Hormetic elevation of taurine restrains inflammaging by deactivating the NLRP3 inflammasome

Abstract

Taurine, the most abundant sulfonic amino acid in humans is largely obtained from diets rich in animal proteins. However, taurine is dietary non-essential because it can be synthesized from cysteine by activation of transsulfuration pathway (TSP) when food consumption is low or if the diet is predominantly plant based. The decline of taurine was proposed as the driver of aging through an undefined mechanism. Here, we found that mild food restriction in humans for one year that resulted in 14% reduction of calorie intake elevated the hypotaurine and taurine concentration in adipose tissue. Therefore, we investigated whether elevated taurine mimics caloric-restriction's beneficial effects on inflammation, a key mechanism of aging. Interestingly, aging increased the circulating and tissue concentrations of taurine suggesting that elevated taurine may serve as a hormetic stress response metabolite that regulates mechanism of age-related inflammation. The elevated taurine protected mice against mortality from sepsis and inhibited inflammasome-driven inflammation and gasdermin-D (GSDMD) mediated pyroptosis. Mechanistically, 'danger signals' including hypotonicity that activate NLRP3-inflammasome, caused upstream taurine efflux from macrophages, which triggered potassium (K⁺) release and downstream canonical NLRP3 inflammasome assembly, caspase-1 activation, GSDMD cleavage and IL-1β and IL-18 secretion that was reversed by taurine restoration. Notably, taurine does not efflux from GSDMD pore and inhibited IL-1β from macrophages independently of known transporters SLC6A6 and SLC36A1. Increased taurine in old mice promotes healthspan by inducing anti-inflammatory pathways previously linked to youthfulness. These findings demonstrate that taurine is an upstream metabolic sensor of cellular perturbations that control NLRP3 inflammasome and lowers age-related inflammation.

Fig.1.. Calorie restriction and aging elevate taurine levels.

(a-c) Unbiased metabolomics analyses of subcutaneous adipose tissue (SAT) of participants in the CALERIA-II trail at baseline and one year after caloric restriction (CR). (a) Volcano plots of the 387 metabolites of SAT from healthy individuals at baseline and 1-year CR. Each dot represents a metabolite identified by LC-MS/MS. The volcano plot shows the fold-change (x-axis) versus the significance (y-axis) of the identified 387 metabolites. The significance (non-adjusted P-value) and the fold change are converted to -Log₁₀(P-value) and log₂(Fold-change). The vertical and horizontal dotted lines show the cut-off of fold-change= ±1.0, and of p-value=0.05. 30 significantly regulated metabolites at 1 year of CR compared with baseline were heighted (up-regulated in red, down-regulated in blue (n=14. P<0.05). (b) Schematic summary of the transsulfuration pathway and metabolites from baseline to 1 year CR that were measured in the healthy human SAT. Blue arrows indicate significantly decreasing, pink arrows indicate significantly increasing metabolites and purple lines indicate not significant changes. (c) Changes in the levels of taurine and hypotaurine measured by LC-MS/MS in human SAT after 1 year CR. (Significance was calculated using paired t-test). (d) The RNA-sequencing analyses of SAT of participants in the CALERIA-II trail at baseline and after 1 year CR. The expression level of Csad, Fmo1 and Fmo2 after CR. Paired t-test was performed (n=8). (e) Circulating taurine concentration from serum in young and aged humans (n=17, 28). (f) Taurine level changes prior and 2 days after influenza vaccine immunization in young and aged human serum (young n=17, aged n=28). (g) Serum taurine levels in different ages of male C57BL/6J mice (n=12, 5, 10, 12, Yale rodent colony), mice are littermates. (h) Serum taurine and hypotaurine levels were determined in 2-month-old and 28-month-old male C57BL/6N mice (n=11/10) by LC-MS/MS. (i) Tissues taurine levels in 2-month-old and 28-month-old C57BL/6J male mice (n=10/group), mice are littermates, by taurine assay kit. Error bars represent the mean ± SEM.

Fig.2.. Taurine inhibits NLRP3 inflammasome activation

(a) Volcano plots of the 290 metabolites identified between the LPS plus ATP challenge group and control group in the bone marrow derived macrophages (BMDMs). Each dot represents a metabolite identified by LC-MS/MS. The volcano plot shows the fold-change (x-axis) versus the significance (y-axis) of the identified 290 metabolites. The significance (non-adjusted p-value) and the fold change are converted to -Log₁₀(p-value) and log₂(fold-change). The horizontal dotted lines show the cut-off of fold-change= ±1.0, and of p-value=0.05. 26 significantly regulated metabolites in response to LPS plus ATP treatment compared with baseline were heighted (up-regulated in red, down-regulated in blue (n=3, P<0.05). The right panel is the top 26 discriminating parameters in descending order of importance from the metabolomics data of BMDMs with LPS, ATP treatment and control group. The colored legend on the right indicates the relative abundance of variables, with red and blue indicating high and low values, respectively, while beige illustrates neutral values. (b) Changes in the levels of taurine and L-cystine in response to LPS and ATP stimulation in BMDMs, measured by LC-MS/MS. (c) Taurine efflux was determined in LPS-primed-BMDMs treated with 10 mM ATP, Hypotonic solution (90 mOsmolarity), 10 μM Nigericin for 45 mins, Ceramide 6, MSU for 6 hours and Imiquimod for 2 hours. (d) Intracellular and culture medium taurine levels in BMDMs primed with LPS and stimulated with ATP. (e) Western blots of cell lysates and supernatants from BMDMs stimulated with LPS and ATP and treated with taurine. These results are representative of five independent experiments. (f) ELISA analysis of IL-1β production from BMDMs stimulated with LPS (1 μg/ml, 4 hours), followed by ATP (5 mM, 45 min) treatment with or without taurine, MCC950 or KCl. (g) Intracellular taurine level following inflammasome activation in BMDMs supplemented with 100 mM taurine, 10 μM MCC950 or 45 mM KCl. (Representative of three experiments). (h-i) Production of IL-18 and LDH from BMDM stimulated with LPS and ATP and treated with taurine as measured by ELISA (h) and LDH assay (i). (j) Intracellular taurine concentration in LPS-primed BMDMs stimulated by hypotonicity in the presence of sodium chloride. (Representative of three experiments). (k) Western blots of cell lysates and supernatants from BMDMs activated with hypotonic solution in the presence of taurine or NaCl. (Representative of three experiments). (l) Production of IL-1β from BMDMs stimulated with LPS and hypotonic solution and treated with taurine. (m) Intracellular taurine level of LPS-primed BMDMs from control and Nlrp3 deficient mice following ATP or hypotonic treatment supplemented with 10 μM MCC950. (Representative of three experiments). (n) Intracellular taurine concentration in thioglycolate elicited macrophages sorted from peritoneal cavity of mice treated with LPS and ATP. These data are representative of three independent experiments. Error bars represent the mean ± SEM.

Fig. 3.. Taurine inhibits NLRP3 inflammasome assembly and GSDMD activation.

(a-c) LPS-primed BMDMs are transfected with Poly(dA:dT) in the presence of taurine. (a) Intracellular taurine levels of BMDMs. (b) Western blots analyses of IL-1β (p17), caspase-1 (p20) in cell lysates and supernatants of AIM2 inflammasome activated BMDMs. (c) Production of IL-1β from these BMDMs, measured by ELISA. These results are representative of three to five independent experiments. (d) Immunoblots of cell lysates and supernatant from BMDMs stimulated with Pam3CSK4 and transfected with LPS in the presence of taurine. Results are representative of three independent experiments. (e) Intracellular taurine levels of BMDMs primed with Pam3CSK4 and transfected with LPS in the presence of taurine. (f) Quantification of intracellular potassium following inflammasome activation with LPS and ATP in the presence of indicated concentration of taurine. Intracellular potassium was detected by potassium indicator, ION Potassium Green-2 AM. (g) Immunoblot of ASC in cell lysates and cross-linked cytosolic insoluble pellets from BMDMs stimulated with LPS and ATP with taurine. Results are representative of three independent experiments. (h) Western blots detection of ASC oligomerization in cross-linked insoluble pellets from BMDMs activated by hypotonic solution in the presence of taurine or sodium chloride. Results are representative of three experiments. (i) Immunoblot of ASC oligomerization in BMDM stimulated with LPS and transfected with poly(dA:dT) in the presence of taurine. These results are representative of three independent experiments. (j) Intracellular taurine levels in control and Gsdmd^−/− BMDMs primed with LPS and treated with ATP, hypotonicity. (k) Immunoblot of cell lysates from BMDM activated by LPS and ATP in presence of taurine. F-GSDMD, full length GSDMD; N-GSDMD, N-terminal GSDMD; C-GSDMD, C-terminal GSDMD. The results are representative of five experiments. (l) Immunblot detection of GSDMD cleavage in BMDM activated by LPS and hypotonicity in presence of taurine. (m) Western blots of IL-1β (p17), caspase-1 (p20) in cell lysates and supernatants of control and Gsdmd^−/− BMDMs stimulated with LPS and ATP with taurine. These results are representative of three independent experiments. (n-o) Transmission electron micrograph of LPS-primed BMDMs and treated with ATP (n) or hypotonicity (o). (n) Resting macrophages containing autophagosomes with clear membrane structure (left). In response to LPS and ATP stimulation, macrophages containing autophagosomes with apparent remnants of degenerating parts (middle). With taurine addition, autophagosome with clear autophagosome vacuoles (right). (o) Hypotonic solution induced cell swelling in LPS-primed BMDMs (left) and in presence of taurine. Scale bar: 2 μm in lower magnification (1,900 ×); 1 μm in higher magnification (4800 ×). Arrows point to the autophagosome. N, nucleus.

Fig. 4.. Taurine inhibits NLRP3 in disease models of cryopyrinopathies independently of Slc6a6 or Slc36a1 transporters.

(a-c) BMDMs from Nlrp3^L351P mice. (a) IL-1β production from these BMDMs stimulated with LPS and treated with taurine as measured by ELISA. These data are representative of three independent experiments carried out in triplicate. (b) Western blots of IL-1β (p17), caspase-1 (p20) in cell lysates and supernatants from BMDMs stimulated with LPS and treated with taurine. These results are representative of three independent experiments. (c) Immunoblot analysis of GSDMD cleavage in cell lysates from BMDM stimulated with LPS and treated with taurine. These results are representative of three independent experiments. (d-f) BMDMs from Nlrp3^A350V mice. (d) IL-1β production from BMDMs stimulated with LPS and treated with taurine. These data are representative of three independent experiments carried out in triplicate. (e) Western blots of IL-1β (p17), caspase-1 (p20) in BMDMs stimulated with LPS and treated with taurine. These results are representative of three independent experiments. (f) Western blot analyses of GSDMD cleavage in BMDM stimulated with LPS and treated with taurine. These results are representative of three independent experiments. (g) qPCR analysis of genes encoding key proteins involved in taurine uptake and synthesis in BMDMs (n=5–6). (h-i) Western blot analyses of IL-1β (p17), caspase-1 (p20) (h) and GSDMD (i) from control and Slc6a6^−/− BMDMs stimulated with LPS and ATP and treated with taurine. These results are representative of three independent experiments. (j) Intracellular taurine levels in control and Slc6a6^−/− BMDMs activated by LPS and ATP and treated with taurine. (k-l) IL-1β production (k) and GSDMD cleavage (l) in control and Slc6a6^−/− BMDMs stimulated with LPS and hypotonicity and treated with taurine. These data are representative of three independent experiments carried out in triplicate. (m and n) 10- to 11-week-old littermate Slc6a6^+/+ and Slc6a6^−/− mice were pretreated with taurine (1000 mg/kg body weight) or vehicle control for 5 days, then given LPS i.p. injection, 4 hours later blood and liver were analyzed (n=17,16,14,14). (m) Taurine levels of the liver from the mice. (n) Serum levels of IL-1β from these mice. (o and p) 13- to 14-week-old littermate Slc6a6^flox/floxLysMCre⁻ or Slc6a6^flox/floxLysMCre⁺ mice were pre-treated with taurine (1000 mg/kg body weight) or vehicle control for 5 days, IL-1β was quantified in blood and liver 4 h post LPS challenge (n=12,12,10,10). Taurine levels in livers (o) and serum IL-1β levels (p) of control and myeloid specific Slc6a6 deficient mice. (q) Production of IL-1β from Cth^+/+ and Cth^−/− BMDMs stimulated with LPS and ATP and treated with taurine. Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate.

Fig. 5.. Elevation of taurine inhibits NLRP3-inflammaging and enhances healthspan.

(a) 8- to 9-week-old C57BL/6J male mice were intraperitoneally injected with taurine at 500 mg/kg body weight or vehicle control for 7 days and then give i.p. LPS injection. 4 hours later, serum levels of IL-1β, TNF-α, and MCP-1 were measured by ELISA (n=3, 8, 4, 9) (b)10- to 11-week-old C57BL/6J female mice were i.p. injected with taurine at 1000 mg/kg body weight or vehicle control for 5 days, and then mice were given i.p. LPS injection for 4 hours. Serum IL-1β was measured by ELISA (n=11, 9). (c and d) 13-week-old C57BL/6J male mice pretreated with taurine (1000 mg/kg body weight) or vehicle control for 5 days, then mice were given LPS for 4 hours followed by ATP treatment for 15 mins (n=6,6,10,10). (c) Levels of IL-1β of the serum and peritoneal lavage were measured by ELISA. (d) body weight changes of these mice. (e) Survival curves of 10- to 11-week-old C57BL/6J male mice pretreated with PBS or Taurine (1000 mg/kg body weight) for 5 days followed by LPS i.p. injection (n=12,15). (f-m) Aged male C57BL/6N mice (20-month-old) given normal water or drinking water with taurine (8,000 mg/kg/day, 4% in water (w/v)) for 4 months. (f) Serum taurine (n=11,8) levels after 4 months of oral taurine treatment. (g) Glucose tolerance test (GTT) and area of the curve (A.O.C.) of mice for 3 months taurine treatment (n=14, 15). (h and i) Rotarod (h) and Grip-strength (i) tests of mice after 3 month of taurine treatment (n=15, 15). (j) UMAP visualizations of the scRNA-sequencing data from stromal vascular faction (SVF) cells of visceral adipose tissue (VAT) in these old mice (n=10, 8 pooled). Identified cells colored by treatment condition (left). Proportions and annotation of selected cell types in control versus taurine treated condition (right). (k) The mouse hallmark gene set enrichment analysis for down-regulated genes in taurine treated group across cell types. Color represents minus log10 adjusted p-values. (l) Dot plots showing significantly up/down-regulated genes (n=10 each) in taurine-treated group versus control group for macrophages, ASPCs and mesothelial cells. The dot colors indicate the log fold changes. Dot sizes indicate the fraction of cells that express the genes. (m) Serum levels of IL-1β and liver levels of IL-1β, IL-18 and IL-6 from mice treated with taurine for 4 months (n=9, 8). Error bars represent the mean ± SEM.