Osteoarthritis (OA) is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA. These attempts involve repolarizing pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype in synovium. In this study, we found that phosphoglycerate mutase 5 (PGAM5) significantly increased in macrophages in OA synovium compared to controls based on histology of human samples and single-cell RNA sequencing results of mice models. To address the role of PGAM5 in macrophages in OA, we found conditional knockout of PGAM5 in macrophages greatly alleviated OA symptoms and promoted anabolic metabolism of chondrocytes in vitro and in vivo. Mechanistically, we found that PGAM5 enhanced M1 polarization via AKT-mTOR/p38/ERK pathways, whereas inhibited M2 polarization via STAT6-PPARγ pathway in murine bone marrow-derived macrophages. Furthermore, we found that PGAM5 directly dephosphorylated Dishevelled Segment Polarity Protein 2 (DVL2) which resulted in the inhibition of β-catenin and repolarization of M2 macrophages into M1 macrophages. Conditional knockout of both PGAM5 and β-catenin in macrophages significantly exacerbated osteoarthritis compared to PGAM5-deficient mice. Motivated by these findings, we successfully designed mannose modified fluoropolymers combined with siPGAM5 to inhibit PGAM5 specifically in synovial macrophages via intra-articular injection, which possessed desired targeting abilities of synovial macrophages and greatly attenuated murine osteoarthritis. Collectively, these findings defined a key role for PGAM5 in orchestrating macrophage polarization and provides insights into novel macrophage-targeted strategy for treating OA.

Super-enhancers (SEs) govern macrophage polarization and function. However, the mechanism underlying the signal-dependent latent SEs remodeling in macrophages remains largely undefined. Here we show that the epigenetic reader ZMYND8 forms liquid compartments with NF-κB/p65 to silence latent SEs and restrict macrophage-mediated inflammation. Mechanistically, the fusion of ZMYND8 and p65 liquid condensates is reinforced by signal-induced acetylation of p65. Then acetylated p65 guides the ZMYND8 redistribution onto latent SEs de novo generated in polarized macrophages, and consequently, recruit LSD1 to decommission latent SEs. The liquidity characteristic of ZMYND8 is critical for its regulatory effect since mutations coagulating ZMYND8 into solid compartments disable the translocation of ZMYND8 and its suppressive function. Thereby, ZMYND8 serves as a molecular rheostat to switch off latent SEs and control the magnitude of the immune response. Meanwhile, we propose a phase separation model by which the latent SEs are fine-tuned in a spatiotemporal manner.

Pathogenic CARD11 mutations cause aberrant nuclear factor κB (NF-κB) activation, which is presumably responsible for multiple immunological disorders. However, whether there is an NF-κB–independent regulatory mechanism contributing to CARD11 mutations related to pathogenesis remains undefined. Using three distinct genetic mouse models, the Card11 knockout (KO) mouse model mimicking primary immunodeficiency, the CARD11 E134G point mutation mouse model representing BENTA (B cell expansion with NF-κB and T cell anergy) disease, and the mouse model bearing oncogenic K215M mutation, we show that CARD11 has a noncanonical function as a negative regulator of the AKT-FOXO1 signal axis, independent of NF-κB activation. Although BENTA disease–related E134G mutant elevates NF-κB activation, we find that E134G mutant mice phenotypically copy Card11 KO mice, in which NF-κB activation is disrupted. Mechanistically, the E134G mutant causes exacerbated AKT activation and reduced FOXO1 protein in B cells similar to that in Card11 KO cells. Moreover, the oncogenic CARD11 mutant K215M reinforces the importance of the noncanonical function of CARD11. In contrast to the E134G mutant, K215M shows a stronger inhibitory effect on AKT activation and more stabilized FOXO1. Likewise, E134G and K215M mutants have converse impacts on B cell development and differentiation. Our results demonstrate that, besides NF-κB, CARD11 also governs the AKT/FOXO1 signaling pathway in B cells. The critical role of CARD11 is further revealed by the effects of pathogenic CARD11 mutants on this noncanonical regulatory function on the AKT-FOXO1 signaling axis.

Introduction: CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg).Method: In this study, we used patients’ samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner.Results and discuss: We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Autoimmune central nervous system (CNS) demyelinating diseases are a major public health burden and poorly controlled by current immunosuppressants. More precise immunotherapies with higher efficacy and fewer side effects are sought. We investigated the effectiveness and mechanism of an injectable myelin-based antigenic polyprotein MMPt (myelin oligodendrocyte glycoprotein, myelin basic protein and proteolipid protein, truncated). We find that it suppresses mouse experimental autoimmune encephalomyelitis without major side effects. MMPt induces rapid apoptosis of the encephalitogenic T cells and suppresses inflammation in the affected CNS. Intravital microscopy shows that MMPt is taken up by perivascular F4/80 ⁺ cells but not conventional antigen-presenting dendritic cells, B cells, or microglia. MMPt-stimulated F4/80 ⁺ cells induce reactive T cell immobilization and apoptosis in situ, resulting in reduced infiltration of inflammatory cells and chemokine production. Our study reveals alternative mechanisms that explain how cognate antigen suppresses CNS inflammation and may be applicable for effectively and safely treating demyelinating diseases.