Title: House dust mite HDM is a common allergen and reactivity is associated with asthma and atopic dermati
1Altered 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
ABSTRACT
RESULTS
Figure 3. Dendritic cell distribution in mite- vs
mite patients.
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
Table 1. Study demographics.
Figure 4. TLR9 mRNA level after CpG stimulation.
Figure 1. Cytokine levels after Der f stimulation.
64.5
PATIENTS AND METHODS
Study group 14 mite positive and 14 mite negative
infants with a history of eczema were recruited.
Subjects were excluded for premature birth (lt36
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-? and IL-1ß, IL-6, IL-10, and IFN-a were
measured using the Multiplex Bead Immunoassays as
per manufacturers 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 Students t-test.
CONCLUSION
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.
Figure 2. Cytokine levels after CpG or poly IC
stimulation.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service
Awards HL080071 (RST) and AI070448 (MHK).
CONTACT INFORMATION
Department of Pediatrics, Division of
Pulmonology, Critical Care and Allergy, Wells
Center for Pediatric Research, Indiana University
School of Medicine, Indianapolis, IN 46202