MicroRNA-206 antagomiR‒enriched extracellular vesicles attenuate lung ischemia‒reperfusion injury through CXCL1 regulation in alveolar epithelial cells

Abstract

Background: Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury.

Methods: The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs.

Results: A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models.

Conclusions: EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury.

Keywords: extracellular vesicles; ischemia‒reperfusion injury; lung transplantation; microRNA; stem cells.

Figure 1.

Identification of miRNAs involved in lung IR injury. A. Increased expression of miR-206 in BAL fluid of patients on day 1 post-lung transplantation compared to day 0 was observed. n=4-5/group; *p=0.007. B. Total RNA obtained from lung tissue of WT mice from the hilar ligation model of IR injury was analyzed by miRNA microarray. Hierarchical cluster analysis of lung IR injury compared to sham is shown by heat map for the highly regulated miRNAs. Each column represents the respective sample, and each row represents a single miR probe. C. The most significant increase was observed in miR-206 expression in the lung tissue after IR compared to sham. n=5/group; ^#p=0.006.

Figure 2.

Correlative analysis of molecular processses associated with altered profile of miRNAs after lung IR injury. A. Biological interpretation of diseases and disorders after Ingenuity Pathway Analysis depicting cellular processes associated with lung IR injury. Threshold value (orange line) indicate -log for a p-value of 0.05. B. List of common miRNAs that are altered after lung IR injury and associated with relevant diseases and disorders are depicted.

Figure 3.

EVs enriched with antagomiR-206 effectively attenuate pulmonary dysfunction after IR injury. A. Schematic of murine lung IR protocol where pulmonary function and injury were measured in WT mice after sham surgery or IR injury. B-D. Significant lung dysfunction occurred after IR as demonstrated by increased airway resistance and pulmonary artery (PA) pressure as well as decreased pulmonary compliance compared to sham controls. Pretreatment with EVs+miR-206 inhibitor (Inh.; antagomiR-206) resulted in significantly reduced lung dysfunction after IR compared to EVs or EVs+miR-206 mimic. n=10/group; *p<0.05 vs. sham; ^#p<0.05 vs. IR, ^§p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Figure 4.

EVs enriched with miR-206 antagomiR decrease neutrophil infiltration and activation as well as lung edema and microvascular permeability after IR injury. A. Representative images showing neutrophil immunostaining in lung sections. Neutrophils are stained red and sections are counterstained with hematoxylin. Scale bars indicate 50pm. B. The number of neutrophils per high power field (HPF) was quantified from immunostained sections. Neutrophil infiltration was significantly attenuated after IR in mice treated with EVs+miR-206 inhibitor (antagomiR-206) compared to EVs or EVs+miR-206 mimic treated mice. C. Myeloperoxidase (MPO) levels in BAL fluid were significantly decreased after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. D. Pulmonary edema (lung wet/dry weight) was significantly decreased after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. E. Pulmonary vascular permeability was significantly elevated in lungs after IR injury, which was attenuated by treatment with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. n=5-8/group. *p<0.05 vs. sham; ^#p<0.05 vs. IR, ^§p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Figure 5.

Lung inflammation after IR is attenuated by antagomiR-206 enriched EVs. A. Proinflammatory cytokine levels (IL-17, TNF-α, HMGB1, MCP-1, IL-6, MIP-1α and RANTES) were significantly attenuated in BAL fluid after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. B-C. Expression of CXCL1 was significantly decreased, and prostaglandin E2 (PGE2) was significantly increased, after treatment with EVs+miR-206 mimic. EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic alone. n=5/group; *p<0.05 vs. sham; ^#p<0.05 vs. IR, ^§p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Figure 6.

EVs enriched with antagomir-206 reduce lung allograft IR injury and inflammation in recipients of DCD lungs. A. Balb/c donor mice were treated with EVs enriched with antagomir-206 (EV-miR-206) or normal saline (NS) immediately upon DCD induction and immediately prior to transplantation (Tx). B. Examination of treated DCD lungs following cold storage but prior to transplantation demonstrated that no significant differences could be seen between treated and untreated lungs. C. Representative histological images 6hrs and 24hrs post transplantation demonstrate decreased diffuse red blood cell alveolar accumulation, neutrophil infiltration, and fibrin deposition in EV-miR206 treated mice compared to untreated controls. n=4-7/group. Scale bars as marked. D. Recipients receiving DCD lungs from EV-miR206 treated donor mice had a significant reduction in IR injury as determined by semi-quantitative histological assessment of injury at both 6hrs and 24hrs, as compared to controls. ^#p=0.006, ^##p=0.002. E. Neutrophil numbers were determined by image analysis of computerized randomly generated high-power fields. Transplanted lungs from DCD donors showed marked diffuse neutrophil infiltration and a significant decrease in neutrophil numbers in EV-miR206 treated recipients as compared to NS controls. ^#p=0.006, ^##p=0.001. F. Arterial blood partial pressure of oxygen (PaO₂) was not significantly different between EV+miR-206 inhibitor treated and control mice at 6 hrs, but PaO₂ was significantly improved in EV-miR206 treated lungs after 24 hrs post-transplantation; ^##p=0.01. G. Analysis of BAL at 24 hrs post-transplantation demonstrated that DCD treatment with EV-miR206 significantly reduced lung edema as demonstrated by albumin levels, and significantly reduced pro-inflammatory cytokine expression; ^##p<0.001.

Figure 6.

EVs enriched with antagomir-206 reduce lung allograft IR injury and inflammation in recipients of DCD lungs. A. Balb/c donor mice were treated with EVs enriched with antagomir-206 (EV-miR-206) or normal saline (NS) immediately upon DCD induction and immediately prior to transplantation (Tx). B. Examination of treated DCD lungs following cold storage but prior to transplantation demonstrated that no significant differences could be seen between treated and untreated lungs. C. Representative histological images 6hrs and 24hrs post transplantation demonstrate decreased diffuse red blood cell alveolar accumulation, neutrophil infiltration, and fibrin deposition in EV-miR206 treated mice compared to untreated controls. n=4-7/group. Scale bars as marked. D. Recipients receiving DCD lungs from EV-miR206 treated donor mice had a significant reduction in IR injury as determined by semi-quantitative histological assessment of injury at both 6hrs and 24hrs, as compared to controls. ^#p=0.006, ^##p=0.002. E. Neutrophil numbers were determined by image analysis of computerized randomly generated high-power fields. Transplanted lungs from DCD donors showed marked diffuse neutrophil infiltration and a significant decrease in neutrophil numbers in EV-miR206 treated recipients as compared to NS controls. ^#p=0.006, ^##p=0.001. F. Arterial blood partial pressure of oxygen (PaO₂) was not significantly different between EV+miR-206 inhibitor treated and control mice at 6 hrs, but PaO₂ was significantly improved in EV-miR206 treated lungs after 24 hrs post-transplantation; ^##p=0.01. G. Analysis of BAL at 24 hrs post-transplantation demonstrated that DCD treatment with EV-miR206 significantly reduced lung edema as demonstrated by albumin levels, and significantly reduced pro-inflammatory cytokine expression; ^##p<0.001.

Figure 7.

EVs enriched with antagomiR-206 inhibit epithelial cell activation after HR. A. HR-exposed iNKT cells produced increased levels of IL-17 compared to normoxia (Norm), and was significantly attenuated by treatment with EVs or EVs+miR-206 mimic. No additional protection was offered by treatment with EVs+miR-206 inhibitor. B. MH-S (alveolar macrophages; AMs) cells were exposed to HR and demonstrated a significant increase in TNF-α production compared to normoxia which was significantly attenuated by treatment with EVs or EVs+mimic miR-206. EVs+miR-206 inhibitor did not offer additional attenuation of TNF-α levels. C. HR-exposed MLE12 (alveolar epithelial; ATM) cells increased the CXCL1 secretion that was significantly attenuated by co-cultures with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. D. Schematic depicting the actions of antagomiR-206 enriched EVs on the lung microenvironment following IR injury leading to decreased ATM cell-secreted CXCL1 and subsequent neutrophil (PMN) infiltration. n=8/group; *p<0.05 vs. sham; ^#p<0.05 vs. IR, ^§p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Figure 7.

EVs enriched with antagomiR-206 inhibit epithelial cell activation after HR. A. HR-exposed iNKT cells produced increased levels of IL-17 compared to normoxia (Norm), and was significantly attenuated by treatment with EVs or EVs+miR-206 mimic. No additional protection was offered by treatment with EVs+miR-206 inhibitor. B. MH-S (alveolar macrophages; AMs) cells were exposed to HR and demonstrated a significant increase in TNF-α production compared to normoxia which was significantly attenuated by treatment with EVs or EVs+mimic miR-206. EVs+miR-206 inhibitor did not offer additional attenuation of TNF-α levels. C. HR-exposed MLE12 (alveolar epithelial; ATM) cells increased the CXCL1 secretion that was significantly attenuated by co-cultures with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. D. Schematic depicting the actions of antagomiR-206 enriched EVs on the lung microenvironment following IR injury leading to decreased ATM cell-secreted CXCL1 and subsequent neutrophil (PMN) infiltration. n=8/group; *p<0.05 vs. sham; ^#p<0.05 vs. IR, ^§p<0.05 vs. IR+EVs or EVs+miR-206 mimic.