NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria

Abstract

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Figure 1. p62, induced on NF-κB activation, suppresses NLRP3-inflammasome dependent IL-1β production

(A) Immunoblot (IB) analysis of autophagy receptors in wild-type (p62^F/F) and p62^ΔMye BMDM stimulated with LPS (200 ng/ml). (B) Wild-type BMDM were incubated with or without LPS in the absence or presence of IKKβ inhibitor ML120b and expression of p62, pro-IL-1β, and NLRP3 was IB analyzed. (C) Survival of p62^F/F or p62^ΔMye mice injected with intraperitoneal (i.p.) LPS (30 mg/kg) without or with anakinra (50 mg/kg), n=11–13. (D, E) 12-weeks old p62^F/F or p62^ΔMye mice were i.p. injected with LPS and their sera collected 3 hrs later and analyzed by ELISA for TNF (D) and IL-1β (E). (F, H) Release of IL-1β (F) or TNF (H) from LPS-primed p62^F/F or p62^ΔMye BMDM that were stimulated with the indicated NLRP3 agonists. Data are averages ± s.d. (n=3). (G) IB analysis of caspase-1 and pro-IL-1β processing in LPS-primed WT (W) or p62-deficient (K) iBMDM stimulated as indicated. Data are representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2. NLRP3 agonists promote mitochondrial damage and p62 recruitment

(A) Intracellular distribution of p62 and mitochondria (Tom20) in LPS-primed BMDM stimulated with NLRP3 agonists examined by confocal microscopy (Scale bars, 5 μm) and (B) quantitated by counting cells with p62 aggregation on mitochondria. Averages ± s.d. represent the ratio of cells with mitochondrial p62 aggregates to total cells in each field. (n=6). (C) Subcellular distribution of p62, LC3II, and Parkin before and after NLRP3-inflammasome activation. Data are representative of three independent experiments. (D) NLRP3 agonist-induced changes in mitochondrial membrane potential (Ψm) in LPS-primed WT (shCtrl) or p62-deficient (shp62) iBMDM were measured by TMRM fluorescence. Data are averages ± s.d.(n=3). (E) Flow cytometric analysis of mitochondrial status in macrophages challenged with NLRP3 agonists. Gates represent cells with damaged mitochondria. (F, G) Electron micrographs of mitochondria in LPS-primed p62^F/F or p62^ΔMye BMDM after incubation with NLRP3 agonists. Shown are representative examples of normal, partially damaged and heavily damaged mitochondria. Scale bars: 500 nm. (G) quantification of damaged mitochondria in (F). Results are averages ± s.e.m. (n=8). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. Mitochondrial p62 recruitment is Parkin dependent

(A) Intracellular distribution of p62, polyubiquitin (poly-Ub) and mitochondria (Mitotracker) in LPS-primed WT BMDM stimulated with NLRP3 agonists determined by confocal microscopy. Scale bars: 5 μm. (B) Mitochondrial poly-Ub decoration examined by confocal microscopy in LPS-primed Park2^+/+ or Park2^−/− BMDM stimulated with NLRP3 agonists. Scale bars: 5 μm. (C) Mitochondrial recruitment of p62 in LPS-primed WT (shCtrl) or Parkin-deficient (shPark2) iBMDM stimulated with NLRP3 agonists. Scale bars: 5 μm. (D) Subcellular distribution of human p62 in LPS-primed, NLRP3 agonist stimulated p62-deficient BMDMs transduced with human p62-full length or p62(ΔUBA) constructs. Scale bars: 5 μm.

Figure 4. Parkin limits accumulation of damaged mitochondria after inflammasome activation

(A) Flow cytometry of mitochondrial status in WT (shCtrl) or Parkin-deficient (shPark2) iBMDM stimulated with NLRP3 agonists. Gates represent cells with damaged mitochondria. (B) NLRP3 agonist-induced changes in mitochondrial membrane potential (Ψm) in LPS-primed WT (shCtrl) or p62-deficient (shp62) iBMDM measured by TMRM fluorescence. Data are averages ± s.d. (n=3). (C) IL-1β release by LPS-primed Park2^+/+ or Park2^−/− BMDM stimulated with NLRP3 agonists. Data are averages ± s.d. (n=3). (D) Caspase-1 activity in LPS-primed WT (shCtrl) or Parkin-deficient (shPark2) iBMDM stimulated with NLRP3 agonists. Results are averages ± s.d. (n=3). (E) IB analysis of cleaved caspase-1 and IL-1β secretion to cell culture supernatants (Sup) or tubulin in cell lysates (Lys) in LPS-primed Park2^+/+ or Park2^−/− BMDM stimulated with NLRP3 agonists. Data are representative of 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 5. Autophagy mediates clearance of p62-bound mitochondria

(A, B) Intracellular distribution of LC3, p62, poly-Ub and mitochondria in LPS-primed LC3-GFP BMDM stimulated with NLRP3 agonists examined by confocal microscopy. (C) Flow cytometric analysis of mitochondrial status in LPS-primed WT (shCtrl) or Atg7-deficient (shAtg7) iBMDM after NLRP3 agonist stimulation. Gates – cells with damaged mitochondria. (D) NLRP3 agonist-induced changes in mitochondrial membrane potential (Ψm) in LPS-primed WT (shCtrl) or Atg7-deficient (shAtg7) iBMDM. Data are averages ± s.d. (n=3). (E) Intracellular distribution of p62 and mitochondria in LPS-primed shCtrl or shAtg7 iBMDM stimulated with NLRP3 agonists. (F) IB analysis of caspase-1 p20 and mature IL-1β in culture supernatants (Sup) and β-actin in cell lysates (Lys) of LPS-primed Atg7^F/F (WT) and Atg7^ΔMye (KO) BMDM stimulated as indicated. Data are representative of three independent experiments. (G) IL-1β secretion by above cells measured by ELISA. Results are averages ± s.d. (n=3). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6. Elimination of mitochondrial signals prevents excessive IL-1β production by p62-deficient macrophages

(A) Relative concentrations of mtDNA in p62^F/F and p62^ΔMye BMDM before and after 4 days of ethidium bromide (EtBr) treatment. Results are averages ± s.d. (n=3). (B–D) TNF (B) IL-1β (C) release and caspase-1 activity (D) in p62^F/F and p62^ΔMye BMDM pre-treated with EtBr or not that were co-stimulated with LPS and different NLRP3 agonists. Results are averages ± s.d. (n=3). (E) IB analysis of pro-IL-1β, NLRP3, ASC, and pro-caspase-1 in lysates of EtBr-pretreated WT BMDM before and after 4 hrs of LPS stimulation. Data are representative of three independent experiments. (F) Relative mtROS amounts determined by MitoSOX staining of LPS-primed WT BMDM stimulated with NLRP3 agonists in the presence of dTPP or Mito-Q. (G,H) Release of IL-1β (F) and TNF (G) from LPS-primed p62^F/F and p62^ΔMye BMDM; treated with Mito-Q or vehicle (dTPP) before addition of NLRP3 agonists. Results are averages ± s.d. (n=3).

Figure 7. p62 inhibits inflammasome-dependent sterile inflammation

(A) Peritoneal IL-1β in p62^F/F or p62^ΔMye mice 4 hrs after i.p. injection of alum or PBS. n=3–6. (B–D) Alum-induced peritoneal infiltration of PEC (B), monocytes (CD11b⁺Ly6C⁺Ly6G⁻) (C) and neutrophils (CD11b⁺Ly6G⁺F4/80⁻) (D) in p62^F/F and p62^ΔMye mice 16 hrs after alum or PBS injection. (E) Representative liver appearance and histology of p62^F/F or p62^ΔMye mice i.p. injected with LPS plus D-galactosamine. (F) Serum ALT in above mice. (G) Fluorescent staining of F4/80, active caspase-1, and DAPI in livers of p62^F/F and p62^ΔMye mice after LPS+ D-gal challenge. *, p<0.05; **, p<0.01; ***, p<0.001. (H) Schematic representation of key findings. NF-κB induces expression of p62, which negatively regulates caspase-1 activation via mitophagic elimination of mitochondria that release NLRP3-inflammasome activating signals.