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detected by using high-resolution metabolomics are presentlyuncharacterized. To this point, we believe there is a criticalneed for a reference database of high-resolution metabolomicdata for healthy subjects. However, there is no consensus onhow a healthy control subject should be defined. Given the degreeof variability present within a nonasthmatic population, webelieve that ‘‘not having asthma’’ would not be sufficientfor such a purpose because issues of confounding related tomultimorbidity, the exposome, the genome, and the proteomewould ultimately remain.

For these reasons and others, our metabolomic study ofsevere asthma2 did not seek to identify biomarkers of asthmasusceptibility but rather to identify biomarkers of asthma severity.Although the former approach implies a case versus controldesign, the latter approach instead was selected and was thereforelimited to an asthma-only population. We believe this approachminimizes some confounding by medication exposure, allergicsensitization, and other factors relevant to the asthma medicalhistory (including genetics). However, despite our best attempts,there are remaining sources of confounding, as recognized byAmaral,1 that cannot be fully addressed with an observationalstudy design. Therefore we can only hope that our preliminaryfindings will lay critical groundwork for future studies. Studiesincorporating a variety of ‘‘-omics’’ (ie, systems biologyapproaches) are of greatest need if we intend to truly advancethe understanding and treatment of asthma. However, such studieswill remain challenging and perhaps be nonreproducible unlessthe issue associated with the identification and characterizationof healthy control populations is solved.

How might investigators in the metabolomics communityidentify and characterize healthy control subjects? We believethe solution to this challenge might rest with children. Theliterature on multimorbidity3 demonstrates that most children arein a predisease state and, furthermore, that they are in a stateof premultimorbidity. Sampling of blood, urine, breath, andother noninvasive biospecimens from a very large number(ie, thousands) of children might help to unravel the metabolomein a state of health, which could then be compared with states ofdisease and, eventually, multimorbidity. Implicit in this notion isthe need for integrated, phenotypic-intensive biospecimen repos-itories with publicly accessible databases, perhaps similar to whatis currently present for genomic research. As pediatric researcherswho routinely struggle with methodological research issues andknowledge gaps in childhood asthma,4 a collaborative pediatricapproach would be of great interest. Given that most studies ofasthma are conducted in adults and the findings are then extrapo-lated to children,4 a large pediatric research initiative, such as thisone, would be a refreshing change of pace as we seek to enhanceunderstanding of the asthma spectrum across the age span.

Anne M. Fitzpatrick, PhDa

Youngja Park, PhDb,c

Lou Ann Brown, PhDa

Dean P. Jones, PhDb

From the Departments of aPediatrics and bMedicine, Emory University School of

Medicine, Atlanta, Ga, and cthe College of Pharmacy, Korea University, Seong

City, Korea. E-mail: anne.fitzpatrick@emory.edu.

The original article associated with this correspondence was funded by grant RO1

NR012021 and the National Center for Advancing Translational Sciences UL1

TR000454.

Disclosure of potential conflict of interest: A. M. Fitzpatrick has consultant

arrangements with MedImmune, Merck, GlaxoSmithKline, Boehringer-Ingelheim,

and Genentech. The rest of the authors declare that they have no relevant conflicts

of interest.

REFERENCES

1. Amaral AFS. Metabolomics of asthma. J Allergy Clin Immunol 2014;133:1497-9.

2. Fitzpatrick AM, Park Y, Brown. LAS, Jones DP. Children with severe asthma have

unique oxidative stress–associated metabolomic profiles. J Allergy Clin Immunol

2014;133:258-61.e8.

3. Barnett K, Mercer SW, Norbury M, Watt G, Wyke S, Guthrie B. Epidemiology of

multimorbidity and implications for health care, research, and medical education:

a cross-sectional study. Lancet 2012;380:37-43.

4. Szefler SJ, Chmiel JF, Fitzpatrick AM, Giacoia G, Green TP, Jackson DJ, et al.

Asthma across the ages: knowledge gaps in childhood asthma. J Allergy Clin

Immunol 2014;133:3-13.

Available online March 29, 2014.http://dx.doi.org/10.1016/j.jaci.2014.02.012

GM-CSF along with IL-4 but not alone isindispensable for the differentiation of humandendritic cells from monocytes

To the Editor:L�opez-Bravo et al1 reported that IL-4 blocks TH1-polarizing/in-

flammatory cytokine expression duringmurinemonocyte-deriveddendritic cell (Mo-DC) differentiation. For their experimental set-up, authors cultured monocytes in the presence of GM-CSF alonefor 24 hours to obtain control Mo-DCs or with IL-4 to deriveIL-4-Mo-DCs. They concluded that IL-4 during murine Mo-DCdifferentiation prevent the ability of LPS-stimulated Mo-DCs toproduce TH1-polarizing and inflammatory cytokines. To obtaintranslational insight on this report, we investigated whether theMo-DC differentiation condition and duration, and conclusionmade by L�opez-Bravo et al, is also pertinent in human Mo-DCs.

We differentiated CD141 human monocytes with GM-CSFalone or with IL-4 for 24 hours (see the Methods section in thisarticle’s Online Repository at www.jacionline.org) as describedby L�opez-Bravo et al. In contrast to mice, human monocytes didnot differentiate into DCs in 24 hours in either of the conditions(Fig 1, A and B). Most of the cells remained positive for CD14,and only 2% to 3% of the cells were CD1a1, a differentiationmarker for human DCs. We then followed the kinetics of Mo-DCdifferentiation over 5 days.2 Monocytes differentiated with GM-CSF and IL-4 progressively lost CD14 and CD16 during thisperiod. At day 5, differentiated DCs were either negative or verylow positive for the above markers and were highly positive forCD1a (Fig 1, C). In contrast, cells differentiated in GM-CSF alonenever acquired the DC phenotype even after 5 days: about 20% ofthe cells retained CD14 and were highly positive for CD16.The expression of CD1a was less than 10% (Fig 1, C). Thus,GM-CSF–differentiated cells exhibited phenotype similar tothose of M1-type macrophages rather than DCs.3 Of note,the expression of CD14 and CD16 was retained on these GM-CSF-differentiated M1-type cells even following LPS stimulationwhile they were negative on GM-CSF and IL-4–differentiatedDCs (Fig 1, D and E).

As in the study by L�opez-Bravo et al, GM-CSF–differentiatedcells produced large quantities of inflammatory cytokines on LPSstimulation (Fig 1, F). But unlike mice, these data only indicatedifferences between human M1 macrophages and DCs but not2 DC subsets in their ability to produce cytokines. Thehigh proportion of inflammatory cytokines produced byGM-CSF–differentiated cells could also be attributed to CD14

FIG 1. Both GM-CSF and IL-4 are indispensable for the human Mo-DC differentiation. A and B, Flow cyto-

metric analysis of cells following 24 hours differentiation of monocytes in GM-CSF or GM-CSF 1 IL-4.

C, Kinetics of expression ofmarkers over 5 days of differentiation (n5 5). Phenotype (D and E) and cytokines

(F) secreted by LPS-stimulated GM-CSF or GM-CSF1IL-4–differentiated cells (n 5 5). G, T-cell–polarizing

ability of GM-CSF1IL-4–differentiated DCs stimulated with ManLAM or ManLAM 1 Rv3812 (n 5 3).

*P < .05, **P < .01, and ***P < .001.

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molecules on these cells because CD14 acts as a coreceptor forLPS.4 However, no major differences were observed in IL-10and IL-12 secretion by these 2 cell types.

Pathogen-associated molecular patterns (PAMPs) havedifferential abilities to polarize T cells on interaction with DCs.5

By using PAMPs of Mycobacterium tuberculosis, that is,

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mannosylated lipoarabinomannan (ManLAM), a glycolipid,6 andRv3812, a cell wall–associated protein belonging to proline-glutamic acid (PE)-polymorphic GC-rich repetitive-sequences(PGRS) family,7 we examined the T-cell–polarizing capacity ofGM-CSF-IL-4–differentiated Mo-DCs. While DCs stimulatedwith ManLAM polarized TH2 responses, this polarization couldbe skewed toward TH1 when DCs were stimulated with a combi-nation of ManLAM and Rv3812 (Fig 1, G). Thus, the presenceof IL-4 during human Mo-DC differentiation does not block theability of DCs to polarize TH1 responses; rather, it is dictated byPAMP-pattern recognition receptor interaction.

To conclude, although murine DCs could be obtained byculturing monocytes with GM-CSF alone for 24 hours, thiscondition is not applicable for humans because it will yieldM1-type macrophages. Thus, GM-CSF and IL-4 are bothindispensable for the differentiation of human Mo-DCs and thesecells retain the ability to polarize TH1 responses depending on thestimuli they receive.

We thank the National Institutes of Health Biodefense and Emerging

Infections Research Resources Repository, National Institute of Allergy and

Infectious Diseases, National Institutes of Health, for kindly providing

ManLAM of Mycobacterium tuberculosis, strain H37Rv, NR-14848.

Sahana Holla, MSca,b*

Meenu Sharma, MSca,c*

Janakiraman Vani, PhDa,b

Srini V. Kaveri, DVM, PhDa,d,e,f

Kithiganahalli N. Balaji, PhDb�Jagadeesh Bayry, DVM, PhDa,d,e,f�

From aUnit�e 1138, Institut National de la Sant�e et de la Recherche M�edicale, Paris,

France; bthe Department of Microbiology and Cell Biology, Indian Institute of

Science, Bangalore, India; cUniversit�e de Technologie de Compi�egne, Compi�egne,

France; dCentre de Recherche des Cordeliers, Equipe 16-Immunopathology and

Therapeutic Immunointervention, Universit�e Pierre et Marie Curie – Paris 6, Paris,

France; eUniversit�e Paris Descartes, Paris, France; and fInternational Associated

Laboratory IMPACT (Institut National de la Sant�e et de la Recherche M�edicale,

France – Indian Council of Medical Research, India), National Institute of

Corrections

With regard to the article in theMarch 2014 issue entitled ‘‘Randragweed sublingual-liquid immunotherapy for allergic rhinocoauthors report that Table I contains an error. The values for ‘‘RWshould be 11.3 6 18.9 for Placebo and 14.6 6 27.3 for RW-SAIconclusions. The authors regret the error.

Erratum to Genetic effect of single-nucleotideairway hyperreactivity in asthmatic patients

[J Allergy Clin Immunol 2014;133(Suppl 2);AB67]

Dr. Jong-Sook Park, MD, Dr. Myung-Sin Kim, Dr. Sung-Woo Pa

Soonchunhyang University Bucheon Hospital, Bucheon, South K

The Publisher regrets that this article is an accidental duplicaAllergy 2011;41:1533-44, http://dx.doi.org/10.1111/j.1365-222withdrawn.

Immunohaematology, Mumbai, India. E-mail: jagadeesh.bayry@crc.jussieu.fr.

Or: balaji@mcbl.iisc.ernet.in.

*These authors contributed equally to this work.

�These authors contributed equally as senior authors.

This study was supported by Institut National de la Sant�e et de la Recherche M�edicale,

Universit�e Pierre et Marie Curie, Universit�e Paris Descartes (S.V.K. and J.B.), Centre

National de la Recherche Scientifique (S.V.K.), Indian Institute of Science,

Department of Biotechnology, Department of Science and Technology, Council for

Scientific and Industrial Research (K.N.B.), a grant from the Indo-French Center

for Promotion of Advanced Research (CEFIPRA, Reference No. 4803-1) (J.B. and

K.N.B.), fellowships from the Indian Institute of Science and CEFIPRA (S.H.), and

a fellowship from CEFIPRA (M.S.).

Disclosure of potential conflict of interest: K. N. Balaji and J. Bayry have

received research support from the Indo-French Center for Promotion of Advanced

Research. The rest of the authors declare that they have no relevant conflicts of

interest.

Editor’s note: There is no accompanying reply to this correspondence.

REFERENCES

1. L�opez-Bravo M, Minguito de la Escalera M, Dom�ınguez PM, Gonz�alez- Cin-

tado L, del Fresno C, Mart�ın P, et al. IL-4 blocks TH1-polarizing/

inflammatory cytokine gene expression during monocyte-derived dendritic

cell differentiation through histone hypoacetylation. J Allergy Clin Immunol

2013;132:1409-19.

2. Trinath J, Hegde P, Sharma M, Maddur MS, Rabin M, Vallat JM, et al.

Intravenous immunoglobulin expands regulatory T cells via induction of

cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells. Blood

2013;122:1419-27.

3. Lacey DC, Achuthan A, Fleetwood AJ, Dinh H, Roiniotis J, Scholz GM, et al.

Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by

in vitro models. J Immunol 2012;188:5752-65.

4. Zanoni I, Ostuni R, Marek LR, Barresi S, Barbalat R, Barton GM, et al. CD14 con-

trols the LPS-induced endocytosis of Toll-like receptor 4. Cell 2011;147:868-80.

5. Walsh KP, Mills KH. Dendritic cells and other innate determinants of T helper cell

polarization. Trends Immunol 2013;34:521-30.

6. Geijtenbeek TB, Van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-

Grauls CM, Appelmelk B, et al. Mycobacteria target DC- SIGN to suppress

dendritic cell function. J Exp Med 2003;197:7-17.

7. Vani J, Shaila MS, Trinath J, Balaji KN, Kaveri SV, Bayry J. Mycobacterium tubercu-

losis cell wall-associated Rv3812 protein induces strong dendritic cell- mediated inter-

feron gamma responses and exhibits vaccine potential. J Infect Dis 2013;208:1034-6.

Available online April 3, 2014.http://dx.doi.org/10.1016/j.jaci.2014.02.021

omized, double-blind, placebo-controlled trial of standardizednjunctivitis’’ (J Allergy Clin Immunol 2014;133:751-8), the-specific IgE (kU/L), mean (SD)’’ in the Treatment columnsL. The error does not affect the integrity of the data or study

polymorphisms in the PPARGC1B gene on

rk, Dr. An-Soo Jang, Dr. Choon-Sik Park, MD

orea

tion of an article that has already been published in Clin Exp2.2011.03801.x. The duplicate article has therefore been

METHODSHuman monocyte differentiation

Human PBMCs were isolated from buffy coats of healthy blood donors

purchased from Centre Necker-Cabanel (Etablissement Francais du Sang,

Paris, France). Ethical committee permission was obtained for the use of buffy

coats of healthy donors (No. 12/EFS/079). Circulating monocytes were

isolated from these PBMCs using CD14 microbeads (Miltenyi Biotec, Paris,

France). The purity was more than 98%. Monocytes were cultured in RMPI-

1640 medium containing 10% FCS for indicated days in the presence of

recombinant human GM-CSF (1000 IU/106 cells) alone or with recombinant

human IL-4 (500 IU/106 cells)E1 (both fromMiltenyi Biotec) and used for sub-

sequent experiments and analysis.

Stimulation of cells with LPSCells were harvested following differentiation of monocytes for 5 days

either in GM-CSF alone or in GM-CSF and IL-4. The cells were washed and

further cultured in respective cytokines and stimulated with LPS (100 ng/mL/

0.5 3 106 cells, from Escherichia coli, Sigma-Aldrich, Lyon, France) for 24

hours. Cell culture supernatants were collected for analyzing cytokines, and

the phenotype of cells were analyzed by flow cytometry.

DC:CD41 T-cell coculturesDCswere derived from differentiation of monocytes for 5 days in GM-CSF

and IL-4. The cells were washed and were cultured (0.53 106 cells/mL) with

GM-CSF and IL-4 alone or GM-CSF, IL-4, 5 mg/mL of ManLAM (BEI

resource, purified from H37Rv, NR-14848) or GM-CSF, IL-4, 5 mg/mL of

ManLAM, 5 mg/mL of Rv3812 for 48 hours. Recombinant Rv3812 protein

was obtained as previously described.E2 Following extensive washings, DCs

were cocultured with CD41 T cells at 1:20 for 96 hours. T-cell cytokines in

DC:T cell cocultures were analyzed in cell-free culture supernatants.

Flow cytometrySurface staining of cells was carried out by using fluorochrome-conjugated

mAbs to CD14, CD16, and CD1a (all from BDBiosciences, Le Pont de Claix,

France). Cells were further processed for flow cytometry wherein 5000 gated

events were recorded for each sample acquired and the data were analyzed

using FACSDiva software (BD Biosciences).

Cytokine analysisCytokines were quantified in cell-free culture supernatants using BD

Cytometric Bead Array human inflammatory cytokine kits and human TH1/

TH2 cytokine kits (BD Biosciences). The data were analyzed using FCAP

Array software (BD Biosciences).

Statistical analysisLevels of significance for comparison between samples were determined

by 2-tailed nonparametric Mann-Whitney test (for data depicted in figures as

the median along with an appropriate range values) or by Student t test (for

data depicted as means and SDs).E3 Values of P < .05 were considered statis-

tically correlated (*P < .05, **P < .01, ***P < .001). All statistical analyses

were performed using Prism 5 software (GraphPad Software, Inc, La Jolla,

Calif).

REFERENCES

E1. Trinath J, Hegde P, Sharma M, Maddur MS, Rabin M, Vallat JM, et al.

Intravenous immunoglobulin expands regulatory T cells via induction of

cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells. Blood

2013;122:1419-27.

E2. Vani J, Shaila MS, Trinath J, Balaji KN, Kaveri SV, Bayry J. Mycobacterium

tuberculosis cell wall-associated Rv3812 protein induces strong dendritic

cell-mediated interferon gamma responses and exhibits vaccine potential.

J Infect Dis 2013;208:1034-6.

E3. Olsen CH. Review of the use of statistics in infection and immunity. Infect Immun

2003;71:6689-92.

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