Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Abstract

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N⁴-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Figure 1

Expression of inflammasome gene modules in older adults and its association with human health and longevity. (a) Gene expression data from the Stanford–Ellison longitudinal cohort^,, (n = 114) were used to find age-associated gene modules that participate in cytokine production and were enriched for inflammasome genes (see Supplementary Figs. 1 and 2). For the determination of significant differences in the expression of inflammasome gene modules 62 and 78, the QuSAGE gene set analysis method was used. Positive fold change values (x-axis) indicate higher expression in aged individuals in samples taken from 2008–2012. P-value for age on combined data for each gene module, 0.001. (b) A logistic regression analysis was conducted on IML (n = 11) or IMH (n = 12) group status and hypertension (shown are regression coefficients for age, sex and IML/IMH status). (c) Seventeen individuals from the year 2011 cohort (the same 8 IML and 9 IMH individuals as in b) were studied to measure the association of IML versus IMH status with the degree of arterial stiffness, as measured by pulse-wave velocity. Multiple regression analysis was performed on the pulse-wave velocity of each individual against their age, sex and IML/IMH status (shown are regression coefficients for each variable). P-values in b,c for each regression coefficient were calculated based on permutation methods (see Online Methods). (d) In the same 17 individuals from the year 2011 cohort, familial longevity was determined on the basis of membership in a family with at least one member over 90 years of age. The P-value was obtained by chi-square test. (e) Association between the expression of inflammasome gene modules 62 and 78 with all-cause mortality. Each point is representative of one individual. The P-value was obtained by Student’s t-test. (f) Serum levels of 62 different cytokines, chemokines and growth factors were compared between IML and IMH subjects using data from year 2013 (IML n = 8, IMH n = 8). Multiple regression analysis on each analyte’s MFI against their age, sex and IML/IMH status was conducted and significance (y-axis) was obtained via permutation tests. (g) IL-1β serum abundance, as shown by longitudinal analysis of data collected during the years 2008–2011 (IML n for 2008, 2009, 2010 and 2011 = 10, 10, 8 and 7, respectively; IMH n 2008, 2009, 2010 and 2011 = 12, 11, 12 and 8, respectively). Whisker bars represent maximum and minimum values.

Figure 2

Metabolites present in IMH individuals induce IL-1β and upregulate the expression of inflammasome genes. (a) Broad-coverage metabolomics profiling was conducted on available serum samples from year 2011 (n = 9 IML, n = 11 IMH). From a total of 692 metabolites analyzed, 67 were differentially expressed (all upregulated) in IMH versus IML at an FDR of Q < 0.2 (by SAM analysis; see Online Methods). Functional annotation and pathway analysis were conducted using MetPA. A significant enrichment for several metabolic pathways was identified for these metabolites (P < 0.05). Darker color indicates higher level of significance. (b) The conversions of cysteine to cystine and of arachidonic acid to 8-isoprostane n the presence of ROS. Circulating levels of (c) cystine and (d) 8-isoprostane are greater in IMH as compared to I ML individuals. (e) Adenine, dl-4-hydroxy-3-methoxymandelic acid (vanillylmandelate) (MMA), scyllo-inositol and N⁴-acetylcytidine (N4A) were selected for further study on the basis of their levels of significance (Q < 0.001; see Supplementary Table 4) and their representation of different metabolic pathways. We assessed each compound’s ability to alter IL-1α and IL-1β levels and the expression of NLRC4 in primary monocytes from four healthy young adults. Results show one representative experiment. Adenosine was used as a positive control. A significant induction of IL-1α and IL-1β was observed when using adenosine and adenine but not the other compounds. (f) The highest dose of each compound (100 µM) was used to determine expression of NLRC4 and NLRP3 by qPCR on the same samples used for cytokine determination. A significant increase in NLRC4 and NLRP3 is shown only for N⁴-acetylcytidine (P < 0.05, by one-sided Student’s t-test). Adenosine treatment upregulated NLRP3 gene expression (P < 0.01 by one-sided Student’s t-test). Expression of GAPDH was used to standardize the samples, and the results are expressed as the normalized ratio in relation to the control. Error bars reflect experimental variability.

Figure 3

Metabolites in IMH individuals activate the NLRC4 inflammasome. (a,b) Differentiated THP-1 cells were treated with ATP (5 mM, 30 min), or they were primed with LPS (1 µg/ml, 4 h) and then pulsed with ATP, or they were treated with the indicated concentrations (mM) of either adenine (Ad) or N⁴-acetylcytidine (N4A) alone (a) or in combination (b) for 6 h. NT, nontreated. They were then either pulsed with ATP or not. Secretion of cytokines IL-1β, IL-18 and TNF-α was measured by ELISA from cell culture supernatants. (c) Differentiated THP-1 cells were treated with compounds as indicated (1 mM N4A; 300 µM adenine (Ad)) for 6 h or with ATP 5 mM for 30 min. Then cells were lysed, and the lysates were immunoblotted with various antibodies to monitor cellular expression of NLRs, caspase-1 (casp1) and pro-IL-1β. (d) Differentiated THP-1 cells were treated with compounds using the same process outlined earlier, and the cell lysates were submitted to immunoprecipitation using biotinyl-YVAD-fmk peptide. Complexes were then recovered by using streptavidin-Sepharose beads and were immunoblotted with anti-caspase-1 p10 antibody. (e) Differentiated THP-1 cells were treated with compounds as before in the presence or absence of Ac-YVAD-fmk or of control DMSO, and then IL-1β secretion was measured. (f) Differentiated wild-type (WT) or stable shTHP-1 cell lines were treated with compounds as before (1 mM N4A; 300 µM Ad) and IL-1β secretion was measured. Right, western blots showing protein expression of NLRC4 in stable shTHP-1 cell lines. Ctrl, control. (g) Bone-marrow-derived macrophages from WT or NLRC4-KO mice were obtained as previously described. Cells were plated in triplicates, treated with combination of compounds (1 mM N4A; 300 µM adenine) and IL-1β secretion was measured. Data in panels a,b,e–g are expressed as the concentration of cytokines (pg/mL) or as the fold increase with respect to baseline as indicated (mean ± s.d.; n = 3 for a,b,e,f; n = 9 for g). P-values were determined by Student’s t-test. *P < 0.05, **P < 0.01.

Figure 4

Metabolites in IMH individuals activate human primary platelets and neutrophils. (a) Primary platelets were isolated from blood of healthy donors and were incubated for 6 h with thrombin, ADP (3 µM) or various concentrations of adenine or N4A. Platelet activation was monitored by measuring the membrane expression of CD61 and CD62P in healthy cells by flow cytometry. Histograms show overlay of CD62P staining within the CD61 positive population for Adenine (0.5 µM) and N4A (100 µM) for each donor. Bar graph summarizes the results obtained from each donor. Student’s t-test with *P < 0.05 applies to each donor. (b) Analysis of immune cell type populations (ImmGen database) show that modules 62 and 78 are predominantly expressed in macrophages, monocytes and granulocytes (orange, red and blue respectively) (P < 10⁻¹⁰). SC, stem cells; BC, B cells; DC, dendritic cells; MP, macrophages; MN, monocytes; GN, granulocytes; ABTC, αβ T cells; GDTC, γδ T cells; STC, stromal cells; IL, innate lymphocytes. (c) Primary neutrophils were isolated from blood of healthy donors and then incubated for 24 h with various concentrations of adenine or N4A either separately or in combination. the concentration of RANK-L⁺ cells present within the CD66b⁺ population was determined. Histograms show overlay of RANK-L staining within the CD66b⁺ population. Graph below summarizes the results of each donor. (d) Primary neutrophils were treated with N4A (1 mM) and/or adenine (1 mM), and the percentage of degranulated population was measured as shown in the scatter plot. Graph at right summarizes results obtained from each donor. (e) Primary neutrophils were treated as described previously with the compounds as indicated in the figure, and IL-1β secretion was measured from the cell culture supernatants obtained from each donor. Data are expressed either as the concentration of cytokines (pg/mL) or as the fold increase with respect to the non-treated (NT) condition as indicated. P-values in a,d,e were determined by Student’s t-test. *P < 0.05, **P < 0.01.

Figure 5

Metabolites in IMH individuals induce high blood pressure in mice. (a) Mice were randomly put into either the control (n = 4) or the treatment (n = 4) group. The treatment group mice were injected with N4A + adenine at 20 mM stock solution of 100 µL per 25 g body weight once daily. Treatment with N4A + adenine had a mild effect with pre-hypertension states as early as 8 d after the first injection (P = 0.02, by one-tailed Student’s t-test). Significant increases were observed after day 20 in the treated mice, with an average systolic blood pressure of 140 (±7) mmHg in that group, as compared to 112 (±3) mmHg in the control group (P = 0.008). *P < 0.05, **P < 0.01 by one-tailed t-test. These experiments were repeated using 10 mice per group with similar results. Filled and empty horizontal lines represent the mean values for the treated and control groups, respectively. Whisker bars represent maximum and minimum values. (b) Increased phosphorylation levels of signaling proteins (FDR Q < 0.05) in blood cells from N4A + adenine-angII treated mice versus control mice (treated with angII alone). Shown are the results of SAM analysis comparing the two groups of mice (see Online Methods); the x-axes represent the FDR or significance (cutoff value of 5%) as a function of score (d) parameter (y-axis), which is equivalent to the t-statistic value of a t-test when comparing two samples. P-values were adjusted for multiple comparisons using SAM, which provides an estimate of FDR. pNF-κB, phosphorylated form of NF-κB (p65 Ser529); pCREB, phosphorylated form of CREB (Ser133); pS6, phosphorylated form of 40S ribosomal protein S6. (c) Higher levels of infiltrating T cells in the kidney, but not the aorta, are observed in the treatment group versus the control group.

Figure 6

Caffeine negatively regulates the NLRC4 inflammasome. Multiple regression analysis was conducted on expression levels of module 62 and 78 (from data collected in year 2008, n = 89) and caffeine intake for each individual in mg/week (adjusted for age, sex and BMI). (a) A significant association was found between caffeine intake and the expression of modules 62 (P < 0.01) and 78 (P = 0.024). (b) Differences in the circulating levels of coffee and coffee-derived metabolites between the IML and IMH groups were computed using a one-tail Student’s t-test, and P-values were combined using a modified generalized Fisher method for combining probabilities from dependent tests (P < 0.01). Whisker bars represent maximum and minimum values. (c) Left: Differentiated THP-1 cells were incubated with various concentrations of caffeine (caff) and were then treated with N4A (at 1 mM) + adenine (at 300 µM) for 6 h. IL-1β secretion was measured from cell culture supernatants. Data are expressed as concentration of cytokines (pg/mL) (mean ± s.d.; n = 3). P-values were determined by Student’s t-test. *P < 0.05. (c) Right: Differentiated THP-1 cells were treated with N4A (at 1 mM) in the presence or absence of caffeine (at 0.5 mM) for 6 h, and expression of various genes as indicated was assessed by qPCR. Cell lysates were submitted to immunoblotting with indicated antibodies for protein expression.