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 This work was supported by Public Health Service Awards HL (RST) and AI (MHK). Study group 14 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 cultures PBMCs 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 analysis Dendritic 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 analysis Patient 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 PCR Cells were collected and total mRNAs were extracted by Trizol reagent. TLR9 mRNA level was measured by realtime PCR. Statistical Analysis Differences 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 Mite IgE- Mite IgE+ P value Gender (male%) Age (month) Total Serum IgE (IU/ml) * * * * * * * * P=0.10 * +43.4% +64.5%