Plasmacytoid Dendritic Cell Deficiency In Neonates Enhances Allergic Airway Inflammation Via Reduced Production Of IFN-α

Background and ObjectiveAsthma is an airway inflammatory syndrome with highly heterogeneity.The most prevalent form of asthma is allergic asthma,which is mainly mediated by type 2 helper T cells（Th2 cells）and type 2 innate lymphoid cells（ILC2 cells）,leading to airway eosinophilia,mucus hyperplasia and airway hyperresponsiveness.Allergic asthma often begins in early life,at a time when immune responsiveness differs substantially from responses in adults.In neonates,adaptive immune responses are often Th2-biased and the underling mechanism remains elusive.DCs are a bridge between innate and adaptive immunity as they govern T cell activation and differentiation.In neonates,the activities of neonatal DCs are likely to play a central role in shaping Th2 skewed responses.Plasmacytoid DCs（pDCs）are a specific subset of DCs,are major type I IFN producers,and play an important role in anti-virus responses.pDCs have been shown to negatively regulate allergic airway responses.In mice,we previously found that there were very few pDCs in neonatal lungs.To elucidate the influence of age difference of pDCs on the tendency of neonatal Th2 immune response,we intend to conduct neonatal and adult mice asthma model to explore the diffence of airway allergic inflammation between age groups and determine the effect and mechanism of pDCs in such process.At last,to provide clinical evidence,we collect sputum samples from children and adult asthmatic patients and compare the number of pDCs between age groups.Methods1)Establishment of neonatal and adult allergic asthma mouse model andmeasurement of allergic responsesEstablishment of OVA-induced model:Neonatal（7 days old）and adult C57BL/6mice（6-8 weeks old）were sensitized by two injections of OVA adsorbed to aluminum hydroxide on days 0 and 7.Control mice were sensitized with saline.On days 15-19,all the mice were challenged by inhalation of OVA generated by a jet nebulizer for 30minutes.24 hours after the final challenge,mice were euthanized for analysis.Measurement of allergic responses:Flow cytometry was used to measure the number of eosinophils,neutrophils,ILC2s and DCs in the lung and BAL fluids;PAS staining of lung sections was used to detect goblet cells metaplasia and mucus hyperplasia;ELISA was used to measure the levels of Th2 cytokines in the culture supernatant of OVA stimulated Parabronchial lymph nodes（PBLN）cells;the concentration of serum total IgE and IgG1 was measured by ELISA.2)pDCs supplementation in neonatal miceTo verify the effect of pDCs on allergic immune response in neonatal mice,neonatal asthma model were treated with Flt3L to induce pDC differentiation during sensitization or adoptive transferred with pDCs before sensitization.Control mice were treated with PBS.After sensitization and challenge,the allergic responses were measured.3)Selective removal of pDCs in adult miceTo verify the effect of pDCs on allergic immune response in adult mice,120G8monoclonal antibody were injected to remove lung pDCs during sensitization and challenge phases in adult asthma model.Control mice were treated with isotype antibody.After sensitization and challenge,the allergic responses were measured.4)Recombinant mIFN-αadministration in neonatal miceTo explore whether IFN-αplays a negative regulatory role in allergic immune responses in neonatal mice,recombinant mIFN-αwas given at the sensitization and challenge phases in neonatal asthma model.The control mice were treated with PBS.After sensitization and challenge,the allergic responses were measured.5)Adoptive transfer of pDCs in wildtype and IFNAR1^-/-neonatal miceTo investigate whether pDC exerts its negative regulation of allergic immune response through IFN-αin neonatal mice,pDCs were transferred into wildtype and IFNAR1^-/-neonatal mice before sensitization.Control mice were treated with PBS.After sensitization and challenge,the allergic responses were measured.6)The interaction between pDCs and airway epithelial cells24 hours after the final challenge,the levels of IL-33,CCL20,and GM-CSF in the lung or BAL fluid were measured by ELISA in the aforementioned models.Airway epithelial cells of neonatal mice were sorted by flow cytometry and stimulated with OVA in vitro for 72 hours.The levels of IL-33 and CCL20 in the supernatant were measured by ELISA.To investigate the interaction of pDCs and airway epithelial cells,recombinant mIFN-αor pDC culture supernatant were added in this culture system in the presence or absence of IFNAR1 blocking antibody,anti-IFNAR1.7)Analysis of sputum samples from asthmatic children and adultsSputum samples from asthmatic children and adult patients were collected and the number of pDCs and the level of IFN-αin the sputum were measured by flow cytometry and ELISA,respectively.Results1)Establishment of neonatal and adult allergic asthma modelAfter OVA sensitization and challenge,allergic airway inflammation,indicated by airway eosinophilia,goblet cell metaplasia,mucus production,higher levels of Th2cytokines in PBLN culture supernatant and higher levels of IgE,IgG1 in the serum,were successfully induced in both age groups.Compared with adult mice,neonatal mice showed more severe allergic airway inflammation after OVA sensitization and challenge.2)The number of lung pDCs in neonatal mice is lower than adult miceThe number of lung pDCs in adult mice significantly increased after OVA sensitization and challenge.In contrast,the number of lung pDCs in neonatal mice was significantly lower than adult mice in negative control group（saline/OVA）,and did not increase upon OVA sensitization and challenge.At steady state,lung pDCs in neonates were hardly detectable and the number increased with age.3)pDCs supplementation reduces allergic inflammatory responses in neonatalmiceIn the neonatal asthma model,the number of lung pDCs significantly increased by Flt3L administration or adoptive transfer of pDCs.Compared with control group,both Flt3L administration and pDCs transfer significantly reduced OVA-induced allergic responses.4)Selective removal of pDCs enhances allergic responses in adult miceIn the adult asthma model,120G8 treatment significantly decreased the number of pDC in the lung compared with control group.120G8 treatment significantly enhanced OVA-induced allergic responses.5)Administration of IFN-αreduces allergic responses in neonatal miceIn the neonatal asthma model,recombinant mIFN-αadministration significantly reduced OVA-induced allergic responses.6)The protective role of pDCs is dependent on expression of IFNAR1In the neonatal asthma model,adoptive transfer of pDCs into wildtype mice significantly reduced OVA-induced allergic responses,but this protection was lost when pDCs were transferred into IFNAR1^-/-mice.7)The regulatory role of pDCs is through suppression of the secretion ofinflammatory cytokines from epithelial cellsIn both age groups,the levels of lung IL-33,CCL20 and GM-CSF were increased following OVA sensitization and challenge.Adoptive transfer of pDCs into neonatal mice before sensitization resulted in significantly lower level of these cytokines.In the in-vitro culture system,the concentrations of IL-33 and CCL20 in OVA-stimulated airway epithelial cell supernatants were significantly higher than in control group.When IFN-αor pDCs culture supernatants were added to the culture,the levels of IL-33 and CCL20 were markedly reduced.This suppressive effect was completely lost with the addition of anti-IFNAR1.8)Sputum pDCs numbers are lower in asthmatic children associated with lowerlevel of IFN-αThe number of pDCs and the concentration of IFN-αin sputum samples from asthmatic children were significantly lower than asthmatic adult patients.ConclusionOur study suggests that pDCs negatively regulate allergic airway inflammation and type 2 immune response in an IFN-α/IFNAR dependant manner.Because of the deficiency of pDCs,neonatal mice demonstrate unrestrained production of IL-33,CCL20 and GM-CSF from airway epithelial cells,which together with increased numbers of ILC2s and CD11b⁺cDCs,result in enhanced Th2 responses.This renders neonatal mice prone to develop more severe allergic airway responses upon allergen exposure.In light of the similar reduction in numbers of pDCs and IFN-αlevels in childhood asthmatics,this study provides a foundation towards better understanding of the mechanisms whereby infants exhibit a greater susceptibility to develop allergic immune responses in the lung.