Dissecting immunoproteasome function in lung regeneration using mouse lung organoid

Immunoproteasomes (IP) are specialized proteasomes that play critical roles in multiple immune responses. Their three catalytic subunits (PSMB8, PSMB9, PSMB10) are expressed at low levels in non-immune cells but are induced when cells encounter virus infection or stimulation with interferons (IFNs) or inflammatory cytokines. It has recently been shown that type I IFN disrupts repair of influenza virus damaged lung epithelial cells. As induction of the IP is part of the canonical response to type I interferon signaling, we here hypothesize that IPs play a role during injury and regeneration of lung. Bioinformatic data revealed that IP is dynamically regulated during lung organoid formation peaking at D14 and reduced at D21. Increased IP genes was observed in cell clusters characterized as intermediates of AT1 differentiation. To validate these data, we established a mouse organoid system and investigated the role of IP for lung regeneration. Taking advantage of the controllable culture, we found that PSMB8, PSMB9 and the AT1 marker PDPN were downregulated under AT2-culture condition compared to freshly isolated AT2 cells. Promoted AT1 differentiation significantly upregulated expression of PSMB8, PSMB9 and PSMB10, as well as PDPN. Of note, inhibition of the IP using LU005i blocked differentiation of AT1 cells from AT2 organoids but did not affect AT2 differentiation suggesting a critical role for the IP in alveolar differentiation. In summary, using organoid culture system allowed detection of the dynamic regulation of IP gene expression following alveolar differentiation. The significance of IP expression for lung regeneration is currently evaluated in depth using IP depletion versus overexpression.