(109) Insights Into the Lung Microenvironment During Chronic Allograft Rejection: The Role of Bal-Evs in Rewiring Respiratory Cells Inflammatory Response

PurposeLittle is known about the pathomolecular signaling in the lung microenvironment that precedes and drives graft rejection, the major cause of graft failure and patients’ death. We previously investigated the immunological messages delivered by BAL-extracellular vesicles (BAL-EVs) in recipient human primary bronchial cells (HBECs; Fig.1A). Now, we analyzed the transcriptome and proteome of HBECs cocultured with BAL-EVs focusing on inflammatory molecules.MethodsBAL-EVs were isolated from patients with stable (CTRL), or chronic (CLAD) rejection and co-cultured with HBECs cells for 48h. Then, cytokine arrays with bronchial cell culture extracts were performed along with a transcriptomic analysis using the nCounter Human Organ Transplant Panel. Data were normalized against controls and expressed as log2. As cut-off for up- or down- modulation we used 0.5.ResultsAt the transcriptional level, HBECs cocultured with BAL-EVs from patients with CLAD show consistent repression of molecules involved in innate immunity and Type-1 interferon signaling whilst upregulating transcripts involved in immune checkpoint signaling and cell-ECM interaction (Fig.1B). Similarly, at the protein level CLAD-EVs cocultured cells showed decreased expression of macrophage chemoattractant molecules (CCL5, MIP-1 and TREM-1) and increased expression of inhibitory factors for macrophages migration (MIF) together with augmented levels of proteins involved in wound healing (Fig. 1C).ConclusionThese data together with previous ones regarding modulation of extracellular cytokines, suggest that bronchial cells activate a compensatory mechanism to dampen innate immune response and macrophages activation during the initial phase of CLAD. This is accompanied by activation of wound repair factors, potentially responsible of tissue fibrosis. This novel knowledge sheds light on the pathomolecular mechanisms behind CLAD. Little is known about the pathomolecular signaling in the lung microenvironment that precedes and drives graft rejection, the major cause of graft failure and patients’ death. We previously investigated the immunological messages delivered by BAL-extracellular vesicles (BAL-EVs) in recipient human primary bronchial cells (HBECs; Fig.1A). Now, we analyzed the transcriptome and proteome of HBECs cocultured with BAL-EVs focusing on inflammatory molecules. BAL-EVs were isolated from patients with stable (CTRL), or chronic (CLAD) rejection and co-cultured with HBECs cells for 48h. Then, cytokine arrays with bronchial cell culture extracts were performed along with a transcriptomic analysis using the nCounter Human Organ Transplant Panel. Data were normalized against controls and expressed as log2. As cut-off for up- or down- modulation we used 0.5. At the transcriptional level, HBECs cocultured with BAL-EVs from patients with CLAD show consistent repression of molecules involved in innate immunity and Type-1 interferon signaling whilst upregulating transcripts involved in immune checkpoint signaling and cell-ECM interaction (Fig.1B). Similarly, at the protein level CLAD-EVs cocultured cells showed decreased expression of macrophage chemoattractant molecules (CCL5, MIP-1 and TREM-1) and increased expression of inhibitory factors for macrophages migration (MIF) together with augmented levels of proteins involved in wound healing (Fig. 1C). These data together with previous ones regarding modulation of extracellular cytokines, suggest that bronchial cells activate a compensatory mechanism to dampen innate immune response and macrophages activation during the initial phase of CLAD. This is accompanied by activation of wound repair factors, potentially responsible of tissue fibrosis. This novel knowledge sheds light on the pathomolecular mechanisms behind CLAD.