© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Correspondence should Correspondence be addressed to D.F. ( University School of Medicine, Stanford, California, USA. California, USA. 7 School of Medicine, Stanford, California, USA. Stanford, California, USA. Translational Medicine, Sidra Medical and Research Center, Doha, Qatar. 1 genes were over elevated consistently some contained inflammasome that modules gene two of levels the that found we data, expression the Stanford–Ellison longitudinal cohort of the large, data immunology-focused use sets that we making have by accumulated on question this investigate to out set we Therefore, disease. age-associated of onset the to contribute they whether and aging human in activated are inflammasomes whether unknown is IL-1 of that was as elevated, was activation masome inflam with that are of associated a expression of number molecules cellular or stress pathogens of presence the by triggered are that protein of or receptors NOD-like (NLRs) the ‘Absent in Melanoma 2’ (AIM2) eases disease IL-1 elevated this people of older blood the in is elevated that cytokine inflammatory IL-1 understood. poorly are inflammation this produce aging with many associated with diseases the associated of been has inflammation chronic Low-grade components may ameliorate chronic inflammation and various other conditions. age-associated the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome production of IL-1 metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 induce inflammasome, the without constitutive expression of IL-1 IL-1 specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of disease or cellular stress, and they trigger the maturation of interleukin-1 producing this inflammation remain unclear. can Inflammasomes drive chronic inflammation in the context of an infectious Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for Vladimir Jojic Jean-François Moreau Edward A Ganio David Furman immunological states stratifies older individuals into two extreme clinical and Expression of geneinflammasome specific modules nature medicine nature Received 2 June 2016; accepted 13 December 2016; published online 16 January 2017; CIRID, CIRID, UMR CNRS 5164, Université Bordeaux 2, Bordeaux Cedex, France. Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, California, USA. b 3 1 , , , nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those 1 3 9 2 , , are one of source IL-1 . Inflammasomes, which are intracellular structures composed , , cancer 9 Department Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina, USA. 1 0 9 , functional decline , functional β 1 , Calvin , J Calvin Kuo

, is linked to an increased risk of cardiovascular cardiovascular of risk increased an to linked is b 2 5 advance online publication online advance , , Junlei Chang , , activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, , , Gabriela K Fragiadakis 4 INSERM INSERM U916 VINCO, Institut Bergonié, Bordeaux Cedex, France. 7 , Garry P , NolanGarry β . . In a recent study of in aging rats, the 3 1 , , Mark M Davis 1 1– [email protected] 3 and various degenerative dis and degenerative various 6 , , Lydia Lartigue 8 7 Institute Institute of Medicine, Cardiovascular Stanford University School of Medicine, Stanford, California, USA. , , but the mechanisms that that mechanisms the but , 15– b , , who lack these Adenine characteristics. and 1 7 . . Starting with the gene 5 11 , , Patrick Blanco Present Present address: Department, Bioinformatics Genentech Inc., South San Francisco, California, USA. 5 β

, , Matthew H Spitzer 1 4 . However, it it However, . β 1 , is a potent potent a is ) ) or B.F. ( 10 4 & Benjamin Faustin , , Christopher R Bolen 3 Department Department of Medicine, Division of Hematology, Stanford University School of Medicine, 8 Department Department of Pediatrics, Division of Infectious Diseases, Stanford University, Stanford, [email protected] 8 - - ; 7 , , Julie Déchanet-Merville has yet to be discovered. Using this approach, we found that of a of that found we approach, this Using discovered. be to yet has function whose those with along a in function involved previously to be known are that genes contain may modules such components; of expression their on the based coordinated into modules organized approach cohort longitudinal Stanford–Ellison the in cells during human aging, we first analyzedimmune from age-related genes of gene expressionexpression the in changes the Toinvestigate Higher expression of inflammasome gene modules in older adults RESULTS with disease. pathogens cardiovascular and inflammation against chronic response immune innate an between link lymphocyte and direct another data provide These in mice. of hypertension kidneys the infiltration induce also could molecules these and that could trigger inflammasome activity individuals in hypertensive elevated that were also DAMP molecules exhibited other comorbidities. Metabolomic data identified two novel also who and hypertensive were who individuals in period 5-year a doi:10.1038/nm.426 5 b , , Isabelle Douchet 5 (IL-1 1 ) ) or M.M.D. ( Department Department of Microbiology and Immunology, Stanford University 8 . . An important feature of this approach is that the genes are 7 b 5 ). ). Here we find that the expression of , 11 , , François Haddad [email protected] N 4 7 -acetylcytidine, nucleotide-derived nucleotide-derived -acetylcytidine, 10 Howard Howard Hughes Medical Institute, Stanford 7 7 , Cornelia , L Cornelia Dekker , , Sophie Daburon 2 Department Department of Systems Biology, Division of ). 6 , , Brice Gaudilliere in vitro 8 , 15– 1 7 s e l c i t r a using a modular modular a using . . In combination, 7 ,

8 ,

5  ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. found a significant association between hypertension and IMH/IML and IMH/IML hypertension between association a found significant ( Table history 2 medication as other such for factors adjusted we confounding addition, In models. regression logistic the included were sex and age large, relatively was cohorts older our of was 51% ( adults older of age that US population general the in rate the to equivalent is which 52%, was cohort older the in individuals the all for rate hypertension The group. IML the in 9%) or (1/11 few very to compared as hypertension), ‘essential’ as ders. However, 75% (9/12) of IMH subjects were hypertensive (known disor or psychiatric for were found diabetes associations significant No history. all on clinical based and of binary was outcomes scoring clinical these The individual. an of disorders psychiatric and sion hyperten diabetes, of history clinical the to respect with phenotype ( combined were 78 and 62 (age range 66–86) or IML (age range 62–90) individuals from modules ( ( correlated highly were individuals examined all across modules two these of levels expression the Furthermore, in (6 each category IMH and 6 IML, 62 and 78 of for modules overlap Wedegree IML. a significant noted 9 78, for and IMH 7 module phenotypes extreme with 16 individuals and and 10 as as IML, IMH 9 62, categorized for module phenotypes extreme with individuals 19 yielded sorting This Methods). Online for each gene module in 3 or more of the 5 years that were analyzed (see (IML) groups if the they were low in the upper module or lower in quartiles, respectively, inflammasome or (IMH) chronicity high module the inflammasome and into assigned magnitude were Subjects 78. and 62 the modules of levels expression both of basis adults the older of on phenotypes extreme the defined first we so, do To cohorts. aging our in phenotype clinical any with associated was 78 Next, we the investigated expression whether of 62 both modules and Inflammasome modules correlate with health and longevity ( modules gene both together, these data sets show a significant age-dependent increase for are considered they When age. with associated is expression module ( study the in appearance first the method QuSAGE using compared was adults years) >89 to 60 (ages older versus years) 20–30 (ages young in modules gene two these of expression ( to were able previ return who ous years from subjects and subjects new both from data of consisted the from collected samples cohort over five years consecutive (2008–2012) blood from data analyzed we 78, and and activity, such as NLRC5 2a Fig. for enrichment same these (FDR modules significant and found tests hypergeometric we conducted result, this analysis functional by indicated as duction, ( respectively) genes, 17 and 82 of composed 78, and 62 (modules two only these, rate (FDR) discovery (false age with correlated were 41 modules, gene 109 total A  ( status Supplementary Fig. 2b Fig. Supplementary

To determine the stability of the age associations for modules 62 62 modules for associations age the of stability the Todetermine We next performed regression analysis to compare IMH and IML IML and IMH compare to analysis regression We performed next s e l c i t r TLR5 ) and body mass index (BMI) (see Online Methods), yet we still and P ). Module 78 contained contained 78 Module ). = 0.002) ( 0.002) = (module 78) ( 78) (module Q IL1B

≤ Supplementary Fig. 1 Fig. Supplementary 0.05, by Benjamini–Hochberg method) by 0.05, Benjamini–Hochberg IL1RN among other genes that are related to inflammasome 2 1 Fig. 1 Fig. . For this analysis, samples from each individual’sfrom each samples . For analysis, this Fig. 1 Fig. , , Supplementary Fig. 3 Fig. Supplementary TLR6 Supplementary Fig. 1 Fig. Supplementary b ). Thus, to improve statistical power, IMH IMH power, statistical improve to Thus, ). 2 ). a 2 n . The rate of hypertension in unclassified in unclassified . of rate The hypertension ; ; = 23) for further analysis. further for 23) = P and < 10 < n NLRC4 = 114) were used to analyze how how analyze to used were 114) = TLR8 Supplementary Table 1 Supplementary −3 P ; see Online Methods). Online see ; -value for -value < enrichment 0.001). ) participate in cytokine pro cytokine in participate ) (module 62), as well as , and module 62 contained contained 62 module and , Q 2 < 0.01) ( 0 ). Because the age the range ). Because ( 8 P ). , R 15 < 0.01). To 0.01). confirm < 2 , 1 = 0.76, 0.76, = 6 . . Each year’s data Supplementary Supplementary Supplementary Supplementary 1 9 , , and among P ), ), and the < 10 < IFAR1 −5 - - - - )

and we conducted a regression analysis to search for associations associations for search to analysis regression a conducted we ( and 2013 in recruited individuals the all in collected samples We measured the abundance of 62 cytokines and chemokines in serum Stable expression of circulating IL-1 ( 62 module for extent, lesser a to true, ( of 2016 as living still those with compared of the study (year 2008) was in higher who when those were deceased beginning the at 78 module of expression that noted we 2009–2016, years the between died or not they to whether of according age years (11%) ( subjects IMH with compared (88%) subjects IML significantly in was age, higher of years 90 over member family with one family least at a in membership by determined as longevity, familial ( individuals fied ( (7.9 group IML the in lower significantly was PWV The testing. (PWV) velocity wave pulse carotid–femoral using complications, vascular cardio for factor risk stable a is which stiffness, arterial determine to study follow-up a conducted we con confounders, other of potential the tribution in and hypertension of history their in differed pantothenate and CoA biosynthesis pathways ( pathways biosynthesis CoA and pantothenate genes of 4a Fig. ( expression pathways purine the and in pyrimidine the in groups involved two the between differences ( Methods) olism (cut-off metab and purine and biosynthesis, CoA pantothenate metabolism, sion levels of the genes involved in metabolism, pyrimidine CoA and pantothenate of ( biosynthesis the and purine, galactose, viduals were associated with the metabolism of pyrimidine, in indi IMH the that were upregulated metabolites that the revealed IML group ( (FDR and IML the groups IMH between different to found be significantly (SAM) array of micro analysis significance Using the spectrometry. by mass uals we analyzed 692 metabolites from the sera process, of 11 IML and either 9 IMH individ influence could that molecules Toidentify tion. func to order in step activation a subsequent and transcription, ate to initi priming, both requires machinery inflammasome main The Metabolic dysfunction and oxidative stress in the IMH group ( groups IML and IMH the with compared when 2010 year for the different not significantly were and 2011 and 2009 2008, years for the intermediate were adults ( 2008–2011 years ( most the als in the IMH group ( that the levels of 17 cytokines were increased in the serum of individu corrected a calculate P to us enabled which resamplings, 500 using analysis permutation by coefficient statistical regression each the of significance estimated we comparisons, multiple for control To groups after for adjusting classified the age and sex of included. those status. IMH IMH and IML of versus subset the from samples IML used we analysis, and this For levels cytokine circulating between P Fig. 1 Fig. P -value for each regression coefficient. At for -value a each coefficient. regression FDR of < 10

On the basis of our observation that the IMH and IML groups groups IML and IMH the that observation our of basis the On ≤ 0.01) ( 0.01) ± 2.4 m/s) than in the IMH group (10.7 (10.7 group IMH the in than m/s) 2.4 Q c −4 , b < 0.2); all were more in in all group IMH < than the the abundant 0.2); ) and was not significantly different from that in unclassi in that from different significantly not was and ) ) ) ( ), but not of genes involved in the the in involved of genes not but ), Fig. 1 Fig. Fig. Fig. 1 Fig. 2 Fig. Fig. 2 Fig. Supplementary TableSupplementary 4 2 3 P analysis (see Online Methods), 67 metabolites were were metabolites 67 Methods), Online (see analysis advance online publication online advance -value < -value 0.01 and pathway > Online impact (see 0.05) f d Supplementary Fig. 3 Fig. Supplementary a ). The differences in IL-1 in differences The ). a ). ). Moreover, when we over individuals classified 85 ). We next analyzed the differences in the expres the in differences the We ). analyzed next Fig. 1 Fig. ). Our analysis revealed statistically significant significant statistically revealed analysis Our ). Supplementary Supplementary Table 3 Supplementary Fig. 3 Fig. Supplementary g ). The levels of IL-1 of levels The ). ). ). Pathway enrichment analysis b P ). Interestingly, self-reported self-reported Interestingly, ). = 0.068) ( 0.068) = in IMH subjects β β P -alanine metabolism or metabolism -alanine were stable during the the during stable were = 0.018). This was also also was This = 0.018). ), ), with IL-1 β ). ± in unclassified older older unclassified in

P 2.1 m/s) ( m/s) 2.1 < 0.05). Genes that that Genes < 0.05). nature medicine nature Fig. 1 Fig. Q Supplementary Supplementary < 0.2, we found e β ). increasing β β P -alanine, -alanine, -alanine -alanine n = 0.02) 0.02) = = 92), 92), = 2 4 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. metabolism dysfunction. metabolism nucleotide of signs exhibit individuals IMH that demonstrate data ( group IMH Fig. 4a the in found species metabolite the with consistent were others, and 1) diphosphohydrolase phosphate homolog), phoribosyltransferase as such kinase), species, (UMP-CMP thymine-derived and uracil generate to (UTP, CTP) triphosphates nucleotide degrade that proteins encode IMH respectively; 7, (IML 2008–2011 years the during collected data of analysis longitudinal by shown as abundance, ( significance and conducted was status IML/IMH and sex age, their against MFI analyte’s (IML 2013 year from data using subjects IMH and IML between compared were factors growth The individual. one of representative is point ( test. chi-square by obtained was the on was year longevity determined the 2011 familial cohort, basis of in membership a family with at least one member over 90 years of age. The P variable). each for coefficients regression are (shown status IML/IMH and sex age, their against individual each of velocity pulse-wave the on performed was analysis regression Multiple velocity. pulse-wave by measured as stiffness, arterial of degree the with status IMH versus IML of association the ( status). IML/IMH and ( IML on conducted was analysis regression 2008–2012. from taken samples in individuals aged in expression method analysis set gene QuSAGE the 78, and 62 modules gene inflammasome of (see genes inflammasome for enriched were and cohort longitudinal Stanford–Ellison the from 1 Figure nature medicine nature ( subjects IML versus IMH -values in in -values We also investigated the presence of markers of oxidative stress in in stress oxidative of markers of presence the We investigated also ). ). Therefore, and of integration gene metabolomics expression

Expression of inflammasome gene modules in older adults and its association with human health and longevity. ( longevity. and health human with association its and adults older in modules gene inflammasome of Expression d c b a b IML/IMH status

, Density c IML/IMH status 0 2 4 6 8 for each regression coefficient were calculated based on permutation methods (see Online Methods). ( Methods). Online (see methods permutation on based calculated were coefficient regression each for High inyoung IMH group IML group –0.2

n Age Sex 2008, 2009, 2010 and 2011 = 12, 11, 12 and 8, respectively). Whisker bars represent maximum and minimum values. minimum and maximum represent bars Whisker respectively). 8, and 12 11, = 12, 2011 and 2010 2009, 2008, advance online publication online advance Age Sex c ) Seventeen individuals from the year 2011 cohort (the same 8 IML and 9 IMH individuals as in in as individuals 9 IMH and 8 IML same (the cohort 2011 year the from individuals ) Seventeen NT5E mod_062 –0.2 0.0 Individuals withfamilymember>90yearsold(% 0.0 2 0 Fig. 2 Fig. log 0.0 (5 0.2 High inold P 2 e P (fold changeold/young) ′ =0.47 4 0 Regression coefficient -nucleotidase), -nucleotidase), ) Association between the expression of inflammasome gene modules 62 and 78 with all-cause mortality. Each Each mortality. all-cause with 78 and 62 modules gene inflammasome of expression the between ) Association =0.66 Regression coefficient 0.2 Pulse-wave velocity b 0.4 ). Using the metabolomics data, data, metabolomics the Using ). Hypertension ENTPD1 0.4 Longevity 6 0 P 0.2 High inyoung =0.32 n = 11) or IMH ( IMH or = 11) 0.6 2008 2012 2011 2010 2009 4 –0.2 P , -value was obtained by Student’s Student’s by obtained was -value 12 P Supplementary Figs. 1 Figs. Supplementary 8 0 , 1 0.8 =0.11 etncesd tri (ectonucleoside 3 ( UPRT 0.0 mod_078 n = 114) were used to find age-associated gene modules that participate in cytokine production production cytokine in participate that modules gene age-associated find to used were = 114) 1.0 0 P Supplementary Supplementary

=0.02 0.4 1.2 (uracil phos (uracil 0.2 n = 12) group status and hypertension (shown are regression coefficients for age, sex sex age, for coefficients regression are (shown hypertension and status group = 12) P P 100 High inold =0.0016 =0.002 ) CMPK1 0.4 P -value for age on combined data for each gene module, 0.001. ( 0.001. module, gene each for data combined on age for -value and and e

- - mod_062 median

f g

2 expresion in year 2008 –0.10 –0.05 ). For the determination of significant differences in the expression expression the in differences significant of determination the For ). olism, metabolic reprogramming of mitochondrial bioenergetics bioenergetics mitochondrial of reprogramming metabolic olism, ( individuals IML with compared as IMH in higher significantly were ( sera available from 8-isoprostane of individuals( IML with compared as IMH in detected were cystine circulating of levels higher Significantly stress. oxidative of marker important an ( (ROS) species oxygen reactive a and cysteine between reaction direct a from generated is compound the IMH and between IML differed product of groups. This cysteine) oxidized (an cystine of circulating levels the whether we determined 0.00 0.05 0.10 0.15 Fig. 2 Fig.

t IL-1 (standardized MFI) -test. ( -test. β FDR –1.5 –0.5 1 0.5 1.5 0.2 0.4 0.6 0.8 1.0 9 was used. Positive fold change values ( values change fold Positive used. was d Alive in ). Therefore, in addition to the defects in nucleotide metab nucleotide in defects the to addition in Therefore, ). –0.4 IML 2016 f ) Serum levels of 62 different cytokines, chemokines and and chemokines cytokines, different 62 of levels ) Serum 2008 y P -axis) was obtained via permutation tests. ( tests. permutation via obtained was -axis) IM Fig. 2 Fig. =0.068 H –0.2 Deceased by n = 8, IMH IMH = 8, 0.0 0.5 1.0 1.5 2.0 c 2016 n ). In addition, we measured the circulating levels levels circulating the measured we addition, In ). for 2008, 2009, 2010 and 2011 = 10, 10, 8 and 8 and 10, = 10, 2011 and 2010 2009, 2008, for Regression coefficient 0.0 IML 2009 IMH n = 8). Multiple regression analysis on each each on analysis regression Multiple = 8). 0.2 mod_078 median

–0.5 expresion in year 2008 0.5 1.0 1.5 2.0 –0.10 –0.05 0.00 0.05 0.10 0.15 0.4 Fig. 2 Fig. IL-17 IML d IL-6 2010 ) In the same 17 individuals from from individuals 17 same the ) In n IM b = 17) and found that these these that found and 17) = Alive in 0.6 ) were studied to measure measure to studied ) were H a b 2016 IFN- ) Gene expression data data expression ) Gene ), and thus its presence is is presence its thus and ), x -axis) indicate higher higher indicate -axis) –1.4 –1.2 –1.0 –0.8 –0.6 –0.4 P γ =0.018 0.8 IL-23 IL-1 Deceased by IML β 2011 s e l c i t r a b 2016 g ) A logistic ) A logistic IM ) ) IL-1 H β serum serum P -value -value  -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. adenine (purine metabolism), metabolism), (purine adenine included compounds These pathways. each metabolic that distinct represent analysis our from identified compounds candidate four ulate higher levels in the sera from IMH in as found compared are that to IML subjects metabolites upreg circulating the whether Todetermine Metabolites in IMH subjects elicit inflammation stress. oxidative chronic subsequent and ROS of levels high constitutive to lead may individuals IMH in variability. experimental reflect bars Error to the control. in ratio relation as the normalized are expressed and the results the samples, to used standardize Student’s one-sided in increase A significant determination. for used cytokine on the samples same ( but compounds. not the other adenine of IL-1 induction A significant control. was as used a positive Adenosine experiment. representative to IL-1 compound’s alter ability ( of significance of levels basis their dl of ( levels of Circulating ROS. in the presence ( MetPAusing conducted were analysis and pathway IML in at IMH an versus FDR of upregulated) (all expressed differentially ( 2011 year from samples serum on available was conducted profiling 2 Figure A  metabolism), tyrosine and phenylalanine (vanillylmandelate; acid P

-4-hydroxy-3-methoxymandelic acid (vanillylmandelate) (MMA), scyllo-inositol and scyllo-inositol (MMA), (vanillylmandelate) acid -4-hydroxy-3-methoxymandelic < 0.05). Darker color indicates higher level of significance. ( of level significance. higher indicates color < Darker 0.05). s e l c i t r NLRC4

Metabolites present in IMH individuals induce IL-1 induce in IMH individuals present Metabolites d a c 8-Isoprostane (pg/mL) Cystine (normalized values) –log(P) gene expression and/or cytokine production, we selected 20 30 40 10 0.8 1.0 1.2 1.4 0 2 4 6 8 0 0.00 metabolism Galactose t -test). Adenosine treatment upregulated upregulated treatment Adenosine -test). metabolis IML β IM -Alanine L P 0.05 =0.01 P Pathway impact =0.02 m α and IL-1 Purine metabolis Q Pantothenate an dl CoA biosynthesi IMH IMH < see 0.001; -4-hydroxy-3-methoxymandelic -4-hydroxy-3-methoxymandelic 0.10 Pyrimidine metabolis f ) The highest dose of each compound (100 (100 of dose ) compound each The highest β levels and the expression of and the expression levels c ) cystine and ( ) cystine m e s d

IL-1α (normalized MFI) IL-1β (normalized MFI) 0.15 3 –1 –0.5 3 Supplementary Table Supplementary 4 0 1 2 3 4 0.0 0.5 1.0 1.5 . A significant enrichment for several metabolic pathways was identified for these metabolites metabolites for these was identified pathways metabolic for several enrichment . A significant Adenosine methionine metabolism Adenosine m Cysteine and * * β d NLRP3 and upregulate the expression of inflammasome genes. ( genes. of inflammasome the expression and upregulate Adenine ) 8-isoprostane are greater in IMH as compared to IML individuals. ( to in IML individuals. IMH as are greater compared ) 8-isoprostane

b Adenine ) The conversions of cysteine to cystine and of arachidonic acid to 8-isoprostane to acid 8-isoprostane and of to arachidonic cystine of cysteine ) The conversions * * gene expression ( expression gene n MMA = 9 IML, IL-1

NLRC4 MMA IL-1 Scyllo-inositol Q β - Scyllo-inositol b < 0.2 (by SAM analysis; see Online Methods). Functional annotation annotation Functional Methods). see Online < 0.2 (by SAM analysis; ) and their representation of different metabolic pathways. We each pathways. assessed metabolic of different ) representation and their 2

α H to to approximately in of one-half the blood the observed concentration used at the same concentration (100 wide range of PAMPs and DAMPs 100 at on chosen the basis of previous showing reports that (used adenosine of or for either adenosine adenine 6 h. was The concentration highest 100 and 30 10, 3, (0, concentrations increasing with incubated and blood fresh from isolated were donors healthy young, four from IL-1 for control positive a as midine metabolism) ( scyllo-inositol (inositol metabolism) and NLRC4 in primary monocytes from four healthy young adults. Results show one show Results adults. young healthy four from monocytes in primary n N H = 11 IMH). From a total of 692 metabolites analyzed, 67 were analyzed, = a 11 From of IMH). total 692 metabolites Cysteine µ S

N N4A M) was used to determine expression of expression M) was to used determine µ and 4 N4A P -acetylcytidine (N4A) were selected for further study on study the for further selected were (N4A) -acetylcytidine O M) can regulate inflammasome activity that is initiated by a by initiated is that activity inflammasome regulate can M) H ROS < 0.01 by one-sided Student’s < by 0.01 one-sided O NLRP3 H f advance online publication online advance NLRC4 mRNA Arachidonic acid

NLRP3 mRNA 0 1 2 3 is shown only for is only shown α

0 1 2 3 Adenosine and IL-1

Supplementary Table 4 Adenosine HO HO H * Adenine O H Adenine β O was observed when using adenosine and adenosine using when was observed N H β 10 S S production O 2 NLRC4 O H N

N MMA 5 HO NLRP3 µ . . The other compounds were also

MMA OH 4 Scyllo-inositol a O H -acetylcytidine ( -acetylcytidine M), M), which for N4A corresponds ) Broad-coverage metabolomics metabolomics ) Broad-coverage t

Scyllo-inositol Cystine -test). Expression of was GAPDH Expression -test). H N 8-Isoprostane 4 -acetylcytidine (N4A; pyri NLRC4 ). Adenosine was included 2 5 . Primary monocytes monocytes Primary . e N4A

) Adenine, ) Adenine,

N4A * and nature medicine nature * P < by 0.05, COOH NLRP3

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. oxide (NO) biosynthesis and platelet degranulation that is secondary that is secondary and degranulation platelet oxide (NO) biosynthesis and systems) overactivity, platelet renin–angiotensin nitric decreased tension neurohumoral (sympathetic include dysfunction, endothelial controls. healthy, disease-free with compared when platelets vated acti of levels high exhibit hypertension with patients that evidence indicates accumulating and origins, non-infectious and infectious conditions of both in occurs inflammatory various Platelet activation N4A and adenine activate platelets and neutrophils activation. inflammasome NLRP3 on stimuli IL-1 ( adenine of from macrophages marrow-derived bone and shNLRC4 express stably that cells THP-1 YVAD-fmk ( inhibitor complete the showing IL-1 compound-mediated of by abrogation demonstrated further was dependency ( compounds two ment with biotinyl-YVAD-fmk frag fromp10 active THP-1the cells immunoprecipitating stimulated with specifically by these confirmed was fragment p10 into active the cleavage procaspase-1 and adenine, IL-1 6 Fig. ( Supplementary analysis of western-blot abundance by the protein NLRP3 in and increase NLRC4 N4A-induced an observed also we mRNAs, NLRP3 and NLRC4 of increase N4A-dependent the with is effect likely on dependent In activation. inflammasome accordance IL-18 was for not TNF- observed of IL-1 on production the of and adenine N4A effect binatorial ATP. com with This cells pulsing by augmented further was which induced a significant increase in IL-1 in increase significant a induced ( ence of N4A induced the secretion of IL-1 cytokines) or TNF- IL-1 of production combination. Neither adenine nor N4A alone significantly influenced or in alone either adenine and N4A with line, cell monocytic human genes. inflammasome of expression ground suggests that there is at least a degree of or upregulate not does treatment adenine That hypothesis. this secretion of IL-1 a second signal for NLRC4 prime may stress), oxidative of marker (a tRNA degradation the in product nucleoside endogenous an N4A, that indicate results These ( observed was scyllo-inositol or acid 3-methoxymandelic NLRC4 treatment trast, with N4A induced the expression of both in increase no However, of expression the increased treatment adenosine uli upregulate the expression of of expression the upregulate uli compounds tested ( ( IL-1 individuals healthy of nature medicine nature Supplementary Fig. Supplementary 5 Fig. 3 Fig. Putative mechanisms that contribute to platelet activation in hyper We then investigated the role of a cells, THP-1 differentiated treated we experiments, parallel In NLRC4 NLRC4 β β β secretion that occurs upon stimulation of THP-1 cells with N4A was observed only for the adenosine and adenine treatments treatments adenine and adenosine the for only observed was secretion, which is consistent with the dependence of these these of dependence the with consistent is which secretion, a expression; in contrast, adenosine and adenine may generate and adenine adenosine in contrast, expression; ( ). Moreover, treating cells with adenine and N4A together together N4A and adenine with cells treating Moreover, ). P Fig. Fig. 3f, significantly reduced the IL-1 but induces IL-1 induces but < 0.01). No significant effect of adenine, adenine, of effect significant No 0.01). < β g . . More studies biochemical are to necessary validate ). shNLRC4 ). had shNLRC4 no on effect the LPS/ATP-induced Fig. 3c Fig. α NLRC4

NLRC4 β Fig. Fig. 2 ( , IL-18 (another member of the IL-1 family of of family IL-1 the of member (another IL-18 , advance online publication online advance Fig. 3 2 Fig. 3 Fig. ). Consistently with the observed increase in in increase observed the with Consistently ). ), ), and no induction was for observed the other 6 . A significant dose-dependent increase in in increase dose-dependent significant A . e , NLRC4 d activation that results in the induction and ). ). We also investigated stim these whether a knockout mice. Knockdown or knockout or knockout Knockdown mice. knockout and and ). However, the addition of ATP in the pres β e production from primary monocytes monocytes primary from production ). α NLRC4 Supplementary Fig. 6 Fig. Supplementary expression was observed. In con In observed. was expression NLRC4 , , which indicates that the observed β β in vivo β and and β and IL-18 levels ( levels IL-18 and secretion induced by N4A + in IL-1 and IL-18 but not of TNF- secretion by the caspase-1 caspase-1 by the secretion NLRP3. priming causing back β secretion by secretion using NLRP3

As expected As dl in vitro ). Caspase-1 Caspase-1 ). -4-hydroxy- Fig. 3 Fig. NLRP3 ( Fig. 3 Fig. Fig. 2 Fig. Fig. 2 Fig. NLRP3 c β and and and and b 2 f f α 5 ). ). ), ), ). ). ------,

population of neutrophils ( neutrophils of population combination of N4A + adenine induced an increase in a degranulated ( lation induced a potent increase in RANKL IL-1 and activation trophil also investigated the effects of N4A + adenine on primary human neu ( Methods Online see entially expressed in monocytes, macrophages and neutrophils ( granulocytes. activated of numbers ing donor) ( each ( effect dose-response significant in resulted a statistically of CD61 tration reach to cells Activation maximal effects. these was monitored by in measuring the concen used previously those than N4A of trations concen lower used we thus and monocytes, primary or cells THP-1 by activated those than sensitive more were platelets N4A-activated (5 ADP chosen for used were concentrations activating adenine standard on of based concentrations tested The adults. young in vitro mine N4A whether and were adenine able to activate human platelets pathway (NLRP3) inflammasome the through shear increased to T regulatory CD4 T regulatory including monocytes, granulocytes, NK cells, CD4 STAT3, STAT1, STAT5, CREB, NF- S6, p38, including proteins, signaling intracellular We also compared the levels of expression of a series of phosphorylatedexpres NF- the the of sion compare to of (CyTOF) end the cytometry at mass group used per We mice study. 10 the of out 6 from samples, aorta and kidney blood, peripheral collected and group per mice 10 using hypertension. sustained induce to compounds these of effect the to pre-hypertensive addition in needed but be may stimuli mice, in pressure blood elevate can adenine ( 112 versus ( (140 group control the to compared when mice treated the in pressure blood systolic average in increase significant a in This resulted 20. day at mice) control the (in (in PBS with compounds or mice) the treated the with combination in angII administered sustained and during jointly stress- act hypertension oxidative response an and inflammatory (angII), dependent II pre-hypertensive angiotensin that as observation such previous stimuli, the of basis the On neutrophils were able to secrete relatively low concentrations of IL-1 significant increases in blood pressure (pre-hypertension) as early early as 8 as d after the first injection(pre-hypertension) ( pressure blood in increases significant Methods). Online (see treated the for in used concentration the pressure times 10 is which mM, blood 1 of final compounds a these in of use concentration to chose We increase below). see the plateau; reached where mice (time d for method cuff 34 tail a using pressure blood in changes monitored blood on effect direct a pressure had adenine and N4A whether study To IMH metabolites induce hypertension and inflammation in mice compared ( to (a untreated 2- cells) to increase 3-fold Computational analysis revealed that modules 62 and 78 were prefer To gain more insight into this effect, we repeated this experiment experiment this repeated we effect, this into insight Tomore gain Treatment with N4A and adenine had a mild effect with borderline- using platelets that were isolated from the blood of two healthy Fig. 4 Fig. in vivo in ± c Fig. Fig. 4 3 3) mmHg; mmHg; 3) ). In addition to promoting expression of RANKL, the the RANKL, of expression promoting to addition In ). 1 , we implanted angII pumps (at 140 ng/kg/min) ng/kg/min) 140 (at pumps angII implanted we , κ + , we injected mice with these compounds daily and and daily compounds these with mice injected we , and CD62P B inhibitor I inhibitor B + T dcells and B cells from both compound-treated compound-treated both and from B T cells dcells 27– a ) with higher doses of adenine inducing increas inducing of doses adenine ) higher with κ 3 B, ERK and MAPKAPK2, in blood cell subsets subsets cell blood in MAPKAPK2, and B, ERK Fig. 4 Fig. 0 . Since viral infection can activate platelets platelets activate can infection viral Since . P β = 0.016) ( 0.016) = Fig. 4 Fig. b secretion. Adenine, in contrast to N4A, N4A, to contrast in Adenine, secretion. P in vitro in + and and κ = 0.04 for group comparison) ( cells by flow cytometry. Only adenine adenine Only by cytometry. flow cells B and the activation marker CD62L. CD62L. marker activation the and B d Supplementary Fig. 7 Fig. Supplementary ). Finally, N4A + adenine-treated adenine-treated + N4A Finally, ). + cells within the CD66b cell popu studies in primary monocytes monocytes primary in studies Fig. 5 Fig. a ). Therefore, N4A and and N4A Therefore, ). 2 8 , we sought to , deter we sought + s e l c i t r a and CD8 Fig. 4 Fig. P ). Thus, we Thus, ). < 0.05 for e P ). + Fig. 5 < 10 T cells, in vivo in µ M). M). ± −10 a 7) 7) ). β  ­ ------;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. secretion was measured. Data was in measured. secretion panels described as were obtained previously of NLRC4 expression in protein blots showing cell ( shTHP-1 stable Ctrl, lines. control. type (WT) or cell shTHP-1 stable as lines were with treated compounds (1 before mM N4A; 300 as with compounds in before the or presence of absence Ac-YVAD-fmk or of DMSO, and control then IL-1 p10 antibody. with anti-caspase-1 beads and were immunoblotted ( by recovered using streptavidin-Sepharose the were earlier, then same outlined Complexes process peptide. using biotinyl-YVAD-fmk and the to cell were lysates submitted immunoprecipitation of and (casp1) pro-IL-1 NLRs, to expression caspase-1 cellular monitor antibodies (1 mM N4A; 300 1 or they were with primed LPS (1 3 Figure in pCREB and pS6 pNF- and of increased and in granulocytes monocytes levels higher by evidenced as mice, treated compound- in activation immune of state a We observed position). and control mice (see A  (mean indicated

β N , , IL-18 and TNF- s e l c i t r 4 -acetylcytidine (N4A) alone ( -acetylcytidine

Metabolites in IMH individuals activate the NLRC4 inflammasome. ( the in activate NLRC4 inflammasome. IMH Metabolites individuals c b a IL-1β secretion (pg/mL) IL-1β secretion (pg/mL) 1,000 1,500 2,000 ATP N4A 1,000 1,500 2,000 ± Ad 500 500 µ s.d.; M adenine M (Ad)) for adenine 6 h or with ATP 5 mM for 30 min. Then cells were with and lysed, various the were lysates immunoblotted 0 α 0 – – – was ( by measured ELISA from cell supernatants. culture Supplementary Table 5 n + – –

= 3 for NT NT ATP + – – LPS/ATP ATP

µ Ad 0.1 g/ml, g/ml, 4 h) and then with pulsed ATP, (mM) or of (Ad) they adenine either were with treated concentrations the indicated + + – Ad 0.3 LPS/ATP a a ,

b N4A 0.1 ) ) or ( in combination Ad 0.3 + – – , + N4A0.1

e N4A 1.0 4 , Ad 1.0 6 f Ad 0.1 + + + ; ; . Cells were plated in triplicates, treated with combination of . (1 with compounds mM treated Cells combination were in plated N4A; 300 triplicates, + ATP a Ad 0.3 n ,

b N4A 0.3 = 9 for

Ad 0.1 * , ** e Actin Pro-lL-1 Casp1 p20 Casp1 p45 AIM2 NLRP6 NLRP3 NLRC4 Ad 0.3

– N4A 1.0 + N4A g Ad 0.1 + ATP are expressed as the concentration of cytokines (pg/mL) or of (pg/mL) as are as cytokines expressed the the concentration with fold to respect as increase baseline

+ ATP N4A 0.3 Ad 0.3 * for antibody panel com * g β Ad 0.1 1

). ). N4A 1.0 Ad 0.3 P -values were determined by were -values Student’sdetermined f d b ) ) for 6 h. NT, They were then with pulsed nontreated. either ATP or of IL- not. Secretion cytokines IL-1β secretion (pg/mL) IL-18 secretion (pg/mL) IL-18 secretion (pg/mL) κ 1,000 1,500 2,000 B in monocytes B in monocytes 100 200 300 400 500 600 100 200 300 400 500 600 500 IP biot-YVAD 0 0 Input lysate 0 Sup NT NT NT ATP N4A ATP Ab /ATP LPS LPS/ATP ATP Ad 0.1 – – – – + – LPS/ATP

N4A Ad 0.3 - N4A 0.1 Ad 0.3 + N4A0.1 a N4A 1.0 c + + – – + – , under acute conditions that the reduction of total I total of reduction the that only is conditions it acute that under fact the by explained be can disagreement apparent This controls. to compared as mice treated in elevated also were els ( controls untreated to compared as mice these in ) Differentiated THP-1 as cells were with treated ) compounds indicated Differentiated Ad b Ad 0.1 Ad 1.0 ) Differentiated THP-1 cells were with treated ) ATPDifferentiated (5 mM, 30 min), or + ATP

β Ad 0.3

. . ( N4A 0.3 N4A

Ad 0.1 * Ad + + + + + – * d shNLRC4 shCtrl WT Ad 0.3 g THIP-1 cells ) Differentiated THP-1 using cells were with treated ) compounds Differentiated

+ N4A N4A 1.0 ) Bone-marrow-derived macrophages from WT macrophages or mice NLRC4-KO ) Bone-marrow-derived ctrl + ATP

sh Ad 0.1

+ ATP N4A 0.3

Ad 0.3 * NLRC4

Casp1 p10 Casp1 p45 Ad 0.1 1 t

sh N4A 1.0

-test. * -test. Ad 0.3 µ advance online publication online advance Actin NLRC4 M M Ad) and IL-1 TNF-α secretion (pg/mL) TNF-α secretion (pg/mL) 10,000 15,000 20,000 10,000 15,000 20,000 P 5,000 5,000 < 0.05, ** e g β 0 0 secretion was measured. ( was measured. secretion Fold increase IL-1β secretion

0.0 0.5 1.0 1.5 2.0 (pg/mL) NLRC4 e 1,000 1,500 NT NT ) Differentiated THP-1 cells were treated ) Differentiated Casp1/11 500 LPS β

P –/– **

0 LPS secretion was measured. Right, western western Right, was measured. secretion ATP < 0.01. LPS/ATP –/– ATP * LPS/ATPNT Ad 0.1 LPS/ATP NLRC4WT Ad 0.3 N4A 0.1 Casp1/11 Ad 0.3 –/– + N4A0.1 N4A 1.0 ** Ad 0.1 Ad 1.0

– + ATP –/– Ad 0.3 ** +YVAD N4A 0.3 +ctrl DMSO N4A+Ad Ad 0.1 WT µ Ad 0.3 N4A 1.0

+ ATP + N4A M adenine) M and adenine) IL-1 f

) Differentiated wild- ) Differentiated Ad 0.1 nature medicine nature N4A 0.3 + ATP

Ad 0.3 5 Fig. Ad 0.1 N4A 1.0 IL-18 IL-1 1 Ad 0.3 κ B tracks with with tracks B β b ). I ). κ B lev B

β -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. of cytokines (pg/mL) or as the fold increase with respect to the non-treated (NT) condition as indicated. indicated. as condition (NT) non-treated the to respect with increase fold the as or (pg/mL) cytokines of IL-1 and figure, ( donor. each from obtained results summarizes right at Graph plot. scatter the in shown as measured was population degranulated of percentage the and mM), (1 adenine and/or mM) (1 N4A with CD66b the within staining RANK-L of overlay RANK-L of concentration the combination. in or separately either N4A ( lymphocytes. ABTC, granulocytes; GN, monocytes; MN, MP, cells; macrophages; dendritic DC, B cells; ( respectively) blue and red (orange, granulocytes and monocytes macrophages, in expressed predominantly are 78 Student’s (0.5 Adenine for population positive CD61 the within staining CD62P of overlay show Histograms cytometry. flow by cells healthy in CD62P and CD61 of expression membrane the measuring (3 ADP thrombin, 6 h with for incubated were and donors 4 Figure pNF- I increased pNF- nature medicine nature t -test. * -test. κ B B signaling following stimulation returns to baseline at a time when κ B levels are still elevated still are levels B P

< 0.05, ** < 0.05, Metabolites in IMH individuals activate human primary platelets and neutrophils. ( neutrophils. and platelets primary human activate individuals IMH in Metabolites t -test with * with -test d c a Count Count Percent of maximum c RANK- CD66b ) Primary neutrophils were isolated from blood of healthy donors and then incubated for 24 h with various concentrations of adenine or adenine of concentrations various h with 24 for incubated then and donors healthy of blood from isolated were neutrophils ) Primary β

SSC κ secretion was measured from the cell culture supernatants obtained from each donor. Data are expressed either as the concentration concentration the as either expressed are Data donor. each from obtained supernatants culture cell the from measured was secretion B levels; under chronic conditions, the initial reduced FS Donor 1 CD62

P L C NT < 0.01. < 0.01. P advance online publication online advance P3 < 0.05 applies to each donor. ( donor. each to applies < 0.05 P 1.7 NT 1 Ad 3 2 Ad 3mM Donor CD62P . P3 2 28 µ N4A +Ad M) and N4A (100 (100 N4A and M) + N4A population. Graph below summarizes the results of each donor. ( donor. each of results the summarizes below Graph population. e N4A Ad ADP Thromb NT ) Primary neutrophils were treated as described previously with the compounds as indicated in the the in indicated as compounds the with previously described as treated were neutrophils ) Primary N4A 3mM P3 b 1.6 ) Analysis of immune cell type populations (ImmGen database) show that modules 62 and and 62 modules that show database) (ImmGen populations type cell immune of ) Analysis + +

% CD61 CD62P cells µ 8 80 20 40 60 M) or various concentrations of adenine or N4A. Platelet activation was monitored by monitored was activation Platelet N4A. or adenine of concentrations various or M) 0 e Fold increase % degranulated µ

M) for each donor. Bar graph summarizes the results obtained from each donor. each from obtained results the summarizes graph Bar donor. each for M) NT IL-1β secretion neutrophils + +

0 1 2 3 4 5 0 2 4 6 8 % CD66b RANKL cells Percent 25 30 10 15 20 Thromb + Ad0.1 0 5 N4A 0.1

NT of maximum

+ ADP cells present within the CD66b the within present cells **

NT Adenine + Ad N4A 0.01 0.1 RANK- + Ad0. N4A 0.1

0.05 * treated mice when compared to the controls ( ( (cortex) Ad 0.3 Ad ( L We also observed significant T cell infiltration in the kidneys kidneys the in infiltration cell T significant observed also We µ 0.1

1 M) 3

N4A 0.5 N4A (mM + Ad1

N4A 3 N4A N4A 3mM N4A 1mM Ad 3mM Ad 1mM NT 0.1 25 Donor Donor P 1 α ( < 0.001) but not the aorta ( aorta the not but 0.001) <

1 µ 50 (mM β M) ) 2 1 T cells; GDTC, GDTC, T cells; a 3 ) Primary platelets were isolated from blood of healthy healthy of blood from isolated were platelets ) Primary 100 ) b SC BC DC MP MN GN ABTC GDTC STC IL

+ 20.00 P population was determined. Histograms show show Histograms determined. was population -values in in -values 5 Module 62 γ

δ 2 T cells; STC, stromal cells; IL, innate innate IL, cells; stromal STC, T cells; d mRNA expression ) Primary neutrophils were treated treated were neutrophils ) Primary 0.5

P 0.2 a < 10 , d

, 0.05 e P were determined by Student’s Student’s by determined were = 0.372) in N4A + adenine- + N4A in 0.372) = −10 20.00 ). SC, stem cells; BC, BC, cells; stem SC, ). Fig. 5 5 Module 78 2 c ). These differences 0.5 s e l c i t r a 0.2 

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. tionnaire consisting of a 15-item survey of dietary and pharmaceu and dietary of survey 15-item a of consisting tionnaire with Toa ques we used expression. this, do associated gene were inflammasome-module levels caffeine whether asked simi next we structural larity, their to due adenosine antagonizes caffeine Since Caffeine inhibits metabolite-induced IL-1 ( mice control in found were that those than mice treated in present infiltration cell T of levels higher with cortex the in trend similar observed a we but medulla, kidney the in significance reach not did S6. ( protein pNF- FDR. to the the Methods); Online (see of the mice two groups comparing of SAM analysis are the results Shown alone). angII with (treated mice control versus mice treated adenine-angII ( values. and minimum maximum represent bars respectively. Whisker groups, and control for the treated the values mean represent lines horizontal and empty Filled results. similar with per mice group to 112 ( compared t ( as as early 8 states d injection the first after Treatment pre-hypertension had with N4A a with + effect mild adenine ( 5 Figure A  of 120 from derived Methods), Online (see caffeine of sources tical -test). Significant increases were observed after day 20 in the treated mice, with an average systolic blood pressure of 140 ( pressure blood systolic an with day average 20 mice, after in the treated observed were increases Significant -test). n P

= 4) group. The treatment group mice were injected with N4A + adenine at 20 mM stock solution of at 100 N4A with + 20 mM solution adenine stock injected were mice group = The treatment 4) group. = 0.09) ( 0.09) = s e l c i t r t -statistic value of value a -statistic

Metabolites in IMH individuals induce high blood pressure in ( mice. pressure blood high induce in IMH individuals Metabolites N4A +Ad c a κ Control B, phosphorylated form of form NF- B, phosphorylated Supplementary Fig. 8 Fig. Supplementary c ) Higher levels of infiltrating T cells in the kidney, but not the aorta, are observed in the treatment group versus the control group. the control T in versus cells the kidney, group in of the infiltrating treatment ) levels are observed but Higher not the aorta, CD3+ cells/field 10 20 30 40 50 0 Blood pressure (mm Hg) ± 160 100 120 140 3) mmHg in the control group ( group 3) in mmHg the control 60 80 Control x N4A +Adenine t -axes represent the FDR or significance (cutoff value of ( value 5%) of (d) as score a (cutoff parameter function the FDR or significance represent -axes -test when comparing two samples. two samples. comparing when -test 5 1 P Kidney <0.001 N4A + ). 1 8 * κ A 1 2 B (p65 Ser529); pCREB, phosphorylated form of CREB (Ser133); pS6, phosphorylated form of 40S ribosomal of form 40S ribosomal phosphorylated pS6, of form (Ser133); CREB phosphorylated pCREB, B Ser529); (p65 PBS Time (d) * 1 4 b

b +

) Increased phosphorylation levels of signaling proteins (FDR (FDR proteins of signaling levels phosphorylation ) Increased CD3 cells/field production 10 15 P 0 5 = 0.008). * = 0.008). 2 8 AngII * Control 2 5 ** 3 9 P Aorta P = 0.372 P -values were adjusted for multiple comparisons using SAM, which provides an estimate of an estimate provides which SAM, using comparisons for multiple adjusted were -values < ** 0.05, ** 2 N4A + 34 ** - - - A

20 P × a < 0.01 by one-tailed < by 0.01 one-tailed feine intake and the expression of modules 62 ( 62 ( modules of expression the and intake caf feine between association BMI-adjusted and sex- age-, significant a We intake. found known caffeine with a associated is that factor BMI, confounding for adjusted also We mg/week). (in intake caffeine year the in multiple ( 2008 individuals all performed Wefrom data 2008. the using year analysis the regression in subjects 91 to istered www.cspinet.org/new/cafchart.ht see Interest; Public the in Science for (Center 2007 in States United the in products caffeinated consumed commonly most the ) Mice were randomly put into either the control ( the control put either into ) randomly were Mice P b = 0.024) ( 0.024) = Cell expression (median) Cell expression (median) Cell expression (median) Score (d) 0.6 0.2 0.3 0.4 0.5 0.6 2.2 2.6 0.4 1.8 3.0 0.0 0.2 1.0 0.8 –4 –3 –2 –1 N = 89) on the expression of modules 62 and 78 and their their and 78 and 62 modules of expression the on 89) = 5 0 N4A + N4A +A N4A +A Granulocytes pCREB Granulocytes pS6 Fig. 6 Fig. Granulocytes IkB SAM analysis 10 advance online publication online advance A FDR 15 a ). t -test. These experiments were repeated using 10 using repeated were experiments These -test. Control Control Control 20 25 µ L L per 25 daily.once g weight body 30 Q < 0.05) in blood cells from N4A from + < in cells 0.05) blood Cell expression (median) Cell expression (median) Cell expression (median) Cell expression (median) m P 1.5 1.0 0.8 1.0 2.0 0.2 2.5 0.4 0.6 18 20 22 24 12 14 26 28 30 13 11 15 = 0.02, by one-tailed Student’s = by 0.02, one-tailed ). The questionnaire was admin was ). questionnaire The N4A + N4A +A N4A +A N4A +A ± Monocytes pCREB Monocytes pNFkB y 7) mmHg in that group, as 7) in mmHg group, that Monocytes pS6 Monocytes IkB -axis), which is equivalent is which equivalent -axis), n A = 4) or the treatment nature medicine nature P Control Control Control Control < 0.01) and 78 78 and 0.01) < http://

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. plasma of coffee drinkers (10–50 (10–50 the drinkers in coffee found of plasma concentrations estimated the matched that trations ( ( group IMH the to compared when metabolites these of sidered jointly without adjusting for multiple comparisons. We 1-methylxanthine, found and that, when theobromine con theophylline, acid, methyluric 1,3,7-tri paraxanthine, metabolites its and caffeine of levels serum compared and generated previously was that data metabolomics the metabolites in the sera from IMH and IML subjects. To do so, we used for expression. protein antibodies with indicated was by assessed to qPCR. Cell were lysates submitted immunoblotting (at caffeine 0.5 mM) for 6 h, of and genes as various expression indicated cells were with treated N4A (at 1 mM) in the or presence of absence by Student’sdetermined (mean of (pg/mL) as cytokines concentration Data are was expressed from cell measured supernatants. secretion culture then with treated N4A (at 1 mM) + (at adenine 300 of and (caff) caffeine were with concentrations cells were various incubated and ( maximum values. minimum represent tests from dependent probabilities combining P IML and IMH using were groups a computed Student’sone-tail the between metabolites of levels the and coffee circulating coffee-derived expression of modules 62 ( ( for for intake in age, each (adjusted mg/week sex individual and BMI). 62 and 78 in (from data collected year 2008, of levels on was analysis module conducted expression regression Multiple 6 Figure nature medicine nature IL-1 of inhibition cant a) Fig. 6 Fig. b a c –0.4 -values were combined using a modified generalized Fisher method for method Fisher using were a -values combined generalized modified We then compared the levels of caffeine and caffeine-derived caffeine-derived and caffeine of levels the compared then We A significant association was found between caffeine intake and was intake caffeine the found between association A significant IL-1β secretion (pg/mL) Metabolite relative levels (log2) 200 15 10 –6 –4 –2 50 0 2 0 0 0

Ca Re –0.2 b Pa

1,3,7 ff Ca gression coef

). We also tested the effect of caffeine caffeine of effect the tested also We ). eine NT Caffeine negatively regulates the NLRC4 inflammasome. the NLRC4 regulates inflammasome. negatively Caffeine raxanthine BM ff Ag -T Se rimeth eine e x I 5 0 0.0 N4A +

3 Theobrylurat Module 62 P 3 P P , , the IML group exhibited elevated significantly levels =0.7 =0.008 0

e =0.8 * Theophomine f 500 icient Ad

1-Meth 0.2 advance online publication online advance Ca ylline t β ylx -test. * -test. ff secretion ( secretion P anthine ( P 0.4 µ < 0.01) and 78 ( <0.0 M) Ca 1,3,7 Pa P ffeine = P raxanthine Normalized abundance -T 1 10 < 0.05. ( 2 0 1 3 r NIR imeth –5 C4 ylaurat NT Theobr –0.4 Fig. 6 Fig. µ NAIP

M) e

N4A Theophomine c –0.2 c NLRP3 Re ) Left: Differentiated THP-1 ) Left: Differentiated 1 Ca ± ) Right: Differentiated THP-1 ) Differentiated Right: 3

3 -Meth P c 4 n s.d.; gression coef 3 ) that is due to a decrease decrease a to due is that ) = 0.024). ( W osre a signifi a observed We . ff = 89) and caffeine (

ylline BMI Ag Se N4A+C ylx eine P DDX58 e anthine x < bars Whisker 0.01). 0.0 n µ PY Module 78 P P af = 3). M) M) for 6 h. IL-1 =0.024 =0.94 in vitro in C f ARD IM IM f 0.2 icient L H

CA NT b P SP1 P ) ) Differences in =0.04 -values were -values – N4A at concen at t P +C 0.4 -test, and -test, =0.003 P af

< 0.01) 0.01) < f NL A β ctin

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activation at the protein level. The regulation of of regulation The level. protein the at activation mod6 cohorts ( of the mice in decline functional and nescence with interact also can response, rats in study recent a by for a previously, role cited to correspond These 78. and 62 modules in identified were two humans, in genes NLR 23 the among example, For extensively. studied been not has inflammation age-related in role their but pathogens, infectious to these modules (62 in and 78 here) are genes known to be a part of the inflammasome response The longevity. familial of lack a and stress oxidative dysfunction, metabolic cytokines, inflammatory of levels chronic stiffness, arterial pressure, blood elevated of occurrence the with correlates individuals older in modules gene inflammasome lar of particu expression persistent and elevated the that Here show we DISCUSSION inflammasome. NLRC4 the ( analysis in molecular pattern molecules (DAMPs) that act together to induce induce to together act that (DAMPs) molecules pattern molecular Therefore, these two maturation. metabolites can be considered danger-associated cytokine and 2) (signal activation inflammasome gers enable to monocytes primes N4A that appears it expression, protein and gene of ATP, presence the in increases not (but adenine) and N4A cells to secrete IL-1 activation able inflammasome to trigger IL-1 and activity to inflammasome contribute may also compounds these that gesting in increased IMH compared with IML) ( enosine, N including and adenosine-derivatives, in adenosine found differences IL-1 and activity inflammasome sustain to known is which adenosine, forms adenine of The ribosylation mice. in hypertension causes actively and adults older in sion of hyperten the occurrence with and N4A correlates adenine bolites data. our from drawn suggestions the corroborate levels serum of cytokines) family IL-1 the of member (another IL-18 increased with occur both affect rs17229943, rs1842893, rs2290414, rs34649619 and rs149451729) that (rs385076, polymorphisms of single-nucleotide existence the tifying Two sion dysfunction. and iden studies vascular recent independent other genes, inflammasome such as to hypertension to were susceptibility linked age suggested in two other studies: first, in a mouse model of kidney dam tial hypertension 62. module of program (see including genes, factor transcription type-I-IFN-dependent signaling IFN type-I on depends sion 1 We find that N4A and when adenine, applied in combination, were meta the of levels the of elevation the that show also results Our In addition to the reported elevation of IL-1 -methyladenosine, NLRC4 3 9 http://cs.un , and second, in humans, where non-coding non-coding where humans, in second, and , 3 NLRP3 ), ), N Q NLRC5 Fig. Fig. 6 NLRC4 gene expression, as indicated by both qPCR and protein protein and qPCR both by indicated as expression, gene 6 = 0.202, NLRC4 -succinyladenosine and 5-methylthioadenosine (all being and 5-methylthioadenosine -succinyladenosine gene was not strongly with associated age in our human d NLRC4 expression may expression promote the inflammasome NLRP3 ). Therefore, caffeine seems to of the seems impair priming ). caffeine Therefore, c.edu/~vjojic/fluy2- β 36– expression (signal 1) and that adenine then trig then adenine that and 1) (signal expression production in the IMH group. IMH the in production β expression and function and that simultaneously simultaneously that and function and expression R 3 and IL-18. Since N4A induces cytokine secretion 8 = 1.91; see , , a role for components inflammasome has been N (module 78) and and 78) (module 6 41 -methyladenosine, 1 , 4 4 NLRC4 . . 2 NLRC5 in patients with coronary artery disease disease artery coronary with patients in http://cs.unc.edu β NLRP3 expression in aging was suggested suggested was aging in expression secretion , which is associated with a viral viral a with associated is which , NLRC4 upd/mod6 3 5 Supplementary Fig. 9 Supplementary , and consistently, we found found we consistently, and , , which triggers immunose triggers which , in in vitro 1 1 . Although the expression expression the Although . , , are involved in hyperten NLRC5 N 2 4 6 β 5 0 -carbamoylthreonylad . Consistently, we also also we Consistently, . . Our data suggest . that Our data suggest in patients with essen , inducing , the inducing THP-1 2 ), in the regulatory regulatory the in ), /~vjojic/fluy2-upd/ s e l c i t r a (module 62). As As 62). (module NLRP3 NLRC5 mutations mutations expres NLRC4 IRF7/9 ), ), sug  ------­

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. B.G., E.A.G., G.P.N., G.K.F. and M.H.S. ordesigned conducted the mass cytometry C.J.K. helped with the design and conducted the hypertension studies in mice; D.F., ordesigned conducted assays; D.F., C.R.B. and V.J. analyzed data; D.F., B.F., S.D., L.L., I.D. and P.B, (Stanford University). D.F. and B.F. conducted stimulation, inhibition and qPCR Gene expression data were generated at the Human Immune Monitoring Core Metabolon Inc. for the metabolite analysis. de la Dordogne, and the Régional Conseil d’Aquitaine. We thank the INCa-Cancéropôle (DEQ20110421287), GSO, Ligue contre le Cancer B.F., J.D-M. and J.F.M. were funded by Fondation pour la Recherche Médicale kindly providing us with material from NLRC4 and knockoutcaspase-1 mice. and VanceR.E. and I. Rauch at the University of Berkeley,California, for and Y. Rosenberg-Hasson (Human Immune Monitoring Core) at Stanford, Medical Institute for the remainder (M.M.D.). We also thank H. Maecker for initial support and to the NIH (U19 AI090019) and the Howard Hughes I. Chang and S. Batra. We also are grateful to The Ellison Medical Foundation Research Clinical Associates A. Goel, T. Quan, K. Span, R. Fleischmann, B. Tse, S. Swope, C. Walsh, S. French, B. Sullivan, S. Cathey, T. Trela and N. Mastman; participation; ManagerProject/Regulatory/Data S. Research Mackey; Nurses We thank the Stanford–Ellison longitudinal cohort volunteers for their online version of the pape Note: Any Supplementary Information and Source Data files are available in the t the in available are references, and codes accession statements of including Methods, and data availability any associated M diseases. aging-associated other of onset potential the and sion, hyperten of development the inflammation, chronic of occurrence nucleotide metabolites, resulting in the activation of immune cells, the pathways eral to may response these be involved in the inflammatory of and upregulation N4A subsequent the of generation the in results which degradation, tRNA in an increase in may result individuals elderly some in stress oxidative subjects show clear signs of oxidative stress, we that speculate a rise in chronic) and acute (both stress oxidative for marker a is degradation tRNA Since ROS-dependent. be also may that nism tion of ROS. Thus, our data suggest the existence of a different mecha far, of no activation However, so 44). (ref. NLRP3 inflammasome of the activation the in of may ROS converge sources and other mitochondrial that showing in inflammation ROS and aging of has been role extensively studied, the with recent and data production, ROS with track angII, elevated hypertension sustained causing and IL-17 by and T the in vessels, kidneys which, cells, turn, infiltrate of IL-1 production the involve proteins These self-proteins. isoketal-modified to response find recent an support inflammatory triggers stress a that of state showing oxidative ings results our subjects, IMH in stress oxidative mortality decreased with correlation its for account may which activation, inflammasome by caused is that inflammation systemic suppress may consumption coffee moderate Thus, adenine. and adenosine antagonizing in role its to addition in of upregulation N4A-mediated on effect antagonistic an has caffeine that indicates group, IML the in levels higher at found are with relates cor negatively caffeine That activation. neutrophil and platelet ing IL-1 A 1 studies F.H. cardiovascular phenotyping supervised data generation; J.-F.M. and AU A he pape he 0 ckn ethods With respect to the link between angII and the higher levels of of levels higher the and angII between link the to respect With

s e l c i t r T H β expression in monocytes and neutrophils, along with enhanc with along neutrophils, and monocytes in expression ow O R R C r . ledgmen O NLRC4 N T RIBU in vitro t gene and protein expression, and its metabolites metabolites its and expression, protein and gene NLRC4 T r s . I O β NS , IL-6 and IL-23 by dendritic cells and IFN- and cells by dendritic IL-23 and , IL-6 studies of monocyte and platelet activation; J.C. and has been reported in to response has reported produc been 3 NLRC4 1 . Pre-hypertensive stimuli, such as as such stimuli, Pre-hypertensive . 4 gene expression. Thus, sev Thus, expression. gene 3 . online version of version online 4 5 , and the IMH IMH the and ,

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7. 8. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial wrote the manuscript. immunological studies and contributed to study design; D.F., M.M.D. and B.F. G.P.N. and M.M.D. provided support, contributed with the planning of the organized and conducted the clinical studies and contributed to study design; J.D.-M. helped with clinical insights and discussion; C.L.D. coordinated, 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. C O

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. buffer was added to the wells. Each sample was measured in duplicate. Platesduplicate. in measured was sample Each wells. the to added was buffer reading the and above as performed was wash temperature, a roomat min 30 as above, and streptavidin-PE was added to it. After samples were incubated for to the plates for 75 min at room temperature with shaking. The plate was inwashed a Biotek ELx405 washer and then a biotinylated detection antibody was added shaker at 500–600 r.p.m. Following the overnight incubation, plates were washed Both cold and room temperature incubation steps were performed on an orbital room temperature for 1 h, followed by overnight incubation at 4 °C to the withplate containing shaking. the mixed antibody-linked beads and were incubated at plate Biotekwasher. a 96-well toa in and ELx405 washed Samples were added recommendations with the modifications as described below. Beads were added which 62 analytes They wereQC. passed accordingused to the manufacturer’s of eBiosciences/Affymetrix, from purchased were kits 2013) year (for 63-plex samples as previously described were added to all wells. per cytokine. Custom assay control beads manufactured by Radix Biosolutions sample per beads 100 of bound lower a with instrument 200 Luminex a using read were Plates duplicate. in measured was sample Each buffer. reading in tional vacuum washes were performed, and then the samples were addi re-suspended two temperature, room at min 40 for incubation After streptavidin-PE. in re-suspended were then and above, as twice washed and filtered then were Samples temperature. room at h 2 for antibody detection biotinylated a with vacuum filtered and washed twice with a wash buffer, and then were incubated werer.p.m. Plates 500–600 at shaker orbital an on performed were steps tion which was followed by overnight incubation at 4 °C. Room temperature h, 2 forincuba temperature room at incubated were and plates filter-bottom 96-well below. Briefly, samples were mixed with polystyrene antibody-linked onbeads described as modifications usage the with manufacturer’srecommendations the to according used and Affymetrix from purchased were kits yeas 2009–2011) (for 51-plex or 2008) year (for 50-plex Human University. Stanford at bead kits. factors. growth and chemokines cytokines, of Determination as though they were drawn from the same distribution. sion between the cell lines/populations. We treated all of the genes in a module across genes in each module. We performed an unpaired (ImmGen) algorithm enrichment geneset the the expression levels of modules 62 and 78 across different populations, we used cells andversus granulocytes other cell populations. To estimate differences in can myeloid in levels expression module gene analysis inflammasome of Estimation tion. expression the Gene in found determination. be chemokine sepa and was before °C cytokine Serum −80 at stored was (below). it and blood, analysis clotted of expression centrifugation by whole rated gene and for venipuncture, used from was obtained blood were samples blood Peripheral draw. blood baseline of day on illness febrile of signs any showed none and of respectively, weeks 6 donors and months 6 or past the within recipients products blood or been blood had volunteers the of none day, vaccination annual each on addition, In medication. immunosuppressive of use current or recent and C or B hepatitis chronic screening, at >150/95 pressure blood liver observed clinically of disease, diabetes mellitus absence treated with insulin, moderatethe to severe renal disease, includes definition This function. of immunologic impairment nor any or known suspected immunodeficiency of history no illness, concurrent serious or systemic acute no had volunteers the enrollment, initial of time Atthe assessment. clinical a by determined as and were healthy, were ambulatory generally All individuals from obtained subjects. all was consent Informed University. Stanford at Office Compliance of for Board study was this the Research approved by Review the Institutional protocol study. The this for done was blinding or randomization no vaccine, samples influenza baseline the with vaccination before Since individuals the all from obtained were NCT#01827462). at registration study 2013 to vaccine (ClinicalTrials.gov 2008 years influenza the an during in Program Vaccine enrolled Stanford–LPCH the were >89) to 20 (ages donors collection. sample and subjects design, Study ONLINE METHODS nature medicine nature II. Magnetic bead kits This assay was performed in the Human Immune Monitoring Center upeetr Methods Supplementary . Serum specimens were collected from the human blood 8 and were frozen in aliquots at −80 °C. Human gn epeso aayi sec analysis expression gene , 4 7 . Available data were analyzed analyzed wereAvailable data . One hundred and fourteen fourteen and hundred One t -test on gene expres

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the measurement of both carotid intima-media thickness (cIMT) and central central and (cIMT) thickness intima-media carotid both of measurement the Institute Biomarker and Phenotypic Core Laboratory. Vascular studies included underwent comprehensive cardiovascular assessment cohort longitudinal at Stanford–Ellison Stanfordthe from unclassified) Cardiovascular 22 + phenotypes phenotyping. Cardiovascular method for combining for years 2008 through 2011 were combined using a modified generalized Fisheryear.that from available was individuals phenotype extreme 14 only from data because analysis this in included not was 2012 from data Cytokine Bartlett’s test. using by homoscedasticity showed data Combined correction. IL-1 or years 2008–2011 the between two groups, using a regression model with IL-1 directly compared wethe levels of IL-1 stable, longitudinally were subjects IL to compared when subjects IH in IL-1 in differences the whether To study resamplings. the significance for each regression coefficient via permutation tests analysis on each analyte’s MFI against the IH/IL status, age and sexlarg and the ( obtained analytes measured of number est with year the was this since 2013 year from data analyzed first we and then used for analysis. further recorded were (MFIs) intensities fluorescence Mean wells. the of all to added were Biosolutions Radix by beads control assay Custom cytokine. per sample were read using a Luminex 200 instrument, with a lower bound of 50 beads per against IML/IMH status after adjusting for age and sex. Significance for each each for Significance sex. and age for adjusting after status IML/IMH against conducted on gene’seach expression using microarray data from year 2008 purine metabolism (PUR) were obtained from KEGG genes. metabolism total of genes metabolism and104 pyrimidine (PYR) 54 genes in participating pyrimidine and purine of expression Differential the manufacturer’s instructions. the Enzyme Immunoassay EIA Kit (Cayman Chemical Company) according to using by 8-isoprostane quantify to used were IMH) 9 and IML (8 phenotypes Determination of circulating isoketals. were used, respectively. topology analyses, a hypergeometric test and the relative-betweeness centrality across more than 874 metabolic pathways.characteristics Fortopological pathway over-representationof analysis andthe pathway with along analyses ment conducted using MetPA metabolites were observed in IML versus IMH individuals. Pathway analysis was an indication of high confidence in a result. A total of 67 differentially regulated model, which included the age and sex as covariates. A microarrays (SAM) value imputation). To determine statistical significance, significance analysis of detection; these values were imputedof limits with the the compound below minimum be (minimum to assumed were values missing anydisplay, data and analyses statistical For control. quality for inspection visual by followed ards automated comparison of ion features to a reference library of chemical stand gas by and mode ion chromatography/mass Metabolites polar (GC/MS). by were identified spectrometry or negative positive, the in (UHPLC/MS) spectrom etry chromatography/mass liquid performance ultrahigh by for analysis aliquots into split were then and extraction methanol to subjected were non-targeted using previously metabolomic profiling described as Metabolon at conducted were analysis. and generation data Metabolomics measurement stiffness arterial reflects diographic distance from the sternal notch to proximal descending aorta. This echocar the minus artery femoral the to notch sternal the from distance the as measured was (D) length path wave; pulse aortic the of time transit the by divided traveled length path the as calculated was artery.PWV carotidAortic lateral and posterior measurements and was averaged for both the right and left for carotid and femoral measurements. The cIMT was the average of aortic the pulse anterior,wave velocity (PWV). We used a 9.0 MHz Philips linear array probe To identify differences between IH and IL individuals in an unbiased fashion, α MFI against IH/IL status, age and sex without multiple hypothesis hypothesis multiple without sex and age status, IH/IL against MFI 2 3 was conducted on the residuals from a multiple regression 5 P 0 2 . Serum samples . fromSerum IMH ( -values -values obtained from dependent tests 4 , which combines several advanced pathway combines which , several enrich A subgroup of subjects ( subjects of subgroup A N α = 62). We 62). = multipleregression conducted and IL-1 4 9 Sera available from a total of 17 extreme . β from the data generated in the Metabolomic data analyses analyses data Metabolomic 5 Q 1 N . Regression analysis was -value <0.05 was used as = 11) and IML ( α and IL-1 and doi: N = 39; 17 extreme extreme 17 39; = 10.1038/nm.4267 3 3 . β 4 observed observed 8 over 500 P -values -values N = 9) 8 A , 1 β 5 - - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. counting the number of positive cells per 10× microscope field. Multiple fields by Histo-Tec Laboratory (Hayward, CA). Infiltrating cells were investigated by Aortic and kidney sectioning and paraformaldehyde. anti-CD3 4% in staining fixed procedures and obtained were were conducted kidneys and tissue Aortic puncture. cardiac control)byversus (treated group per mice 6/10 from blood once with 10 mice per group. In the second experiment, we collected peripheral raphy (Vistech System BP-2000, Apex, NC, USA). The experiment was repeated were measured every other day in conscious mice by use of tail-cuff plethysmog anesthesia (2% oxygen, 2.5% isoflurane). under mice The of systolic neck the aroundblood side pressures dorsal the on of subcutaneously theimplanted mice pump (Durect Corporation, Cupertino, CA, USA). The osmotic pump was then µ another 2 weeks.for MO) AngII Louis, (140 St. ng per Sigma-Aldrich, kg body #A9525, weight (angII, per II min) angiotensin was human dissolvedof in 100 PBS or N4A + Adenine, mice from both groups were administered an infusion of injections daily receiving continued they while treatment, of weeks 3 After chosen so that the of body weight, retro-orbital injection, once size daily) or PBS. This concentration sample was a with estimated we mice), control 4 mice (statistical in power = short- 92%, treatment to due angII (pre-hypertension term 10) = and (s.d. mmHg mmHg >140 120 of of average value sample expected or an treatment For to measurement. blinded pressure not blood was investigator ( The PBS). treated-mice in dissolved compound nine into or placed PBS randomly were groups: weeks) two 12–18 (aged mice WT C57BL/6 male mice. in studprifeies Hypertension ratio of this expression relative to the control. used to standardize the samples, and the results are expressed was as GAPDH the of normalizedexpression The Biosciences). (Applied system PCR time real StepOne a on Technologies), run were(Life plates assays andexpression gene NLRP3 expression was measured by quantitative PCR using pre-design TaqMan VILO SuperScript® following the manufacturer’s recommendations. cDNA was prepared using the of coffee drinkers (10–50 500 and 50 (0, concentrations several test to chose we caffeine, of studies the For (100 tion concentra highest The DAMPs. and PAMPs of range wide a by initiated is 100 at used (when adenosine highest concentration was chosen on the basis of previous reports showing that profiles dose-dependent significant of identification the for allows and experiments dose-response or time-series for methods clustering uses which (STEM), miner expression time-series short used we experiments, response in the supernatants ( ( dose-responses significant 100 and 30 10, 3, (0, concentrations increasing at tested growth and chemokines factors cytokines, using the of 63-plex Luminex quantification system, as for described above. Compounds Stanford were at Core MonitoringHumanImmune the to transferred then were Samples °C. −80 at bated for 6 h at 37 °C. Supernatants were collected, frozen3 × immediately10 and stored facturer’s recommendations. were Cells plated on plates96-well at a density of #15068, Stemcell Technologies, Vancouver,(cat Cocktail Enrichment BC, Monocytes HumanCanada) accordingRosetteSep the to using the manuenriched was purchased from Santa TX). Cruz Biotechnology (Dallas, and MO) Louis, St. (Sigma-Aldrich, Sigma form purchased assays. adenine, qPCR and secretion cytokine treatment, Compound test. A ferentially expressed were subjected to enrichment analysis by hypergeometric regression coefficient was obtained via permutation tests doi: L L 20 mM (N4A + Adenine) or in 100 RNA was extracted from cell pellets using the RNeasy micro kit (Qiagen) (Qiagen) kit micro RNeasy the using pellets cell from extracted was RNA Whole blood was obtained from venipuncture (30 mL), and monocytes were 10.1038/nm.4267 µ N M) M) that match the estimated concentrations that are found in the plasma −5 P 4 -acetylcytidine and adenine (stock solution 20 mM each, 100 100 each, mM 20 solution (stock adenine and -acetylcytidine cells in 200 -value -value < 0.05 was used as an indication of high confidence in a result. dl µ M) was also used for all other compounds and stimulation assays. stimulation and compounds other all for used also was M) -4-hydroxy-3-methoxymandelic acid and scyllo-inositol were all all were scyllo-inositol and acid -4-hydroxy-3-methoxymandelic in vivo TM µ Supplementary Fig. 5 L LGM-3 serum-free media (Lonza), and plates were incu cDNA Synthesis Kit (Life Technologies). NLRC4 and NLRC4 Technologies). (Life Kit Synthesis cDNA concentration reaches 1 mM of circulating compound. µ M). P < 0.01) in the concentration of cytokines found cytokines of concentration the in 0.01) < α µ = 0.01). Compound-treated mice were injected M) can regulate inflammasome activity that that activity inflammasome regulate can M) yetnin tde i mc. Adult mice. in studies Hypertension µ L L PBS and loaded into a small osmotic ). To assess significance for the dose- N 4 aeyctdn + ade + -acetylcytidine 4 8 . Genes that were dif µ M) and displayed displayed and M) N 4 -acetylcytidine -acetylcytidine Adenosine, Adenosine, µ 5 L/25 g L/25 2 . The The . ------

species andspecies applications is provided on the manufacturer’s website. 3510-1H-20; respectively). Validation for antibody reactivity against the relevant IFN- measure to kits ELISA Biosciences. LI-COR from purchased were antibodies and ATP were purchased from SIGMA. Fluorescent dye-conjugated secondary (L6529) 055:B5 serotype Coli from E. LPS (ALX-260-098). Sciences Life Enzo from from biotinyl-VAD-fmk and 218746), purchased VI; inhibitor was (caspase-1 Calbiochem Ac-YVAD-fmk inhibitor Caspase-1 (OAEB01074). Clinisciences from AIM2 anti-human and (ABF29); Millipore Merck from anti-humanNLRP6 (AB115537); Abcam from anti-humanNLRC4 (#12242); anti-human (#3866); IL-1 Signaling Cell from antibody (D7F10) p20 caspase-1 anti-human (sc-515); Biotechnology Sciences (ALX-804-819); anti-human caspase-1 p10 antibody from Santa Cruz Reagents and antibodies. mass cytometry (CyTOF) section. of regulationsethical for animal studies of Stanford University. described below. This study was conducted in compliance with the requirements by time-of-flight was used to characterize the activation status of blood cells as cat # MTS1P) in a 5-tube base station (Smart Tube Inc. cat # PBS05). Cytometry SmartTubes using Tubefixed (Smart temperatureatwas roomit before h Inc. for tubes 2–4 collection kept was heparinized in Mousetion. blood peripheral were evaluated, and representative pictures were taken from 5–6 fields per condi de Vectorologie” at Bordeaux University, and THP-1 cells were transduced in Vectorologie”in de transduced University,were Bordeaux atTHP-1cells and “Plateforme the by generated then were lentiviruses Recombinant shRNAs). (MISSION Sigma from purchased were gene NLRC4 human the silence that infections. Lentivirus overnight at 37 °C. 5.10of PBS and finally were washed in complete RPMI medium at a final concentration °C),wereat4washedmin oncelysis7 inbuffer, werewashedsecond timeina between 5–15 min later. Neutrophils were then isolated by centrifugation (460 KHCO hypotonicpH7NH mL lysismMbuffer50suspended(155 and in blood samples of healthy donors by using a Ficoll gradient.Isolationneutrophils.humanprimaryof The pellet was retained derived macrophages were used for stimulation assays with N4A + adenine. marrow-bone 8, dayAtcultures. cell the to added was BMM ml 10 4, dayAt were resuspended in 30 ml BMM media and grown in 150 × 15 mm petri dishes. Supernatant was then centrifuged for 10 minutes at 4 °C at 1,000 r.p.m. and cells were then gently centrifuged for 1 minute at 4 °C at 500 r.p.m.mechanically. Cells marrowto remove bone disruptingdebris. by and tibia and femur through fetal calf serum, 1% glutamine, 5% 3T3-CSF-cellby flushing conditionedpre-chilled Bone mediaMarrow andMedia antibiotics)(BMM) (RPMI, were 10% obtainedheat-inactivated from bones from wild type, NLRC4 and Casp1/11 knockout mice, Isolation and growth of bone marrow-derived macrophages. subunit of processed human caspase-1 (Santa Cruz Biotechnology, sc515). lyzed by SDS-PAGE/immunoblotting using an antibody thatthe beads. Beads weredetects then heated thefor 5 min p10and pelleted.small Supernatant was ana NaF, 0.3% NP-40, 0.1 mM Na washed 3 times in cold IP buffer (50 mM Tris-HCl, wereandcentrifugation°C)by were pelletedmin/4pHrpm/10 (3000 Beads °C. 4 7.4, 150 mM NaCl, 50 mM streptavidin-Sepharosesuspension 250per mLof cell supernatant overnight at recoveredstreptavidin-Sepharoseusingby 30 adding(Sigma), beads fragment p10 of caspase-1. The biotinyl-VAD-fmk/caspase-1 p10 complex was VAD-fmk(30 treated with one of the four compounds of interest were incubated with biotinyl- ofactive caspase-1, cellculture supernatants ofadherent THP-1 cellsthat were 50 ng/mL PMA (phorbol 12-myristate 13-acetate) for 12 h at 37 °C. For recovery formed as described previously Immunoblottingofand activerecovery caspase-1. Mass cytometry experiments are described in the γ and TNF- and 5 3 cells/ml. Then,12-wellcellswerea platein seeded and wereincubated , 0.1 mM EDTA), and the lysis of red blood cells was finished on ice on finished was cells blood red of lysis the EDTA),and mM 0.1 , µ M) overnight at 4 °C in order to precipitateorderto in cleavedactive,°Covernight the 4M) at α secretion were purchased from Mabtech (3420-1H-20 and (3420-1H-20 Mabtech from purchased were secretion etvrl lsi cntut epesn shRNAs expressing constructs plasmid Lentiviral Anti-human NLRP3 was purchased from Enzo Life 3 5 VO 3 . THP-1 cell lines in suspension were treated with 4 ) before adding SDS loading buffer on top of β (3A6) antibody from Cell Signaling Signaling fromCell antibody (3A6) Neutrophils isolatedthewere from Immunoblotting wasper Supplementary Methods nature medicine nature Bone marrow cells 4 Cl, 10 mM 10 Cl, µ L of 1:1 of L g - - - , ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. buffer (134 mM NaCl, 12 mM NaHCO mM 12 NaCl, mM (134 buffer (10 min at 2000 r.p.m. without a break), the supernatant din E1 was(Sigma, cat # P8908) was toadded the plate.removed After gentle centrifugation and Tyrode at centrifugation min 20 a 1000 r.p.m. After without tubes. a break, EDTAthe supernatant in was harvested venipuncture and 1 by donors contamination. mycoplasmafor tested were ATCC(R).Cells by identified and from obtained were lines Cell 3310-9). # cat (Mabtech, peroxidase streptavidin-horseradish and anti-IL18-biotin capture cat(MBL, antibodies and # adding D045-6) then usingmeasured a custom-made was ELISA combining IL-18 anti-IL18 coating respectively). (MBL, cat # 3510-1H-20 D044-3) # cat and 3415-1H-20, # IL-1 cat using by supernatants culture cell from IL-1 cytokines of secretion ATP. without The or with either pulsed were cells incubation the h, 6 After concentrations. indicated the at sc-222023) # cat Cruz, (Santa acetylcytidine cat # 1852; 5 mM, 30 min) or the compounds Adenine (Sigma, cat # A2786)/ ATPand h) (Sigma, 4 ng/ml, 1 L6529; # cat (Sigma, LPS agonists with treated (10 ng/ml). The day after, adherent cells were washed with fresh media and were ated overnight with phorbol 12-myristate 13-acetate (PMA; Sigma, cat # P1585) RPMI media (supplemented with 10% Fetal Bovine Serum) and were differenti in plates 6-well in cultured (ATCC,were lines TIB202) cell THP-1monocytic platelets. human blood THP-1 with and primary Experiments cells using anti-human NLRC4 antibody. and were amplified to test the silencing of gene expression by western-blots by 1 on selected were cells infected the post-infection, d 2 At plate. 6-well a in 30) and 10 MOIs(1, various at virus each with suspension a nature medicine nature Human primary platelets were obtained from the whole blood of healthy healthy of blood whole the from obtained were platelets primary Human β , IL-18, and TNF- and IL-18, , 3 , 2.9 mM KCl, 0.34 mM Na mM 0.34 KCl, mM 2.9 , α β was then measured by ELISA ELISA by measured then was n TNF- and µ α g/mL puromycin g/mL kits (Mabtech, (Mabtech, kits µ M prostaglan Human 2 HPO N 4 4 - - - ,

52. 51. 50. 49. 48. 47. SDY31 ImmPort, Portal Analysis and Database Immunology the on deposited was 2012 2008– availability. Data Coulter, (Beckman anti-CD62-PE and cat population) # 1759U) (marker of activation involved in aggregation). platelet of (marker of membrane Coulter, markers (Beckman anti-CD61-PC7 with cat IM3716) # immunostaining using cytometry flow by and monitored was adenine, activation or platelet N4A of concentrations indicated the with or A2754) # cat then stimulated with thrombin (Sigma, cat # T9326; at 0.5 U/ml), ADP (Sigma, MgCl mM 1 53.

Ernst, J. & Bar-Joseph, Z. STEM: a tool for the analysis of short time series gene series time short of analysis the for tool a STEM: Z. Bar-Joseph, & J. Ernst, Kanehisa, M. S.Y. Shin, wave pulse of Determinants Collaboration. Stiffness’ Arterial for Values Reference and practical a rate: discovery false the Controlling Y. Hochberg, & Y. Benjamini, T.S. Heng, Faustin, B. Faustin, in KEGG. in Genet. Nat. factors: values’. risk cardiovascular reference (2010). of and presence the normal in ‘establishing and people healthy in velocity (1995). 289–300 testing. multiple to approach powerful cells. immune in expression of caspase-1 activation. caspase-1 of data. expression 2 , , SDY31 https://immport.n 2 Nucleic Acids Res. Acids Nucleic , 10 mM HEPES, pH 7.4) was added to the plate. Platelets were Platelets plate. the to added was 7.4) pH HEPES, mM 10 , & Painter, M.W. The immunological genome project: networks of gene of networks project: genome immunological The M.W.Painter, &

et t al. et 46 al. et al. 1 , 543–550 (2014). 543–550 , , , SDY11 Reconstituted NALP1 inflammasome reveals two-step mechanism two-step reveals inflammasome NALP1 Reconstituted Accession codes: Raw gene expression data for years years for data expression gene Raw codes: Accession BMC Bioinformatics BMC An atlas of genetic influences on human blood metabolites. blood human on influences genetic of atlas An Data, information, knowledge and principle: back to metabolism 2 Mol. Cell Mol. and iaid.nih.go Nat. Immunol. Nat.

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