M2 Macrophages Promote Collagen Expression and Synthesis in Laryngotracheal Stenosis Fibroblasts.
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ABSTRACT: Objective
Macrophages exhibit distinct phenotypes and are dysregulated in a model of iatrogenic laryngotracheal stenosis (iLTS). Increased populations of alternatively activated or M2 macrophages have been demonstrated. However, the role of these macrophages is unknown. The aims of this study are: 1) define the macrophage population in iLTS in the context of classically activated or M1 and M2 macrophage phenotypes, and 2) characterize the effect of monocyte-derived M1 and M2 macrophages on normal airway and LTS-derived fibroblasts (FBs) in vitro.Study design
Comparative analysis; in vitro controlled study.Methods
Immunohistochemical analysis of human iLTS and control specimens was performed to define the macrophage population. In vitro, M1, and M2 macrophages were polarized using M-CSF + Interferon-gamma and lipopolysaccharide or Interleukin-4, respectively. FBs isolated from laryngotracheal scar (LTS-FBs) and normal tracheal airway (NA-FBs) in eight patients with iLTS were cocultured with polarized macrophages. Fibrosis gene expression, soluble collagen production, and proliferation were assessed.Results
Immunohistochemical analysis revealed increased CD11b + cells (macrophage marker) in laryngotracheal scar specimens (18.3% vs. 8.5%, P = .03) and predominant CD206 (M2) costaining versus CD86 (M1) (51.5% vs. 9.8%, n = 10, P = .001). In vitro, NA-FBs cultured with M2 macrophages demonstrated a 2.41-fold increase in collagen-1 expression (P = .05, n = 8) and an increase in soluble collagen (9.98 vs. 8.875, mean difference: 1.11 95%, confidence interval 0.024-2.192, n = 8, P = .015).Conclusion
Increased populations of CD11b cells are present in iLTS specimens and are predominantly CD206+, indicating an M2 phenotype. In vitro, M2 macrophages promoted collagen expression in airway FBs. Targeting macrophages may represent a therapeutic strategy for attenuating fibrosis in iLTS.Level of evidence
NA Laryngoscope, 131:E346-E353, 2021.
SUBMITTER: Motz K
PROVIDER: S-EPMC7844402 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundLaryngotracheal stenosis (LTS) is a life-threatening disease that commonly results in airway obstruction in children. Traditional treatments such as laryngotracheal reconstruction and balloon dilation all have the risk of laryngotracheal restenosis. It is of great importance to spare patients the morbidity of LTS and risks of restenosis associated with these treatments. Laboratory and clinical trials have focused on fibrosis, the crucial pathological process of LTS. This study was undertaken to investigate the function of CXC chemokine receptor-7 (CXCR7) in the fibroblasts derived from LTS.MethodsRNA sequencing was performed on acquired human LTS and normal trachea tissues to analyze differentially expressed genes. Fibroblasts from LTS and normal trachea tissues were isolated and cultured. CXCR7 knockdown was performed using specific small interfering RNAs (siRNAs) and activated by CXCR7 agonist VUF11207. The assessment of cell proliferation and migration was conducted using EdU proliferation, wound healing, and transwell assays. The assessment of cell proliferation and migration was conducted using EdU proliferation, wound healing, and transwell assays. The expressions of CXCR7, E-cadherin and NF-κB signaling pathway were analyzed by quantitative polymerase chain reaction (qPCR), western blotting, immunohistochemistry, and immunofluorescence.ResultsRNA sequencing showed that CXCR7 was among the most differentially expressed genes. LTS had an increased CXCR7 expression but decreased E-cadherin expression in vivo. CXCR7 agonist stimulated the migration of LTS derived fibroblasts significantly in vitro, with no significant influence on the cell proliferation and apoptosis. CXCR7 agonist inhibited the expression of E-cadherin by activating the NF-κB signaling pathway. The effects of CXCR7 on cell migration and E-cadherin expression were blocked by CXCR7 siRNA.ConclusionsLTS had an increased CXCR7 expression but decreased E-cadherin expression. CXCR7 activation inhibited E-cadherin expression by NF-κB signaling pathway and thereby promoted the migration of LTS derived fibroblasts.
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Project description:OBJECTIVE/HYPOTHESIS:This prospective controlled human and murine study assessed the presence of inflammatory cells and cytokines to test the hypothesis that immune cells are associated with fibroproliferation in iatrogenic laryngotracheal stenosis (iLTS). METHODS:Inflammation was assessed by histology and immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), and flow cytometry of cricotracheal resections of iLTS patients compared to normal controls. An iLTS murine model assessed the temporal relationship between inflammation and fibrosis. RESULTS:iLTS specimens showed increased inflammation versus normal controls (159/high power field [hpf] vs. 119/hpf, P?=?0.038), and increased CD3?+?T-cells, CD4?+?cells, and CD3+/CD4?+?T-helper (TH ) cells (all P?<?0.05). The inflammatory infiltrate was located immediately adjacent to the epithelial surface in the superficial aspect of the thickened lamina propria. Human flow cytometry and qRT-PCR showed a significant increase in interleukin (IL)-4 gene expression, indicating a TH 2 phenotype. Murine IF revealed a dense CD4?+?T-cell inflammatory infiltrate on day 4 to 7 postinjury, which preceded the development of fibrosis. Murine flow cytometry and qRT-PCR studies mirrored the human ones, with increased T-helper cells and IL-4 in iLTS versus normal controls. CONCLUSION:CD3/CD4?+?T-helper lymphocytes and the proinflammatory cytokine IL-4 are associated with iLTS. The association of a TH 2 immunophenotype with iLTS is consistent with findings in other fibroinflammatory disorders. The murine results reveal that the inflammatory infiltrate precedes the development of fibrosis. However, human iLTS specimens with well-developed fibrosis also contain a marked chronic inflammatory infiltrate, suggesting that the continued release of IL-4 by T-helper lymphocytes may continue to propagate iLTS. LEVEL OF EVIDENCE:NA Laryngoscope, 129:177-186, 2019.
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