Canonical Nlrp3 inflammasome links systemic low-grade inflammation to functional decline in aging

Abstract

Despite a wealth of clinical data showing an association between inflammation and degenerative disorders in the elderly, the immune sensors that causally link systemic inflammation to aging remain unclear. Here we detail a mechanism by which the Nlrp3 inflammasome controls systemic low-grade age-related "sterile" inflammation in both periphery and brain independently of the noncanonical caspase-11 inflammasome. Ablation of Nlrp3 inflammasome protected mice from age-related increases in the innate immune activation, alterations in CNS transcriptome, and astrogliosis. Consistent with the hypothesis that systemic low-grade inflammation promotes age-related degenerative changes, the deficient Nlrp3 inflammasome-mediated caspase-1 activity improved glycemic control and attenuated bone loss and thymic demise. Notably, IL-1 mediated only Nlrp3 inflammasome-dependent improvement in cognitive function and motor performance in aged mice. These studies reveal Nlrp3 inflammasome as an upstream target that controls age-related inflammation and offer an innovative therapeutic strategy to lower Nlrp3 activity to delay multiple age-related chronic diseases.

Figure 1. The Nlrp3 inflammasome controls metabolic inflammation and glucose intolerance in aging

(A) LPS primed WT and Nlrp3^−/− BMDMs were stimulated with multiple aging-relevant DAMPs and supernatants were analyzed for active IL-1β (p17) by immunoblotting. (B) Caspase-1 immunoblot analysis in epididymal adipose tissue of 2- and 15-month old WT, and 15-month old Nlrp3^−/− mice maintained on control chow diet. Age-related increase in caspase-1 activation (p20) subunit of caspase-1 is substantially reduced in absence of Nlrp3. (C) Real time PCR analysis of proinflammatory cytokines Il1b in liver and epididymal fat pad. (n = 4–6/age group/genotype). (D) Serum IL-18 and (E) IL-6 levels in 2-month and 23-month old WT, Nlrp3^−/− and Asc^−/−, Casp11^−/− mice (n = 6–8 mice per age group/strain). (F) Glucose tolerance test in 14- and 19- and (G) 23month old WT, Nlrp3^−/− mice maintained on control chow diet (n =7–9/age group/genotype). (H, I, J) Realtime PCR analysis of proinflamamtory mediators complement C3, Tnf and Il10 in epididymal adipose tissue and (K) glucose tolerance test in 20 month old WT and Il1r^−/− mice (n = 9/group). All data are presented as Mean (SEM)* P<0.05. See also Figure S1 and S2.

Figure 2. Ablation of Nlrp3 inflammasome reduces age-related thymic involution and effector T cell expansion in an IL-1 independent mechanism

(A) Thymi of 23mo old WT and Nlrp3 mice. The FFPE section of 23mo old Nlrp3^−/− mice display reduced ectopic lipid, maintenance of cortical-medullary junctions and lower thymic involution as measured by increased thymic mass and total thymocyte counts in Nlrp3 mutant mice. (B, C) FACS analysis depicting (CD4/CD8⁺CD62L⁺CD44⁻) and effector-memory E/M (CD4/CD8⁺CD62L⁻CD44⁺) T cells from 2-month (left), and 23-month (right hand) old WT and age-matched Asc^−/− and Casp11^−/− mice. (n = 6–8) are shown. (D) Immunoblot analyses and quantification of dock2 in spleen and bone marrow derived macrophages (BMDMs) of WT and Asc^−/− mice. (E) Thymic weight and total thymocyte counts 20 month old WT and Il1r^−/− mice (n = 9/group). (F) The FACS analysis of splenocytes revealed that compared to 20month old WT mice, the Il1r^−/− mice do not show any significant difference in CD4 or CD8 naïve and effector-memory frequency (n = 9/group). All data are presented as Mean (SEM)* P<0.05.

Figure 3. Nlrp3 inflammasome regulates age-related caspase1 activation and astrogliosis in brain

(A) Brain cryosections from WT and Nlrp3 mutant mice (23mo old) were stained with anti-Iba antibody-Alexa fluor 488 (green) to identify microglial morphology in dentate gyrus region of hippocampus. Representative confocal Z stack images revealed reduce microglia activation in aged Nlrp3^−/− mice (n=5). Real time PCR analysis of proinflammatory cytokines (B) Il1b, (C) Tnf in hippocampus and cerebral cortex in young (2mo) and old (23mo) WT, Nlrp3^−/− and Asc^−/− mice. (n = 4–6 per age group/strain). (D) Brain cryosections from WT and Nlrp3^−/− mice (23mo old) were stained with anti-GFAP antibody. Hippocampal astrogliosis was evident in aged DG (dentate gyrus), Mol (molecular layer of DG) while Nlrp3^−/− mice displayed lower GFAP immunoreactivity in DG and regions. (E) The confocal Z stack analysis of anti-GFAP stained cryosection of 24month old WT and Nlrp3^−/− mice in PoDG and Mol layer of hippocampus revealed that loss of Nlrp3 protects against age-related astrogliosis and change in astrocytic morphology (n = 4/strain repeated thrice). (F) Quantification of mean fluorescence intensity of GFAP immunoreactivity in hippocampus of WT and Nlrp3^−/− mice maintained on 60% HFD for 14months. (G) The caspase-1 immunoblot analysis of Glast1⁺ astrocytes primed with LPS and stimulated with ATP reveals presence of active p20 subunit of capsase-1. (H, I) Caspase-1 and (J) IL-1β quantification by immunoblot analysis in hippocampus of 2mo, and 23mo old WT and 23mo old Nlrp3^−/− mice. All data are presented as Mean (SEM)* P<0.05. See also Figure S3 and S4.

Figure 4. Nlrp3 inflammasome regulates age-dependent alterations in hippocampal transcriptome

(A) The age-related changes in gene expression profiling on hippocampal tissue obtained from WT as well as Nlrp3 and Asc mutant mice. (B) A set of 298 gene probes that respond to aging, and are modulated by loss of Nlrp3 and Asc. (C) Examination of the direction of change revealed that nearly all (295 of 298) gene probes with expression differences between old and young WT (blue bars) show expression changes in the opposite direction when comparing old mutant to old WT. (D, E) Pathway analyses of genes with age-dependent expression differences reveal a strong enrichment of genes related to cell death as well as to inflammatory disease in WT mice compared to Nlrp3 and Asc mutants. Similar differences in association were found for NF-kB, IL-1, and IL-8 signaling, where the respective pathways were found to be strongly associated with age-dependent expression differences in WT mice compared to Nlrp3^−/− and Asc^−/− animals. (F) The list of genes (p<0.05, 1.5 fold change) that are involved in cell death and inflammation and regulated by both Nlrp3 and Asc during aging. This pattern suggests that these group of genes are characterized by age-dependent gene expression changes which is regulated by the absence of the Nlrp3 inflammasome activity. See also Figure S5.

Figure 5. Ablation of canonical Nlrp3 inflammasome reduces innate immune activation in CNS

The hippocampi from young, old WT and old (23m) Nlrp3^−/− and Casp11^−/− was used to confirm the pathways identified from microarray profiling. Realtime PCR analysis of (A) IL-1 signaling (B) astrogliosis pathway (C) interferon pathway and (D, E) complement pathway and (F) gene implicated in cognitive function. All data are presented as Mean (SEM)* P<0.05.

Figure 6. Ablation of IL-1 signaling partially protects against age-related functional decline

(A) The hippocampi from separate cohort of young (3 month), old (20 month) WT and Il1r^−/− mice (n= 6–12/group) were analyzed for complement, interferon and Nlrp3-dependent inflammatory genes using realtime-PCR. (B) The Stone T-maze test in 20-month old WT mice and age-matched Il1r^−/− mice (n = 12/group). Mice were given 15 trials in the T-maze with each trial having a maximum length of 300 sec and number of errors during each trial block was recorded. (C) Bone mineral content of femorae of 20-month old WT and Il1r^−/− mice (n=12) is measured in grams of calcium hydroxypatite; bone mineral density represents the mineral in bone per area i.e. areal bone mineral density. (D) The mean latency to fall from a rotating rod (Rotarod test) in 20-month old WT and Il1r^−/− mice (n=12). (E, F) The treadmill test showing total running time and total distance run by 20-month old WT and Il1r^−/− mice (n =12). All data are presented as Mean (SEM)* P<0.05.

Figure 7. Reduction in Nlrp3 inflammasome activation enhances healthspan and reduces age-related functional decline

(A) The Stone T-maze test in 18-month old WT mice and age-matched Nlrp3^−/− mice (n = 8/group). Mice were given 15 trials in the T-maze with each trial having a maximum length of 300 sec. During each trial, the number of errors committed were recorded (B) Mean latency to reach the goal box during multiple trials of 18-month old WT and Nlrp3^−/− mice (n = 8/group). (C) To confirm improved memory and cognitive performance, an additional cohort of WT littermates and Nlrp3^−/− mice were aged for 24 months (n=6–8/strain) and total number of errors in a Stone T-maze test were recorded. (D) Compared to WT mice and age-matched Nlrp3^−/− mice (18mo old) displayed improved performance on the rotarod (tested in 2 separate cohorts of n = 8 each, 4+4 males and females). (E, F) At 24-month of age additional old WT littermates and Nlrp3^−/− mice were also tested for running distance and time using a treadmill test (n = 6–9/group) (p = 0.05). (G) The same cohort of WT littermates and Nlrp3^−/− mice were tested for lens opacity in both eyes by an ophthalmologist using a slit lamp and scored on a scale of normal (0), punctuate (1), incipient (2), diffuse (3) and complete (4). The ophthalmologist was blinded to the group identity to reduce investigator bias. (H, I) Representative microcomputed tomographic images of cortical bone cross sections from the mid-femur diaphysis of 24 mol female WT (n = 5) and ^Nlrp3−/− mice (n = 8). The female WT and Nlrp3^−/− mouse femurs were imaged by microcomputed tomography. (J) The microcomputed tomographic scans of trabecular bone mass and (K) trabecular thickness in 24-month old male WT and Nlrp3^−/− femorae. (L, M) Bone mineral content of 24-month old male WT and Nlrp3^−/− mice (n=13) is measured in grams of calcium hydroxypatite; bone mineral density represents the mineral in bone per area i.e. areal bone mineral density. The coefficient of variation for repetitive scanning ex vivo is approximately 2.4%. Similar to male mice, the 24-month old female Nlrp3^−/− mice displayed significant increase in (N) total bone area, (O) cortical area and (P) moment of inertia (MOI). All behavioral and functional analysis in WT and mutant mice were conducted in investigator blinded fashion. All data are presented as Mean (SEM)* P<0.05. See also Figure S6, S7 and S8.