The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance

Abstract

The emergence of chronic inflammation during obesity in the absence of overt infection or well-defined autoimmune processes is a puzzling phenomenon. The Nod-like receptor (NLR) family of innate immune cell sensors, such as the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (Nlrp3, but also known as Nalp3 or cryopyrin) inflammasome are implicated in recognizing certain nonmicrobial originated 'danger signals' leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) and IL-18 secretion. We show that calorie restriction and exercise-mediated weight loss in obese individuals with type 2 diabetes is associated with a reduction in adipose tissue expression of Nlrp3 as well as with decreased inflammation and improved insulin sensitivity. We further found that the Nlrp3 inflammasome senses lipotoxicity-associated increases in intracellular ceramide to induce caspase-1 cleavage in macrophages and adipose tissue. Ablation of Nlrp3 in mice prevents obesity-induced inflammasome activation in fat depots and liver as well as enhances insulin signaling. Furthermore, elimination of Nlrp3 in obese mice reduces IL-18 and adipose tissue interferon-γ (IFN-γ) expression, increases naive T cell numbers and reduces effector T cell numbers in adipose tissue. Collectively, these data establish that the Nlrp3 inflammasome senses obesity-associated danger signals and contributes to obesity-induced inflammation and insulin resistance.

Figure 1. Reduction of Nlrp3 and IL-1β expression is associated with improvement of insulin-sensitivity

Positive correlation of the visceral fat mRNA expression of (a) Il-1β and (b) Nlrp3 with body weight of C57BL/6 mice (n = 32), Pearson's correlations are r = 0.364, P = 0.0178 for Il-1β and r = 0.672, P < 0.0001 for Nlrp3 respectively. (c) Il-1β, (d) Nlrp3 and (e) Asc mRNA in visceral and subcutaneous adipose tissue from AL fed control and calorie restricted 12 month old mice, n = 6; *P < 0.01, ** P < 0.005. Representative H&E staining showing adipocyte size in (f) visceral and (g) subcutaneous fat tissue from AL fed control and calorie restricted 12 month old C57BL/6 mice. (h) IL-1β (left) and NLRP3 (middle) and ASC (right) gene expression, as examined by qRT-PCR in human SAT in obese T2D patients before and after 1-year weight loss. (i) Positive correlation of changes in gene expression of IL-1β and NLRP3 in human abdominal subcutaneous fat with changes in fasting glucose level from baseline to 1-year post intervention; Pearson's correlations are r = 0.53, P = 0.12 for IL-1β and r = 0.69, P = 0.03 for NLRP3 respectively. Relative gene expression levels are depicted as means ± SEM. n = 10; *P < 0.01, **P < 0.001. All samples analyses were performed in a blinded fashion.

Figure 2. Elimination of Nlrp3 signaling prevents obesity induced caspase-1 cleavage and IL-1β/IL-18 activation

(a) Immunofluorescent staining of epididymal fat (eFat) tissue sections stained with antibodies against F4/80 (red), and Nlrp3 (green). Merge of images with nuclear stain DAPI shows co–localization of Nlrp3 with ATM (yellow arrow heads). Negative control staining with addition of antibody to Nlrp3 together with antibody to F4/80 in adipose tissue of Nlrp3^−/− mice displayed reduced Nlrp3 specific immunostaining. (b-c) Quantitative real time–PCR analysis of Nlrp3 and Asc mRNA in purified ATM, SVF derived from SAT and VAT of 6 month old DIO mice and mature 3T3L1 adipocytes (Adip). (d) Immunoblot analysis showing kinetics of caspase-1 (p20) cleavage and active IL-1β (p17) in adipose tissue of mice at different stages of diet–induced obesity. (e) Western blot analysis of activated caspase-1 (p20) in VAT, SAT and liver tissues from 9 month old DIO–WT (C57BL/6) and DIO– Nlrp3^−/− mice. Results shown are representative of three independent experiments. (f) Western blot analysis of activated caspase-1 (p20) in kidney from 9 month old DIO–WT and DIO – Nlrp3^−/− mice. (g) Western blot analysis of IL-1β activation in adipose tissue of 6 and 7 month old DIO mice. (h) Serum IL-18 concentration in age–matched WT and Nlrp3^−/− mice fed chow and 60% HFD for 4 months (6 month old DIO) and 7 months (9 month old DIO). All data are presented as means ± SEM, n = 6–10 mice; *P < 0.05.

Figure 3. Nlrp3 inflammasome regulates insulin–sensitivity and steatohepatitis in obesity

(a) Insulin tolerance test (ITT) and glucose tolerance test (GTT) in male WT and Nlrp3^−/− DIO mice fed 60% HFD for 6 weeks. (b) The ITT and GTT in 6 month and (c) 9 month old WT and Nlrp3^−/− DIO mice. The ITT graphs display the area under the curve (green for WT and blue for Nlrp3^−/−) with trend lines (n = 5–7 per group). (d) The total area under the curve for ITT in 6 and 9 month old WT and Nlrp3^−/− DIO mice. (*P < 0.01). (e) Adipocyte size in visceral fat tissue from 8 month old DIO–WT (Nlrp3^+/+) and Nlrp3^−/− mice (hematoxylin and eosin staining). The cross–sectional size (area, μM²) of adipocytes (100 cells per mouse) were determined in VAT using Image J software. Representative micrographs and the quantified results are shown. n = 6; *P < 0.001. (f) Akt, IRS-1 and MAPK signaling in vivo as determined by western blotting in VAT, SAT, liver, and muscle from the 8 month old DIO Nlrp3^+/+ and DIO Nlrp3^−/− mice. (g) Immunohistochemistry (hematoxylin and eosin staining) of section from liver tissues from 9 month old DIO WT and Nlrp3^−/−. (h) Fatty acid oxidation and fatty acid synthesis gene expression in liver tissue of Nlrp3^+/+ and Nlrp3^−/− mice analyzed by quantitative RT–PCR. ΔΔCt of the genes are shown means ± SEM. (n = 5), * P < 0.05, **P < 0.001.

Figure 4. Nlrp3 senses ceramide to induce IL-1β and regulates adipose tissue macrophage activation in obesity

(a) Western blot analysis of a large (∼20kD) catalytic subunits of caspase-1 in BMDM from Nlrp3^+/+ and Nlrp3^−/− mice (n = 6–9). The BMDMs were primed with LPS and treated with C2 Ceramide (100 μM). (b) IL-1β secretion in BMDM culture supernatants determined by ELISA. Results are representative of three separate experiments. (c) Epididymal adipose tissue (eAT) explants from 9 month old Nlrp3^+/+ and Nlrp3^−/− DIO mice were cultured for 24h in presence of LPS and/or ceramides. Caspase-1 activation was determined by immunoblot analysis (d) M1 and M2 associated gene expression, was examined by qRT–PCR in macrophages originating from VAT and SAT of 9 month old DIO–Nlrp3^+/+ and DIO– Nlrp3^−/− mice. The mRNA expression was normalized to Gapdh and shown as fold change (ΔΔCt). All data are presented as means ± SEM mean, *P < 0.01, **P < 0.001.

Figure 5. Ablation of Nlrp3 inflammasome reduces adipose tissue effector T cells without affecting T_reg cells in visceral fat of obese mice

(a) The FACS plot of SVF cells isolated from VAT of 9 month old WT and Nlrp3^−/− obese mice. First dot plots depict Forward and side scatter (FSC/SSC) and gating strategy for analysis of ATMs and T cells. Gate1 (upper panel) of larger cells shows presence of F4/80⁺ cells (histogram) and expression of macrophage markers CD206 and CD11c on ATMs. Gating of smaller cells (gate 2, lower panel) reveals absence of ATMs in this population of SVF. (b) The gate 2 (lymphoid gate) presenting CD4, CD8 T cells in SVF. CD4 and CD8 cells were evaluated for naïve T cells (CD62L⁺CD44⁻, blue box) and effector memory E/M (CD62L⁻CD44⁺, red box) CD4 and CD8 T cells. The FACS analysis was repeated in three independent pooled SVF fractions from a total of 12-14 mice. (c) Gated percentage and absolute numbers (in million cells) of naïve (CD62L⁺CD44⁻) and E/M (CD62L⁻CD44⁺) CD4 and CD8 T cells. (d) Number of stromal vascular cells per gram of fat tissue (n = 4–6) in 3 and 9 month old WT and Nlrp3^−/− DIO mice. (e) Representative FACS plots showing CD4⁺CD25⁺Foxp3⁺ T regulatory cells in VAT of 9 month old WT and Nlrp3^−/− DIO mice. (f) Gated percentage of T_reg cells in VAT and SAT of 9 month old WT and Nlrp3^−/− DIO mice (n = 6 per group). All data are presented as means ± SEM mean, *P < 0.05.

Figure 6. Elimination of Nlrp3 inflammasome reduces obesity–induced macrophage-T cell activation in adipose tissue

(a) The representative FACS plots of SVF cells from SAT of 6 month and 9 month old WT and Nlrp3^−/− DIO mice stained with CD206 and CD11c. (b) The gated percentage of CD11c⁻CD206⁺ M2 cells in SAT of 9 month old obese mice. (c) Dot plots showing naive (CD62L⁺CD44⁻, blue box) and effector memory (CD62L⁻CD44⁺, red box) CD4 and CD8 T cells in SVF from 9 month old obese WT and Nlrp3^−/− mice. (d) Gated percentage and absolute numbers (in million cells) of naïve (CD62L⁺CD44⁻) and effector memory (CD62L⁻CD44⁺) CD4 and CD8 T cells. (e) mRNA level of Th1 cytokine Ifnγ in VAT and SAT of 9 month old obese WT and Nlrp3^−/− mice as determined by quantitative RT–PCR. (f) IFNγ (19kD) in VAT of 9 month old WT and Nlrp3^−/− DIO mice as examined by western blotting. (g) IP10/CXCL10 and MCP–1/CCL2 levels in the serum of 9 month old lean WT and obese WT and Nlrp3^−/− mice (n = 5). The data shown are mean ± SEM and *, P < 0.05. (h) Hypothetical model of Nlrp3 inflammasome activation in obesity. In absence of ‘danger signals’ in healthy lean state, the tissue resident macrophages and T cells may participate in the maintenance of adipose tissue function. Nlrp3 inflammasome senses the obesity–associated ‘danger signals’ such as ceramides leading to caspase-1 autoactivation and IL-1β and IL-18 production from ATMs. Secondary signals from activated ATMs to effector adipose T cells sustain the reciprocal proinflammatory feed–forward cascade in obesity leading to insulin–resistance.