IL-10 Mediated Immunomodulation Limits Subepithelial Fibrosis and Repairs Airway Epithelium in Rejecting Airway Allografts

Abstract

Interleukin-10 plays a vital role in maintaining peripheral immunotolerance and favors a regulatory immune milieu through the suppression of T effector cells. Inflammation-induced microvascular loss has been associated with airway epithelial injury, which is a key pathological source of graft malfunctioning and subepithelial fibrosis in rejecting allografts. The regulatory immune phase maneuvers alloimmune inflammation through various regulatory modulators, and thereby promotes graft microvascular repair and suppresses the progression of fibrosis after transplantation. The present study was designed to investigate the therapeutic impact of IL-10 on immunotolerance, in particular, the reparative microenvironment, which negates airway epithelial injury, and fibrosis in a mouse model of airway graft rejection. Here, we depleted and reconstituted IL-10, and serially monitored the phase of immunotolerance, graft microvasculature, inflammatory cytokines, airway epithelium, and subepithelial collagen in rejecting airway transplants. We demonstrated that the IL-10 depletion suppresses FOXP3⁺ Tregs, tumor necrosis factor-inducible gene 6 protein (TSG-6), graft microvasculature, and establishes a pro-inflammatory phase, which augments airway epithelial injury and subepithelial collagen deposition while the IL-10 reconstitution facilitates FOXP3⁺ Tregs, TSG-6 deposition, graft microvasculature, and thereby favors airway epithelial repair and subepithelial collagen suppression. These findings establish a potential reparative modulation of IL-10-associated immunotolerance on microvascular, epithelial, and fibrotic remodeling, which could provide a vital therapeutic option to rescue rejecting transplants in clinical settings.

Keywords: TSG-6; immunotolerance; interleukin-10; subepithelial fibrosis.

Figure 1

IL-10 is sufficient to establish immunotolerance in rejecting allograft. Flow cytometry analysis of Tregs from peripheral blood of transplants at d10 post-transplantation: (A,B) Percentage of gated lymphocytes and semi-quantitative analysis of FOXP3⁺ T cells in a given gated lymphocyte population. (C,D) Semi-quantitative analysis and immunofluorescent staining of CD4⁺FOXP3⁺ co-expression at d10 post-transplantation. Yellow arrows highlight CD4⁺FOXP3⁺ Treg cells. Data are presented as means with SE of 16 transplants/time point/experiment. * p < 0.05. Original magnification, ×40.

Figure 2

IL-10 is sufficient to augment TSG-6 deposition. (A) Immunofluorescent staining and (B) Semiquantitative analysis for subepithelial deposition of TSG-6 in control and IL-10 treated allografts at d10 post-transplantation. Data are presented as means with SE of 16 transplants/time point/experiment. * p < 0.05. Original magnification, ×40.

Figure 3

IL-10 is sufficient to suppress pro-inflammatory cytokines. Quantitative analysis (A) to validate the IL-10 depletion (−) and IL-10 reconstitution (+); (B–H) serum cytokines in control and IL-10-treated allografts at d10 post-transplantation. Data are presented as means with SE of 8–12 transplants/time point/experiment. * p < 0.05.

Figure 4

IL-10 is sufficient to restore graft oxygenation and microvascular blood flow. (A) Tissue pO₂ (mean ± SE, mmHg) and (B) Blood perfusion units (mean ± SE, units) were plotted over different time points (d9–d28). (C) Lectin binding assay in control and IL-10-treated allografts at d9, d10, d12, d14, and d28 post-transplantation. Original magnification, ×20. Data are presented as means with SE of 16 transplants/time point/experiment. * p < 0.05.

Figure 5

IL-10 is sufficient to augment airway epithelial repair. (A,B) H&E staining of graft transverse sections and (C) Subepithelial infiltrating mononuclear cells in control and IL-10-treated allografts at d10 and d28 post-transplantation. ‘SE’ designates subepithelial areas in the graft section. Data are shown as means with SE 16 transplants/time point/experiment. * p < 0.05. Original magnification, ×40.

Figure 6

IL-10 is sufficient to suppress subepithelial fibrosis. (A,B) Collagen staining of graft transverse sections and (C) Semiquantitative analysis of subepithelial deposition of collagen in control and IL-10-treated allografts at d10 and d28 post-transplantation: Blue bands represent subepithelial collagen deposition, and semi-quantitative analysis of blue collagen bands was performed using the ImageJ program. ‘SE’ designates subepithelial areas in the graft section. Data are shown as means with SE 16 transplants/time point/experiment. * p < 0.05. Original magnification, ×40.