An Acetylation Switch of the NLRP3 Inflammasome Regulates Aging-Associated Chronic Inflammation and Insulin Resistance

Abstract

It is well documented that the rate of aging can be slowed, but it remains unclear to which extent aging-associated conditions can be reversed. How the interface of immunity and metabolism impinges upon the diabetes pandemic is largely unknown. Here, we show that NLRP3, a pattern recognition receptor, is modified by acetylation in macrophages and is deacetylated by SIRT2, an NAD⁺-dependent deacetylase and a metabolic sensor. We have developed a cell-based system that models aging-associated inflammation, a defined co-culture system that simulates the effects of inflammatory milieu on insulin resistance in metabolic tissues during aging, and aging mouse models; and demonstrate that SIRT2 and NLRP3 deacetylation prevent, and can be targeted to reverse, aging-associated inflammation and insulin resistance. These results establish the dysregulation of the acetylation switch of the NLRP3 inflammasome as an origin of aging-associated chronic inflammation and highlight the reversibility of aging-associated chronic inflammation and insulin resistance.

Keywords: NLRP3; SIRT1; SIRT2; SIRT3; SIRT4; SIRT5; SIRT6; SIRT7; caspase 1; inflammasome.

Figure 1.. NLRP3 is modified by acetylation in macrophages and is deacetylated by SIRT2.

(A) NLRP3-Flag was immunopurified from NG5 cells treated with control or SIRT2 siRNA and analyzed by mass spectrometry. Acetylation sites are marked with *. (B) Domain structure of NLRP3. Acetylation sites are marked with *. (C) NLRP3-Flag was immunopurified from NG5 cells followed by Western analyses. NLRP3 KO cells were used as a negative control. (D) NLRP3-Flag was immunopurified from NG5 cells treated with SIRT2 or control siRNA followed by Western analyses. (E) The acetylation sites on NLRP3 are conserved across species. Sequence alignment of NLRP3 from various mammalian species is shown. Acetylated lysine residues are labeled with *. (F, G) Bone marrow derived macrophages (BMDM) isolated from WT or SIRT2 KO mice were primed with LPS and then stimulated with nigericin or ATP (NLRP3 inducers), flagellin (NLRC4 inducer), or dsDNA (AIM2 inducer). Cell lysates were used for Western analyses for pro IL-1β and pro caspase 1 and culture supernatants were used for p17 IL-1β and p20 caspase 1 Western analyses (F). IL-1β in culture supernatants was quantified using ELISA (G). Error bars represent SE. * p<0.05. ns: p>0.05. Student’s t test. See also Figure S1.

Figure 2.. Acetylation of NLRP3 enhances the NLRP3 inflammasome assembly and activity.

(A) NG5 cells were primed with LPS and treated with ATP or dsDNA. NLRP3-Flag was immunopurified followed by Western analyses. (B, C) NLRP3 KO macrophages were reconstituted with NLRP3 mutants and WT NLRP3 control by retroviral transduction and were stimulated with LPS and ATP. Cell lysates were used for Western analyses for pro IL-1β. Culture supernatants were used for p17 IL-1β Western analyses. n.s., non-specific band. (D) Position of K21, K22 and K24 residues in NLRP3 PYD domain structure (PDB code: 2NAQ). (E, F) K21 and K22 are involved in Type I interactions in modeled NLRP3-NLRP3 interaction (E) and NLRP3-ASC interaction (F). (G) Sequence alignment of the PYD domain of NLRP3 and ASC indicates conserved charged residues (green box) and less conserved hydrophobic residues (orange box) in Type I interface. See also Figure S2 and Table S1.

Figure 3.. Young SIRT2 KO mice fed a chow diet are metabolically normal.

Young WT and SIRT2 KO mice fed a chow diet for 6 months were compared. (A) Tissue weight. (B) Plasma IL-18. (C) Plasma glucose. (D, E) Glucose tolerance test. Error bars represent SE. n.s.: p>0.05. Student’s t test. See also Figure S3.

Figure 4.. SIRT2 prevents aging-associated chronic inflammation and insulin resistance.

2-year-old WT and SIRT2 KO mice fed a chow diet were compared. (A) Plasma glucose. (B) Plasma insulin. (C) Glucose tolerance test. (D) Area under the curve for (C). (E) Western blots of AKT and phosphorylated AKT in the livers and muscles after the mice were infused with insulin. (F) Plasma IL-18. (G) Glucose tolerance test for aged WT, SIRT2 KO (T2 KO), caspase 1 KO (C1 KO), and SIRT2/caspase 1 double KO mice (DKO). *: comparing WT and SIRT2 KO. #: comparing WT and Caspase 1 KO. &: comparing SIRT2 KO and DKO. (H) Area under the curve for (G). (I) Body Weight for mice used in (G). Error bars represent SE. *, #: p<0.05. **, ##, &&: p<0.01. ***, &&&: p<0.001. n.s.: p>0.05. Student’s t test. See also Figure S4.

Figure 5.. A cell-based system that models aging-associated inflammation.

(A) SIRT2 mRNA levels in bone marrow derived macrophages (BMDMs) isolated from young and old mice were quantified by qPCR. (B, C) BMDMs were isolated from young and old mice (B). Myeloid progenitors were isolated from bone marrow of young and old mice, immortalized with ER-Hoxb8, and differentiated into macrophages (C). Macrophages were primed with LPS, and stimulated with nigericin, ATP, flagellin, or dsDNA. Cell lysates were used for Western analyses for pro IL-1β and pro caspase 1. Culture supernatants were used for p17 IL-1β and p20 caspase 1 Western analyses. Error bars represent SE. ***: p<0.001. Student’s t test.

Figure 6.. SIRT2 and NLRP3 deacetylation reverse aging-associated inflammation and insulin resistance.

(A) Experimental design. (B, C) Immortalized myeloid progenitors from old WT mice were transduced with control or SIRT2 lentivirus (B), WT or constitutively deacetylated mutant NLRP3 virus (C), selected, differentiated into macrophages, and treated for inflammasome activation. Cell lysates were used for Western analyses for pro IL-1β and pro caspase 1. Culture supernatants were used for p17 IL-1β and p20 caspase 1 Western analyses. (D, E, F) Macrophages derived from immortalized myeloid progenitors from young or old mice with specified transduction were co-cultured with a piece of intact white adipose tissue from young or old mice as indicated, stimulated for NLRP3 inflammasome activation, followed by insulin signaling activation. The insulin signaling in the white adipose tissues was assessed by Western analyses.

Figure 7.. NLRP3 Deacetylation Improves Aging-associated Glucose Homeostasis in vivo.

Hematopoietic stem cells from aged WT mice were transduced with WT or K21/22/24R mutant NLRP3 and transplanted into lethally irradiated aged WT mice to reconstitute their hematopoietic system. Mice were analyzed 6 weeks post-transplantation for glucose tolerance tests (A, B), plasma IL-18 (C), and body weight (D). Error bars represent SE. *: p<0.05. n.s.: p>0.05. Student’s t test.