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The role of TNFAIP3 and NF-κB signalling in childhood asthma development and environment-mediated protection
The role of TNFAIP3 and NF-κB signalling in childhood asthma development and environment-mediated protection
Asthma has become one of the most common chronic diseases among children in developed Western countries, with its prevalence still rising in other parts of the world. Distinct microbial exposures encountered in traditional farming environments early in life have been associated with robust protection from developing the disease. The practical relevance of this protective ‘farm-effect’, however, remains limited as long as the causal mediators and molecular mechanisms have not been fully identified. In this context, recent findings in mouse models of asthma have suggested a key role for the enzyme TNFAIP3, which attenuates NF-kB dependent inflammation, but equivalent immunological studies in humans are missing. The objective of the present study was therefore to unravel the role of the anti-inflammatory regulator TNFAIP3 and related innate immune signalling pathways in the development of allergic asthma and its environmentally mediated protection in children. The study aimed to investigate target gene and protein expression in peripheral blood mononuclear cells (PBMCs) of allergic asthmatic children during disease manifestation. It further aimed to examine the effect of in vitro stimulation with farm dust extracts and bacterial lipopolysaccharide (LPS) on the expression of these target molecules. Moreover, the study aimed to assess whether children with continuous early life in vivo farm exposure showed modified TNFAIP3 pathway gene expression in immune cells at the age of 4.5, 6 and 10 years. Therefore, peripheral blood samples of asthmatic and healthy children from urban and farm environments from two distinct study population cohorts were analysed. As part of the cross-sectional CLARA/CLAUS study, a representative nested study sample comprising allergic asthmatic (AA) and healthy control (HC) school-age children (n = 48) was recruited. PBMCs were isolated from whole blood samples and stimulated in vitro with four different farm-derived dust extracts (from Germany, Finland, China and from the Amish farming environment) as well as LPS for 24 hours. Furthermore, from the prospective birth cohort study PASTURE/EFRAIM, a nested sample of farm (F) and nonfarm (NF) children (n = 100) was selected. Unstimulated and LPS-stimulated whole blood samples were analysed at the children’s age of 4.5, 6 and 10 years. For both cohorts, gene expression and protein levels of TNFAIP3 and NF-kB signalling-associated candidate molecules (TLR4, MyD88, TRAF6, TNIP2, TAX1BP1, MALT1, CD80, CD86, CD274, PDCD1) were assessed by qPCR and Western Blot analysis respectively. The following results were achieved and are presented in this thesis: (i) Analyses in unstimulated PBMC samples revealed significantly reduced expression of the antiinflammatory TNFAIP3 gene and protein in allergic asthmatic children compared to healthy controls, while expression of the pro-inflammatory pathway genes TLR4 and MyD88 was increased in allergic asthmatic subjects. (ii) In vitro stimulation with LPS and all four farm dust extracts except the Chinese sample restored TNFAIP3 expression of asthmatic patients to healthy levels and significantly reduced the expression of the pro-inflammatory TLR4-pathway-related target genes (TLR4, MyD88, TRAF6). The observed anti-inflammatory effects were accompanied by significant downregulation of dendritic cell associated co-stimulatory molecule CD86 and upregulation of the co-inhibitory molecule CD274 upon stimulation. (iii) Unstimulated whole blood samples of farm children at ages 4.5, 6 and 10 years exhibited decreased expression of both pro- and anti-inflammatory genes compared to their non-farming peers. However, the capacity to induce anti-inflammatory TNFAIP3 and CD274 expression and to downregulate pro-inflammatory TLR4 levels upon LPS and farm dust stimulation was preserved at all ages. The results of this study in children’s peripheral immune cells indicate that farming exposures in vitro and in vivo may inhibit critical inflammatory processes in human asthma development by activating regulatory TNFAIP3-dependent processes and shaping innate immune pathways. These findings increase the potential for novel farm-derived microbe-based treatment and prevention strategies, and thus may ultimately pave the way for improving asthma management on a global scale.
childhood asthma, farm effect, A20, TNFAIP3, LPS, innate immunity, DCs
Rehbach, Katharina
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Rehbach, Katharina (2021): The role of TNFAIP3 and NF-κB signalling in childhood asthma development and environment-mediated protection. Dissertation, LMU München: Faculty of Medicine
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Abstract

Asthma has become one of the most common chronic diseases among children in developed Western countries, with its prevalence still rising in other parts of the world. Distinct microbial exposures encountered in traditional farming environments early in life have been associated with robust protection from developing the disease. The practical relevance of this protective ‘farm-effect’, however, remains limited as long as the causal mediators and molecular mechanisms have not been fully identified. In this context, recent findings in mouse models of asthma have suggested a key role for the enzyme TNFAIP3, which attenuates NF-kB dependent inflammation, but equivalent immunological studies in humans are missing. The objective of the present study was therefore to unravel the role of the anti-inflammatory regulator TNFAIP3 and related innate immune signalling pathways in the development of allergic asthma and its environmentally mediated protection in children. The study aimed to investigate target gene and protein expression in peripheral blood mononuclear cells (PBMCs) of allergic asthmatic children during disease manifestation. It further aimed to examine the effect of in vitro stimulation with farm dust extracts and bacterial lipopolysaccharide (LPS) on the expression of these target molecules. Moreover, the study aimed to assess whether children with continuous early life in vivo farm exposure showed modified TNFAIP3 pathway gene expression in immune cells at the age of 4.5, 6 and 10 years. Therefore, peripheral blood samples of asthmatic and healthy children from urban and farm environments from two distinct study population cohorts were analysed. As part of the cross-sectional CLARA/CLAUS study, a representative nested study sample comprising allergic asthmatic (AA) and healthy control (HC) school-age children (n = 48) was recruited. PBMCs were isolated from whole blood samples and stimulated in vitro with four different farm-derived dust extracts (from Germany, Finland, China and from the Amish farming environment) as well as LPS for 24 hours. Furthermore, from the prospective birth cohort study PASTURE/EFRAIM, a nested sample of farm (F) and nonfarm (NF) children (n = 100) was selected. Unstimulated and LPS-stimulated whole blood samples were analysed at the children’s age of 4.5, 6 and 10 years. For both cohorts, gene expression and protein levels of TNFAIP3 and NF-kB signalling-associated candidate molecules (TLR4, MyD88, TRAF6, TNIP2, TAX1BP1, MALT1, CD80, CD86, CD274, PDCD1) were assessed by qPCR and Western Blot analysis respectively. The following results were achieved and are presented in this thesis: (i) Analyses in unstimulated PBMC samples revealed significantly reduced expression of the antiinflammatory TNFAIP3 gene and protein in allergic asthmatic children compared to healthy controls, while expression of the pro-inflammatory pathway genes TLR4 and MyD88 was increased in allergic asthmatic subjects. (ii) In vitro stimulation with LPS and all four farm dust extracts except the Chinese sample restored TNFAIP3 expression of asthmatic patients to healthy levels and significantly reduced the expression of the pro-inflammatory TLR4-pathway-related target genes (TLR4, MyD88, TRAF6). The observed anti-inflammatory effects were accompanied by significant downregulation of dendritic cell associated co-stimulatory molecule CD86 and upregulation of the co-inhibitory molecule CD274 upon stimulation. (iii) Unstimulated whole blood samples of farm children at ages 4.5, 6 and 10 years exhibited decreased expression of both pro- and anti-inflammatory genes compared to their non-farming peers. However, the capacity to induce anti-inflammatory TNFAIP3 and CD274 expression and to downregulate pro-inflammatory TLR4 levels upon LPS and farm dust stimulation was preserved at all ages. The results of this study in children’s peripheral immune cells indicate that farming exposures in vitro and in vivo may inhibit critical inflammatory processes in human asthma development by activating regulatory TNFAIP3-dependent processes and shaping innate immune pathways. These findings increase the potential for novel farm-derived microbe-based treatment and prevention strategies, and thus may ultimately pave the way for improving asthma management on a global scale.