Interferons and Toxoplasma gondii shape PD-L1 regulation in retinal barrier cells: The critical role of proteases

Benjamin GEILLER¹Camila Cevallos¹Iuliia Tsybenko¹Lydia Arnoux¹Marie-Paule Felder-Schmittbuhl²Alexander W Pfaff^1,3*

• ¹UR 3073 Pathogens Host Arthropod Vectors Interface, Toxoplasmosis Immunology Team, Université de Strasbourg, Strasbourg, France
• ²Centre National de la Recherche Scientifique, Institut des Neurosciences Cellulaires et Intégratives (UPR 3212), Université de Strasbourg, Strasbourg, France
• ³Service de Parasitologie et Mycologie Médicale, Hôpitaux Universitaires de Strasbourg, Strasbourg, Alsace, France

The apicomplexan parasite Toxoplasma gondii establishes chronic infection in the central nervous system, including the retina, causing ocular toxoplasmosis (OT). This persistence relies on a fine balance between inflammatory and immunomodulatory mechanisms, especially in the immune-privileged ocular environment. We previously described the immunologic interactions between retinal cells, and particularly the roles of type I and III interferons. In this study, we investigated the regulatory dynamics of PD-L1, a central immunomodulatory receptor on immune cells.We first investigated the mechanisms of PD-L1 regulation and the roles of type I and III interferons in an in vitro T. gondii infection model using mono-and co-culture systems of human microglia, astrocytes, and Müller cells. We also assessed PD-L1 expression in an outer blood-retina barrier model (oBRB) of differentiated retinal pigmented epithelial (RPE) cells.Additionally, we looked at retinal cell activation, PD-L1 expression and the roles of these interferons in a mouse model of OT.Our findings reveal new roles for type I and III interferons in regulating glial cell activation and PD-L1 expression in RPE, Müller, astrocytes and microglial cells. Notably, Müller cells, the most abundant glial cells in the retina, showed the highest baseline PD-L1 expression at both the mRNA and protein levels, and responded robustly to interferon stimulation. This points to a more prominent immunoregulatory role for Müller cells in the retina than previously recognized. Furthermore, we identified a parasite protease-dependent mechanism that reduces PD-L1 expression in our in vitro oBRB model potentially contributing to immune evasion and inflammation during OT. Finally, in a murine model of OT, we demonstrated that PD-L1 expression reached its peak on day 7 post-infection and that interferon neutralization plays a crucial role in regulating both PD-L1 expression and glial activation. The parasite T. gondii orchestrates the IFN type I and III dependent retinal immune interaction and downregulates PD-L1 in the oBRB by a protease-dependent mechanism, potentially contributing to immune evasion and inflammation in retinal infection. Our results can pave the way to fully elucidate retinal immune networks and PD-L1 regulation mechanisms, offering potential targets for therapeutic interventions in OT and other retinal inflammatory diseases.