crucial role in the inductionof tolerogenic responses to commensal bacteria (1). We recentlyshowed that commensal bacteria-reactive, effector Th1 and Th17CD4+ T cells exist in normal human LP. Furthermore, we identifieda subset of resident LP DCs expressing CD1c that mediated theexpansion of bacteria-reactive effector T cell responses in vitro (5).These findings suggest a complex role for LP DCs in mediating

homeostatic responses to commensal bacteria, as well as in-flammatory responses to intestinal pathogens.DCs typically detect the presence of pathogens through pattern-

recognition receptors, especially those of the TLR family thatrecognize conserved microbial components (6). TLR ligandstimulation of DCs promotes their maturation and the inductionof proinflammatory or regulatory responses, depending on thecytokine profiles induced (7). The manner in which intestinalDCs respond to recognition of TLR ligands expressed by path-ogenic and commensal microbes within the intestinal environ-ment is of crucial importance to understanding the immunore-gulatory nature of these cells.In the steady state, resident intestinal DCs are likely condi-

tioned by intestinal epithelial cell-derived factors to become tol-erant to commensal microbes or to induce regulatory or Th2-type Tcell responses (1, 8–10). It was postulated that DCs capable ofresponding to microbial stimuli in a proinflammatory manner in-vivo consist of newly recruited DCs that have yet to be exposedto various conditioning factors or are resident DCs that arerefractory to suppressive conditioning (1). Debate exists as towhether the conditioning effects on resident LP DCs are subsetdependent, are permanent or require constant exposure to in-hibitory factors, or whether suppressive effects of conditioningapply only to commensal bacteria but not pathogenic microbes.Even less is known about how intestinal DCs respond to viralstimuli and whether such interactions alter intestinal homeostasis.This is particularly relevant in the context of HIV-1 infection, inwhich HIV-1 replication, CD4+ T cell depletion, intestinal epi-thelial barrier dysfunction, and the translocation of microbialproducts from the lumen into LP and systemic circulation havebeen reported (11, 12). It was also recently shown that small

*Department of Medicine and †Department of Surgery, University of Colorado Den-ver, Aurora, CO 80045

Received for publication January 7, 2010. Accepted for publication April 3, 2010.

This work was supported by National Institutes of Health Grants R01 AI065275 andK24 AI07434 (to C.C.W) and was facilitated by the infrastructure and resourcesprovided by the Colorado Center for AIDS Research (AI054907).

Address correspondence and reprint requests to Dr. C.C. Wilson, Department ofMedicine, University of Colorado Denver, P.O. Box 6511, Aurora, CO 80045. E-mailaddress: cara.wilson@ucdenver.edu

Abbreviations used in this paper: CM, complete medium; DC, dendritic cell; GI,gastrointestinal; LP, lamina propria; LPMC, lamina propria mononuclear cell; mDC,myeloid dendritic cell; MFI, mean fluorescence intensity; PB, peripheral blood; pDC,plasmacytoid dendritic cell; PGN, peptidoglycan; TLR2L, TLR2 ligand; TLR4L,TLR4 ligand; TLR5L, TLR5 ligand; TLR7/8L, TLR7/8 ligand.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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intestinal DCs express HIV-1 coreceptors and are capable oftransporting HIV-1 through the mucosa to transmit the virus tointestinal T cells (13).HIV-1 ssRNA encodes for numerous ligands that bind and ac-

tivate APCs, such as DCs, via TLR7/8 in vitro (14–17), and there isevidence that HIV-1 replicates at high levels in the gut, providingample viral RNA to serve as these ligands (18). It is unknownwhether innate sensing by LP DCs to the combination of repli-cating virus and translocating commensal bacteria contributes tointestinal inflammation and T cell depletion. Further, the linkingof innate and adaptive immunity through TLR ligation makes TLRligands ideal candidates as vaccine adjuvants (19, 20). Given theunique environment of human intestinal mucosa, it is imperativethat these types of mucosal-specific innate immune responses areinvestigated in a near physiological environment.In the current study, a systematic approach was taken to address

the effect of gastrointestinal (GI) tract conditioning on human LPDCs by comparing maturation phenotype and innate function of“unconditioned” peripheral blood (PB) DCs with DCs isolatedfrom the LP of normal small and large bowel. Cytokine responsesof DCs to a range of bacterial-derived TLR ligands and to a syn-thetic viral TLR ligand were measured using multicolor flow cy-tometry. Additionally, we evaluated the response of LP DCs to theconcurrent exposure of a viral and bacterial TLR ligand. Thesestudies shed light on the mechanisms by which viruses, such asHIV-1, might induce intestinal inflammation and potentially alterthe homeostatic response to commensal flora.

Materials and MethodsStudy participants

Human intestinal biopsies (n = 7 jejunum, n = 10 colon) were obtainedfrom patients undergoing elective abdominal surgery and representedotherwise discarded tissue from surgical anastomic junctions that wasconsidered macroscopically normal. Patients with a history of inflam-matory bowel disease or those receiving chemotherapy, radiation, or otherimmunosuppressive drugs were excluded from the study. This cohortconsisted of 7 females and 10 males with a median age of 56 y (range, 21–72 y). PB samples were obtained from 22 healthy adults (12 females and10 males) with a median age of 35 y (range, 22–58 y) who voluntarily gavewritten informed consent to participate. A statistical difference was notedin the median age of the two cohorts (p = 0.0004). Collection of PBsamples was approved by the Colorado Multiple Institutional ReviewBoard at the University of Colorado Denver.

Collection and preparation of human LP mononuclear cells

LP mononuclear cells (LPMCs) were isolated using techniques describedin detail elsewhere (5). Briefly, tissue specimens were trimmed of fat andincubated in 1.67 mM DTT (Sigma-Aldrich, St. Louis, MO) in HBSS toremove additional mucus; the epithelial layer was subsequently removedwith 1 mM EDTA solution supplemented with 1% BSA (Sigma-Aldrich).The remaining tissue was treated with collagenase D (1–2 mg/ml, Roche,Nutley, NJ) in RPMI 1640 (Invitrogen, Carlsbad, CA) + 1% penicillin/streptomycin/L-glutamine (Sigma-Aldrich) with or without 500 mg/ml pi-peracillin/tazobactam (Wyeth, Madison, NJ) (complete media [CM]) +10% FBS (Sigma-Aldrich) or 0.1% BSA + 10 mg/ml DNase I (Sigma-Aldrich). All released LPMCs were cryopreserved in complete media +10% FBS + 10% DMSO (Fisher Scientific, Pittsburgh, PA) and stored inliquid nitrogen. LPMCs were thawed in CM + 10% FBS + 10 mg/mlDNase I prior to use. The overall viability of thawed LPMC samples was73.4%6 4.0% (mean 6 SEM), based on exclusion of a viability dye (live/dead fixable dead cell stain; see below).

Collection and preparation of human PBMCs

PBMCs were isolated from heparinized blood by standard Ficoll-Hypaque(Amersham Biosciences, Picataway, NJ) density-gradient centrifugation,as described previously (5, 21). In some instances, PBMCs were treatedwith collagenase D, as detailed for the isolation of LPMCs. PBMCs werecryopreserved in 10% DMSO in CM containing 10% human AB serum(Gemini Bio-Products, Woodland, CA) and stored in liquid nitrogen.

Abs for flow cytometry

Various combinations of the following Abs were used: CD19 (PE-Cy5),HLA-DR (allophycocyanin-Cy7), CD83 (PE), CD86 (FITC) purifiedCCR7, biotinylated anti-mouse IgG, streptavidin PE-Texas Red, IL-12p40/p70 (PE), TNF-a (FITC), IL-10 (PE) (all from BD Biosciences, San Jose,CA); biotinylated TLR4 and IL-6 (FITC) (both from eBioscience, SanDiego, CA); CD1c ([BDCA-1], allophycocyanin) and CD303 ([BDCA-2],FITC) (both from Miltenyi Biotec, Auburn, CA); TLR5 (PE; Imgenex, SanDiego, CA); biotinylated CD40 (Ancell, Bayport, MN); and streptavidin-PE-Texas Red (Beckman Coulter, Fullerton, CA).

In all cases, the recommended, appropriate isotype-control Abs wereused, and FcR-blocking reagent (Miltenyi Biotec) was included in allinitial incubations to limit nonspecific Ab binding through FcRs.

Flow cytometry protocol for surface and intracellular Abs

Standard flow-cytometric staining protocols for surface markers are detailedelsewhere (5, 21). Eight-color flow cytometry was performed on LPMCsand PBMCs immediately after thawing (baseline) or after culture for as-sessment of intracellular cytokine production using a FACSAria or anLSRII flow cytometer (BD Biosciences). Cells were washed in cold Dul-becco’s PBS (Invitrogen) containing 1% BSA and supplemented with2 mM EDTA (FACS buffer). Cells were incubated with appropriate surfaceAbs (detailed above) for 20 min at 4˚C and washed in cold PBS. For CCR7,a three-step staining protocol was used with purified CCR7 + biotinylatedanti-mouse IgG + streptavidin-PE-Texas Red, whereas CD40 staining wasachieved with a two-step protocol using biotinylated CD40 + streptavidin-PE-Texas Red. TLR4 staining was performed using biotinylated TLR4 andstreptavidin-PE-Texas Red. Cells were washed twice in FACS buffer beforethe final incubation with the streptavidin-labeled fluorochrome, after whichcells were washed in cold PBS as for the single-Ab staining proceduredetailed above. At the completion of surface Ab incubations, all cells werestained with a Live/Dead Fixable Dead Cell Stain (Aqua Fluorescent re-active dye; Invitrogen) for 30 min at 4˚C. Cells were then washed inDulbecco’s PBS, and, for surface-only staining, cells were resuspended in4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature,washed, and resuspended in FACS buffer prior to acquisition.

For intracellular cytokine staining, cells were washed in PBS at thecompletion of the surface-staining and live/dead cell-staining protocol,fixed and permeabilized in Cytofix/Cytoperm buffer (BD Biosciences) for15 min, washed in Perm/Wash buffer (BD Biosciences), resuspended inPerm/Wash buffer containing the appropriate cytokine Abs, and incubatedfor 15 min at 4˚C. After an additional wash in Perm/Wash buffer, cells wereresuspended in 0.5% paraformaldehyde prior to acquisition.

In vitro stimulation of LPMCs and PBMCs

Thawed LPMCor PBMC samples were resuspended at 0.9–23 106 cells/mlin CM + 10% human AB serum. Cells were cultured at 37˚C in a humidified5% CO2 atmosphere without exogenous stimuli or were stimulated with 10mg/ml peptidoglycan (PGN; TLR2 ligand [TLR2L]; Sigma-Aldrich), 10mg/ml LPS from Salmonella minnesota (TLR4 ligand [TLR4L]; Sigma-Aldrich), 0.1 mg/ml S. typhimurium flagellin (TLR5 ligand [TLR5L]; In-vivoGen, San Diego, CA), 5 mg/ml CLO97, a derivative of the imidazo-quinoline compound R848 (TLR7/8 ligand [TLR7/8L]; InvivoGen), ora combination of TLR4L and TLR7/8L. For the assessment of intracellularcytokines, cells were cultured for 30 min, 1 mg/ml Golgi Plug (brefeldin A,BD Biosciences) was added, and cultures were incubated for an additional17–24 h. For the assessment of secreted cytokines, culture supernatants werecollected after 18–28 h of culture in the absence of Golgi Plug. Prior toevaluation of cytokine production, 10 mg/ml DNase I was added to allcultures for 5 min at 37˚C to dissociate cell clumps.

In vitro stimulation of DC-depleted LPMC cultures

Total LPMCs were stained with fluorescent-labeled CD1c and CD19, asdescribed above, and CD1c+CD192 myeloid DCs (mDCs) were depletedfrom total LPMCs by FACS using a FACS ARIA. Collected LPMCs were99.99% depleted of mDCs (data not shown). Total LPMCs (also labeledwith CD1c and CD19 Abs but not flow sorted) or DC-depleted LPMCswere stimulated with or without TLR7/8L, as described above, and culturesupernatants were collected after 23–24 h of culture. Highly enrichedpopulations of LP CD1c1CD192mDCs (.85% purity, range 86–95%)were obtained from four donors by flow-sorting and stimulated with orwithout TLR7/8L for 24 h prior to collection of culture supernatants.

Detection of IL-23 and -12p70 within culture supernatants

IL-23 and -12p70 (both from eBioscience) ELISAs were run using culturesupernatants from stimulated LPMC and PBMC cultures following the

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manufacturer’s recommended protocols. The lower detection limits were15 pg/ml for IL-23 and 4 pg/ml for IL-12p70.

Data analysis

All flow-cytometry data analysis was performed using DIVA software (BDBiosciences). Analysis was performed if $100 DCs were acquired. Forstatistical analysis of surface marker and cytokine expression, Mann–Whitney analysis was performed comparing PBMCs with LPMCs. TheWilcoxon signed-rank test was used to compare PBMCs before and aftercollagenase treatment and to compare the difference in cytokine pro-duction before and after the addition of exogenous stimuli. The Friedmantest was used for matched-paired comparisons across multiple groups.Statistical analysis was performed using GraphPad Prism Statistical Soft-ware, version 4.0 (GraphPad Software, San Diego, CA). No significantdifferences were observed between CD1c+ mDCs isolated from the jeju-num or the colon for phenotypic or functional analyses (Mann-Whitney ttest); therefore, results are shown using pooled samples, with the specifictissue location highlighted where possible.

ResultsCD1c+ mDCs, but not CD303+ plasmacytoid DCs, are readilyidentifiable within normal human LP

Previous investigations identified human intestinal mDCs by theexpression of HLA-DR and CD11c in the absence of the expressionof a range of lineagemarkers (CD3,CD14, CD16, CD19,CD20, andCD56) (22), a combination of Abs traditionally used to identifyblood mDCs. The majority of blood mDCs can also be directlyidentified by the expression of CD1c and exclusion of CD19 (23),whereas plasmacytoid DCs (pDCs), a second subset of blood DCs,specifically express CD303 (23). Thus, we focused on identifyingLP DCs based on the expression of CD1c and CD303, with com-parisons made with profiles of DCs observed within PBMCs(Fig. 1A). Much like B cells within the blood (23), a subset of LP B

cells also expressed CD1c and was excluded from the CD1c+ mDCpopulation by the inclusion of CD19 in the Ab mixture. As in theblood, these LP CD1c+CD192 DCs were identified as a subset ofthe previously identified human CD11c+Lineage2HLA-DR+ LPDCs (22) (data not shown).In comparing DCs from the blood (n = 11) with those from LP

samples (n = 9) of nonautologous donors, a significantly higherfrequency of CD1c+ mDCs was observed in LP preparations (Fig.1B), with CD1c+ mDCs making up 0.76% (range, 0.40–1.13%) ofviable LPMCs. Conversely, pDCs were virtually undetectable innormal LP (Fig. 1B). Thus, additional investigations focused onthe CD1c+/CD192 mDC population in LP (hereafter referred to asCD1c+ DCs).

LP CD1c+ DCs display a more activated phenotype than PBCD1c+ DCs

The level of activation and the maturation state of mDCs arecritical to their function as APCs (3), yet few studies have ad-dressed the maturation status of human DCs exposed to localenvironmental factors present in the intestinal LP relative to PBDCs, which are unlikely to have been exposed to the same tissue-specific factors.Expression of a panel of typical DC maturation markers CD40,

CD83, CD86, HLA-DR, and CCR7 was compared between PB(n = 11) and LP (n = 7–9) CD1c+ DCs (Fig. 2, Table I). LPCD1c+ DCs expressed significantly higher levels of CD40,CD83, CD86, and HLA-DR (Table I) compared with PB CD1c+

DCs. Additionally, the lymph node-homing chemokine receptorCCR7 was expressed at significantly higher levels on LP CD1c+

DCs (Table I).

FIGURE 1. CD1c+ DCs are readily identifiable within the LP. A, A representative example is shown of the gating strategy used to identify DCs within PB

(PBMC) or LP (LPMC). Viable cells are initially gated using a viable cell dye (not shown), and cellular debris is removed using a forward/side scatter gate.

mDCs were identified based on the expression of CD1c in the absence of CD19 (CD1c+ DCs), whereas pDCs expressed CD303. B, A higher frequency of

CD1c+CD192 DCs was measured in the LP (n = 9) compared with similarly identified DCs within the PB (n = 11), whereas fewer pDCs were identified in

the LP compared with PB. Frequencies of CD1c+ DCs and CD303+ pDCs from jejunum (n = 5) and colon (n = 4) are shown and are expressed as the

percentage of total viable cells. Lines represent median values. Statistical analysis comparing frequencies of CD1c+ DCs or CD303+ pDCs within the LP

with those detected in PB was performed using the Mann–Whitney t test.

6614 PROINFLAMMATORY HUMAN LAMINA PROPRIA DCS

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CD103 is an integrin found on a subpopulation of murine DCsthat are thought to play a crucial role in inducing gut-homingreceptors on T cells and Foxp3+ T cell differentiation in vitro (24–26). It was recently identified on a subpopulation of humanmesenteric lymph node DCs and shown to induce the gut-homingmolecule CCR9 on allogeneic CD8+ T cells (27). Low levelsof CD103 (net mean fluorescence intensity [MFI]: median, 153;range, 35–495; n = 9) were detected on a very small fraction of LPCD1c+ DCs (net percentage of CD103+ CD1c+ DCs: median,3.8%; range, 0–18.5%), although LP DC expression of CD103was higher than that expressed on PB CD1c+ DCs (net MFI:median, 4; range, 0–31; net percentage of CD103+ CD1c+ DCs:median, 0.04%; range, 0–0.74%; n = 11). It remains to be de-termined whether other human LP DC subsets express higherlevels of CD103 than CD1c+ DCs.To evaluate the possible effects of the enzymatic-digestion step

required for the isolation of LPMCs on the DC phenotype, PBMCswere treated with a similar collagenase-digestion protocol andcomparisonsweremadewithuntreatedPBMCsfrommatcheddonors(n = 5–7). Exposure of PBMCs to collagenase induced increasedexpression of CD86 (2.67-fold increase over untreated DCs) andCD83 (32.9-fold increase over untreated DCs) on CD1c+ DCs.However, the expression levels of CD86 and CD83 on LP CD1c+

DCs (Table I) remained significantly higher than on collagenase-treated PB CD1c+ DCs (p = 0.008 and p = 0.005, respectively; datanot shown). No significant increases in HLA-DR, CD40, or CCR7expressionwere noted on PBCD1c+ DCs after collagenase exposurecompared with untreated PB CD1c+ DCs. Thus, even after ac-counting for a potential effect of the LPMC-isolation procedure onthe expression levels of CD86 and CD83, LP CD1c+ DCs remainedconsistently more mature than blood CD1c+ DCs.Despite the greater expression of CD40, CD86, CD83, HLA-DR,

and CCR7, a phenotype typical of DC maturation and activation,LP CD1c+ DCs were not in a fully mature state, as indicated bytheir ability to further upregulate the expression of maturation

markers after overnight culture with or without specific stimula-tion (data not shown).

LP CD1c+ DCs constitutively produce more IL-6 and -10 andTNF-a than PB CD1c+ DCs

To assess the cytokines that LP CD1c+ DCs produce in vivo underhomeostatic steady-state conditions, total LPMCs or PBMCs werecultured overnight in the absence of exogenous stimuli, and theconstitutive production of IL-12p40/p70, -10 and -6 and TNF-a byLP or PB CD1c+ DCs was determined by an intracellular cytokinestaining assay (Fig. 3A). IL-12p40/p70–producing CD1c+ DCswere virtually undetectable within PBMC or LPMC cultureswithout exogenous stimulation (Fig. 3B), and no significant dif-ference between the groups was observed. However, significantlymore IL-10+ and TNF-a+ and, to a lesser extent, IL-6+ CD1c+

DCs were identified within unstimulated LPMC compared withPBMC cultures (Fig. 3B). No significant differences in cytokineproduction were observed in CD1c+ DCs from PBMCs versuscollagenase-treated PBMCs (n = 5; data not shown), suggestingthat increased basal cytokine production in LP CD1c+ DCs did notresult from effects of the tissue-digestion process.

LP CD1c+ DCs have a blunted cytokine response to TLR4Land TLR5L but a similar response to TLR2L and TLR7/8Lrelative to PB CD1c+ DCs

We next evaluated the cytokine profiles of CD1c+ DCs in responseto viral and bacterial TLR ligands. Because HIV-1 ssRNA encodesfor multiple TLR7/8Ls (14–17), we used a synthetic TLR7/8L tomodel these potential innate interactions of HIV-1 with LP CD1c+

DCs. Stimulation with TLR4L and TLR5L induced significantincreases in the frequencies of TNF-a+ and IL-6+ PB CD1c+ DCs,but the same stimuli failed to significantly increase the frequenciesof cytokine-producing LP CD1c+ DCs above constitutive levels(Table II). TLR2 stimulation of PBMCs and LPMCs resulted inincreased frequencies of IL-10+, TNF-a+, and IL-6+ CD1c+ DCs

FIGURE 2. Representative exam-

ple showing expression of various

maturation/activation markers on

CD1c+ DCs from PB (PBMC) and

LP (LPMC) samples. Multiparameter

flow-cytometry techniques were used

to assess the expression of matura-

tion/activation markers (CD86, CD83,

CD40, HLA-DR and CCR7; shaded

graph) on CD1c+ DCs, identified as

described in Fig. 1A. Background

staining was assessed using isotype

controls (open graph).

Table I. Expression of maturation markers by PB and LP CD1c+ DCs

PB (Net MFI: Median [Range]) LP (net MFI: Median [Range]) Fold Increase LPMC/PBMCa

CD86 6,813 (4,340–8,231) 25,190 (13,154–41,594) 3.70*CD83 11 (0–45) 1,867 (268–4,123) 169.7*CD40 4,783 (2,641–7,751) 37,744 (28,263–46,623) 7.89*HLA-DR 16,008 (9,682–20,160) 48,626 (30,873–59,697) 3.04**CCR7 371 (240–965) 1,746 (103–5,210) 4.71***

Expression of various maturation markers were determined on CD1c+ DCs (as defined in Fig. 1A) from PB (n = 10–11) andLP (n = 7–9). Values shown are net MFI, determined by removing background staining using appropriate isotype controls.Increased expression levels on LP CD1c+ DCs are shown as fold increase over PB CD1c+ DC expression levels.

aStatistical analysis comparing expression levels on LP CD1c+ DCs with PB CD1c+ DCs was performed using the Mann-Whitney t test.

pp # 0.001; ppp # 0.0001; pppp # 0.05.

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but no change in IL-12p40/p70+ CD1c+ DC frequencies (Table II).In fact, TLR2L was the only stimulus that induced significantincreases in the frequencies of IL-10–producing PB and LPCD1c+ DCs (Table II). In contrast to the other stimuli, only TLR7/8 stimulation significantly increased the frequency of IL-12p40/p70+ CD1c+ DCs in PB and LP, along with increased frequenciesof TNF-a+ and IL-6+ CD1c+ DCs (Table II). Focused comparisonsof TLR7/8L-induced IL-12p40/p70 responses showed similarfrequencies of IL-12p40/p70+ CD1c+ DCs in PB (median, 8.6%;range 0–31.1%; n = 11) and LP (median, 13.2%, range, 5.0–28.1%; n = 11; p = 0.54) in response to TLR7/8 stimulation.

LP CD1c+ DCs express less TLR4 but more TLR5 than PBCD1c+ DCs

TLR expression levels may, in part, determine the response patternof DCs to microbial products, and low TLR expression on gutDCs was postulated to play a role in the tolerance of commensalorganisms (28–30). Given that we observed blunted cytokine re-sponses by LP CD1c+ DCs to TLR4 and TLR5 stimulation, wecompared the surface expression of TLR4 and TLR5 on CD1c+

DCs in LP (n = 6–7) versus PB (n = 9). Although TLR4 wasexpressed at very low levels on PB and LP DCs, a statisticallylower frequency of CD1c+ DCs in LP expressed TLR4 comparedwith PB CD1c+ DCs (Fig. 4). Expression of TLR5 was low toundetectable on PB CD1c+ DCs, and a statistically greater fre-quency of LP CD1c+ DCs expressed TLR5 (Fig. 4). No differ-ences were observed in TLR4 or TLR5 expression on CD1c+

DCs in comparisons of PBMCs with collagenase-treated PBMCs(n = 4; data not shown).

LP CD1c+ DCs produce IL-23 but not IL-12p70 in response toTLR7/8 stimulation

Given the finding that only TLR7/8 stimulation induced signif-icant increases in the frequency of LP CD1c+ DCs producing

IL-12p40/p70 and that the p40 subunit of IL-12 also forms part

of the IL-23 complex (31), we next evaluated whether the TLR7/

8L-induced IL-12p40/p70 responses reflected IL-12p70 or -23

production. Because of the paucity of IL-23–specific Abs ap-

propriate for intracellular detection, IL-23 released into culturesupernatants following TLR stimulation was assessed by ELISA.

In accord with the increase in IL-12p40/p70+ CD1c+ DCs ob-

served in the intracellular cytokine staining flow cytometry as-

say, TLR7/8 stimulation induced significant amounts of IL-23

from LPMCs, whereas stimulation of the same LPMCs with

bacterial TLRs resulted in limited IL-23 production (Fig. 5A).Minimal IL-12p70 production was detected for any stimulation

condition (Fig. 5A). To evaluate the contribution of LP CD1c+

DCs to the total IL-23 production observed by ELISA within

LPMC cultures, CD1c+ DCs were first depleted from LPMCs by

flow sorting, and CD1c+ DC-depleted and nondepleted LPMCs

were cultured with or without TLR7/8L. Levels of measurableIL-23 were reduced by 92.7% (range: 41.4–100%) in TLR7/8L-

stimulated cultures following depletion of CD1c+ DCs, in-

dicating that the majority of IL-23 measured in LPMC cultures

following TLR7/8 stimulation was produced by or dependent

upon CD1c+ DCs (Fig. 5B).To determinewhether LPCD1c+ DCs produced IL-23 in response

to direct stimulation by TLR7/8L, experiments were performed

FIGURE 3. Constitutive production of IL-6, -10, and -12p40/p70 and TNF-a by LP and PB CD1c+ DCs. LPMCs (LP) or PBMCs (PB) were cultured for

17–24 h in the absence of exogenous stimulation, and the frequencies of 12p40/p70+, IL-10+, TNF-a+, and IL-6+ CD1c+ DCs were determined using an

intracellular cytokine staining assay. A, A representative example of intracellular cytokine levels within viable jejunal LP CD1c+ DCs with quadrants

established using appropriate isotype controls for each cytokine. B, Frequencies of cytokine-producing CD1c+ DCs as the percentage of total CD1c+ DCs

were compared between PB (n = 11) and LP (n = 9–12), with results from jejunum (n = 5–6) and colon (n = 4–6) delineated. Lines represent median values,

and statistical analysis comparing LP CD1c+ DCs with PB CD1c+ DCs was performed using the Mann–Whitney t test.

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using sort-purified LP CD1c+ DCs. Using the bulk of total LPMCsavailable from intestinal samples from four donors, LP CD1c+ DCswith .85% purity (range, 86–95%) were obtained by flow sorting.Following overnight stimulation of sorted LP CD1c+ DCs (6,400–36,500 CD1c+ DCs per condition) with TLR7/8L versus mediaalone, low levels of IL-23 were measured in stimulated culture su-pernatant from three of four samples (1.39–7.47 pg IL-23 per 1 3104 CD1c+ DCs) but not in unstimulated cultures, confirming that

direct stimulation of LP CD1c+ DCs induced IL-23 production.Given that the levels of IL-23 induced were low, likely due in part tothe small numbers of CD1c+ DCs obtained by sorting, which ne-cessitated plating low cell concentrations, it is difficult to concludethat direct stimulation of LP CD1c+ DCs by TLR7/8L accounted forthe majority of IL-23 production observed in TLR7/8-stimulatedLPMCs. Thus, DC-dependent production of IL-23 may also occurthrough indirect mechanisms.

Table II. Frequency of cytokine-producing CD1c+ DCs in response to TLR ligand stimulation

PB CD1c+ DCs (%) Cytokine+

(Net) (Median [Range]) TrendaLP CD1c+ DCs (%) Cytokine+

(Net) (Median [Range]) Trenda

TLR4LIL-12p40/p70 0 (0–1.4) ↔ 0.15 (0–1.8) ↔TNF-a 25.4 (13.2–41.2) ↑* 10.1 (22.8–41.9) ↔IL-6 23.9 (12.1–36.9) ↑* 7.7 (23.7–25.3) ↔IL-10 0.7 (21.7–3.1) ↔ 0.9 (21.3–8.5) ↔

TLR5LIL-12p40/p70 0 (0–1.4) ↔ 0.6 (0–5.2) ↔TNF-a 3.1 (21.7–11.1) ↑** 6.6 (22.6–11.8) ↔IL-6 4.9 (2.4–13.9) ↑*** 0.6 (20.6–10.7) ↔IL-10 0.3 (20.7–3.4) ↔ 0.6 (23.4–4.0) ↔

TLR2LIL-12p40/p70 0 (0–0.8) ↔ 0.2 (21.9–1.6) ↔TNF-a 31.8 (24.5–39.4) ↑*** 17.9 (24.0–34.1) ↑**IL-6 4.7 (20.7–22.7) ↑** 10.8 (21.9–21.1) ↑**IL-10 1.7 (20.2–9.1) ↑** 9.4 (2.2–13.0) ↑***

TLR7/8LIL-12p40/p70 8.6 (0–31.1) ↑*** 13.2 (5.0–28.1) ↑*TNF-a 48.8 (27.1–73.7) ↑* 34.5 (3.1–76.7) ↑*IL-6 61.8 (21.3–73.9) ↑* 26.5 (23.9–44.0) ↑***IL-10 1.1 (20.6–7.5) ↔ 4.2 (25.8–16.1) ↔

PBMCs (PB; n = 8–11) or LPMCs (LP; n = 6-11) were stimulated with LPS (TLR4L; 10 mg/ml), S. typhimurium flagellin(TLR5L; 0.1 mg/ml), PGN (TLR2L; 10 mg/ml), or a derivative of the imidazoquinoline compound R848 (TLR7/8L; 5 mg/ml)for 17–24 h, and intracellular levels of IL-6, -10, and -12p40/p70 and TNF-a within PB CD1c+ DCs or LP CD1c+ DCs weredetermined (as shown in Fig. 3A). Values are expressed as the difference between frequencies of cytokine+ CD1c+ DCs instimulated cultures and those in cultures without exogenous stimulation (net).

aOverall change between cytokine+ CD1c+ DC frequencies in TLR-stimulated and unstimulated conditions; Wilcoxonsigned-rank test. ↑, increase; ↔, no change.

pp # 0.001; ppp # 0.05; pppp # 0.01.

FIGURE 4. Expression of TLR4 and TLR5 by LP

and PB CD1c+ DCs. Surface expression of TLR4 and

TLR5 were determined within PB (n = 9) and LP [(n =

6–7); jejunum (n = 3–4); colon (n = 3)] CD1c+ DC

populations using flow-cytometry techniques. Values

are expressed as net MFI (top panels) or as net per-

centage positive (bottom panels) by removing back-

ground fluorescence based on isotype controls. Lines

represent median values, and statistical analysis com-

paring levels of expression and frequencies of TLR-

expressing CD1c+ DCs within the LP with those de-

tected in PB was performed using the Mann–Whitney

t test.

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Combined TLR7/8 and TLR4 stimulation synergisticallyincreases IL-23 production in LPMCs

Because altered epithelial integrity during mucosal viral infectionsmay expose LP DCs to translocated bacterial products, as well asviral products, we evaluated the impact of combined TLR4 andTLR7/8 stimulation on IL-23 and -12p70 production in a subset ofLPMC samples (n = 7; Fig. 5C). Despite the absence of significantIL-23 production following TLR4 stimulation alone, combinedstimulation with TLR4L and TLR7/8L resulted in a synergisticenhancement of IL-23 production in LPMCs (Fig. 5C). Previousstudies showed that combined TLR stimulation is required forIL-12p70 production by blood mDCs (32, 33); however, a signif-icant induction of IL-12p70 was not observed following com-bined TLR4L and TLR7/8L stimulation within LPMC cultures(p = 0.13; Fig. 5C).Next, the relative levels of IL-23 and -12p70 induced in response

to combinatorial TLR stimulation in PBMCs relative to LPMCcultures was evaluated. Similar levels of IL-23 were observed inPBMC and LPMC cultures following stimulation (p = 0.84; Fig.5D). In agreement with previous reports (32, 33), a significantincrease in IL-12p70 production in response to combined TLRstimulation was observed in PBMC cultures (Fig. 5D). The ob-

servation that combined TLR4 and TLR7/8 stimulation failed toinduce significant IL-12p70 production within LPMC cultures(Fig. 5C, 5D) suggests that LP CD1c+ DCs selectively produceIL-23, even in the presence of stimulatory conditions that inducedIL-12p70 production from PBMCs.

DiscussionTo evaluate the effects of intestinal conditioning on the innatefunction of DCs obtained from the human GI tract, we directlycompared unconditioned CD1c+ DCs in PB with CD1c+ DCsobtained from the LP of small and large bowel. LP CD1c+ DCsexpressed higher levels of markers typical of DC activation thandid blood CD1c+ DCs, even after accounting for possible effectsof the tissue-digestion process. Importantly, LP CD1c+ DCs stillhad the potential to upregulate these activation markers further, anobservation in keeping with the upregulation of maturationmarkers observed in LP CD11c+ DCs that had migrated out ofhuman colonic biopsies (22). Thus, resident LP CD1c+ DCs thathave been exposed to the intestinal environment are more acti-vated or mature than their counterparts in PB. This activation ofLP DCs may result from their exposure to intestinal microbes,from soluble factors in the mucosal environment, or from contact

FIGURE 5. IL-23 production by LP CD1c+ DCs. A, To detect IL-23 and -12p70 within LPMC cultures (n = 6–13), total LPMCs were stimulated with

LPS (TLR4L; 10 mg/ml), PGN (TLR2L; 10 mg/ml), S. typhimurium flagellin (TLR5L; 0.1 mg/ml), or TLR7/8L (5 mg/ml) for 17–28 h, and levels of IL-23

and -12p70 in culture supernatants were evaluated by ELISA. Values represent the net amount of cytokine (picogram) per 1 3 106 total LPMCs within

TLR-stimulated cultures after subtraction of cytokine amounts (per 1 3 106 total LPMCs) detected in cultures without exogenous stimuli (line = median).

Statistical analysis was performed on paired samples with and without each specific TLR stimulation using the Wilcoxon signed-rank test. B, To determine

the contribution of LP CD1c+ DCs to IL-23 production observed in total LPMC cultures with TLR7/8 stimulation, LPMCs from a subset of samples (n = 3)

were depleted of CD1c+ DCs by flow-sorting techniques, cultured with or without TLR7/8L (5 mg/ml) for 23–24 h, and levels of IL-23 were determined by

ELISA. Comparisons of total IL-23 detected within CD1c+ DC-depleted cultures were made to parallel cultures of matched total LPMCs similarly

stimulated with or without TLR7/8L. The values (median, range) represent the net amount of cytokine (picogram) per 1 3 106 LPMCs determined by

subtracting the amount of cytokine detected in cultures without exogenous stimuli from that within TLR7/8-stimulated cultures. C, To detect IL-23 and

-12p70 within LPMC cultures (n = 7) in response to combined viral and bacterial TLR ligand stimulation, total LPMCs were stimulated with TLR4L (10

mg/ml), TLR7/8L (5 mg/ml), or a combination of both TLR ligands for 24–28 h, and IL-23 and -12p70 in culture supernatants were evaluated by ELISA.

Values represent the net amount of cytokine (picogram) per 13 106 total LPMCs within TLR-stimulated cultures after subtraction of cytokine amounts (per

1 3 106 total LPMC) detected in cultures without exogenous stimuli (line = median). Statistical analysis was performed using the Friedman test. D, Levels

of IL-23 and -12p70 in PBMC (PB; n = 6) or LPMC (LP; n = 7) culture supernatants were evaluated by ELISA 24–28 h after stimulation with a com-

bination of TLR4L (10 mg/ml) and TLR7/8L (5 mg/ml). Values represent the net amount of cytokine (picogram) per 13 106 total LPMCs or PBMCs within

stimulated cultures after subtraction of cytokine amounts (per 1 3 106 total LPMCs or PBMCs) detected in cultures without exogenous stimuli (line =

median). Statistical analysis was performed on paired samples with and without stimulation using the Wilcoxon signed-rank test.

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with other cell populations in the intestinal mucosa. Interestingly,it was reported that CD3+ TCR g/d T cells, a subset of T cellsresident in the intestinal mucosa, could induce DC maturation ina CD1c-restricted manner (34). The presence of DCs residing inthe LP in a state of partial activation may allow for a more rapidimmune response in the event of microbial invasion.Higher frequencies of LP CD1c+ DCs constitutively producing

IL-6 and -10 and TNF-a also likely reflect the more activated stateof these cells compared with blood CD1c+ DCs. Spontaneous pro-duction of IL-10 and a concurrent lack of IL-12p40/p70 productionwere observed in human colonic CD11c+ DCs (35) However, incontrast to our study, Hart et al. (35) did not observe IL-6–producingCD11c+ DCs in the normal intestine. Constitutive production of IL-10 and -6 by CD1c+ DCs may reflect activation by commensalbacterial signals and aid in maintaining an anti-inflammatory envi-ronment within the LP through the induction of regulatory T cells,Th2 cells (36), and the induction of IgA-secreting B cells (37).Additionally, these cytokines were shown to play a role in main-taining epithelial barrier integrity (38, 39). The constitutive pro-duction of TNF-a is more intriguing given that it has typically beendefined as a proinflammatory cytokine. However, a number ofstudies demonstrated the potential of TNF-a to be anti-in-flammatory through specific regulation of IL-12 and -23 productionby blood-derived macrophages and DCs (40, 41). It is possible thatthe combined production of these cytokines, in addition to otherlocal factors, contributes to resident CD1c+ DCs existing in a state ofinflammatory anergy that is necessary for GI tract homeostasis (42).The effect of GI tract conditioning on innate responses by CD1c+

DCs was investigated using bacterial and viral TLR ligands.TLR4L and TLR5L stimulation of blood CD1c+ DCs led to in-creased production of IL-6 and TNF-a, despite the low expressionof TLR4 and minimal expression of TLR5. Blood CD1c+ DCswere shown to express TLR5 using RT-PCR (43). Thus, it ispossible that the flow cytometry techniques and specific Abs usedin the present study lacked the required sensitivity to detect verylow levels of surface protein adequate to allow stimulation witha TLR5L. Because these experiments were conducted using totalPBMCs, it is also possible that DC-specific TLRs are upregulatedin a bystander fashion during TLR stimulation in vitro, which maymore accurately reflect the in vivo situation.In contrast, TLR4L and TLR5L stimulation failed to signifi-

cantly increase the frequency of cytokine-producing LP CD1c+

DCs over steady-state levels, even though LP CD1c+ DCs ex-pressed significantly higher levels of TLR5 compared with theirblood counterparts. Lower frequencies of TLR4-expressing LPCD1c+ DCs compared with blood CD1c+ DCs is in agreementwith earlier observations of TLR expression on intestinal CD11c+

DCs (35). Notably, the actual levels of expression of TLR4 werevery low on blood and LP CD1c+ DCs. Although a lack of IL-12p40 production by LP DCs in response to TLR4L contrasts withthe results from one study of human CD11c+ intestinal DCs (44),the observation is consistent with other studies showing thatmurine LP CD11c+ DCs and rat lymph DCs were hyporesponsiveto LPS, likely related to lower TLR4 expression (28–30, 45, 46).To our knowledge, this is the first study to show that human LP

CD1c+ DCs express TLR5, yet they fail to respond to stimulationwith TLR5L. Tolerance in human monocytes induced by priorexposure to flagellin did not alter TLR5 expression, but it altereddownstream signaling proteins (47). It is possible that a similaralteration of TLR5 signaling occurs in human intestinal CD1c+

DCs as a result of the specific microenvironment to limit re-sponses against commensal bacteria and that additional signalingis required from virulence factors to induce a proinflammatoryresponse (48). The inability of TLR5L stimulation to induce cy-

tokine frequencies above constitutive levels differs with somereports in murine models, in which a subset of LP DCs expressingTLR5 mRNA produced IL-12p70 in response to stimulation withflagellin (46, 49). Differences in the experimental approach andthe specific DC subset evaluated, whether isolated DCs or DCs inwhole LPMCs were evaluated, and species differences may ac-count for these observed differences in TLR responses betweenstudies. Importantly, we showed that in contrast to blood CD1c+

DCs, intestinal-conditioned human LP CD1c+ DCs did not sig-nificantly alter cytokine production profiles in response to TLR4or TLR5 stimulation, indicating a form of anergy or tolerance tothese two specific microbial products in vitro.Stimulation with TLR2L induced a distinct cytokine profile in

DCs from blood and LP, characterized by the production of IL-10and -6 and TNF-a and a lack of IL-12p40/p70 upregulation. Theobservation that blood and LP CD1c+ DCs produced IL-10 inresponse to TLR2L may indicate a strong propensity of this TLRsignaling pathway to lead to the generation of regulatory re-sponses, independent of intestinal conditioning. Indeed, TLR2Lstimulation of systemic DCs was reported to promote T regulatoryresponses through regulation of the MAPK pathway (50, 51).The finding that TLR7/8 activation of LP CD1c+ DCs resulted in

the potent induction of IL-12p40/p70 to frequencies comparableto those observed with similarly stimulated blood CD1c+ DCssuggest that this response was not significantly altered by theintestinal environment. This TLR7/8L-induced increase in IL-12p40/p70 by LP CD1c+ mDCs was associated with CD1c+ DC-dependent production of IL-23 rather than IL-12p70. Furthermore,the synergistic enhancement of IL-23 production, but not ofIL-12p70, following combined TLR4 and TLR7/8 stimulationsuggests a greater propensity toward IL-23 production within theintestinal tract (52). The mechanism behind synergistic IL-23production by LP CD1c+ DCs in response to bacterial and viralTLR ligand costimulation is under investigation, but it may relateto regulation of TLR expression (53).The novel finding that human LP CD1c+ DCs produced IL-23,

a proinflammatory cytokine involved in the expansion of Th17and Th1 cells (54), only in response to a viral TLR ligand addsa crucial component to our understanding of the role that DCs mayplay in the intestinal innate immune response and their contribu-tion to intestinal homeostasis, host defense, and inflammation.Given that we observed anergic-type responses by this samepopulation of DCs to TLR4 and TLR5 stimulation, and a typicalanti-inflammatory response was generated in response to TLR2stimulation, the ability of this DC subset to respond in a proin-flammatory manner is likely to be TLR ligand specific. This hassignificant implications for the development of effective mucosalvaccines, with the recent focus on the use of TLR ligands asvaccine adjuvants (19, 20).Importantly, because resident LPCD1c+DCs in our invitromodel

were presumably conditioned in the gut in vivo, these results in-dicate that a population of DCs with the potential to respond ina proinflammatory manner exists in normal human LP. Constitutiveproduction of IL-23 by murine terminal ileum DCs was reported(55), and Denning et al. (56) showed that a subset of murine in-testinal DCs promoted Th17 production, suggesting that DC subsetswith a nonregulatory function also exist within the GI tract of ro-dents. A CD14+ population of macrophages, recently identified inhuman LP with a macrophage/DC phenotype, were capable ofproducing greater amounts of proinflammatory cytokines, such asIL-23, compared with CD142 cells, an observation particularlynoted in patients with Crohn’s disease (57). Interestingly, thispopulation of cells did not express CD1c, suggesting the existence ofmultiple populations of cells within the LP that are capable of

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producing proinflammatory responses, with the potential for exac-erbation of these responses when disruption of the GI tract occurs.We previously demonstrated the existence of commensal bac-

teria-reactive Th1 and Th17 effector CD4+ T cells in normal hu-man LP (5). In the context of increased IL-23 production by LPCD1c+ DCs in response to ssRNA viruses and concomitant ex-posure to bacteria, these bacteria-reactive T cells would likelybe induced to expand in vivo. This scenario may be particularlyrelevant in the setting of HIV-1 infection, in which a compro-mised epithelial barrier could lead to increased contact of HIV-1–exposed LP CD1c+ DCs to bacteria, potentially resulting inenhanced IL-23 production, activation and infection of bacteria-reactive Th1 and Th17 cells, and persistent HIV-1 replication. Thisproinflammatory response would create a vicious cycle, leading tofurther disruption of intestinal homeostasis, local immune acti-vation, T cell depletion, and increased microbial translocation.There are several limitations to this study. First, a statistical

difference in donor age was noted between the unmatched PBMCand LPMC donors. Age-related changes in blood DC function werereported, i.e., increased spontaneous cytokine production and de-creased responses to TLR stimulation (58). No significant asso-ciations between age and CD1c+ DC frequency, phenotype, orcytokine responses to TLR stimulation were observed in our studysubjects. However, a statistically significant correlation betweenage and spontaneous TNF-a production by PB CD1c+ DCs wasnoted (Spearman’s test: r = 0.60, p = 0.05). Therefore, it possiblethat the age differences between cohorts may account for some ofthe differences in DC function that were observed in blood versusintestinal samples. An additional limitation of this study may bethe wide range of DC cytokine responses to stimulation observedbetween the different donors. This type of variability is inherentin human-based research; in a larger cohort of healthy donors,Lombardi et al. (53) noted that at least one in three donors couldbe classified as a high responder with respect to blood DC cyto-kine production in response to combined TLR stimulation. Withthis in mind, we only concluded that differences existed betweenLP and PB CD1c+ DCs in either phenotype or function whenconfirmed by appropriate statistical analysis.In summary, we describe a novel mechanism by which ssRNA

viruses, through innate stimulation of CD1c+ DCs via TLR7/8,may subvert the normal homeostatic mechanisms in place in theintestinal mucosa to induce inflammation. As such, these findingshave important implications for understanding viral pathogenesisin the intestinal mucosa. A better understanding of the pro- andanti-inflammatory responses of intestinal DCs to different micro-bial stimuli may also facilitate the development of more effectivemucosal vaccines.

AcknowledgmentsWe thank all of the subjects for their generous participation in our study. We

thank Dr. Ricardo Gonzalez and all of the members of the surgical teams of

the Department of Surgery, University of Colorado Hospital, Denver, CO,

for assistance with the collection of tissue samples. We acknowledge the

Colorado Center for AIDS Research Immunology Core for assistance with

flow cytometry and the Clinical Investigation Core for assistance with the

recruiting of subjects.

DisclosuresThe authors have no financial conflicts of interest.

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