Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages

Abstract

The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes involved in shaping adaptive immune responses, but their role in innate immune signaling is still elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages, highly specialized tissue macrophages of the alveolar lung surface. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits, low molecular mass protein 2 (LMP2), LMP7 and multicatalytic endopeptidase complex-like 1, which was accompanied by increased immunoproteasome activity in M1 cells. Deficiency of LMP7 had no effect on the LPS/IFNγ-triggered M1 profile indicating that immunoproteasome function is dispensable for classical alveolar macrophage activation. In contrast, IL-4 triggered alternative (M2) activation of primary alveolar macrophages was accompanied by a transcriptionally independent amplified expression of LMP2 and LMP7 and an increase in immunoproteasome activity. Alveolar macrophages from LMP7 knockout mice disclosed a distorted M2 profile upon IL-4 stimulation as characterized by increased M2 marker gene expression and CCL17 cytokine release. Comparative transcriptome analysis revealed enrichment of IL-4-responsive genes and of genes involved in cellular response to defense, wounding and inflammation in LMP7-deficient alveolar macrophages indicating a distinct M2 inflammation resolving phenotype. Moreover, augmented M2 polarization was accompanied by amplified AKT/STAT6 activation and increased RNA and protein expression of the M2 master transcription factor interferon regulatory factor 4 in LMP7 − / − alveolar macrophages. IL-13 stimulation of LMP7-deficient macrophages induced a similar M2-skewed profile indicative for augmented signaling via the IL-4 receptor α (IL4Rα). IL4Rα expression was generally elevated only on protein but not RNA level in LMP7 − / − alveolar macrophages. Importantly, specific catalytic inhibition with an LMP7-specific proteasome inhibitor confirmed augmented IL-4-mediated M2 polarization of alveolar macrophages. Our results thus suggest a novel role of immunoproteasome function for regulating alternative activation of macrophages by limiting IL4Rα expression and signaling. The immunoproteasome is a specialized type of proteasome and constitutively active in immune cells, whereas standard proteasomes are expressed in all non-immune cells. Similar to the standard 20S proteasome, immunoproteasomes are composed of two outer α-rings and two inner β-rings of seven subunits each 1 but contain a different set of IFNγ-inducible

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy

“…They showed that inhibition of LMP7 increased M2 alternative activated macrophages by increasing their responsiveness to IL-4, while classical M1 macrophages were relatively unaltered. 49 These findings could open interesting perspectives in DMD while an important macrophagic component is always present within muscle fibers. Furthermore, a shift in macrophages polarization M1-M2 is observed from acute phase of 4-weeks to chronic stage, 12 weeks of age, where M2 macrophages are supposed to promote muscle repair by enhancing satellite cells proliferation.…”

Macrophages: Their role, activation and polarization in pulmonary diseases

“…Thus, M1phenotype prompts all the three subunits of immunoproteasome. On the other hand, alternatively activated M2 macrophages increases immunoproteasome activity by aggravating expression of LMP2 and LMP7 subunits only (Chen et al, 2016).…”

Proteasome function shapes innate and adaptive immune responses

“…These cells express high levels of immunoproteasomes. While the proinflammatory IFN-␥/LPS-induced M1 phenotype was not changed in LMP7-deficient primary macrophages, IL-4 treatment of wild-type or LMP7-deficient cells resulted in augmented M2 polarization marker gene expression, increased M2 signaling (via STAT6 and AKT), and an increase in IL-4 receptor-␣ expression already at baseline (23). Catalytic inhibition of LMP7 with the immunoproteasome-specific inhibitor ONX-0914 led to similar results.…”

“…Special interest and conflicting data exist on the role of immunoproteasomes in NF-B signaling, which might reflect cell type-specific effects or the outcome of different experimental settings (44,49,72). Our own and partially unpublished data indicate that NF-B signaling is not affected by deletion of the immunoproteasome subunits LMP2 or LMP7: NF-B promotor-driven reporter gene as well as NF-B target gene expression were unchanged in alveolar macrophages of LMP2-deficient mice after LPS and IFN-␥induced macrophage polarization (23). Several mutations in the human PSMB8 and PSMB9 genes encoding the LMP7 and LMP2 immunoproteasome subunits, respectively, have been discovered in autoinflammatory disorders (1,4,17,62,70,74).…”