Original Basic Science-GeneralBackground. There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time-sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods. Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 h and 10 d after transplantation, and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using antihuman CD63 antibody quantum dot on the nanoparticle detector and via GFP trigger on the nanoparticle flow cytometer. Results. In syngeneic controls, steady-state levels of circulating donor exosomes were detected at all posttransplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Conclusions. Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.
Objective
Chronic lung allograft dysfunction (CLAD) limits the survival after lung transplantation (Tx). CLAD is characterized by progressive fibrosis of small airways and lung parenchyma. No effective therapy is available that reverses or prevents CLAD. CD26 is a molecule with enzymatic activity also playing a key role in the progression of fibrotic diseases. Here, we analyzed the inhibitory effect of CD26 on fibroblast activity in vitro and the role of CD26-inhibition on allograft rejection in lung transplant patients.
Methods
Profibrogenic mRNA and protein levels were analyzed in vitro on the CD26-expressing fibroblast cell line Wi-38 using RT-qPCR and Western blot. CD26 was inhibited by Vildagliptin. Migration and proliferation activity of activated fibroblasts were analyzed by Incucyte® and Celltiter-Glo®. Characteristics of patients undergoing lung Tx between 2004 and 2021 were reviewed. Lung biopsies were analyzed by immunohistochemistry (IHC) for CD26.
Results
In vitro, the expression of profibrogenic genes (αSMA, FAPα, IGFBP7, Collagen 3 and Fibronectin) was significantly reduced in activated lung fibroblasts by Vildagliptin treatment. Also, migration and proliferation activity were attenuated by Vildagliptin. In 221 patients analyzed, CLAD was absent in 34 patients treated with the CD26-inhibitor Sitagliptin vs. an incidence of 18% in patients without Sitagliptin intake (p=0.02). Five-year survival in patients on Sitagliptin was significantly improved vs. patients without CD26-inhibitor intake (80% vs. 58%, p=0.006). Likewise, the incidence of acute cellular rejection (ACR) was significantly reduced in patients on Sitagliptin (7% vs. 35%, p=0.01). IHC of patient lung biopsies showed expression of CD26 in perifibrotic areas of CLAD lesions. Additional clinical data from University Hospital Zurich and from University Hospital Padua confirmed the finding that Sitagliptin intake correlated with the absence of acute and chronic allograft rejection.
Conclusion
CD26-inhibition attenuates key pro-fibrotic mediators and fibroblast activity in vitro. Impressively, patients on CD26-inhibitor did not show any CLAD. Moreover, ACR was significantly reduced. Gliptins which are in routine clinical use for the treatment of type II diabetes therefore seem to have great potential to be repurposed for a novel clinical application against lung allograft rejection.
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