2011Low neonatal Toll-like receptor 4-mediated interleukin-10 production is
doi:10.1111/j.1365-2222.2011.03857.x Clinical&Experimental Allergy,42,66–75 ORIGINAL ARTICLE Clinical Mechanisms in Allergic Disease?2011Blackwell Publishing Ltd Low neonatal Toll-like receptor4-mediated interleukin-10production is associated with subsequent atopic dermatitis
M.E.Belderbos1,E.F.Knol2,M.L.Houben1,G.M.van Bleek1,B.Wilbrink3,J.L.L.Kimpen1,M.Rovers4and L.Bont1
1Department of Pediatrics,University Medical Center Utrecht,Utrecht,The Netherlands,2Department of Dermatology and Allergology,University Medical Center Utrecht,Utrecht,The Netherlands,3Laboratory of Infectious Diseases and Perinatal Screening,National Institute of Public Health and the Environment,Bilthoven,The Netherlands and4Julius Center for Health Sciences and Primary Care,University Medical Center Utrecht,Utrecht,The Netherlands
Clinical& Experimental
Allergy Correspondence:
L.Bont,Room KE04.133.1, Wilhelmina Children’s Hospital,PO Box85090,3508AB Utrecht,The Netherlands.
E-mail:l.bont@umcutrecht.nl
Cite this as:M.E.Belderbos,E.F. Knol,M.L.Houben,G.M.van Bleek, B.Wilbrink,J.L.L.Kimpen,M.Rovers and L.Bont,Clinical&Experimental Allergy,2012(42)66–75.Summary
Background Atopic dermatitis(AD)and respiratory syncytial virus lower respiratory tract infection(RSV LRTI)are common diseases during early life.Impaired Th1-cell polarizing Toll-like receptor(TLR)responses play an important role in the pathogenesis of both dis-eases.Neonatal TLR-mediated production of Th1-type cytokines is decreased at birth,but rapidly increases during the?rst month of life.
Objective To determine whether decreased TLR-mediated production of Th1-polarizing cytokines,at the age of1month is associated with subsequent AD or RSV LRTI. Methods A prospective healthy birth cohort study was performed.Whole blood concen-trations of innate immune cells and TLR-mediated cytokine responses were measured at the age of1month in291neonates.AD was determined by a physician questionnaire at the age of1year and RSV LRTI was de?ned as parent-reported respiratory symptoms and presence of RSV RNA in a nose–throat specimen.
Results Of participating neonates,45(15%)developed AD and41(14%)developed RSV LRTI.Risks of AD and RSV LRTI were not associated(v2,P=1.00).AD was associated with decreased concentrations of basophils(7.6vs.14.09106/mL,P=0.002)and plas-macytoid dendritic cells(17.0vs.20.59106/mL,P=0.04),increased concentrations of NK-cells(79.7vs.45.19106/mL,P=0.03),and twofold lower TLR4-mediated IL-10 production(P=0.001).In contrast,RSV LRTI was associated neither with neonatal concentrations of innate immune cells,nor with TLR-mediated TNF-a,IL-12p70,IL-10or IFN-a production.
Conclusions and Clinical Relevance Atopic dermatitis,but not RSV LRTI,is associated with distinct pre-symptomatic differences in the innate immune system.We hypothesize that decreased neonatal IL-10-mediated immune regulation during early life might play a causal role in the initiation of AD.
Keywords allergic disease,atopy,bronchiolitis,endotoxin,hygiene hypothesis,newborn, eczema,Toll-like receptor
Submitted22April2011;revised17July2011;accepted18July2011
Introduction
Toll-like receptors(TLR)are key pathogen recognition receptors of the innate immune system that initiate the early antimicrobial response and orchestrate the ensu-ing adaptive response[1].We and others have shown that neonatal TLR responses are characterized by decreased production of Th1-polarizing cytokines (IL-12p70,TNF-a,IFN-a)and increased production of the regulatory cytokine IL-10compared with adult responses[2–7].Polarized neonatal cytokine responses are thought to prevent potentially harmful allo-immune responses towards maternal antigens in utero,and to facilitate microbial colonization after birth[3].How-ever,impaired generation of Th1-polarizing responses leaves the neonate highly susceptible to infection[8]. In addition,bias towards Th2-type immune responses at the neonatal and infant age has been associated
with increased risk of allergic disease in later life [9–11].
Atopic dermatitis(AD)is a chronic in?ammatory skin disorder,with a lifetime prevalence of20%in western-ized countries[12].AD occurs in the?rst6months of life in45%of children and before the age of one in 60%[12].Hyper-reactive Th2-type immune responses play a central role in the pathogenesis of AD[13]. Acute eczematous skin lesions are in?ltrated with Th2-cells expressing high levels of IL-4,IL-5and IL-13[14]. In addition,patients with AD have elevated IL-13-expressing T cells in the peripheral blood[14],increased eosinophils and high serum IgE[15].This Th2-bias might be dictated by the innate immune system,as both genetic and functional defects in TLRs and their down-stream signalling molecules are associated with AD [16–18].
Respiratory syncytial virus(RSV)is the most common cause of childhood acute lower respiratory tract infec-tion world-wide,affecting90%of children before the age of2years[19,20].Respiratory syncytial virus dis-ease severity varies from mild upper respiratory tract infection to lower respiratory tract infection(LRTI)and respiratory insuf?ciency necessitating hospitalization. Speci?c patterns of TLR-mediated cytokine responses might impact disease severity[21–23].Whereas most respiratory viruses induce robust Th1-type immune responses,immune responses in infants with acute RSV infection are characterized by low levels of Th1-type cytokines IFN-c and IL-12p70[21,22].In addition, infants with severe RSV bronchiolitis have an increased IL-10/IL-12mRNA ratio in nasal lavage?uid and ele-vated IL-4/IFN-c mRNA ratios in nasal lavage?uid and in stimulated PBMC[23]compared with infants with upper respiratory tract infection.Decreased serum con-centrations of IFN-c were observed in RSV-infected infants,but not in infants infected with parain?uenza-virus or adenovirus[22],suggesting that insuf?cient production of pro-in?ammatory or Th1-type cytokines is a speci?c feature of severe RSV infection[24,25]. We have previously identi?ed the?rst month of life as a critical period for the development of the TLR system.During this period,TLR-mediated cytokine res ponses rapidly mature from a Th2-biased pro?le to wards increased Th1-type responses characteristic of later life[26].Post-natal maturation of TLR responses is probably guided by exposure to innate immune stimuli, which might thus modulate the risk of subsequent dis-ease[27].In the current study,we hypothesized that neonates with delayed maturation of TLR responses during the?rst month of life,resulting in persistent impairments in Th1-immunity,may have increased susceptibility to RSV LRTI and AD in infancy and child-hood.We therefore studied the impact of different TLR responses at the age of1month on the subsequent risk of AD and RSV LRTI in the?rst year of life in a healthy birth cohort.
Methods
Study design and population
A prospective observational birth cohort study was performed in two urban hospitals in Utrecht,the Netherlands,from March2006to February2010.Study design and inclusion criteria have been published previ-ously[28].Eligible subjects were healthy neonates born after uncomplicated pregnancy of!37weeks through either vaginal delivery or elective caesarean section. Data on clinical characteristics were collected from hos-pital charts and standardized parental questionnaires [29].At the age of1month,neonatal venous blood was obtained in sterile sodium heparin tubes.Samples were processed within5h(range0.5–5)of blood collection. None of the participants had any signs or symptoms of infectious disease(e.g.runny nose,fever)or AD in the 2weeks prior to sampling.The study was approved by the ethical review boards of the University Medical Center Utrecht and the Diakonessen Hospital Utrecht. All parents provided written informed consent for study participation.
Innate immune cellularity
Whole blood concentrations of innate immune cells were determined as previously described[5].Concentra-tions of neutrophils,eosinophils and basophils were obtained from differential leucocyte count.Concentra-tions of monocytes,natural killer(NK)-cells,myeloid dendritic cell(mDC)and plasmacytoid dendritic cells (pDC)were determined by?ow cytometry.Monocytes were identi?ed as HLA-DR+/CD14+,NK-cells as CD3à/ CD16+/CD56+,mDC as HLA-DR+/lineageà/CD11c+and pDC as HLA-DR+/lineageà/CD123+(Table S1)[5]).To obtain absolute concentrations,the percentage of cells in the lymphocyte/monocyte gate(as determined by forward-sideward scatter properties)was multiplied by the total numbers of lymphocytes and monocytes from the differential leucocyte count.
Toll-like receptor stimulation
In vitro TLR stimulation was performed using optimal concentrations of TLR agonists and incubation times for cytokine measurements,as titrated in pilot experiments and described previously(Table S2)[5].To study cyto-kine responses to multiple TLR agonists in limited blood volume,whole blood was diluted1:14in serum-free RPMI prior to in vitro TLR stimulation.After a4-h (TNF-a)or24-h(IFN-a,IL-12p70,IL-10)incubation at
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Low neonatal TLR4-mediated IL-10production predisposes to atopic dermatitis67
37°C and5%CO2,samples were centrifuged at1000g for5min.Supernatants were collected and stored at à80°C,until further analysis.
Cytokine protein detection
Cytokine readouts were chosen based upon their key Th1-(IL-12p70,IFN-a)or Th2-polarizing capacity(IL-10).The TLR2-mediated TNF-a was included because previous reports related impairments in TLR2-mediated TNF-a to increased risk of neonatal infection[3,30]. Cytokine concentrations in culture supernatants were determined by ELISA according to the manufac-turer’s instructions:TNF-a,IL-10(both from Sanquin/ CLB,Amsterdam,the Netherlands),IL-12p70(Diaclone Research,Besanc?on,France)and IFN-a(Bender Medsys-tems,Burlingame,CA,USA,detects all natural isoforms of human IFN-a,except for IFN-a1).The lower limits of detection were 1.0pg/mL(TNF-a and IL-10)and 2.0pg/mL(IL-12p70and IFN-a).For samples in which the cytokine concentration was below the detection limit,the concentration was arbitrarily de?ned as half of the detection limit.
Clinical outcomes
The primary outcome of RSV LRTI was de?ned as (i)parent-reported LRTI symptoms,and(ii)simulta-neous presence of RSV RNA in a nose–throat specimen. Throughout the?rst year of life,parents completed prospective logs to record presence and severity of respiratory symptoms.LRTI was de?ned by two inde-pendent researchers,using strict criteria:moderate or severe cough or wheeze of any severity lasting for at least2days.At every respiratory episode,parents obtained a nose–throat swab[31].Samples were sent to the researchers in viral transport medium and upon receipt,frozen at80°C.The presence of RSV RNA was determined by real-time polymerase chain reaction (PCR)as described previously[32].Secondary outcomes were‘physician-attended RSV infection’and‘atopic dermatitis’.Physician-attended RSV infection was de?ned as an episode of respiratory tract symptoms for which the general practitioner or paediatrician was visited,and simultaneous presence of RSV RNA in a nose–throat swab.At the age of1year,general physi-cians of all participants were contacted to answer a standardized,four-item questionnaire that was based on the international classi?cation of primary care codes (ICPCC).AD was assessed by the question‘Was this par-ticipant diagnosed with one or multiple episodes of “atopic eczema”(ICPCC S87)in his/her?rst year of life?’For sensitivity analysis,parent-reported AD was derived from validated parental questionnaire at the age of1year[29],and de?ned as a positive answer to the question‘Did your child experience any eczema or pruritic skin rash in the?rst year of life?’
Statistical analysis
Whole blood concentrations of innate immune cells and TLR-mediated cytokine responses were logarithmically transformed to obtain normal distribution.Means of logarithmic transformed variables were compared be tween groups using two-sided Student’s t-test.Differ-ences between dichotomic variables were determined using the v2-test.Logistic regression analysis was used to determine the effect of innate immune determinants on outcome variables,while adjusting for possible confounding factors.Based on results from univariate analysis,‘birth weight’,‘birth season’and‘day care’were considered potential confounders for the associa-tion between innate immune variables and RSV LRTI. Similarly,‘birth season’,‘day care’and‘parental atopy’
?2011Blackwell Publishing Ltd,Clinical&Experimental Allergy,42:66–75 68M.E.Belderbos et al
were considered potential confounders for the associa-tion between innate immune variables and AD. Results
During the study period,1298neonates were eligible for study participation(Fig.1).Of these,402gave informed consent.Due to missed appointments(n= 47)or technical reasons(e.g.failure in blood collec-tion,blood clotting,n=64),innate immune parame-ters at the age of1month were not available for111 (28%)of participants,resulting in a?nal cohort of 291neonates.Baseline characteristics did not differ between participating neonates and non-participants (Table S3).At the age of1year,45participants had AD[absolute risk(AR)15%,Table1].Baseline charac-teristics were similar between neonates with and with-out AD.In addition,41participants had experienced an episode of RSV LRTI(AR14%,Table1),and22 participants had physician-attended RSV infection(AR 7%).Neonates who developed RSV LRTI had a higher birth weight and gestational age compared with those who did not,as observed previously[28].There was no relationship between risks of AD and RSV LRTI(v2, P=1.00).Atopic dermatitis
Distinct neonatal concentrations of innate immune cells are associated with subsequent AD.Whole blood concen-trations of innate immune cells at the age of1month were compared between neonates with and those without subsequent AD(Fig.2).Neonates with subsequent AD had decreased concentrations of basophils(7.6vs.
14.09106/L,P=0.002)and pDCs(17.0vs.20.59106/ L,P=0.04),and increased concentrations of NK-cells (79.7vs.45.19106/L,P=0.03)compared with neonates without subsequent AD.All associations remained signi?cant after adjustment for potential con-founders(Table2)and an additional borderline signi?-cant association was found between whole blood concentrations of eosinophils and subsequent risk of AD (P=0.05).
Decreased TLR4-mediated IL-10production is associated with increased risk of subsequent atopic dermatitis.We next investigated whether TLR-mediated cytokine responses at the age of1month were associated with prospective susceptibility to AD(Fig.3).Neonates who developed physician-diagnosed AD had1.8-fold lower TLR4-mediated IL-10responses at the age of1month
Table1.Characteristics of study population
RSV LRTI Atopic dermatitis
Yes(n=41)No(n=250)P-value Yes(n=45)No(n=246)P-value
Gestational age,weeks(SEM)40.3(0.2)39.9(0.1)0.0140.1(0.1)39.9(0.1)0.27 Birth weight,g(SEM)3834(73)3574(29)0.0013653(71)3603(29)0.52 Gender,n(%)0.610.87 Male18(44)123(49)21(47)120(49)
Female23(56)127(51)24(53)126(51)
Delivery mode,n(%)0.09 1.00 Vaginal39(95)212(85)39(87)212(86)
Elective caesarean section2(5)38(15)6(13)34(14)
Birth season,n(%)0.060.12 Winter8(20)62(25)5(12)65(26)
Spring9(22)67(27)16(37)60(24)
Summer17(41)55(22)11(26)61(25)
Fall7(17)66(26)13(30)60(24)
Siblings,n(%)25(61)161(66)0.7329(64)157(64) 1.00 Pets,n(%)12(29)80(33)0.7216(36)65(33)0.73 Feeding,n(%)
Ever breastfed24(85)211(87)0.8137(84)209(85)0.63 Exclusive breastfeeding>1mo24(59)130(54)0.6120(46)134(54)0.25 Age at start formula,mo(SEM) 4.3(0.8) 4.2(0.3)0.93 4.8(0.9) 4.1(0.3)0.38 Day care attendance,n(%)30(79)128(64)0.0931(78)127(51)0.07 Maternal atopy*,n(%)12(29)93(38)0.3015(34)90(37)0.74 Parental atopy*,n(%)23(56)137(56) 1.0024(55)136(55)0.87 Maternal atopic dermatitis,n(%)10(24)46(19)0.409(20)47(19)0.84 Parental atopic dermatitis,n(%)68(28)14(34)0.4617(39)65(26)0.15 Parental smoking,n(%)4(10)69(28)0.108(18)65(26)0.26
*Parental atopy was de?ned as parent-reported physician-diagnosed asthma,atopic dermatitis or hayfever.SEM,standard error of the mean.
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Low neonatal TLR4-mediated IL-10production predisposes to atopic dermatitis69
compared with those who did not(533pg/mL vs. 954pg/mL,P=0.003).Logistic regression analysis, adjusting for birth season,parental atopy and day care as potential confounders,showed similar results [B=0.13(95%CI:0.04–0.41),P<0.001](Table2). Sensitivity analyses using‘parent-reported atopic der-matitis’(de?ned as eczema or pruritic skin rash,derived from parental questionnaire at the age of1year)ren-dered similar?ndings,both in univariate analysis (588pg/mL vs.995pg/mL,P=0.001)and in multi variate analysis[B=0.24(0.10–0.62),P=0.003].No signi?cant correlations were found between concentra-tions of NK-cells,pDC,basophils,eosinophils and TLR4-mediated IL-10production.
Respiratory syncytial virus lower respiratory tract infection
Neonatal whole blood concentrations of innate immune cells are not associated with the development of RSV LRTI.The primary outcome of community-based RSV
Table2.Independent associations between concentrations of innate
immune cells and TLR-mediated cytokine responses at the age of
1month and the subsequent risk of atopic dermatitis
OR(95%CI)P-value
pDC0.19(0.40–0.91)0.04
NK cells 1.83(1.12–3.02)0.02
Eosinophils 3.00(1.00–8.97)0.05
Basophils0.28(0.12–0.71)0.01
TLR4-mediated IL-100.13(0.04–0.41)<0.001
Multivariable logistic regression analysis was performed comparing
concentrations of innate immune cells and TLR-mediated cytokine
responses between neonates with subsequent atopic dermatitis
(n=45)and those without(n=246),while adjusting for birth season,
parental atopy and day care as potential confounders.Only signi?cant
associations are shown.
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LRTI in the?rst year of life was analysed in relation to neonatal whole blood concentrations of innate immune cells(Fig.2).There were no differences in concentra-tions of mDCs,pDCs,neutrophils and basophils at the age of1month between neonates with and those with-out subsequent RSV LRTI.Neonates who developed RSV LRTI had increased concentrations of monocytes (468vs.3639106/L,P=0.05)and eosinophils(437 vs.3639106/L,P=0.05)compared with those without RSV LRTI,but signi?cance was lost after adjustment for potential confounders.No differences in cell concentrations were found in relation to the sec-ondary outcome physician-attended RSV(data not shown).Analyses using cell percentages instead of absolute concentrations(e.g.eosinophils as a percent-age of total leucocytes)rendered similar results(data not shown).
Neonatal TLR-mediated cytokine responses are not associated with the development of RSV LRTI.No differences in cytokine responses to agonists for TLR2, TLR3,TLR4,TLR7and TLR9were found between
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Low neonatal TLR4-mediated IL-10production predisposes to atopic dermatitis71
neonates who developed RSV LRTI in the?rst year of life and those who did not(Fig.3).The secondary out-come,‘physician-attended RSV infection’,was associ-ated with higher TLR3-mediated IL-12p70production at the age of1month(676pg/mL vs.316pg/mL, P=0.015).This remained statistically signi?cant after adjustment for potential confounders[B=7.48 (1.67–33.4,P=0.021)].
Discussion
This prospective unselected birth cohort study demon-strates that AD is preceded by marked differences in the innate immune system at the age of1month.Neonates who subsequently developed AD had decreased TLR4-mediated IL-10production,decreased concentrations of basophils and pDC and increased concentrations of NK-cells compared with those who did not.On the contrary,except for a modest association with increased TLR3-mediated IL-12p70production,RSV LRTI was associated with pre-symptomatic differences neither in innate immune cellularity nor in TLR-mediated cytokine responses at the age of1month,
Dysregulated TLR-mediated cytokine responses during early life may play an important role in the pathogenesis of AD[33].At birth,cord blood mononu-clear cells(CBMC)from atopic mothers produce higher TLR2-,TLR3-and TLR4-mediated IL-12and IFN-c compared with those from non-atopic mothers[33].In addition,in high-risk neonates,increased TLR2-,TLR4-and TLR5-mediated IL-6and TNF-a production by CBMC is associated with increased risk of childhood AD [33].Thus,cord blood TLR hyperresponsiveness,either due to genetic factors or environmental exposures,may play a causal role in the pathogenesis of AD. Disrupted post-natal development of the TLR system might also contribute to AD https://www.wendangku.net/doc/a33298737.html,ck of early life exposure to innate immune stimuli,includ-ing the TLR4agonist endotoxin,contributes to the subsequent development of asthma,allergic sensitiza-tion and AD[27,34,35].In addition,atopic children demonstrate an inverse pattern of innate immune maturation from non-atopic children,decreasing, rather than increasing,TLR-mediated production of TNF-a,IL-12,IL-6and IL-1b,from birth to the age of ?ve[36].Impaired production of Th1-polarizing innate immune signals may contribute to the persistent Th2-skewed allergen-speci?c immune responses(i.e. increased IL-4,IL-13,decreased IFN-c)observed in atopic children[37,38].
In summary,disrupted early life TLR development might induce persistent dysfunction of innate and adaptive immunity,thus predisposing to atopy.Previ-ously,we identi?ed the?rst month of life as a period of rapid development of the TLR system,providing a
potential window of opportunity for interventions aimed at preventing atopy[5].The association between TLR4-mediated IL-10at the age of1month and subsequent risk of AD suggests that strategies aimed at increasing IL-10production during this highly sensitive period might restore immune balance and protect from subsequent AD.
Several explanations may account for the association between low neonatal TLR4-mediated IL-10production and AD.First,IL-10is an important regulatory cytokine [39].In mice,intradermal injections of IL-10plasmid DNA suppress the development of AD skin lesions[40]. In humans,subcutaneous allergen-speci?c immunother-apy increases IL-10expression in AD skin,coinciding with clinical improvement[41].Thus,de?cient IL-10-mediated immune regulation might predispose to AD. Second,decreased neonatal IL-10production to TLR4 stimulation might re?ect a general dysregulation of responses mediated by TLR4.Several allergens associ-ated with AD[42,43]show structural and functional homology to components of the TLR4receptor complex [44–46].Similarities in the structure and biological functions of LPS and allergens might account for the association between neonatal TLR4-mediated IL-10and AD.However,we found no association between TLR4-mediated IL-12p70production and AD,suggesting that other mechanisms also play a role.Third,differences in microbial?ora between atopic and non-atopic neonates might account for the association between neonatal innate immune responses and AD[3].Atopic dermatitis patients have distinct microbial?ora of their skin[47] and gut[48]compared with non-atopic subjects.Differ-ences in gut colonization precede the development of clinical AD[48].suggesting that dysregulated micro-bial-induced immune development might play a causal role in the initiation of AD.
Atopic dermatitis was also associated with decreased whole blood concentrations of basophils and(in regres-sion analysis)increased concentrations of eosinophils.
A potential cause of this preferential differentiation of eosinophil/basophil precursor cells towards the eosino-phil lineage is the key Th2-cytokine IL-5[49].Previous studies strengthen this hypothesis,demonstrating that increased IL-5production during the neonatal and infant period is associated with elevated concentrations of eosinophils and total IgE[50]and increased risk of atopy at the age of1year[51].
Why are neonatal innate immune responses associated with AD,but not RSV LRTI?The lack of associations between neonatal innate immune parame-ters measured in this study and subsequent RSV LRTI might indicate that the immune dysfunction observed during severe RSV infection[21–23]is the result,rather than the cause,of severe RSV disease.Alternatively, dysregulation of TLR-mediated cytokine responses ?2011Blackwell Publishing Ltd,Clinical&Experimental Allergy,42:66–75
72M.E.Belderbos et al
might be speci?c for infants with hospital-admitted(as opposed to community-based)RSV infection.Insight into the reciprocal interactions between RSV and the host immune response is highly needed for treatment of severe RSV bronchiolitis and prevention of post-bronchiolitis wheeze.
This study has several strengths,including(i)we were able to follow a large cohort of healthy neonates born at term after uncomplicated gestation and delivery prospectively;(ii)venous blood was collected at the age of1month,preventing any potential artefacts present in cord blood(e.g.hypoglycaemia,low pH,circulating maternal cells),and(iii)all respiratory symptoms sus-pected to arise from RSV infection were con?rmed by PCR.Potential limitations also deserve discussion.First, we did not identify the cell type responsible for TLR-mediated cytokine production.Consequently,the observed association between TLR4-mediated IL-10and AD might be secondary to differences in innate immune cell concentrations.However,the cell types that were relatively decreased in atopic neonates either lack TLR4 (pDC)[52]or are incapable of responding to LPS in vitro (basophils)[53],and adjustment for concentrations of innate immune cells rendered a similar signi?cant asso-ciation between TLR4-mediated IL-10and AD(data not shown).Second,the in vitro whole blood model is very different from the in vivo response in AD skin or RSV-infected airways.Third,our comprehensive character-ization of neonatal innate immunity includes multiple comparisons.To minimize the risk of false discovery, we focused on associations that were highly signi?cant, that remained signi?cant after adjustment for potential confounders and that were present for multiple related outcomes(e.g.physician-diagnosed AD and parent-reported AD).No attempt was made to correct for multiple testing,and future studies will be needed to validate our?ndings.
In summary,this study demonstrates that atopic dermatitis,but not RSV LRTI,is associated with dis-tinct pre-symptomatic TLR-mediated cytokine respon ses at the age of1month.Future studies aimed at characterizing the basic mechanisms and clinical fac-tors modulating neonatal TLR4-mediated IL-10produc-tion might identify potential targets for prevention of AD.
Acknowledgements
We thank the obstetricians,midwives and participants for their contributions to this study.This article is funded by grants from the Netherlands Asthma Foundation(grant no.
3.2.07.001)and the Alexandre Suerman foundation of the University Medical Center Utrecht.None of the authors has any?nancial relationship to disclose with a biotechnology and/or pharmaceutical manufacturer that has interest in the subject matter or materials discussed in the submitted manuscript.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Table S1.Markers used to identify innate immune cells by?ow cytometry
Table S2.Agonists used for in vitro TLR stimulation Table S3.Baseline characteristics of study population compared to all eligible neonates
Please note:Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors.Any queries(other than miss-ing material)should be directed to the corresponding author for the article.
?2011Blackwell Publishing Ltd,Clinical&Experimental Allergy,42:66–75
Low neonatal TLR4-mediated IL-10production predisposes to atopic dermatitis75