Fig 1.
Protocol for acute HDM exposure and progressive accumulation of inflammatory cells in BALF of inbred C57BL/6 mice.
(A) Eight- to 10-week-old (W8-10) inbred C57BL/6 female mice were intranasally challenged with daily consecutive doses of 40 μg of HDM extract in 20 μl of PBS (2 mg/ml) or equal volume of PBS. BALF and lungs were collected 24 h after the last exposure on days [D] 3, D5 or D7. (B-C) Representative images of BALF cytospin preparations (scale bar: 20 μm) and total and differential cell counts in BALF from PBS- or HDM-treated inbred C57BL/6 mice at D3, D5 and D7. Data are expressed as mean ± SEM (n = 4 animals per group). *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). HDM, house dust mite; PBS, phosphate buffered saline.
Fig 2.
Progressive increase in airway inflammation and remodeling after acute HDM exposure in inbred C57BL/6 mice.
Representative images of proximal airways showing inflamed lung areas (left) (scale bar: 0.5 mm), and airway thickness (orange bars in insets) (center left), mucus-producing cells per epithelium length (red arrowheads in insets) (center right) and collagen content (right) (scale bars: 50 μm) in PBS- or HDM-treated C57BL/6 mice at D3, D5 and D7. Bottom graphs represent quantification of the abovementioned parameters. Data are expressed as mean ± SEM (n = 4 animals per group). **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). H&E, Hematoxilin and eosin; PAS, Periodic Acid Schiff; AW, airway; HDM, house dust mite; PBS, phosphate buffered saline.
Fig 3.
Expression of airway inflammation and remodeling markers after acute HDM treatment in inbred C57BL/6 mice.
(A) Lung tissue mRNA expression levels of dendritic cell activators (Il33 and Tslp), T-lymphocyte marker (Cd4), Th2 cytokines (Il4, Il10 and Il13), eosinophil (Ccl11 and Ccl5), macrophage (Ccl2) and neutrophil (Cxcl1) chemoattractants and Th1 cytokines (Tnf and Il1b); (B) bronchoconstriction (Acta2), goblet cell hyperplasia (Muc5ac) and collagen deposition (Col1a1) markers, and (C) CCL11 protein levels in lung homogenates in PBS- or HDM-treated inbred C57BL/6 mice at D3, D5 and D7. Data are expressed as mean ± SEM (n = 3–4 animals per group). *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). HDM, house dust mite; PBS, phosphate buffered saline.
Fig 4.
Protocol for prophylactic induction of Igf1r deficiency and HDM treatment, bone marrow cell counts and circulating levels of allergy-related markers.
(A) Igf1rfl/fl (controls) and UBC-CreERT2; Igf1rfl/fl female mice were treated with tamoxifen (TMX) for five consecutive days at four weeks of age (W4) to induce a postnatal Igf1r gene deletion [14]. Then, eight- to 10-week-old (W8-10) Igf1rfl/fl and UBC-CreERT2; Igf1rΔ/Δ (CreERT2) female mice were intranasally challenged with seven daily consecutive doses of 40 μg of HDM extract in 20 μl of PBS (2 mg/ml) or equal volume of PBS. Bone marrow, serum, BALF and lungs were harvested 24 h after last dose on D7. (B-C) Representative images and total, neutrophil and eosinophil (red arrowheads) counts in bone marrow (BM) cytospin preparations (Scale bar: 10 μm; n = 4 animals per group) and (D-E) serum levels of IL10, IL13, IL33, CCL11 and IgE (n = 3–6 animals per group) in PBS- or HDM-exposed Igf1r-deficient vs. Igf1fl/fl mice. Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). HDM, house dust mite; PBS, phosphate buffered saline.
Fig 5.
Igf1r deficiency decreases airway inflammation and remodeling after HDM exposure.
(A) Total and differential cell counts performed on cytospin preparations of BALF (n = 8 animals per group) and (B) representative images of proximal airways showing inflamed lung areas (left) (scale bar: 0.5 mm); airway thickness (orange bars in insets) (center left), mucus-producing cells per epithelium length (red arrowheads in insets) (center right) and collagen content (right) (scale bars: 50 μm; n = 5–6 animals per group) in PBS- or HDM-exposed Igf1r-deficient vs. Igf1fl/fl mice. Bottom graphs represent quantification of the abovementioned parameters. Data are expressed as mean ± SEM *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). H&E, Hematoxilin and eosin; PAS, Periodic Acid Schiff; AW, airway; HDM, house dust mite; PBS, phosphate buffered saline.
Fig 6.
Expression of IGF genes and airway inflammation and remodeling markers in HDM-exposed Igf1r-deficient lungs.
Lung tissue mRNA expression levels of (A) IGF-system genes (Igf1r, Igf1, Insr, Igfbp3 and Igfbp5); (B) dendritic cell activator (Il33), T-lymphocyte marker (Cd4), Th2 cytokines (Il4, Il5 and Il13), eosinophil (Ccl11), macrophage (Ccl2) and neutrophil (Cxcl1) chemoattractants and Th1 cytokines (Tnf and Il1b); (C) bronchoconstriction (Acta2 and Ptgs2), goblet cell hyperplasia (Foxm1 and Muc5ac) and collagen deposition (Col1a1) markers, and (D) IL10, IL13, IL33 and CCL11 protein levels in lung homogenates in PBS- and HDM-exposed Igf1r-deficient and Igf1fl/fl mice. Data are expressed as mean ± SEM (n = 5–6 animals per group). *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). HDM, house dust mite; PBS, phosphate buffered saline.
Fig 7.
Protocol for therapeutic induction of Igf1r deficiency, HDM treatment and circulating levels of allergy-related markers.
(A) Eight- to 10-week-old (W8-10) Igf1rfl/fl and UBC-CreERT2; Igf1rfl/fl female mice were intranasally challenged with seven (first set of animals non-treated with TMX and sacrificed at D7) or fourteen (second set of animals receiving five consecutive intraperitoneal TMX injections between D7 and D11 to induce Igf1r deletion in UBC-CreERT2; Igf1rfl/fl mice to generate CreERT2 mice) daily consecutive doses of 40 μg of HDM extract in 20 μl of PBS (2 mg/ml). Serum, BALF and lungs were collected 24 h after the last exposure. (B-C) Serum levels of IL10, IL13, IL33, CCL11 and IgE (n = 3–4 animals per group) in HDM-exposed CreERT2 vs. Igf1fl/fl mice after the TMX treatment (D14) (+ TMX). Note that the CreERT2 term used at D7 (- TMX) refers to UBC-CreERT2; Igf1rfl/fl mice. Data are expressed as mean ± SEM. *p<0.05; **p<0.01 (Mann-Whitney U and Dunn-Sidak multiple comparison tests). HDM, house dust mite; PBS, phosphate buffered saline; TMX, tamoxifen.
Fig 8.
Therapeutic targeting of Igf1r reduces airway inflammation and remodeling features after HDM exposure.
(A) Total and differential cell counts performed on cytospin preparations of BALF (n = 4–5 animals per group). (B) representative images of proximal airways showing inflamed lung areas (left) (scale bar: 0.5 mm) and airway thickness (orange bars in insets) (center left), mucus-producing cells per epithelium length (red arrowheads in insets) (center right) and collagen content (right) (scale bars: 50 μm) in HDM-exposed CreERT2 vs. Igf1fl/fl mice non-treated with TMX (D7) (- TMX) or after the TMX treatment (D14) (+ TMX). Note that the CreERT2 term used at D7 (- TMX) refers to UBC-CreERT2; Igf1rfl/fl mice. Bottom graphs represent quantification of the abovementioned parameters (n = 5 animals per group). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). H&E, Hematoxilin and eosin; PAS, Periodic Acid Schiff; AW, airway; HDM, house dust mite; PBS, phosphate buffered saline; TMX, tamoxifen.
Fig 9.
Therapeutic Igf1r-gene targeting diminishes expression of airway inflammation and remodeling-related markers after HDM exposure.
Lung tissue mRNA expression levels of (A) Insulin-like growth factor 1 receptor (Igf1r), dendritic cell activator (Il33), T-lymphocyte marker (Cd4), Th2 cytokines (Il4 and Il13), eosinophil (Ccl11), macrophage (Ccl2) and neutrophil (Cxcl1) chemoattractants and Th1 cytokines (Tnf and Il1b) and (B) bronchoconstriction (Acta2 and Ptgs2), goblet cell hyperplasia (Scgb1a1 and Muc5ac) and collagen deposition (Col1a1) markers, and (C) IL33, IL10, IL13 and CCL11 protein levels in lung homogenates (n = 3–5 animals per group). Quantifications were performed in HDM-exposed CreERT2 vs. Igf1fl/fl mice non-treated with TMX (D7) (- TMX) or after the TMX treatment (D14) (+ TMX). Note that the CreERT2 term used at D7 (- TMX) refers to UBC-CreERT2; Igf1rfl/fl mice. Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 (Dunn-Sidak multiple comparison test). HDM, house dust mite; PBS, phosphate buffered saline; TMX, tamoxifen.
Fig 10.
Proposed mechanism for reduced susceptibility to airway inflammation in Igf1r-deficient mice upon HDM-challenge.
Following HDM exposure Igf1r deficiency counteracts collagen deposition, smooth muscle thickening and mucus secretion. The airway epithelium is known to be a major source of IL33 and its delayed differentiation by Igf1r deficiency could diminish IL33 levels after HDM treatment, reducing the induction of Th2 immunity and particularly IL13 expression. After HDM exposure, IL13 normally stimulates goblet cell differentiation in the airway epithelium which leads to goblet cell hyperplasia and mucus hyperproduction in addition to triggering the release of CCL11. Delayed differentiation of the airway epithelium caused by Igf1r deficiency together with diminished IL13 levels may inhibit differentiation of goblet cells and CCL11 production, reducing mucus secretion and eosinophil recruitment to the lung. Additionally, decreased eosinophilopoiesis in bone marrow of Igf1r deficient mice can also substantially contribute to reduced eosinophil presence in the lung. The proposed mechanism illustrated in this figure is supported by results from the present study and additional reports [12,20,28,29,36,38].