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Immune responses to lipopolysaccharide in relation to allergic disease: a TLR4 gene polymorphism and endotoxin exposure
Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Allergic diseases have increased during the last decades, particularly in affluent countries, possibly due to a reduced and/or altered microbial exposure during infancy. Activation of the immune system by microbes early in life is probably required for accurate maturation of the immune system and tolerance development. It is not fully understood how microbial exposure is associated with the development of allergic diseases, however. Genetic factors may influence microbial induced immune responses. A certain polymorphism, in the gene coding for the Toll-like receptor 4, i.e. (TLR4 Asp299Gly), has been suggested to alter the immunological responsiveness to bacterial lipopolysaccharide (LPS).

Aim: The aim of this thesis was to study the interplay between LPS induced immune responses, LPS signalling related genetic polymorphisms, allergic disease and endotoxin exposure.

Subjects: The thesis is based on the results obtained from individuals in three different study groups, i.e. Estonian and Swedish children followed prospectively from birth up to five years of age, Swedish school-children eight and 14 years of age and young adults.

Methods: The study subjects were clinically evaluated regarding allergic diseases with skin prick tests, circulating IgE levels, validated questionnaires and clinical examinations by paediatricians or research nurses. The gene polymorphisms TLR4 Asp299Gly and CD14/-159 were analysed. Peripheral blood mononuclear cells were isolated from blood and cultured with LPS from two Gram negative bacterial strains, i.e. Salmonella enterica serotype Typhimurium (Serotype Typhimurium) and Escherichia coli (E. coli). Cytokine and chemokine secretions were analysed with Luminex or ELISA technique. Receptor expression of circulating peripheral blood monocytes was analysed with flow cytometry. The phosphorylation of intracellular proteins involved in LPS signalling pathways was analysed with Luminex technique. mRNA expression of proteins involved in LPS signalling pathways and of markers for T regulatory cells were analysed with realtime-PCR.

Results: In school-children and young adults, the TLR4 Asp299Gly gene polymorphism was associated with reduced LPS induced IκBα phosphorylation, IL-10 and IL-12 cytokine secretion. Interestingly, these findings were observed only when the cells were cultured with LPS from Serotype Typhimurium but not with LPS from E. coli. The polymorphism was positively associated with asthma, especially atopic asthma.

Several differences in immunological responses to LPS were observed between allergic and non-allergic individuals. Asthma in school-children was associated with reduced LPS induced cytokine production of IL-10 and IL-12. The phosphorylation of IκBα was lower in adult allergic compared to non-allergic individuals. Swedish children who had developed allergic disease at five years of age had lower TLR2 mRNA expression at birth compared to children who remained healthy.

Estonian children displayed generally lower LPS induced cytokine and chemokine production as compared to Swedish children both at birth and at 3 and 6 months of age. The mRNA expression of the T regulatory associated markers Foxp3 and Ebi3 were higher in the Estonian compared to the Swedish children at birth.

Conclusion: Polymorphisms in genes coding for pattern recognition receptors can alter the immune responsiveness of the host to microbial components and may be of importance for the development of asthma. Lower LPS induced cytokine response and higher expression of T regulatory associated markers were seen in children from Estonia as compared to Sweden, suggesting an increased capacity for early immune regulation among infants from a country with a low prevalence of allergic disease.

Abstract [sv]

Bakgrund: Under de senaste decennierna har förekomsten av allergiska sjukdomar ökat i västvärlden. En av möjliga förklaringar kan vara en minskad eller förändrad mikrobiell exponering under uppväxttiden. Mikrobiella stimuli under de första levnadsåren tros vara viktiga för utmognaden av immunsystemet och utveckling av tolerans. Exakt hur mikroorganismers påverkan på immunförsvaret är kopplat till utvecklingen av allergiska sjukdomar är dock ännu okänt. Genetiska polymorfier kan påverka immunsvar mot mikrobiella komponenter. En sådan polymorfi, TLR4 Asp299Gly, har observerats i genen som kodar för receptorn TLR4 som känner igen lipopolysackarid (LPS) från Gramnegativa bakteriers cellvägg, och har föreslagits vara associerad med en förändrad förmåga att svara immunologiskt mot LPS.

Syfte: Syftet med studierna var att studera immunsvar mot LPS i relation till specifika genetiska polymorfier, allergisk sjukdom samt mikrobiellt tryck i form av endotoxinnivåer.

Studiepopulationer: Denna avhandling baseras på resultat från tre studiegrupper: estniska och svenska barn som är följda från födseln upp till fem års ålder, en grupp svenska skolbarn 8 och 14 år gamla samt en grupp unga vuxna.

Metoder: Två genetiska polymorfier, TLR4 Asp299Gly och CD14/-159, analyserades. Mononukleära celler isolerades från perifert blod och odlades tillsammans med LPS från två olika Gramnegativa bakteriestammar, Salmonella enterica serotype Typhimurium (Serotype Typhimurium) och Escherichia coli ( E. coli). Cytokin- och kemokinsekretion analyserades i cellsupernatanter med Luminex eller ELISA. Ytmarkörer på monocyter i helblod studerades med flödescytometri. Intracellulära signaleringsproteiner, som är inblandade i TLR4s signalvägar analyserades med Luminexteknik. mRNA uttryck av proteiner som är relaterade till LPS signalering och markörer för regulatoriska T celler analyserades med realtids-PCR.

Resultat: TLR4 Asp299Gly polymorfin var associerad med lägre fosforylering av det intracellulära signaleringsproteinet IκBα och lägre utsöndring av cytokinerna IL-12 och IL-10 efter cellstimulering med LPS hos skolbarn och unga vuxna. Skillnader i cellsvar mellan individer med och utan polymorfin kunde påvisas när cellerna odlats med LPS från Serotype Typhimurium men inte med LPS från E. coli. Polymorfin var också associerad med astma och särskilt atopisk astma.

Flera skillnader i immunsvar mot LPS observerades mellan allergiska och ickeallergiska individer. Skolbarn med astma hade lägre LPS inducerad IL-10 och IL-12 cytokinproduktion. Vuxna allergiker hade lägre LPS inducerad IκBα fosforylering. Svenska barn som vid fem års ålder hade utvecklat allergisk sjukdom hade lägre mRNA uttryck av TLR2 vid födseln.

Estniska barn hade generellt lägre LPS inducerade cytokinsvar än svenska barn vid födseln och vid 3 och 6 månaders ålder. mRNA uttrycket av de T-regulatoriskt associerade markörerna Foxp3 och Ebi3 var vid födseln högre hos de estniska jämfört med de svenska barnen.

Slutsats: Genetiska förutsättningar kan påverka immunsvar mot LPS och kan möjligen ha en betydelse för utveckling av astma. De generellt lägre LPS inducerade cytokinsvaren och högre uttryck av markörer för Treg celler hos estniska jämfört med svenska barn skulle kunna bero på att deras uppväxtmiljö med ett högre mikrobiellt tryck påverkar den tidiga utvecklingen av immunförsvaret och kan möjligen vara en bidragande förklaring till den lägre allergifrekvens som ses i Estland jämfört med Sverige.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 73 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1098
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-16783ISBN: 978-91-7393-704-7 (print)OAI: oai:DiVA.org:liu-16783DiVA: diva2:173893
Public defence
2009-03-05, Berzeliussalen, Campus US, Linköpings Universitet , Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-02-18 Created: 2009-02-18 Last updated: 2009-05-13Bibliographically approved
List of papers
1. A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children
Open this publication in new window or tab >>A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children
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2004 (English)In: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 114, no 3, 561-567 p.Article in journal (Refereed) Published
Abstract [en]

Background

Bacterial signals play an important role in the maturation of the immune system. Polymorphisms in genes coding for receptors to bacterial components can alter the immune responsiveness of the host to microbial agents and may indicate the development of aberrant immune responses that are associated with immune-mediated diseases such as atopic diseases.

Objective

The study's objective was to investigate the relationship between TLR4 and CD14 gene polymorphisms, the LPS responsiveness of PBMCs, and the presence of asthma and allergic rhinoconjunctivitis in children.

Methods

The TLR4 (Asp299Gly) and CD14/−159 polymorphisms were determined in 115 Swedish children aged 8 and 14 years. LPS-induced IL-12(p70), IL-10, and IFN-γ responses of PBMCs from 69 of the children were analyzed by means of ELISA. The levels of soluble CD14 in serum samples were analyzed by means of ELISA, and the total IgE levels were analyzed by means of UniCAP Total IgE (Pharmacia Diagnostics, Uppsala, Sweden).

Results

Decreased LPS-induced IL-12(p70) and IL-10 responses were associated with the TLR4 (Asp299Gly) polymorphism and independently with asthma, especially atopic asthma. The TLR4 (Asp299Gly) polymorphism was associated with a 4-fold higher prevalence of asthma in school-aged children (adjusted odds ratio 4.5, 95% CI 1.1-17.4) but not to allergic rhinoconjunctivitis.

Conclusion

A TLR4 polymorphism modifies innate immune responses in children and may be an important determinant for the development of asthma. This may influence the outcome of intervention studies that use microbial stimuli as immune modulators.

Keyword
asthma; TLR4; lipopolysaccharide; IL-12; IL-10 sCD14, Soluble CD14; SPT, Skin prick test; Th, T-helper; TLR, Toll-like receptor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16779 (URN)10.1016/j.jaci.2004.04.050 (DOI)
Available from: 2009-02-18 Created: 2009-02-18 Last updated: 2017-12-13Bibliographically approved
2. Lipopolysaccharide-Induced Immune Responses in Relation to the TLR4(Asp299Gly) Gene Polymorphism
Open this publication in new window or tab >>Lipopolysaccharide-Induced Immune Responses in Relation to the TLR4(Asp299Gly) Gene Polymorphism
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2008 (English)In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 15, no 12, 1878-1883 p.Article in journal (Refereed) Published
Abstract [en]

Altered microbial exposure is a possible explanation for the increase of allergies in the Western world. However, genetic factors influence microbially induced immune responses. We have investigated the TLR4(Asp299Gly) gene polymorphism and its possible association with receptor expression of circulating peripheral blood monocytes and the in vitro cytokine responses and phosphorylation of intracellular signaling proteins in peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) from Escherichia coli and Salmonella enterica serotype Typhimurium. We studied 34 of the predominant haplotype TLR4 Asp299 (AA) and 8 heterozygote Asp299Gly (AG) individuals. TLR4 expression levels were similar in the two genotype groups. Serovar Typhimurium LPS induced interleukin-12p70 from PBMC, and the degree of phosphorylation of the intracellular signaling protein I kappa B alpha in PBMC was lower in the AG than the AA group (P = 0.03 and P = 0.04, respectively). These results were not seen, however, when PMBC were stimulated with E. coli-derived LPS. Based on these results, we propose that TLR4(Asp299Gly) gene polymorphism and the bacterial origin of LPS should be considered when environmental LPS exposure is evaluated in disease risk or protection.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16220 (URN)10.1128/CVI.00241-08 (DOI)
Available from: 2009-01-12 Created: 2009-01-09 Last updated: 2017-10-31Bibliographically approved
3. Lower LPS responsiveness in Estonian than Swedish infants associates with less allergy development and high endotoxin exposure
Open this publication in new window or tab >>Lower LPS responsiveness in Estonian than Swedish infants associates with less allergy development and high endotoxin exposure
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(English)Manuscript (Other academic)
Abstract [en]

Background: Allergic diseases have increased in the last decades, particularly in countries with an affluent lifestyle, possibly due to a reduced or altered microbial exposure during infancy.

Objective: The aim of this study was to follow lipopolysaccharide (LPS) induced immune responses prospectively in infants from Estonia and Sweden, i.e. two countries with different frequencies of allergic disease and a different domestic endotoxin exposure. '

Methods: The study included 14 Estonian and 36 Swedish infants who were followed prospectively from birth up to two years of age regarding development of allergy using questionnaires, clinical examinations and skin prick tests. Isolated cord blood mononuclear cells (birth) and peripheral blood mononuclear cells (obtained at 3, 6, 12 and 24 months) were cultured for 24h with LPS in combination with IFN-γ. The secretion of IL-6, CXCL8 (IL-8), IL-10, IL12p70, IL-17, IL-1β, CCL2 (MCP-1), CCL4 (MIP-1β) and TNF was analysed with a Luminex assay.

Results: The Swedish, as compared to Estonian children, had higher levels of LPS/IFN-γ induced CCL4 and IL-6 at birth and of IL-1β, IL-12p70, IL-17 and TNF at 3 months as well as IL-6 at 6 months. Also, the levels of CCL2 at 3 and 6 months of age and CCL4 and TNF at 6 months of age were higher in Swedish than Estonian infants in unstimulated cultures. Sensitised Swedish infants had higher levels of LPS/IFN-γ induced IL-10 at 3 and 12 months of age compared to non-sensitised Swedish infants.

Conclusion: The enhanced LPS/IFN-γ induced cytokine and chemokine secretion in Swedish, compared to Estonian infants may support a less rapid induction of immune regulation in an affluent society. This could possibly be due to a reduced microbial pressure on Swedish children during early childhood.

Keyword
Lipopolysaccharide, cytokines, chemokines, childhood, allergy, endotoxin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16781 (URN)
Available from: 2009-02-18 Created: 2009-02-18 Last updated: 2010-01-14Bibliographically approved
4. Expression of toll-like receptors and immune-regulatory markers during early infancy and allergy development in Estonian and Swedish infants
Open this publication in new window or tab >>Expression of toll-like receptors and immune-regulatory markers during early infancy and allergy development in Estonian and Swedish infants
(English)Manuscript (Other academic)
Abstract [en]

Background: Allergies have increased during the last decades with the highest prevalences in countries with a Westernised lifestyle. As microbial activation might be required for an accurate maturation of the immune system, a reduced or altered microbial exposure during infancy has been suggested as a possible cause for the increase of allergic diseases in affluent countries.

Aim: The aim of this study was to investigate the gene expression of TLR2, TLR4, Foxp3, Ebi3 and SOCS3 involved in LPS signaling pathways and immune regulation in two prospectively followed birth cohorts, one from Estonia and one from Sweden, in relation to domestic endotoxin levels and allergy development.

Material and methods: Twenty-three children from Estonia and 52 from Sweden were followed from birth to five years of age regarding allergy development using questionnaires, clinical examinations and skin prick tests. RNA was isolated from unstimulated peripheral blood mononuclear cells (PBMC) in samples collected at birth, 3, 6, 12 and 24 months of age. mRNA expression of TLR2, TLR4, SOCS3, Ebi3 and Foxp3 was analysed with real-time RTPCR. The mRNA expression of the genes were compared to previously obtained data of LPS/IFN-γ induced cytokine and chemokine PBMC secretion at the corresponding time-points and also to domestic endotoxin levels.

Results: The Estonian infants had higher mRNA expression of the two regulatory T cell (Treg) associated markers Foxp3 and Ebi3 at birth than the Swedish infants. The Foxp3 and Ebi3 expression correlated at all time-points. The mRNA expression of TLR2, TLR4 and SOCS3 was similar in Estonian and Swedish infants at all ages. None of the genes were associated with endotoxin levels.TLR4 mRNA expression correlated positively with cytokines that were upregulated by LPS/IFN- γ stimulation, but was negatively correlated to CCL2 secretion which was downregulated by the stimulation. Low expression of TLR2 mRNA at birth was found in Swedish children who were allergic at 5 years of age. Sensitisation up to 5 years of age was associated with low Ebi3 expression at birth.

Conclusion: Estonian children are born with enhanced Ebi3 and Foxp3 expression compared to Swedish children, suggesting an increased capacity for early immune regulation among infants from a country with a low prevalence of allergic disease. An increased early capacity to respond to TLR2 ligands, i.e. Gram positive bacteria, may protect against allergy development.

Keyword
Regulatory T cells, TLR2, TLR4, SOCS3, foxp3, Ebi3, allergy, LPS, endotoxin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16782 (URN)
Available from: 2009-02-18 Created: 2009-02-18 Last updated: 2010-01-14Bibliographically approved

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