Asthmatic Eosinophils Promote Contractility and Migration of Airway Smooth Muscle Cells and Pulmonary Fibroblasts In Vitro

Ieva Janulaityte, Andrius Januskevicius, Virginija Kalinauskaite-Zukauske, Jolita Palacionyte, Kestutis Malakauskas, Ieva Janulaityte, Andrius Januskevicius, Virginija Kalinauskaite-Zukauske, Jolita Palacionyte, Kestutis Malakauskas

Abstract

Enhanced contractility and migration of airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) are part of airway remodeling in asthma. Eosinophils are the central inflammatory cells that participate in airway inflammation. However, the role of asthmatic eosinophils in ASMC and PF contractility, migration, and differentiation to contractile phenotype has not yet been precisely described. A total of 38 individuals were included in this study: 13 steroid-free non-severe allergic asthma (AA) patients, 11 severe non-allergic eosinophilic asthma (SNEA) patients, and 14 healthy subjects (HS). For AA patients and HS groups, a bronchial allergen challenge with D. pteronyssinus was performed. Individual combined cell cultures were prepared from isolated peripheral blood eosinophils and immortalized ASMC or commercial PF cell lines separately. The migration of ASMC and PF was evaluated using wound healing assay and contractility using collagen gel assay. Gene expression of contractile apparatus proteins, COL1A1, COL5A1, and FN, in ASMC and PF was evaluated using qRT-PCR. We found that contractility and migration of ASMC and PF significantly increased after incubation with asthmatic eosinophils compared to HS eosinophils, p < 0.05, and SNEA eosinophils demonstrated the highest effect on contractility of ASMC and migration of both cell lines, p < 0.05. AA and SNEA eosinophils significantly increased gene expression of contractile apparatus proteins, COL1A1 and FN, in both cell lines, p < 0.05. Furthermore, the allergen-activated AA eosinophils significantly increased the contractility of ASMC, and migration and gene expression in ASMC and PF, p < 0.05. Thus, asthmatic eosinophils change ASMC and PF behavior by increasing their contractility and migration, contributing to airway remodeling.

Trial registration: ClinicalTrials.gov NCT03388359.

Keywords: airway smooth muscle cell; asthma; contractility; eosinophil; extracellular matrix; migration; pathogenesis; pulmonary fibroblast.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flowchart of the study design. (A)—Recruitment of study subjects, clinical examination of allergic asthma patients and healthy subjects. (B)—Experimental workflow. AA—allergic asthma; ASMC—airway smooth muscle cells; HS—healthy subjects; PF—pulmonary fibroblasts; qRT-PCR—quantitative reverse transcription polymerase chain reaction.
Figure 2
Figure 2
The detailed experimental plan. ASMC—airway smooth muscle cells; PF—pulmonary fibroblast.
Figure 3
Figure 3
Collagen gel assay. (A)—ASMC after 24 h incubation with HS eosinophils; (B)—ASMC after 24 h incubation with SNEA patients’ eosinophils; (C)—ASMC after 24 h incubation with AA patients’ eosinophils; (D)—collagen gel contraction percentage from control ASMC after 1, 2, and 3 h incubation with subjects’ eosinophils, AA n = 3, SNEA n = 3, HS n = 3; (E)—collagen gel contraction percentage from control PF cells after 1, 2, and 3 h incubation with subjects’ eosinophils, AA n = 3, SNEA n = 3, HS n = 3. AA—allergic asthma patient; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast; SNEA—severe non-allergic eosinophilic asthma patients. Data presented as mean ± SEM.
Figure 3
Figure 3
Collagen gel assay. (A)—ASMC after 24 h incubation with HS eosinophils; (B)—ASMC after 24 h incubation with SNEA patients’ eosinophils; (C)—ASMC after 24 h incubation with AA patients’ eosinophils; (D)—collagen gel contraction percentage from control ASMC after 1, 2, and 3 h incubation with subjects’ eosinophils, AA n = 3, SNEA n = 3, HS n = 3; (E)—collagen gel contraction percentage from control PF cells after 1, 2, and 3 h incubation with subjects’ eosinophils, AA n = 3, SNEA n = 3, HS n = 3. AA—allergic asthma patient; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast; SNEA—severe non-allergic eosinophilic asthma patients. Data presented as mean ± SEM.
Figure 4
Figure 4
Wound healing assay. (A)—ASMC migration after 0 h, 24 h, 48 h, 72 h incubation with eosinophils; (B)—PF migration after 0 h, 24 h, 48 h, 72 h incubation with eosinophils. AA n = 5, SNEA n = 5, HS n = 5. Presented as a cell-covered area, as a percentage of control cells. AA—allergic asthma; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast; SNEA—severe non-allergic eosinophilic asthma patients. Data presented as mean ± SEM.
Figure 5
Figure 5
(A)—The effect of subjects’ eosinophils and/or serum on the ASMC-induced collagen gel disk shrinkage; (B)—The effect of subjects’ eosinophils and/or serum on the PF-induced collagen gel disk shrinkage. Data presented as percentage of control cells that were not incubated with eosinophils, as mean ± SEM. Added blood serum of each investigated subject to individual combined cultures: 2% v/v. AA—allergic asthma; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast; SNEA—severe non-allergic eosinophilic asthma. α p < 0.05 compared to the only serum effect; β p < 0.05 compared to eosinophil and serum combined effect. A—* p < 0.001 compared to control ASMC; B—* p < 0.01 compared to control PF. AA n = 12; SNEA n = 11; HS n = 10. Statistical analysis between investigated groups—Kruskal-Wallis test and post hoc Mann–Whitney two-sided U-test (independent data); within one study group—Friedman test for multiple comparison within the group and the post hoc Wilcoxon matched-pairs signed-rank two-sided test (dependent data). Significant differences were found in the eosinophil effect on ASMC and PF–induced collagen gel shrinkage between investigated groups by Kruskal-Wallis test, respectively, χ2 = 21.49, df = 2, p < 0.0001 and χ2 = 16.55, df = 2, p = 0.0003. Significant differences of ASMC-induced collagen gel shrinkage were found in AA and SNEA groups by Friedman test, respectively χ2 = 15.24, df = 2, p < 0.0001; χ2 = 11.89, df = 2, p = 0.0011. Significant differences of PF-induced collagen gel shrinkage were found in AA and HS groups by Friedman test, respectively, χ2 = 12.51, df = 2, p = 0.0007; and χ2 = 16.76; df = 2, p = 0.0002. Lines connect comparison groups with a p-value denoting the significant difference and pair-wise comparisons.
Figure 6
Figure 6
(A)–Migration of ASMC after incubation with eosinophils and/or subjects’ serum. (B)–Migration of PF after incubation with eosinophils and/or subjects’ serum. Data presented as cell covered area, as a percentage of control ASMC. Added blood serum of each investigated subject to individual combined cultures: 2% v/v. AA n = 13; SNEA n = 11; HS n = 14. AA—allergic asthma; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast; SNEA—severe non-allergic eosinophilic asthma. α p < 0.05 compared to the only serum effect; β p < 0.05 compared to the eosinophil and serum combined effect; A—* p < 0.001 compared to control ASMC, B—* p < 0.001 compared to control PF. Statistical analysis between investigated groups—Kruskal-Wallis test and post hoc Mann–Whitney two-sided U-test (independent data); within one study group—Friedman test for multiple comparison within the group and the post hoc Wilcoxon matched-pairs signed-rank two-sided test (dependent data). Significant differences were found in the eosinophil effect on ASMC and PF migration between investigated groups by Kruskal-Wallis test, respectively, χ2 = 22.33, df = 2, p < 0.0001 and χ2 = 25.93, df = 2, p < 0.0001. Significant differences of ASMC migration were found in AA and SNEA groups by Friedman test, respectively χ2 = 19.08, df = 2, p < 0.0001; χ2 = 16.55, df = 2, p < 0.0001. Significant differences of PF migration were found in AA and SNEA groups by Friedman test, respectively, χ2 = 19.08, df = 2, p < 0.0001; and χ2 = 14.60, df = 2, p = 0.0007. Lines connect comparison groups with a p-value denoting the significant difference in pair-wise comparisons.
Figure 7
Figure 7
(A)–Gene expression in ASMC after incubation with eosinophils; (B)–Gene expression in PF after incubation with eosinophils. Data presented as folds over the HS eosinophil effect as mean ± SEM. AA—allergic asthma patient; SNEA—severe non-allergic eosinophilic asthma patient; ASMC—airway smooth muscle cells; PF–pulmonary fibroblast; HS—healthy subject; COL1A1—collagen I α 1; COL5A1—collagen V α 1; FN—fibronectin; α-sm-actin—α smooth muscle actin; sm-MHC—smooth muscle myosin heavy chain; SM22—transgelin; sm-MLCK—smooth muscle myosin light chain kinase. * p < 0.01 compared to HS eosinophils; ** p < 0.001 compared to HS eosinophils. AA n = 11; SNEA n = 10; HS n = 8. Statistical analysis between investigated groups—Mann–Whitney two-sided U-test (independent data); Wilcoxon signed-rank test was used for gene expression analysis against ASMC or PF control. Lines connect comparison groups with a p-value denoting the significant difference in pair-wise comparisons.
Figure 8
Figure 8
(A)–Contraction of collagen gel disk with ASMC after incubation with eosinophils and/or subjects’ serum 24 h after bronchial allergen challenge with D. pteronyssinus; (B)–Contraction of collagen gel disk with PF after incubation with eosinophils and/or subjects’ serum 24 h after bronchial allergen challenge with D. pteronyssinus allergen. Data presented as a percentage of control ASMC that were not incubated with eosinophils. Added blood serum of each investigated subject to individual combined cultures: 2% v/v. ASMC—airway smooth muscle cells; HS—healthy subject; AA—allergic asthma; PF—pulmonary fibroblast. α—p < 0.05 compared to results 24h after allergen challenge; β—p < 0.05 compared to the only serum effect at the same visit; χ—p < 0.05 compared to the eosinophil and serum combined effect at the same visit; A—* p < 0.001 compared to control ASMC; B—* p < 0.01 compared to control PF; ** p < 0.001 compared to control PF. AA n = 10; HS n = 10. Statistical analysis within one study group—Friedman test for multiple comparison within the group and the post hoc Wilcoxon matched-pairs signed-rank two-sided test (dependent data). Significant differences of ASMC-induced collagen gel shrinkage were found in AA group 24 h after bronchial allergen challenge by Friedman test, χ2 = 12.96, df = 2, p = 0.0005. Significant differences of PF-induced collagen gel shrinkage were found in HS group 24 h after bronchial allergen challenge by Friedman test, χ2 = 9.929, df = 2, p = 0.0038. The results from Figure 5A,B of AA and HS patients were re-used as the baseline (before allergen challenge) result.
Figure 9
Figure 9
(A)–Migration of ASMC after incubation with allergen-activated eosinophils and/or subjects’ serum. (B)–Migration of PF after incubation with allergen-activated eosinophils and/or subjects’ serum. Data presented as cell covered area, as a percentage of control cells. Added blood serum of each investigated subject to individual combined cultures: 2% v/v. AA n = 13; HS n = 14. AA—allergic asthma; ASMC—airway smooth muscle cells; HS—healthy subject; PF—pulmonary fibroblast. α—p < 0.05 compared to results 24h after allergen challenge; β—p < 0.05 compared to the only serum effect at the same visit; χ—p < 0.05 compared to the eosinophil and serum combined effect at the same visit; A—* p < 0.001 compared to control ASMC; B—* p < 0.01 compared to control PF cells. Statistical analysis within one study group—Friedman test for multiple comparison within the group and the post hoc Wilcoxon matched-pairs signed-rank two-sided test (dependent data). Significant differences of ASMC and PF migration were found in AA group 24 h after bronchial allergen challenge by Friedman test, respectively, χ2 = 17.23, df = 2, p = 0.0002; χ2 = 18.00, df = 2, p = 0.0001. The results from Figure 6A,B of AA and HS patients were re-used as the baseline (before allergen challenge) result.
Figure 10
Figure 10
(A)–The change in gene expression in ASMC 24 h after bronchial allergen challenge with D. pteronyssinus, (B)–The change in gene expression in PF cells 24 h after bronchial allergen challenge with D. pteronyssinus. Data presented as folds over baseline visit result as mean ± SEM. Added blood serum of each investigated subject to individual combined cultures: 2% v/v. AA—allergic asthma patient; ASMC—airway smooth muscle cells; PF—pulmonary fibroblast; HS—healthy subject; COL1A1—collagen I α 1; COL5A1—collagen V α 1; FN—fibronectin; α-sm-actin—α smooth muscle actin; sm-MHC—smooth muscle myosin heavy chain; SM22—transgelin; sm-MLCK—smooth muscle myosin light chain kinase. AA n = 11; HS n = 8. * p < 0.05 compared to control cells; ** p < 0.01. Statistical analysis between investigated groups—Mann–Whitney two-sided U-test (independent data); Wilcoxon signed-rank test was used for gene expression analysis against ASMC or PF control. Lines connect comparison groups with a p-value denoting the significant difference in pair-wise comparisons.

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