The TGFβ1 Promoter SNP C-509T and Food Sensitization Promote Esophageal Remodeling in Pediatric Eosinophilic Esophagitis

<div><p>Background</p><p>Eosinophilic esophagitis (EoE) is a chronic antigen mediated disease associated with substantial esophageal remodeling and fibrosis. The functional TGFβ1 promoter SNP C-509 associates with renal fibrosis and asthma. The effect of TGFβ1 genotype and EoE severity or potential gene-environment interactions have not been previously reported in EoE.</p><p>Methods</p><p>Genotype at TGFβ1 C-509T and remodeling was analyzed in 144 subjects with EoE. The severity of remodeling and inflammation was analyzed in the context of IgE sensitization to food antigens and C-509T genotype.</p><p>Results</p><p>The TGFβ1 promoter C-509 genotypes CC, CT, and TT were 35%, 52%, and 13%, respectively. Sixty-six percent of subjects were sensitized to foods by positive skin prick test (SPT) or serum specific IgE. TT genotype subjects had significantly more TGFβ1 (CC subjects = 1300 per mm²; TT = 2250 per mm²) (p<0.05) and tryptase (CC subjects = 145 per mm²: TT = 307 per mm²) (p<0.05) positive cells and higher epithelial remodeling scores (2.4 vs 3.7, p<0.001) than CC subjects. The differences in TGFβ1 and tryptase positive cells as well as fibrosis were significantly increased when there was concurrent food sensitization. Food sensitization alone did not associate with any parameters of inflammation or remodeling.</p><p>Conclusions</p><p>Our data support a gene-environment interaction between food and genotype at C-509 that modulates disease severity in EoE. Since EoE subjects often continue to consume foods to which they are sensitized, these findings may have clinical relevance for disease management.</p></div

Mast cells and remodeling parameters in subjects by genotype with concurrent food sensitization.

<p>Tryptase positive cells by genotype in subjects with serum and/or skin prick positive food specific IgE (A) or food sensitization only on skin prick testing (B). TGFβ1 (C) and pSmad2/3 (D) positive cells and vWF positive blood vessels (E) by genotype with coexistent food sensitization</p

Lamina propria remodeling features by genotype.

<p>TT subjects have the highest numbers of TGFβ1 positive cells (A, B). Representative image of TGFβ1 positive cells in TT versus CC genotype subjects (B). pSmad2/3 positive cells by genotype (C), fibrosis score by genotype (D), and vWF (E) and VCAM (F) positive vessels by genotype.</p

Inflammatory cells and epithelial remodeling by genotype.

<p>CT genotype subjects have the highest numbers of epithelial (A) and lamina propria (LP) eosinophils (B) but TT genotype subjects have the highest numbers of tryptase positive mast cells (C) and epithelial remodeling (D). Bars represent mean with standard error.</p

Genotype TT subjects have higher fibrosis.

<p>Fibrosis score by genotype among subjects with skin prick test positivity (A) and within the TT genotype between subjects with and without food sensitization (B)</p

Bone resorption is increased in NOMID mice.

<p>Femoral sections from P13 mice were stained with H&E (<i>A</i>) or for TRAP activity (<i>B</i>–<i>D</i>), and OC number (<i>C</i>) or surface (<i>D</i>) per bone surface was determined. NOMID mice exhibited a larger bone marrow cavity in relation to overall bone size compared to WT mice (<i>A</i>). Abundant OC (stained in red, <i>B</i>) were present in the primary spongiosa and on the endocortical bone surface of NOMID mice. There were fewer trabeculae (T) and thinner cortical bone (bracket) in NOMID compared to WT mice. Original magnification: ×2 (<i>A</i>), ×10 (<i>B</i>, trabecular region), ×20 (<i>B</i>, cortical region). Serum was collected for the measurement of CTX-1 levels (<i>E</i>), and supernatants were collected from centrifuged bone marrow for the measurement of IL-1β (F). CTX-1 and IL-1β levels were 3- to 4-fold higher in NOMID compared to WT mice. Quantitative data were obtained from 5 mice/genotype and expressed as the mean ± S.D. (<i>C</i>, <i>D</i> and <i>F</i>) or mean ± S.E.M. (<i>E</i>). *P<0.05; **P<0.007.</p

Unfractionated NOMID bone marrow cells proliferate and survive significantly less than their WT counterparts.

<p>Unfractionated bone marrow cells (<i>A</i>–<i>C</i>) or BMM (<i>D</i> and <i>E</i>) were cultured in media containing M-CSF for the indicated times. Proliferation (<i>A</i> and <i>D</i>), metabolic activity (<i>B</i> and <i>E</i>) and Western blot analysis of PARP cleavage (<i>C</i>) were carried out. While unfractionated NOMID cells proliferated and survived less than WT cells, no differences were seen in BMM proliferation and survival between genotypes. PARP cleavage was higher in NOMID than in WT cells. Determinations were performed in triplicate and expressed as the mean ± S.D. Results are representative of three independent experiments. *P<0.05; **P<0.007 over the control (Ctl).</p

OC differentiation is increased in NOMID cells.

<p>Unfractionated bone marrow cells (<i>A</i>) or BMM previously cultured for 3 days in the presence of M-CSF (<i>B</i>) were induced to differentiate into OC in the presence of M-CSF and 100 ng/ml RANKL. (<i>C</i>) Co-cultures of WT or NOMID (NOM) BMM and WT BMSC were carried out in the presence of 10 nM dexamethasone and 1 nM 1,25(OH)₂ vitamin D₃ for 5–7 days. The cultures were stained for TRAP activity, and the number of OC (cells stained in red with ≥3 nuclei, arrow) were counted manually. The pictures were taken at the same magnification (×4) for both genotypes. The data show that NOMID cells formed more OC than WT cells. Determinations were performed in triplicate and expressed as the mean ± S.E.M. Results are representative of at least three independent experiments. **P<0.007.</p

NOMID mice exhibit disorganized growth plates.

<p>Femoral sections from P13 (<i>A</i>–<i>C</i>) or P8 (<i>D</i> and <i>E</i>) mice were used for safranin O (<i>A</i>) and H&E (<i>B</i>, <i>D</i> and <i>E</i>) staining or for TUNEL (<i>C</i>). Original magnification: ×20 (<i>A</i> and <i>C</i>), ×10 (<i>B</i>, <i>D</i> and <i>E</i>). The spike (arrowhead) and early morphological changes (D, arrowhead) were observed only in NOMID mice. NOMID cells showed a high degree of apoptosis. hz, hypertrophic zone.</p

NOMID mice develop leukocytosis associated with high levels of inflammatory mediators.

<p>Complete blood cell counts (<i>A</i>) and serum cytokine analysis (<i>B</i>) were carried out on samples harvested from P12–13 mice (n = 4–7). NOMID mice developed neutrophilia, lymphopenia, thrombocytosis and anemia and produced higher levels of several inflammatory mediators. G-CSF values were extrapolated beyond the standard range. IL-6 and TNF-α levels were near statistical significance between genotypes. Data are expressed as mean ± S.E.M. *P<0.05, **P<0.007. WBC, white blood cells; RBC, red blood cells.</p