House dust mite (HDM) is a common allergen and reactivity is associated with asthma and atopic dermatitis. The development of these allergic diseases depends upon T-helper type 2 (Th2) cells and dendritic cells (DCs). To study the phenotype and functional characteristics of HDM-specific T cells and DCs in both HDM-reactive (mite IgE+) and non-reactive (mite IgE-) children with atopic dermatitis, we examined the responses of recombinant Dermatophagoides farinae (Der f) proteins and toll-like receptor (TLR) ligands on peripheral blood mononuclear cells (PBMC) from 14 HDM-reactive and 14 matched HDM-non-reactive children, respectively. We found that incubation with Der f proteins induced a significant increase in Th2 cytokine production in cultures of PBMC from HDM-reactive compared to non-reactive children. Interestingly, TLR ligand treatment stimulated greater IL-6, IL-10, IFN-alpha, and IL-1beta production in cultures of PBMCs from HDM-reactive but not in HDM-non-reactive children. Further studies demonstrated that HDM-reactive children had higher percentages of plasmacytoid dendritic cells (pDC) in the peripheral blood and expressed more TLR9 mRNA in PBMCs. Our data suggested that PBMCs from children reactive to Der f had higher Th2 cytokine production and had altered DC responses to TLR ligands, suggesting that in an atopic environment, re-programmed DC responses may contribute to continuing allergic responses.

Key words: HDM, TLR9, PBMC, DC

PBMCs from children reactive to Der f had higher Th2 cytokine production and had altered DC responses to TLR ligands, suggesting that in an atopic environment, re-programmed DC responses may contribute to continuing allergic responses.

Department of Pediatrics, Division of Pulmonology, Critical Care and Allergy, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

This work was supported by Public Health Service Awards HL080071 (RST) and AI070448 (MHK).

Study group14 mite positive and 14 mite negative infants with a history of eczema were recruited. Subjects were excluded for premature birth (<36 weeks gestation), congenital malformations of the cardio-respiratory system, history of lower respiratory illness or wheezing. The Institutional Review Board approved the study and informed consent was obtained from parents. All subjects were evaluated at James Whitcomb Riley Hospital for Children, Indianapolis, Indiana.PBMC culturesPBMCs were specifically activated with recombinant (r)Der f. protein and TLR ligands, respectively. (r)Der f. treatment PBMC will be cultured for 7 days and TLR treatment PBMC will be cultured for 24 hours. Flow cytometric analysisDendritic cells type 1 (mDC1), mDC2 and Plasmocytoid DC (pDC) were stained using a blood dendritic cell enumeration kit following manufacturer instructions (Miltenyi Biotec) and analyzed in a FACScalibur cytometer (Becton Dickinson, San Jose, CA). Cytokine multiplex analysisPatient cultured supernatants were collected and levels of IL-4, IL-5, IL-13, IFN-γ and IL-1β, IL-6, IL-10, and IFN-α were measured using the Multiplex Bead Immunoassays as per manufacturer’s protocol (Millipore, Billerica, MA). Realtime PCRCells were collected and total mRNAs were extracted by Trizol reagent. TLR9 mRNA level was measured by realtime PCR. Statistical AnalysisDifferences between mite negative and positive subjects were compared using Student’s t-test.

ABSTRACT

PATIENTS AND METHODS

RESULTS

CONCLUSION

ACKNOWLEDGEMENTS

CONTACT INFORMATION

Figure 1. Cytokine levels after Der f stimulation.

Figure 2. Cytokine levels after CpG or poly I:C stimulation.

Figure 3. Dendritic cell distribution in mite- vs mite+ patients.

Figure 4. TLR9 mRNA level after CpG stimulation.

Table 1. Study demographics.

Altered Dendritic Cell Responses in Infants with Atopic Reactivity to Dermatophagoides Farinae

Weiguo Yao, Robert S. Tepper and Mark H. Kaplan

Department of Pediatrics, Division of Pulmonology, Critical Care and Allergy, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202

Mite

IgE-

Mite

IgE+

P

value

Gender (male%) 57 71 0.39

Age (month) 24.1 24.4 0.86

Total Serum IgE (IU/ml) 104 464 0.03*

IL4 and IL5 level after 7day stimulation

0

5

10

15

20

25

30

IL4 IL5

pg/m

l

no Der f (mite-)

no Der f (mite+)

Der f (mite-)

Der f (mite+)

*

IL13 and IFN-Y level after 7d stimulaiton

0

200

400

600

800

1000

1200

IL13 IFN-Y

pg/m

l

*

IL-6 level after 24h stimulation

0

1000

2000

3000

4000

5000

control CpG poly I:C

pg

/ml

mite -

mite +

IL-10 level after 24h stimulation

0

100

200

300

400

control CpG poly I:C

pg

/ml

IFN-alpha level after 24h stimulation

0

200

400

600

control CpG poly I:C

pg

/ml

*

*

*

* *

P=0.10

IL-1beta level after 24h stimulation

0

200

400

control CpG poly I:C

pg/m

l

*

Dendritic cell distribution in mite- vs mite+ patients

0

0.2

0.4

0.6

0.8

1

1.2

1.4

pDC(BDCA2+) mDC1(BDCA1+) mDC2(BDCA3+)

perc

en

tag

e

mite-

mite+

+43

.4%

TLR9 mRNA level after CpG stimulation

0

2

4

6

8

mite- mite+

RQ

+64

.5%