of May 29, 2017.This information is current as

Infection Reflect Lower CCR1 ExpressionDendritic Cells in Patent Human Filarial Expanded Numbers of Circulating Myeloid

Siddhartha MahantySekou F. Traoré, Amy Klion, Thomas B. Nutman and

Diallo,Dramane Sanogo, Salif Seriba Doumbia, Abdallah A. Michel E. Coulibaly, Lamine Soumaoro, Siaka Y. Coulibaly,Dembele, Siaka Konate, Simon Metenou, Housseini Dolo, Roshanak Tolouei Semnani, Lily Mahapatra, Benoit

ol.1001605http://www.jimmunol.org/content/early/2010/10/18/jimmun

published online 18 October 2010J Immunol 

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The Journal of Immunology

Expanded Numbers of Circulating Myeloid Dendritic Cellsin Patent Human Filarial Infection Reflect Lower CCR1Expression

Roshanak Tolouei Semnani,* Lily Mahapatra,* Benoit Dembele,† Siaka Konate,†

Simon Metenou,* Housseini Dolo,† Michel E. Coulibaly,† Lamine Soumaoro,†

Siaka Y. Coulibaly,† Dramane Sanogo,† Salif Seriba Doumbia,† Abdallah A. Diallo,†

Sekou F. Traore,† Amy Klion,* Thomas B. Nutman,* and Siddhartha Mahanty*

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infec-

tion. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived

dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in

humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c2CD123lo) in peripheral blood of infected

microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based

on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c+CD123lo) in filaria-

infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs,

monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers

between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals

had a lower percentage of circulating CCR1+ mDCs and a higher percentage of circulating CCR5+ mDCs and pDCs. Finally, live

microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their

calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC

migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations

have major implications for regulation of immune responses to these long-lived parasites. The Journal of Immunology, 2010, 185:

000–000.

Antigen presenting cell dysfunction has been one mech-anism used to explain the profound parasite-specificT cell hyporesponsiveness seen in chronic, patent lym-

phatic filariasis (1), although the mechanisms by which the filariaeinduce this have only been partially elucidated. Previously, wehave shown that live microfilariae (mf) of Brugia malayi modulatedendritic cell (DC) function by two different mechanisms: 1) byaltering TLR3 and TLR4 expression and function; and 2) by in-ducing apoptotic DC cell death (2, 3).Two subsets of DCs have been identified in human blood (4)

based on the pattern of expression of CD11c and CD123, withmyeloid DCs (mDCs) being CD11c+CD123lo and plasmacytoid

DCs (pDCs) being CD11c2CD123+ (5, 6). Although the functionof these circulating DCs is not well understood, it is thought thatthese cells are in transit from either the bone marrow to peripheraltissues or from tissue to lymph nodes or spleen.Following Ag/pathogen recognition, DCs mature and migrate

from peripheral tissues to secondary lymphoid organs, where theypresent the Ag to lymphocytes (7, 8). This process of DC traf-ficking and migration is tightly regulated by chemokine receptorexpression on these cells and their response to chemokine ligands(9). To this end, it has been shown that immature mDCs expressfunctional CCR1 and CCR5, can respond to their ligands (MIP-1aand RANTES for both CCRs and MCP3, a chemokine that signalsthrough CCR1 and CCR2), and migrate to sites of inflammation.During the process of maturation and in response to inflammatorystimuli, DCs downregulate their cell surface expression of thesechemokine receptors and upregulate their expression of CCR7(reviewed in Ref. 9).Our present study addresses whether apoptosis observed in vitro is

reflected in the number of circulating mDCs in the peripheral bloodof microfilaria-positive, filaria-infected individuals (Fil+). By flowcytometry, we show that Fil+ have a significantly higher numberof circulating mDCs compared with their uninfected counterparts(Fil2). Furthermore, the number of other APCs (pDCs, monocytes[MC], and macrophages [MF]) was not different between the twogroups. Filarial infection was associated with a lower percentage ofCCR1-expressing mDCs that could be modeled in vitro by micro-filaria–DC interactions. These findings collectively suggest that mfare capable of altering mDC migration through changes in chemo-kine receptor-mediated signaling, thereby altering DC homeostasis.

*Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892; and †Filariasis Unit, Faculty ofMedicine, Pharmacy and Odontostomatology, University of Bamako, Bamako, Mali

Received for publication May 18, 2010. Accepted for publication September 16,2010.

This work was supported by the Intramural Research Program of the Division ofIntramural Research, National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health.

Address correspondence and reprint requests to Dr. Roshanak Tolouei-Semnani andDr. Siddhartha Mahanty National Institute of Allergy and Infectious Diseases, Na-tional Institutes of Health, 4 Center Drive, Room 4/126, Bethesda, MD 20892. E-mailaddresses: rsemnani@niaid.nih.gov and smahanty@niaid.nih.gov

The online version of this article contains supplemental material.

Abbreviations used in this paper: DC, dendritic cell; Fil+, filaria-infected individual;Fil2, filaria-uninfected individual; GM, geometric mean; GMFI, geometric meanfluorescence intensity; iGMFI, integrated geometric mean fluorescence intensity;MF, macrophages; MC, monocytes; mDC, myeloid dendritic cell; mf, microfilariae;pDC, plasmacytoid dendritic cell; RFU, relative fluorescence unit.

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Materials and MethodsStudy populations

The study village, N’Tessoni, located ∼385 km southeast of Bamako, is ina region of Mali endemic for the filarial parasitesWuchereria bancrofti andMansonella perstans. The immunological study described in this paper waspart of a larger study (http://www.ClinicalTrials.gov trial identification:NCT00341666) investigating clinical and immunological differences be-tween Fil+ and Fil2 subjects with respect to symptomatic malaria that wasconducted in 2006 to 2007. Prior to the study, community permission for thestudy was obtained from village elders, and the study was approved by theethical review committees (Institutional Review Boards) of the Faculty ofMedicine, Pharmacy, and Dentistry at the University of Bamako (Bamako,Mali) and the National Institute of Allergy and Infectious Diseases (Bethesda,MD). Adults aged 18–65 y old were screened for this study, and written in-formed consent was obtained from all participants. Individuals who receivedtreatment for filarial infections (albendazole or ivermectin) previously wereexcluded at screening.

Two study populations were identified for this immunological substudy:1) individuals with confirmed active filarial infection as defined by thepresence of circulating filarial Ag and/or mf of W. bancrofti and/or mf ofM. perstans (Fil+; n = 9); and 2) individuals with no evidence of activeW. bancrofti orM. perstans infection (circulating filarial Ag and mf negative,Fil2; n = 9). All microfilaremic individuals identified by our screening in thevillage were included in this study. Following our study, all residents of thevillage received ivermectin and albendazole treatment for lymphatic underthe Malian Program for Elimination of Lymphatic Filariasis.

Study procedures

Each subject had a focused clinical evaluation for symptoms and signs offilarial infection and provided a blood sample that was transported toa laboratory in Bamako for processing. Hematologic analyses (includinghemoglobin levels and total and differential WBC counts) were performedon all subjects. Additional laboratory analysis of the blood samples includedblood smears for malaria parasites and mf (W. bancrofti and M. perstans)and quantitation of circulating filarial Ag (TropBio, Townsville, Australia).

PBMC separation

PBMCs from the blood of Malian volunteers were isolated by Ficoll dia-trizoate density centrifugation as described previously (1, 10).

Flow cytometry

Sample staining. PBMCs were isolated from study volunteers, immediatelyfixed in 4% paraformaldehyde, and cryopreserved as previously described(11). Staining of cells with Abs was carried out according to standard pro-tocols as follows. Cells were incubated with 10 ml human IgG (10 mg/ml;Sigma-Aldrich, St. Louis, MO) for 10 min at 4˚C to inhibit nonspecific bind-ing through FcgRs. Cells were then incubated with marker-specific mAb

conjugated with FITC, Alexa Fluor 700, APC, PE-Cy7, or PE at saturatingconcentrations for 30 min at 4˚C and washed twice with FACS medium. Themouse anti-humanmAbs used in this study are listed in Supplemental Table I.mAbs specific for lineage markers Lin 1 (Supplemental Table II), HLA-DR, CD11c, and CD123 were used in all samples to gate on mDCs andpDCs. Cells were also stained for surface expression of the chemokine re-ceptors (CCR, CCR1, CCR5, CCR6, and CCR7). Monocyte cell populationswere identified as cells that were positive for both CD14 and HLA-DR andmacrophage populations as CD14+HLA-DR+CD68+.

Flow cytometric analysis. For flow cytometric analysis, 50,000 events wereacquired per tube using a BD LSRII flow cytometer (BD Biosciences, SanJose, CA). Compensation was performed in every experiment using BDCompBeads (BD Biosciences) for single-color controls and unstained cellsas negative controls. The flow cytometry setup for these experiments isshown in Supplemental Table II. Data were analyzed using FlowJo Soft-ware (Tree Star, Ashland, OR). The gating strategy used in this study issummarized in Fig. 1. Nonviable cells and granulocytes were excludedfrom our analysis on the basis of forward and side scatter. We calculatedintegrated geometric mean fluorescence intensity (iGMFI) for CCR1 andCCR5 by multiplying the percentage of CCR1+ or CCR5+ cells in eachpopulation (mDCs or pDCs) by the corresponding GMFI of the chemokinereceptors.

Normalization of the number of mDCs and pDCs in 1 ml blood

For analysis of DC and macrophage populations within the PBMC pop-ulations, we used gates that included both lymphocytic and mononuclearcell populations identified by side and forward scatter (Fig. 1). The fre-quencies of DCs and MC, calculated from the percentage of target cellsand the total number of mononuclear cells in the PBMCs analyzed, werenormalized for the number of mononuclear cells per milliliter of wholeblood from which the PBMCs were purified, using the total and differentialcounts of the blood (enumerated by a Coulter counter [Beckman Coulter,Fullerton, CA]). For example, the frequency of the monocyte population(HLA-DR+/CD14+ cells) in a given individual per milliliter of blood wascalculated by multiplying the percentage of this population by the totalnumber of MC and lymphocytes per 1 ml blood.

Cytokine measurement

Levels of cytokines/chemokines in the serum of Fil+ and Fil2 were mea-sured by Searchlight proteome arrays (Aushon Biosystems, Billerica, MA).All samples were tested in duplicate, and the results are expressed as themean of the replicates.

Calcium flux measurement

The response of cells to MCP3, MIP-1b, and RANTES was investigatedby measuring intracellular calcium concentration changes using the FLIPRcalcium 3 assay kit (PeproTech, Rocky Hill, NJ) according to the manu-facturer’s protocol. Briefly, DCs and mf-exposed DCs were harvested,

Table I. Clinical and parasitological characteristics of the study populations

Parameter Fil2 Fil+ p Value

N 9 9Gender [n (%)]Male 2 (22.3) 5 (55.6) NSFemale 7 (77.8) 4 (44.4) NS

Age (y), median (range) 38 (18–56) 54 (30–63) 0.019Clinical signs of filariasis [n (% positive)]Hydrocele 0 1 (11.1) NSArm/leg edema 1 (11) 3 (33) NSLymphangitis 2 (22) 0 NSElephantiasis 0 0 NSCellulitis 1 (11) 0 NS

Laboratory parameters (mean 6 SD)WBC count (per ml) 5.4 6 0.4 6.2 6 0.5 NSEosinophil count (% of WBC) 11.4 6 2.7 9.8 6 1.4 NS

MicrofilaremiaW. bancrofti

n (% positive) 0 9 (100)mf per 60 ml [GM (range)] – 252 (83–917)

M. perstansn (% positive) 0 8 (80)mf per 60 ml [GM (range)] – 234 (33–950)

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washed, and plated on poly-L-lysine (Sigma-Aldrich) precoated 96-wellblack-wall clear plates at 1 3 105 cells/well in HBSS (Invitrogen, Carls-bad, CA). The plate was centrifuged at 2000 rpm for 5 min. The supernatantwas discarded, and the calcium dye was diluted at 1:1 in HBSS and addedto the cells (100 ml/well). The plate was incubated for 20 min at37˚C in 5% CO2. Cells were then centrifuged once for 5 min. Agonists wereprepared in HBSS at several dilutions down to a final concentration of 100nM. The plates were transferred directly to a FlexStation for measurementof fluorescence, and the average fluorescence values of the duplicates wereused for each data set. Maximum 2 baseline fluorescence values, withnegative control subtraction from each well, were calculated and graphed.

Preparation of mf

Live B. malayi mf were provided by Dr. Ray Kaplan (University of Georgia,Athens, GA) as described previously (12). Briefly, live mf were collected byperitoneal lavage from infected jirds and separated from peritoneal cells byFicoll diatrizoate density centrifugation. The mf were then washed threetimes in RPMI 1640 with antibiotics and cultured overnight at 37˚C in 5%CO2.

In vitro generation of DCs

CD14+ peripheral blood-derived MC were isolated from WBCs collectedfrom healthy donors by counterflow centrifugal elutriation. MC were cry-opreserved in liquid nitrogen at 5 3 107/vial and thawed for culture insix-well tissue culture plates (Costar, Cambridge, MA) at 2 to 3 3 106/ml incomplete RPMI 1640 (BioWhittaker, Walkersville, MD) supplementedwith 20 mM glutamine (BioWhittaker), 2% heat-inactivated human ABserum (Gemini Bioproducts, West Sacramento, CA), 100 IU/ml penicillin,and 100 mg/ml streptomycin (Biofluids, Rockville, MD). Recombinanthuman IL-4 and recombinant human GM-CSF (PeproTech) were added tothe culture at 50 ng/ml on days 1, 4, and 6 of culture. Live mf were added onday 6 at a final concentration of 5 3 104/well (per 1 to 2 3 106 DCs). Cells

were harvested at day 8 of culture after Versene/EDTA treatment (Biofluids)and washed twice with PBS (without Ca2+/Mg2+). Viability and cell countswere determined by trypan blue exclusion and used for flow cytometricanalysis or other functional studies. DCs harvested at day 8 were repeatedlyshown to be CD1a+, HLA-DR+, CD86+, CD40+, CD32, CD142/lo, CD192,and CD562 by flow cytometry (FACSCalibur, BD Biosciences).

Real-time RT-PCR

RNA (1 mg) from an infected individual (patient 6 from Table I) was usedto generate cDNA and then assessed by standard multiplex TaqMan assays(Applied Biosystems, Foster City, CA). This individual was the onlysubject with sufficient RNAyields to perform this analysis on both pre- andposttreatment samples.

For RT-PCR, random hexamers were used to prime RNA samples forreverse transcription using MultiScribe reverse transcriptase (AppliedBiosystems), after which PCR products for HSPA4, HSPA8, HSPCA, andHSPCB, as well as endogenous 18s rRNA controls, were assessed in triplicatewells using TaqMan assay reagents developed by the manufacturer (AppliedBiosystems). These include PCR forward and reverse primers and a FAMdye-labeled TaqMan MGB (minor groove binder) probe, which crosses intron/exon boundaries to amplify only mRNA. For endogenous control, a set ofprimer and a VIC-labeled MGB 18S probe was used. Real-time quantitativeRT-PCR was performed on an ABI 7700 sequence detection system (AppliedBiosystems).

Statistical analysis

The nonparametric Mann–Whitney U test and the nonparametric Wilcoxonsigned-rank test were used as indicated in each figure legend. Correlationswere assessed using the Spearman rank correlation test. Statistical analyseswere performed with StatView 5 (SAS Institute, Cary, NC) or GraphPadPrism Version 5.0 (GraphPad, San Diego, CA). In all comparisons, pvalues ,0.05 were considered statistically significant.

FIGURE 1. Subtyping of mDCs and pDCs. mDC and pDC subpopulations were evaluated in fixed PBMC from Fil+ and Fil2 individuals. Gated PBMCs

(A) negative for expression of Lin markers and positive for expression of HLA-DR (B) were stained for expression of CD11c and CD123. HLA-DR+/Lin2

populations that are CD11c+ and CD123lo are considered to be mDCs; those that are CD11c2 and CD123hi are considered to be pDCs (C). The monocyte

population was identified based on gated PBMC (A) that were positive for expression of CD14 and HLA-DR (D) and the macrophage population based on

the HLA-DR+/CD14+ cells (D) that were positive for expression of CD68 (E).

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ResultsStudy populations

Fil+ and Fil2 residents of a single village were randomly selectedafter a screening visit during which blood samples were collected.Based on predefined selection criteria (see Materials and Meth-ods), we identified nine Fil+ and nine Fil2 individuals for thisstudy. The two groups (Fil+ and Fil2) did not differ significantlyin gender composition or clinical signs related to their filarialinfections (Table I). The Fil+ group had a significantly highermedian age (54 y; range 30–63) than the Fil2 group (38 y; range18–56; p = 0.019), not unexpected in this setting, as filarialinfections increase in prevalence with age in all endemic regions.WBC counts and eosinophil counts were also statistically equiv-alent in the two groups. All of the Fil+ individuals had circulatingW. bancrofti mf (GM mf count 252/ml; range 83–917), and eightout of nine (89%) also had circulating mf of M. perstans (GM mfcount 234/ml; range 33–950) (Table I). All individuals in the studypopulation (Fil+ and Fil2) were seropositive for antifilarial IgG,reflecting ongoing exposure to filarial parasites in this village lo-cated in a region endemic for lymphatic filariasis.

Fil+ individuals have a higher number of circulating mDCscompared with Fil2 individuals

Previously, we have shown that live mf of B. malayi induce apo-ptotic cell death in human mDCs (2). To assess whether our in vitrofindings were reflected by alterations in circulating mDC numbersin vivo, PBMCs were immediately fixed and cryopreserved. PBMCsfrom the two groups (Fil+ and Fil2) were then examined for numberof circulating mDCs (Lin2DR+CD11c+CD123lo), pDCs (Lin2DR+

CD11c2CD123hi) based on established criteria (see Fig. 1 for thegating strategy), MC, and MF. Based on these analyses, we dem-onstrated a significant increase in the number of mDCs in Fil+ com-pared with Fil2 individuals (Fig. 2A), with the GM for Fil+ being87,794 cells/ml compared with 47,000 cells/ml for Fil2 (p = 0.002).

Although slightly increased in the Fil+ group compared with Fil2,the number of pDCs did not differ significantly (Fig. 2B). In contrast,there was no difference in the number of circulating MC (HLA2

DR+/CD14+) or MF (HLA2DR+/CD14+/CD68+) between the twogroups (Fig. 2C, 2D). There was no relationship between the numberof circulating mf and the number of circulating mDCs (p = 0.538;r2 = 0.066) or pDCs (p = 0.12, r2 = 0.352) in Fil+ individuals (datanot shown).

Fil+ individuals have a lower percentage of circulating CCR1+

mDCs and a higher percentage of circulating CCR5+ mDCsand pDCs compared with Fil2 individuals

To address whether the increased number of circulating mDCsobserved in Fil+ is due to changes in subpopulations of DCs ex-pressing particular CCRs known to mediate transendothelial mi-gration in DCs, we assessed the expression of CCR1, CCR5, CCR6,and CCR7 on both mDCs and pDCs. Although there were no dif-ferences in the percentages of CCR6+ and CCR7+ cells in bothmDC and pDC populations between the two groups (Figs. 3C, 3D,4C, 4D), there was a significant decrease in the percentage of cir-culating CCR1+ mDCs in Fil+ compared with Fil2 individuals(p = 0.01) (Fig. 3A). The percentage of CCR1+ pDCs was alsoslightly (but not statistically significant) lower in Fil+ individuals(GM 24.8 in Fil2 versus 14.5 in Fil+) (Fig. 4A). In contrast, thepercentages of circulating CCR5+ mDCs (p = 0.007) and pDCs(p = 0.01) were significantly higher in Fil+ compared with Fil2

individuals (Figs. 3B, 4B). Furthermore, the per-cell surface ex-pression of CCR1 as measured by iGMFI was also significantlylower on mDCs (p = 0.048) (Fig. 3E) but not on pDCs (Fig. 4E),and the per-cell surface expression of CCR5 was significantlyhigher in both mDCs and pDCs in the Fil+ as compared with theFil2 group (Figs. 3F, 4F).We next assessed whether the difference in the percentage of

CCR1+ or CCR5+ mDCs between Fil+ and Fil2 were related to the

FIGURE 2. Filaria-infected individuals have

a higher number of circulating mDCs com-

pared with uninfected individuals. The num-

ber of circulating mDCs (A), pDCs (B), HLA-

DR+/CD14+ (C), or HLA-DR+/CD14+/CD68+

(D) from either filaria-infected (n; n = 9) or

-uninfected (d; n = 8–9) individuals were

normalized to 1 ml blood (see Materials

and Methods). The Mann–Whitney U test was

used in A–D.

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serum levels of their ligands (chemokines). We did not detect anyMCP3 (CCR1 ligand) and very low levels of MCP2 (CCR1,CCR2, and CCR5 ligand) in the sera of both Fil+ and Fil2 indi-viduals (GM 0.9 pg/ml). In addition, whereas MIP-1b (a CCR5-specific ligand) was detected in both groups, there were no dif-ferences in the levels of these chemokines between the groups.Furthermore, there were no differences between the groups inserum levels of eotaxin, MIP-3a, MIP-3b, IL-8, RANTES, orCCL16 (Fig. 5A, 5B).

Live mf of B. malayi downregulate expression of CCR1 onmonocyte-derived DCs in vitro

Given that Fil+ individuals had a significant decrease in not onlythe percentage of circulating CCR1+ mDCs but also in the per-cell expression level of CCR1, we assessed the role of mf in di-rectly mediating the decreased expression level of CCR1 using anin vitro model of mf–DC interaction. Ags and mf of B. malayihave been used extensively in place of W. bancrofti for in vitrostudies of immune responses to filariae because propagation andmaintenance of W. bancrofti is not feasible in nonhuman animals.In addition, the genetic makeup in the most genes products thathave been identified to date for W. bancrofti and B. malayi are.90% homologous at the protein level.As shown in Fig. 6, DCs exposed to live mf for 48 h showed

diminished cell-surface expression of CCR1 (p = 0.03) comparedwith the mf-unexposed cells, whereas there was no difference in

CCR5 expression. Thus, our in vitro data suggest that live mf candirectly regulate the expression of CCR1 but not CCR5 or CCR2,CCR6, or CCR7 (data not shown) on mDCs.

Exposure to mf reduces CCR1 ligand-driven calcium flux inmDCs

To assess whether the diminished expression of CCR1 on DCs isreflected in diminished signaling through CCR1, we measuredcalcium flux in these cells in response to MCP3, a ligand that bindsCCR1 (but not CCR5). We found that mf-exposed DCs exhibitedsignificantly diminished calcium flux in response to MCP3 at 100nM (DC/mf: maximum 2 baseline [relative fluorescence unit(RFU)]; range 0–19) (Fig. 7A, 7B) as well as at 20 nM (data notshown) compared with the unexposed cells (DCs: maximum 2baseline [RFU]; range 3–63). Furthermore, whereas there wasa minimal increase in the intracellular calcium concentration ofDCs in response to MIP-1b (a ligand that binds and signalsthrough CCR5 but not CCR1), exposure to mf did not alter thisresponse in 8 out of 10 donors (Fig. 7B). Interestingly, mf-exposedDCs showed a significant decrease in calcium flux in response toRANTES, a ligand that works through both CCR1 and CCR5, at100 nM (DC/mf: maximum 2 baseline [RFU]; range 2–22) (Fig.7B) and at 1 nM (data not shown), compared with unexposed DCs(DCs: maximum 2 baseline [RFU], range 5–123), suggesting thatan alteration in expression of CCR1 (and not CCR5) on DCs bymf resulted in a diminished ability of these cells to flux calcium in

FIGURE 3. Filaria-infected individuals have a lower

percentage of circulating CCR1+ and a higher per-

centage of circulating CCR5+ mDCs compared with

uninfected individuals. The percentage of CCR1+ (A),

CCR5+ (B), CCR6+ (C), and CCR7+ (D) mDCs in

filaria-infected (d; n = 6–7) and -uninfected (n; n = 9)

individuals. iGMFI calculated for CCR1 (E) and CCR5

(F) show the per-cell expression of these receptors. The

Mann–Whitney U test was used to test for statistical

significance.

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response to ligands (MCP3 and RANTES) that work throughCCR1. Importantly, there was no statistically significant differencein calcium flux between mf-exposed DCs and unexposed DCs intheir responses to ATP (Fig. 7B).

DiscussionThe mechanisms of Ag-specific hyporesponsiveness observed inFil+ individuals with mf have been the focus of intense investi-gation, and our previous studies have shed light on the role of APCdysfunction and mf in this phenomenon. We have shown that livemf of B. malayi can disrupt normal functioning of human monocyte-derived DCs in at least two ways: 1) by inducing a caspase-dependent apoptotic cell death (2); and 2) by negatively regulatingtheir TLR expression and signaling (3), thereby inhibiting Ag-pre-senting function. In other human population studies, it has also beenshown that when MC from mf individuals are stimulated in vitro

with filarial Ag, there is diminished upregulation of TLR expressionand diminished cytokine production in response to some TLR li-gands (13). Our study was designed to test whether the apoptotic celldeath observed in vitro (2) extend to Fil+ individuals with activefilarial infection in vivo, resulting in a lower number of circulatingmDCs. Unexpectedly, our data demonstrated a significant increase(rather than a decrease) in the number of circulating mDCs inmicrofilaremic individuals (Fig. 2A). Importantly, there was no dif-ference between the groups in the number of other circulating APCincluding pDCs, MC, and MF (Fig. 2C, 2D). Although there was anincrease in the frequency of circulating mDCs, there was no re-lationship between the level of microfilaremia, the levels of parasiteAg (representing the adult worm load), and the number of these cells(data not shown), suggesting this increase in mDCs is not a directconsequence of the number of mf circulating in the infected indi-viduals. Similarly, the frequency of CCR1+ DC subpopulations also

FIGURE 4. Filaria-infected indi-

viduals have a higher percentage of

circulating CCR5+ pDCs compared

with -uninfected individuals. The per-

centage of CCR1+ (A), CCR5+ (B),

CCR6+ (C), and CCR7+ (D) pDCs

in filaria-infected (n; n = 6–9) and

-uninfected (d; n = 6–9) individuals.

iGMFIs for CCR1 (E) and CCR5 (F)

were measured to show the per-cell

expression of these receptors. Mann–

Whitney U test was used to measure

statistical significance.

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did not correlate with either microfilaremia or parasite Ag levels inthe blood (data not shown).Although our in vivo observations differ from what we would

have predicted from our in vitro findings, there are several possibleexplanations for what we found in vivo. For example, the expansionin mDC population that we observed in microfilaremic individualscould reflect an increase in DC cell death in compartments such asthe bone marrow, resulting in increased turnover of mDCs withmore precursor cells moving through the circulation. Alternatively,an increase differentiation of MC to mDCs could be occurring inthe periphery or a failure of DCs to migrate through the endothelial

barrier based on trafficking defects such as altered chemokine/chemokine receptor homeostasis.Chemokines and chemokine receptors play an important role in

directing the migration of DCs (14–16). mDCs and pDCs isolatedfrom human blood differ in their capacity to migrate to chemotacticstimuli (17), and the expression patterns of chemokine receptors onthese cell populations play a key role in directing migration of DCsinto specific tissues because tissue migration follows specific che-mokine gradients (18–20). DCs perform widely different functionsat different stages of maturation and traffic to specific microenvi-ronments as dictated by the local chemokine milieu (19). Optimalresponses to different sets of chemokines are generally regulated bychanges in the patterns of CCR expression. For example, immatureDCs expressing a specific pattern of CCR, that include CCR1,CCR5, and CCR6, are able to home to peripheral tissues, whereasmature DCs express CCR7, a key molecule for migration from theperiphery to T cell areas of secondary lymphoid tissues (9).To investigate the possibility that DC migration was impaired

in Fil+ individuals, we studied the expression of CCR1, CCR5,CCR6, and CCR7 in both Fil+ and Fil2 study populations. Ourfindings indicate that although there was a very small percentageof circulating mDCs expressing CCR1 in both groups, this sub-population of mDCs was significantly smaller in Fil+ individuals(Fig. 3A); expression of CCR1 per cell on mDCs as measured byiGMFI was also lower in Fil+ individuals (Fig. 3E). In contrast, thepercentage of circulating CCR5+ mDCs and pDCs and per-cellexpression of CCR5 was significantly higher in Fil+ individualscompared with Fil2 individuals (Figs. 3B, 4B), with no significant

FIGURE 5. A and B, Serum levels of CCR1 or CCR5

ligands do not differ between the filaria-infected and

-uninfected groups. Chemokine levels in sera of filarial-

infected (n; n = 9) and -uninfected individuals (d; n =

9) were measured using proteome arrays. Each circle

or box represents an individual. The Mann–Whitney U

test was used to measure statistical significance.

FIGURE 6. Live B. malayi microfilariae downregulate cell-surface ex-

pression of CCR1 on monocyte-derived DCs. Cell surface expression of

CCR1 (A) and CCR5 (B) on in vitro-generated DCs either unexposed (d)

or after 48 h exposure to live microfilariae of B. malayi (n) in six donors.

Each line represents an independent experiment (n = 6). The non-

parametric Wilcoxon signed-rank test was used to measure significance.

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differences seen in either CCR6 or CCR7. Interestingly, we didnot see a corresponding difference in the serum levels of MCP2,CCL16 (ligands for CCR1), MIP-1b, RANTES, or eotaxin (ligandsfor CCR5) between the two groups (Fig. 5). Together with theobservation that these changes were unrelated to concentration ofcirculating chemokines (Fig. 5) or expression of CCR6 and CCR7,these data would suggest that mDCs in Fil+ individuals have a re-duced capacity to migrate into tissues. These findings also raisea question as to whether the expanded size of the mDC populationreflects redistribution of these cells into the circulatory compartmentor is a compensatory increase in the bone marrow in response toincreased DC turnover. Redistribution of blood DCs has beenobserved in autoimmune diseases such as systemic lupus eryth-ematosus, in which reduced numbers of blood DCs with corre-sponding accumulation of DCs in the inflammatory sites have beenreported (21).Although there is limited information on the role of chemokine

receptor expression in DC homeostasis in humans, redistributionof other cell populations has been linked to expression of thesereceptors. For example, in Chagas disease and schistosomiasis,a change in the expression of chemokine receptors on PBMCshas been associated with progression of the disease (22, 23). Inthe case of severe chronic chagasic cardiomyopathy, a seriouscomplication of Trypanosoma cruzi infection, diminished CCR5

and CXCR4 expression on PBMCs of infected individuals wasassociated with the onset of heart failure (22). In Schistosomamansoni infections, unbalanced chemokine receptor activationwith increased expression of CCR1 relative to CCR5 correlatedwith a severe form of the disease (23). Chemokine receptor ex-pression can also be altered in viral infections such as chronichepatitis C infection, in which reduced surface expression ofCCR1 and CCR5 on peripheral blood CD8+ T lymphocytes resultsin lower CC chemokine responsiveness (24), which, in turn, mayaffect T cell recruitment to the infected liver, an important step inclearing the virus. The relationship between chemokine receptorsand cell distribution has also been shown in the wart, hypo-gammaglobulinemia infection, and myelokathexis syndrome, a rarecombined (B and T cell) immunodeficiency associated with ab-normal retention of neutrophils in the bone marrow that is linked toCXCR4 dysfunction (25, 26).Evidence for defects in the expression of certain chemokine

receptors has also been reported in individuals with lymphaticfilariasis. Among Fil+ individuals, those with chronic lymphaticpathology have an augmented expression of CCR9 and diminishedexpression of CXCR1 and CXCR3 compared with Fil2 individ-uals (27). Furthermore, CCR9 expression on these cells correlatedwith the grade of lymphedema in these patients (27). Finally, ina murine model of onchocerciasis (river blindness), it has been

FIGURE 7. mf-exposed DCs have a reduced mag-

nitude of calcium flux in response to MCP3 and

RANTES compared with unexposed cells. A, A repre-

sentative Ca2+ flux plot (1 of 10 donors) showing DCs

and mf-exposed DCs in response to MCP3. B, Increase

in intracellular calcium concentration was measured in

response to MCP3, MIP-1b, RANTES, and ATP in

unexposed monocyte-derived DCs (circles) and DCs

that were exposed to live B. malayi microfilariae for

48 h (squares). Responses shown are the peak increase

in fluorescence units minus basal (RFU maximum 2baseline). The graph represents averages of duplicate

samples in each experiment. Each line represents an

independent experiment (n = 6–10 donors).

8 CIRCULATING DENDRITIC CELL POPULATIONS IN FILARIASIS

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demonstrated that CXCR2 expression is critical for neutrophilinfiltration to the cornea (28).We wanted to determine the direct role of mf on the differences

in chemokine receptor expression observed between infected anduninfected populations. Using an in vitro model, live mf signifi-cantly downregulated the cell-surface expression of CCR1 but didnot alter the expression of CCR5 on DCs (Fig. 6), suggesting thatmf may directly influence chemokine expression (and respon-siveness) in mDCs in vivo. These data found functional correlatesin the diminished signaling induced by CCR1 ligands.Chemokine receptor-induced calcium signaling is not com-

pletely understood, although it has been used as a surrogate of CCRsignaling. It has been shown that calcium channel blockers as wellas phospholipase C and protein kinase inhibitors affect calcium fluxin response to some of the CCR ligands (29). In this study, wemeasured the capacity of mf-exposed DCs to induce calcium fluxin response to some CCR ligands. Our data indicate that mf-exposed DCs had a diminished ability to induce calcium flux inresponse to the CCR1 ligand MCP3 as well as to RANTES, aligand that signals through both CCR1 and CCR5. Whereas therewas a minimal increase in the intracellular calcium concentrationof DCs in response to MIP-1b (a ligand that binds and signalsthrough CCR5 but not CCR1), exposure to mf did not alter thisresponse, suggesting that a specific alteration in expression ofCCR1 (but not CCR5) on DCs by mf resulted in diminished abilityof these cells to trigger a response through ligands such as MCP3and RANTES that signal through this receptor. These data col-lectively suggest that mf downmodulate the cell-surface expres-sion of CCR1 on DCs and thereby alter the levels of receptor-mediated signaling to CCR1 ligands.In conclusion, our results suggest that Fil+ individuals have

a significantly increased number of circulating mDCs comparedwith Fil2 individuals from the same endemic region. Our studiesalso reveal an important finding that this mDC redistribution inFil+ patients may be due to lower levels of CCR1 expression.Finally, we demonstrated the potential relevance of this mechanismin human infections by showing that live mf negatively regulate theexpression and function of CCR1 on monocyte-derived DCs, whichin turn may be one mechanism for the aberrant redistribution ofthis important cell population.

AcknowledgmentsWe thank the residents of N’Tessoni for their cooperation and participation

in the clinical study and extend heartfelt thanks to Dr. Richard Sakai (Mali

International Center of Excellence in Research, National Institute of Al-

lergy and Infectious Diseases, Bamako, Mali) and members of the Filari-

asis Unit of the University of Bamako for invaluable logistical support

for conduct of the study under difficult conditions in a remote area of

Mali. We also thank Drs. Philip Murphy, Sunny Yung, Jiliang Gao, Theresa

Ojode, and William Kovacs for providing equipment and generous assis-

tance for the calcium flux assays, Brenda Rae Marshall for editorial help,

and Abhi Kole (Laboratory of Parasitic Diseases, National Institute of

Allergy and Infectious Diseases) for assistance with filarial Ag measure-

ment by ELISA.

DisclosuresThe authors have no financial conflicts of interest.

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