Dendritic cell and T cell responses in children with food allergy.

BACKGROUND: Food allergy (FA) and eosinophilic oesophagitis (EE) are increasingly common clinical problems. Dendritic cells (DCs) are key regulators of the sensitization and effector phases of allergic immune responses, but their role in these diseases is largely unknown. OBJECTIVE: To evaluate for alterations in the phenotype and function of DCs in children with IgE-mediated milk allergy or EE compared with their non-affected siblings. METHODS: Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were prepared from peripheral blood of children with milk allergy (FA), EE, and non-affected siblings (CON). Purified pDCs and mDCs were cultured alone or with autologous CD4(+) lymphocytes. Cytokine levels in plasma, or culture supernatants following stimulation, were measured using multiplex array immunoassay. Cell-surface molecule expression was determined by flow cytometry. RESULTS: DCs from FA subjects produced greater levels of pro-inflammatory cytokines (IL-6, TNF-α), granulocyte macrophage-colony forming factor, and mDC-derived IL-10 compared with controls following allergen exposure. T(H) 2 but not T(H) 1 cytokines were spontaneously produced in DC-CD4(+) T cell co-cultures from children with FA and were not significantly increased after stimulation with milk extract, suggesting an ongoing activation in vivo. This hypothesis was further supported by evidence for elevated IL-5 and IL-13 protein in the plasma of children with both FA and EE. The only significant DC phenotypic differences were: (1) reduced levels of CD80 in EE subjects and (2) FcɛRI expression that correlated with serum IgE levels in both groups of subjects. CONCLUSION: This study suggests that DCs from children with FA and EE produce more pro-inflammatory cytokines, and that their CD4(+) T cells are spontaneously activated to produce T(H) 2 cytokines in the presence of FcɛRI-bearing DCs.

Subcutaneous allergen immunotherapy restores human dendritic cell innate immune function.

BACKGROUND: We recently reported that human blood dendritic cells from allergic subjects have impaired IFN-alpha production following toll-like receptor 9 (TLR9)-dependent innate immune stimulation. It is not known how subcutaneous allergen immunotherapy (SCIT) affects dendritic cell immune responses. OBJECTIVE: The aim of this study is to determine how SCIT affects human dendritic cell function. METHODS: Peripheral blood mononuclear cell (PBMC) and plasmacytoid dendritic cells (pDCs) were isolated from the blood of seven dust mite allergic subjects at baseline and upon reaching a standard SCIT maintenance dose that included dust mite and other aeroallergens. Cells were stimulated with various adaptive and innate immune receptor stimuli, or media alone for 20 h with secreted cytokine levels determined by ELISA. A portion of the cells were used to measure intracellular signalling proteins by flow cytometry. Humoral immune responses were measured from plasma. RESULTS: SCIT resulted in a threefold increase in PBMC production of IFN-alpha in response to CpG at 100 nM (P=0.015) and at 500 nM (P=0.015), n=7. The predominant cell type known to produce IFN-alpha in response to CpG (CpG ODN-2216) and other TLR9 agonists is the pDC. As expected, a robust innate immune response from isolated pDCs was re-established among allergic subjects undergoing SCIT resulting in a fivefold increase in IFN-alpha production in response to CpG at 500 nM (P=0.046), n=7. In contrast, IL-6 production was unaffected by SCIT (P=0.468). Consistent with published reports, IgG4 blocking antibody increased 10-fold with SCIT (P=0.031), n=7. There was no significant increase in the frequency of pDCs or the expression of TLR9 that would account for the rise in IFN-alpha production. CONCLUSIONS: Allergen immunotherapy increases dendritic cell TLR9-mediated innate immune function, which has previously been shown to be impaired at baseline in allergic subjects.

Pulmonary allergic responses augment interleukin-13 secretion by circulating basophils yet suppress interferon-alpha from plasmacytoid dendritic cells.

BACKGROUND: Allergic inflammatory processes may have the capacity to propagate systemically through the actions of circulating leucocytes. Consequently, basophils from allergic individuals are often 'primed', as evidenced by their hyperresponsiveness in vitro. IFN-alpha secreted predominantly by plasmacytoid dendritic cells (pDCs), suppresses basophil priming for IL-13 production in vitro. OBJECTIVE: This study sought in vivo correlates arising during experimental allergen challenge that support an 'axis-interplay' between basophils and pDCs. METHODS: Using segmental allergen challenge (SAC) in the lung, the immune responses of both cell types from the blood were investigated in volunteers (n=10) before and 24 h after allergen exposure. These responses were then correlated with inflammatory parameters measured in bronchoalveolar lavage fluids (BALF). RESULTS: In the blood, SAC significantly augmented IL-13 secretion by basophils induced by IL-3 (P=0.009), yet reduced IFN-alpha secreted by pDCs stimulated with CpG (P=0.018). Both parameters were negatively correlated (P=0.0015), at least among those subjects that secreted the latter. Circulating basophil IL-13 responses further correlated with post-SAC bronchoalveolar lavage (BAL) parameters including IL-13 protein (P=0.04), basophil (P=0.051), eosinophil (P=0.0018), and total cell counts (P<0.003). Basophil and IL-13 levels in BAL correlated likewise (P=0.0002). CONCLUSIONS: These results support a mechanism of immune regulation whereby an allergen reduces innate immune responses and IFN-alpha production by pDCs, resulting in an enhanced inflammation and basophil cytokine production at sites of allergen exposure.

Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon-alpha via Toll-like receptor 9.

BACKGROUND: High-affinity IgE receptor (Fc epsilon RI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro-inflammatory cytokines (IL-6, TNF-alpha) following Fc epsilon RI stimulation - a mode of activation that simultaneously reduces expression of Toll-like receptor 9 (TLR9). Whether or not TLR9 and/or Fc epsilon RI levels and their function on dendritic cells relate to allergic status is unknown. OBJECTIVE: The aim of this study is to compare the innate (TLR9-mediated) immune response of human pDCs to TLR9 and Fc epsilon RI alpha receptor expression in allergic and non-allergic subjects. METHODS: Basophil-depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non-allergic subjects. Intracellular TLR9 and surface Fc epsilon RI alpha expression in blood dendritic cell antigen-2-positive cells were determined by flow cytometry. Activating anti-IgE antibody, anti-Fc epsilon RI alpha antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA. RESULTS: No difference in the frequency of pDCs was detected among allergic (n=9) vs. non-allergic (n=11) subjects (P=0.261). While there was also no difference in the baseline expression of TLR9, pDCs from allergic subjects produced sixfold less IFN-alpha when stimulated with CpG (P=0.002). Conversely, there was higher Fc epsilon RI alpha expression (P=0.01) on the pDCs of allergic subjects. CONCLUSIONS: Impaired TLR9-dependent immune responses in human pDCs are associated with allergic status and inversely correlated with Fc epsilon RI alpha expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG-based immunotherapy.