3D Bioprinting of Pig Macrophages and Human Cells Discovered the P2Y14 Receptor as a Mediator of Xenogenic Immune Responses.

Background: The survival rate of pig lung xenotransplantation (PLXTx) recipients is severely limited by intense xenogenic immune responses, necessitating further insights into xenogeneic immunity and the development of models to study the PLXTx immune response.

Methods: We identified regulators of PLXTx immune response Using Gene ontology analysis. We assessed the metabolic changes and protein levels in 3D4/31 pig alveolar macrophages (PAMs) through flow cytometry and immunoblotting. To induce a xenogenic immune response, we co-cultured 3D4/31-PAMs with A549 human alveolar epithelial cells and evaluated cytokine expression using qRT-PCR.

Results: Gene ontology analysis identified STAT1 and alveolar macrophages as contributors to lung autoimmunity and transplant rejection. In 3D4/31-PAMs, phorbol myristate acetate-induced glycogen accumulation and cyclooxygenase-2 expression were inhibited by the P2Y₁₄ inhibitor PPTN. Co-culturing 3D4/31-PAMs with A549 human alveolar epithelial cells via 3D bioprinting resulted in a more pronounced inflammatory response than 2D co-culture, with increased expression of genes related to the P2Y₁₄ cascade and inflammation. This inflammatory gene expression was prevented by PPTN treatment.

Conclusion: Based on these results, we propose alginate bioprinting as an in vitro model for PLXTx and suggest that P2Y₁₄ is a key regulator of xenogeneic immune responses in PAMs.