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In Silico Analyses Reveal Putative Regulatory Elements Upstream of the Thymic Stromal Lymphopoietin Receptor Gene
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TLR4 Deficiency Exacerbates Allergen-Induced Atopic Dermatitis E. B. Brandt, A. M. Gibson, S. Bass, M. Lindsey, G. K. Khurana Hershey; Cincinnati Children’s Hospital Medical center, Cincinnati, OH. RATIONALE: Despite its presence on resident skin cells, the role of TLR4 in skin diseases remains poorly understood. We aim to establish the contribution of TLR4 to atopic dermatitis (AD). METHODS: TLR4-deficient and wild type mice were epicutaneously exposed to Aspergillus fumigatus (Asp). Skin barrier function was assessed by trans-epidermal water loss (TEWL). Inflammation was determined by immunohistochemistry and RT-PCR. Gene expression data of RNA samples derived from acutely exacerbated AD skin (publically available on NCBI Gene Expression Omnibus) were analyzed. RESULTS: The absence of TLR4 resulted in enhanced TEWL, epidermal thickness, AD symptom scores, epicutaneous sensitization, and decreased skin filaggrin expression. However, TLR4 deficiency had no impact on nascent Th2-driven skin inflammation (eosinophil, mast cell and IL-4 mRNA skin levels). IL-17A and TNFa skin levels were increased, and IFNg levels decreased in Asp-exposed TLR4 deficient mice, but their levels did not correlate with TEWL. Levels of S100A8/A9 (calprotectin, an endogenous TLR4 ligand) were significantly increased and correlated directly with TEWL and skin thickness. Similarly, acutely exacerbated skin samples from AD patients revealed decreased TLR4 skin levels associated with increased S100A8/A9 and impaired skin barrier. CONCLUSIONS: Signaling through TLR4 limits allergen-induced skin barrier dysfunction and AD severity. TLR4 deficiency is associated with increased skin expression of S100A8/A9 in an experimental AD model and in human AD patients. S100A8/A9 skin levels correlate with skin barrier dysfunction suggesting that calprotectin may be a useful biomarker of AD disease severity. In Silico Analyses Reveal Putative Regulatory Elements Upstream of the Thymic Stromal Lymphopoietin Receptor Gene M. J. Romeo, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: We previously reported the unique propensity for dendritic cells from patients with atopic dermatitis (AD) to upregulate thymic stromal lymphopoietin receptor (TSLPR) following allergen stimulation. The objective was to interrogate DNA sequences upstream of the TSLPR gene for transcription factor binding sites that could provide candidate regulatory elements relevant to AD. METHODS: Bioinformatics approaches were used to identify candidate DNA sequences located 5’ to the TSLPR gene coding region that may contain transcription factor binding sites. Databases including MatrixCatch, UCSC’s Genome Browser, TRANSFAC and NCBI Entrez were queried using either the TSLPR gene name (CRLF2), accession number (AB052639), or DNA sequence obtained from the NCBI human genome database. RESULTS: Clustering of transcription factor binding sites for NF-AT, NFkappaB, PU.1 and IRF proteins was identified in a 5,000 nucleotide span immediately upstream of the TSLPR gene transcription start site. This region contained NF-AT, PU.1 and IRF sites proximal to (and NFkappaB sites distal to) the transcription start site. MatrixCatch uniquely identified the presence of several possible PU.1/IRF composite sites which are known to influence the transcriptional regulation of an array of immunomodulatory genes including CIITA, which controls MHCII expression during dendritic cell maturation. Molecular cloning of this putative promoter region into a luciferase reporter vector was successful using genomic DNA isolated from AD patients. CONCLUSION: In silico analyses identified a putative TSLPR gene promoter containing regulatory elements that could yield new insight into the genetic mechanisms underlying allergen-responsiveness in dendritic cells from AD subjects.
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Filaggrin Deficiency Impairs Viral Containment in Mice Cutaneously Inoculated with Vaccinia Virus (VV) R. S. Geha, M. K. Oyoshi; Children’s Hospital Boston, Boston, MA. RATIONALE: Eczema Vaccinatum (EV) is a life threatening complication of exposure to smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of VV in skin and internal organs. Filaggrin deficiency is present in up to 48% of patients with AD, and filaggrin null mutations are associated with eczema herpeticum. We examined the effects of filaggrin deficiency on the response of mice to cutaneous VV inoculation. METHODS: Unsensitized or ovalbumin (OVA)-sensitized shaved unstripped skin of filaggrin-deficient ft/ft mice on BALB/c background or WT controls was inoculated with 107 plaque-forming units of VV by scarification. Viral load and cytokine mRNA expression were assessed by quantitative PCR, and skin histology was examined by H&E staining. RESULTS: VV inoculation in unsensitized skin resulted in significantly increased size of primary lesions, number of satellite lesions, viral loads in skin and internal organs, dermal cellular infiltration and mRNA expression of IL-17, IL-4, IL-13, and IFN-g in ft/ft mice compared to WT controls, which showed minimal response to VV inoculation. Inoculation of VV at sites of OVA application to unstripped skin accentuated all the aforementioned changes in ft/ft mice, but had no detectable effect in WT controls. The number of satellite lesions and the viral loads in internal organs were significantly decreased in ft/ft mice bred onto the IL-17 null background after VV inoculation in unsensitized skin, as well as in OVA-sensitized skin. CONCLUSIONS: Filaggrin deficiency may predispose to EV, particularly if VV is introduced at sites of cutaneous antigen sensitization. Blocking IL-17 may attenuate EV in filaggrin deficiency.
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Sequencing Of The Flg2 Gene In Patients With Atopic Dermatitis And Eczema Herpeticum In A Population Of European Descent. N. M. Rafaels1, D. Y. Leung2, L. Beck3, R. Lewis1, L. Huang1, P. Gao1, M. Boguniewicz2, T. Hata4, L. Schneider5, J. Hanifin6, R. Gallo4, L. Gao1, R. A. Mathias1, K. C. Barnes1; 1Johns Hopkins School of Medicine, Baltimore, MD, 2Department of Pediatrics, National Jewish Health, Denver, CO, 3Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 4Division of Dermatology, University of California, San Diego, CA, 5Division of Immunology, Children’s Hospital Boston, Boston, MA, 6Department of Dermatology, Oregon Health & Science University, Portland, OR. RATIONALE: Atopic dermatitis (AD) is characterized by disseminated viral skin infections, particularly eczema herpeticum (ADEH). Previously, we genotyped 8 common (>1% minor allele frequency) non-synonymous coding single nucleotide polymorphisms in FLG2, a gene on the epidermal differentiation complex locus associated with ADEH, at the conclusion of the 2011 AAAAI meeting. Currently we are sequencing the entire gene to discover additional rare and novel variants that may be associated with ADEH. METHODS: We are currently sequencing 15kb of the FLG2 gene using Agilent’s targeted deep-resequencing platform on 112 ADEH- and 125 ADEH+ European ancestry patients. To date a subset (100 ADEH-, 100 ADEH+) has been sequenced on exon 2 and 600 base pairs of exon 3 nearest exon 2, using Sanger sequencing. Genetic associations for risk of ADEH and associated traits were determined by the Cochran-Armitage trend test. RESULTS: Sequencing so far has uncovered three previously known SNPs, and two novel variants in exon 3 not previously documented in dbSNP or the Thousand Genomes Project. Tests for association on single SNPs and a collapsed test on all rare variants did not reveal any associations with ADEH. We also performed analyses adjusting for FLG mutations R501X and 2282del4, but did not observe significant associations with ADEH. CONCLUSIONS: Interim sequence data on FLG2 has not yielded novel associations with ADEH; however, we anticipate additional novel variants to be identified by targeted deep-resequencing of the entire gene, and will perform additional burden tests collapsing on all newly identified rare functional variants previously un-captured by any tagging genotyping strategy.
SATURDAY
Abstracts AB37
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
TLR4 Deficiency Exacerbates Allergen-Induced Atopic Dermatitis E. B. Brandt, A. M. Gibson, S. Bass, M. Lindsey, G. K. Khurana Hershey; Cincinnati Children’s Hospital Medical center, Cincinnati, OH. RATIONALE: Despite its presence on resident skin cells, the role of TLR4 in skin diseases remains poorly understood. We aim to establish the contribution of TLR4 to atopic dermatitis (AD). METHODS: TLR4-deficient and wild type mice were epicutaneously exposed to Aspergillus fumigatus (Asp). Skin barrier function was assessed by trans-epidermal water loss (TEWL). Inflammation was determined by immunohistochemistry and RT-PCR. Gene expression data of RNA samples derived from acutely exacerbated AD skin (publically available on NCBI Gene Expression Omnibus) were analyzed. RESULTS: The absence of TLR4 resulted in enhanced TEWL, epidermal thickness, AD symptom scores, epicutaneous sensitization, and decreased skin filaggrin expression. However, TLR4 deficiency had no impact on nascent Th2-driven skin inflammation (eosinophil, mast cell and IL-4 mRNA skin levels). IL-17A and TNFa skin levels were increased, and IFNg levels decreased in Asp-exposed TLR4 deficient mice, but their levels did not correlate with TEWL. Levels of S100A8/A9 (calprotectin, an endogenous TLR4 ligand) were significantly increased and correlated directly with TEWL and skin thickness. Similarly, acutely exacerbated skin samples from AD patients revealed decreased TLR4 skin levels associated with increased S100A8/A9 and impaired skin barrier. CONCLUSIONS: Signaling through TLR4 limits allergen-induced skin barrier dysfunction and AD severity. TLR4 deficiency is associated with increased skin expression of S100A8/A9 in an experimental AD model and in human AD patients. S100A8/A9 skin levels correlate with skin barrier dysfunction suggesting that calprotectin may be a useful biomarker of AD disease severity. In Silico Analyses Reveal Putative Regulatory Elements Upstream of the Thymic Stromal Lymphopoietin Receptor Gene M. J. Romeo, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: We previously reported the unique propensity for dendritic cells from patients with atopic dermatitis (AD) to upregulate thymic stromal lymphopoietin receptor (TSLPR) following allergen stimulation. The objective was to interrogate DNA sequences upstream of the TSLPR gene for transcription factor binding sites that could provide candidate regulatory elements relevant to AD. METHODS: Bioinformatics approaches were used to identify candidate DNA sequences located 5’ to the TSLPR gene coding region that may contain transcription factor binding sites. Databases including MatrixCatch, UCSC’s Genome Browser, TRANSFAC and NCBI Entrez were queried using either the TSLPR gene name (CRLF2), accession number (AB052639), or DNA sequence obtained from the NCBI human genome database. RESULTS: Clustering of transcription factor binding sites for NF-AT, NFkappaB, PU.1 and IRF proteins was identified in a 5,000 nucleotide span immediately upstream of the TSLPR gene transcription start site. This region contained NF-AT, PU.1 and IRF sites proximal to (and NFkappaB sites distal to) the transcription start site. MatrixCatch uniquely identified the presence of several possible PU.1/IRF composite sites which are known to influence the transcriptional regulation of an array of immunomodulatory genes including CIITA, which controls MHCII expression during dendritic cell maturation. Molecular cloning of this putative promoter region into a luciferase reporter vector was successful using genomic DNA isolated from AD patients. CONCLUSION: In silico analyses identified a putative TSLPR gene promoter containing regulatory elements that could yield new insight into the genetic mechanisms underlying allergen-responsiveness in dendritic cells from AD subjects.
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Filaggrin Deficiency Impairs Viral Containment in Mice Cutaneously Inoculated with Vaccinia Virus (VV) R. S. Geha, M. K. Oyoshi; Children’s Hospital Boston, Boston, MA. RATIONALE: Eczema Vaccinatum (EV) is a life threatening complication of exposure to smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of VV in skin and internal organs. Filaggrin deficiency is present in up to 48% of patients with AD, and filaggrin null mutations are associated with eczema herpeticum. We examined the effects of filaggrin deficiency on the response of mice to cutaneous VV inoculation. METHODS: Unsensitized or ovalbumin (OVA)-sensitized shaved unstripped skin of filaggrin-deficient ft/ft mice on BALB/c background or WT controls was inoculated with 107 plaque-forming units of VV by scarification. Viral load and cytokine mRNA expression were assessed by quantitative PCR, and skin histology was examined by H&E staining. RESULTS: VV inoculation in unsensitized skin resulted in significantly increased size of primary lesions, number of satellite lesions, viral loads in skin and internal organs, dermal cellular infiltration and mRNA expression of IL-17, IL-4, IL-13, and IFN-g in ft/ft mice compared to WT controls, which showed minimal response to VV inoculation. Inoculation of VV at sites of OVA application to unstripped skin accentuated all the aforementioned changes in ft/ft mice, but had no detectable effect in WT controls. The number of satellite lesions and the viral loads in internal organs were significantly decreased in ft/ft mice bred onto the IL-17 null background after VV inoculation in unsensitized skin, as well as in OVA-sensitized skin. CONCLUSIONS: Filaggrin deficiency may predispose to EV, particularly if VV is introduced at sites of cutaneous antigen sensitization. Blocking IL-17 may attenuate EV in filaggrin deficiency.
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Sequencing Of The Flg2 Gene In Patients With Atopic Dermatitis And Eczema Herpeticum In A Population Of European Descent. N. M. Rafaels1, D. Y. Leung2, L. Beck3, R. Lewis1, L. Huang1, P. Gao1, M. Boguniewicz2, T. Hata4, L. Schneider5, J. Hanifin6, R. Gallo4, L. Gao1, R. A. Mathias1, K. C. Barnes1; 1Johns Hopkins School of Medicine, Baltimore, MD, 2Department of Pediatrics, National Jewish Health, Denver, CO, 3Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 4Division of Dermatology, University of California, San Diego, CA, 5Division of Immunology, Children’s Hospital Boston, Boston, MA, 6Department of Dermatology, Oregon Health & Science University, Portland, OR. RATIONALE: Atopic dermatitis (AD) is characterized by disseminated viral skin infections, particularly eczema herpeticum (ADEH). Previously, we genotyped 8 common (>1% minor allele frequency) non-synonymous coding single nucleotide polymorphisms in FLG2, a gene on the epidermal differentiation complex locus associated with ADEH, at the conclusion of the 2011 AAAAI meeting. Currently we are sequencing the entire gene to discover additional rare and novel variants that may be associated with ADEH. METHODS: We are currently sequencing 15kb of the FLG2 gene using Agilent’s targeted deep-resequencing platform on 112 ADEH- and 125 ADEH+ European ancestry patients. To date a subset (100 ADEH-, 100 ADEH+) has been sequenced on exon 2 and 600 base pairs of exon 3 nearest exon 2, using Sanger sequencing. Genetic associations for risk of ADEH and associated traits were determined by the Cochran-Armitage trend test. RESULTS: Sequencing so far has uncovered three previously known SNPs, and two novel variants in exon 3 not previously documented in dbSNP or the Thousand Genomes Project. Tests for association on single SNPs and a collapsed test on all rare variants did not reveal any associations with ADEH. We also performed analyses adjusting for FLG mutations R501X and 2282del4, but did not observe significant associations with ADEH. CONCLUSIONS: Interim sequence data on FLG2 has not yielded novel associations with ADEH; however, we anticipate additional novel variants to be identified by targeted deep-resequencing of the entire gene, and will perform additional burden tests collapsing on all newly identified rare functional variants previously un-captured by any tagging genotyping strategy.
SATURDAY
Abstracts AB37
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2