The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan.

A icle The Se 7 Lysine Me hyl ans e ase Regula es Plas ici y in Oxida i e Phospho yla ion Necessa y o T ained Immuni y Induced by b-Glucan G aphical Abs ac Highligh s dSe 7 egula es enhanced cy okine p oduc ion in ained immuni y in i o dSe 7 knockou mice a e unable o moun ained immuni y agains endo oxin challenge dSe 7 modula es cellula espi a ion in b-glucan- ained mac ophages dSe 7-dependen his one me hyla ion egula es MDH2 and SDHB in ained cells Au ho s Samuel T. Kea ing, Laszlo G oh, Cha lo e D.C.C. an de Heijden, ..., Leo A.B. Joos en, Mihai G. Ne ea, Niels P. Riksen Co espondence niels. iksen@ adboudumc.nl In B ie Using a combina ion o pha macological and gene ic app oaches, Kea ing e al. show ha he Se 7 me hyl ans e ase is a egula o o ained immuni y induced by b-glucan. Ac i a ion o Se 7 inc eases oxida i e phospho yla ion in ained cells ia his one lysine me hyla ion a gene enhance s o key enzymes o he TCA cycle. Kea ing e al., 2020, Cell Repo s 31, 107548 Ap il 21, 2020 ª2020 The Au ho (s). h ps://doi.o g/10.1016/j.cel ep.2020.107548 Cell Repo s A icle The Se 7 Lysine Me hyl ans e ase Regula es Plas ici y in Oxida i e Phospho yla ion Necessa y o T ained Immuni y Induced by b-Glucan Samuel T. Kea ing, 1 Laszlo G oh, 1 Cha lo e D.C.C. an de Heijden, 1 Hanah Rod iguez, 2 Je ´ssica C. dos San os, 1 S ephanie Fanucchi, 3,4 Jun Okabe, 2 Ha ik ishnan Kaipananickal, 2,5 Jelme H. an Pu elen, 1,6 Leonie Helde , 1 Ma lies P. Noz, 1 Vasiliki Ma za aki, 1 Yang Li, 1,7 L. Cha lo e J. de B ee, 1,8,9 Vale ie A.C.M. Koeken, 1 Simone J.C.F.M. Moo lag, 1 Ve a P. Mou i s, 1 Jo ge Domı ´nguez-And e ´s, 1 Ma ije Oos ing, 1 Elianne P. Bul huis, 10 We ne J.H. Koopman, 10 Musa Mhlanga, 3 Assam El-Os a, 2,5,11 Leo A.B. Joos en, 1,12 Mihai G. Ne ea, 1,13 and Niels P. Riksen 1,14, * 1 Depa men o In e nal Medicine and Radboud Ins i u e o Molecula Li e Sciences (RIMLS), Radboud Uni e si y Medical Cen e , Nijmegen, he Ne he lands 2 Epigene ics in Human Heal h and Disease, Depa men o Diabe es, Monash Uni e si y, Melbou ne, VIC, Aus alia 3 Di ision o Chemical, Sys ems and Syn he ic Biology, Depa men o In eg a i e Biomedical Sciences, Facul y o Heal h Sciences, Ins i u e o In ec ious Disease and Molecula Medicine, Uni e si y o Cape Town, Cape Town, Sou h A ica 4 Gene Exp ession and Biophysics G oup, CSIR Biosciences, P e o ia, Sou h A ica 5 Depa men o Clinical Pa hology, The Uni e si y o Melbou ne, Melbou ne, VIC, Aus alia 6 Depa men o Heal h E idence, Radboud Uni e si y Medical Cen e , Nijmegen, he Ne he lands 7 Depa men o Compu a ional Biology o Indi idualised In ec ion Medicine, Cen e o Indi idualised In ec ion Medicine, Helmhol z Cen e o In ec ion Resea ch, Hanno e Medical School, 30625 Hanno e , Ge many 8 Resea ch Cen e o Vi amins and Vaccines, Bandim Heal h P ojec , S a ens Se um Ins i u , Copenhagen, Denma k 9 Odense Pa ien Da a Explo a i e Ne wo k, Uni e si y o Sou he n Denma k/Odense Uni e si y Hospi al, Odense, Denma k 10 Depa men o Biochemis y, Radboud Ins i u e o Molecula Li e Sciences (RIMLS), Radboud Uni e si y Medical Cen e , Nijmegen, he Ne he lands 11 P ince o Wales Hospi al, The Chinese Uni e si y o Hong Kong, Hong Kong Ci y, Hong Kong SAR 12 Depa men o Medical Gene ics, Iuliu Ha ieganu Uni e si y o Medicine and Pha macy, Cluj-Napoca, Romania 13 Depa men o Genomics and Immuno egula ion, Li e and Medical Sciences Ins i u e (LIMES), Uni e si y o Bonn, Bonn, Ge many 14 Lead Con ac *Co espondence: niels. iksen@ adboudumc.nl h ps://doi.o g/10.1016/j.cel ep.2020.107548 SUMMARY T ained immuni y con e s a sus ained augmen ed esponse o inna e immune cells o a seconda y challenge, ia a p ocess dependen on me abolic and ansc ip ional ep og amming. Because o i s p e ious associa ions wi h me abolic and an- sc ip ional memo y, as well as he impo ance o H3 his one lysine 4 monome hyla ion (H3K4me1) o inna e immune memo y, we hypo hesize ha he Se 7 me hyl ans e ase has an impo an ole in ained immuni y induced by b-glucan. Using pha macological s udies o human p ima y mono- cy es, we iden i y ained immuni y-speci ic immunome abolic pa hways egula ed by Se 7, including a p e iously un epo ed H3K4me1- dependen plas ici y in he induc ion o oxida- i e phospho yla ion. Recapi ula ion o b-glucan aining in i o addi ionally iden i ies Se 7-depen- den changes in gene exp ession p e iously asso- cia ed wi h he modula ion o myelopoiesis p o- geni o s in ained immuni y. By e ealing Se 7 as a key egula o o ained immuni y, hese indings p o ide mechanis ic insigh in o sus ained me a- bolic changes and unde sco e he impo ance o cha ac e izing egula o y ci cui s o inna e immune memo y. INTRODUCTION A se ies o ecen disco e ies has unco e ed how cells o he inna e immune sys em such as monocy es and mac ophages unde go unc ional ep og amming o moun a de ac o immune memo y o an in ec ious o in lamma o y inju y by a p ocess called ained immuni y, which acili a es augmen ed esponses o subsequen pa hogenic encoun e s (Ne ea e al., 2020). In he con ex o in ec ions o accina ion, ained immuni y p o ides bene icial he e ologous e ec s by he enhanced cy okine esponse o s imula ion wi h non- ela ed pa hogens. P o o ypi- cal s imuli o ained immuni y include he ungal cell wall compo- nen b-glucan (Quin in e al., 2012) and he bacillus Calme e- Gue ´ in (BCG) accine (Kleinnijenhuis e al., 2012). Recen a en- ion has also u ned o endogenous d i e s o in lamma ion as in- duce s o ained immuni y (Bekke ing e al., 2014; an de Valk e al., 2016). These s imuli shape inna e immunological mem- o ies by ep og amming me abolic and ansc ip ional p o iles (A s e al., 2016a; Cheng e al., 2014). Pos ansla ional me hyla ion o p o eins con eys in o ma- ion o cellula pa hways, including hose ha egula e gene Cell Repo s 31, 107548, Ap il 21, 2020 ª2020 The Au ho (s). 1 This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Figu e 1. Se 7 Is Associa ed wi h T ained Immuni y Induced by b-Glucan (A) G aphical ou line o in i o aining me hods. Adhe en monocy es (Mo) we e s imula ed wi h 1 mg/mL b-glucan o s anda d cul u e medium (RPMI) o 24 h ( i s s imulus), allowed o di e en ia e o mac ophages (M4) o 5 days, and es imula ed o 24 h wi h LPS o RPMI on day 6. (legend con inued on nex page) 2Cell Repo s 31, 107548, Ap il 21, 2020 exp ession. In i o expe imen s wi h pan-me hyl ans e ase inhibi o s e ealed he pi o al impo ance o his chemical modi ica ion o ained mac ophages (Cheng e al., 2014; Quin in e al., 2012). Changes in his one lysine me hyl modi i- ca ions (H3 his ones monome hyla ed [H3K4me1] o ime hy- la ed [H3K4me3] a lysine 4) unde lie b-glucan-induced ained immuni y (No ako ic e al., 2016). Signaling ac o s de i ed om local issue en i onmen s play key oles in de e mining mac ophage a e, and e idence poin s o he ole o enhance elemen s in shaping specialized mac ophage popula ions (Denisenko e al., 2017; Gosselin e al., 2014). H3K4me1 is a ch oma in signa u e o enhance s (Hein zman e al., 2007). This modi ica ion was shown o pe sis a decommissioned dis al elemen s, indica ing ha H3K4me1 p o ides a mecha- nism o epigene ic memo y in ained immuni y in mac o- phages (Saeed e al., 2014). Despi e his, he mechanisms linking immunological signals induced by mic obial s imuli o accines o ch oma in-dependen changes in ained immuni y a e unclea . Mo eo e , he iden i ies o he ch o- ma in-modi ying enzymes c i ical o hese p ocesses emain obscu e. One enzyme ha w i es he H3K4me1 modi ica ion o ansc ip ionally ac i a ing o poised genomic egions is he Se 7 lysine me hyl ans e ase (Wang e al., 2001)(also called Se 9 [Nishioka e al., 2002], Se 7/9 [Tamu a e al., 2018], o KMT7 [Allis e al., 2007], and encoded by SETD7). Se 7 w i es a pe sis en H3K4me1 signa u e pe ain- ing o ascula endo helial in lamma o y signaling (B asacchio e al., 2009). The impo ance o Se 7 o media ing H3K4me1 signa u es a enhance s associa ed wi h endo helial gene exp ession was demons a ed using an unbiased epige- nome-wide app oach (Kea ing e al., 2014). Al hough Se 7 has no been s udied in he speci ic con ex o ained immu- ni y, ou p e ious analysis o mac ophages ained wi h b-glucan iden i ied ele a ed le els o SETD7 exp ession (Quin- in e al., 2012). The cu en s udy explo ed he ole o Se 7 in b-glucan- induced ained immuni y. Using gene ic and pha macological app oaches, we demons a e ha Se 7 is c i ical o he induc- ion o ained immuni y in i o and in i o. We iden i y a ole o Se 7 in immunome abolic pa hways, including he induc ion o oxida i e phospho yla ion (OXPHOS). Cha ac e iza ion o epigene ic ne wo ks is key o a deepe unde s anding o egula- o y mechanisms suppo ing ained immuni y, and could iden- i y s a egies o modula e p o-in lamma o y ci cui s o he inna e immune sys em. RESULTS Se 7 Exp ession and Ac i i y A e Inc eased in Human P ima y Monocy es and Mac ophages S imula ed wi h b-Glucan To in es iga e he ole o Se 7, we adop ed a p e iously desc ibed in i o model o ained immuni y using he ungal cell wall componen b-glucan (Cheng e al., 2014). Adhe en hu- man p ima y monocy es we e incuba ed wi h cul u e medium o b-glucan (1 mg/mL) o 24 h. Cells we e washed and incuba ed in no mal cul u e condi ions o a u he 5 days, du ing which ime hey di e en ia ed in o mac ophages. On day 6, he cells we e es imula ed wi h he Toll-like ecep o 4 ligand lipopolysaccha- ide (LPS) (10 ng/mL) o 24 h and p o-in lamma o y cy okine p o- duc ion was measu ed (Figu e 1A). Tumo nec osis ac o alpha (TNFa) and IL-6 we e measu ed as unc ional eadou s o ained immuni y (A s e al., 2016a; Cheng e al., 2014). Cells s imula ed wi h b-glucan exhibi ed enhanced TNFaand IL-6 p oduc ion ollowing LPS es imula ion (Figu e 1B). We alida ed ou p e i- ous ansc ip ome da a showing ha SETD7 mRNA exp ession was signi ican ly inc eased on day 6 o he in i o aining p o o- col in mac ophages ained wi h b-glucan. Cu en ly, he e is no gold s anda d es o Se 7 ac i i y in p ima y cells. Howe e , a ecen s udy iden i ied he ibosomal p o ein Rpl29, a compo- nen o he 60S ibosomal subuni , as a non-his one me hyla ion subs a e o Se 7 (Hamidi e al., 2018), and dime hyla ed Rpl29 (Rpl29k5me2) was shown o se e as a eliable bioma ke o Se 7 ac i i y in cance cells. We obse ed ha Rpl29k5me2 le els co ela ed wi h Se 7 p o ein exp ession in mac ophages ained wi h b-glucan (Figu e 1C). To u he in es iga e he associa ion o Se 7 o ained immu- ni y, we conduc ed a gene ic s udy o pe iphe al blood mononu- clea cells (PBMCs) isola ed om 267 heal hy olun ee s o Wes e n Eu opean ances y. Adhe en PBMCs om all olun- ee s we e incuba ed wi h ained wi h cul u e medium o b-glucan (1 mg/mL) o 24 h as desc ibed abo e. We es ed o associa ions among common single-nucleo ide polymo phisms (SNPs) and a ia ion in he magni ude o b-glucan- ained cy o- kine esponses o indi idual subjec s and iden i ied wo SNPs sugges i ely associa ed (p < 9.99 310 3 ) wi h adap i e changes in cy okine p oduc ion mapped wi hin SETD7. In onic a ian s s7680948 (loca ed in in on 4 o SETD7 [p < 0.004]) and s56183115 (loca ed in in on 3 o SETD7 [p = 0.004]) we e asso- cia ed wi h he po en ia ion o TNFaand IL-6 p oduc ion, espec- i ely, upon induc ion o ained immuni y by b-glucan (Fig- u e 1D). To in es iga e Se 7 in ained immuni y induced by (B) P oduc ion o p o-in lamma o y cy okines TNFaand IL-6 by ained mac ophages ollowing es imula ion (n = 6 heal hy olun ee s pe g oup). (C) Exp ession o SETD7 mRNA p io o es imula ion (6 d) (n = 7 heal hy olun ee s pe g oup). Rep esen a i e wes e n blo analysis o di e en ia ed mac o- phages pe o med on day 6, p io o es imula ion. Se 7 encoded by he SETD7 gene; Rpl29k5me2 as a ma ke o Se 7 ac i i y; b-ac in was used as loading con ol. (D) Single-nucleo ide polymo phisms (SNPs) wi hin SETD7 sugges i ely associa ed wi h ained esponses o b-glucan in pe iphe al blood mononuclea cells (n = 267). Age- and sex-co ec ed TNFaand IL-6 changes a e shown as boxplo s o s7680948 and s56183115, espec i ely. (E) SNPs nea SETD7 sugges i ely associa ed wi h ained esponses o he bacillus Calme e-Gue ´ in accine in pe iphe al blood mononuclea cells. Age- and sex-co ec ed TNFaand IL-6 changes a e shown as boxplo s o s795971 (n = 213) and s6816973 (n = 248), espec i ely. (F) SNPs nea SETD7 sugges i ely associa ed wi h ained esponses o he oxidized low-densi y lipop o ein in pe iphe al blood mononuclea cells. Age- and sex- co ec ed TNFaand IL-6 changes a e shown as boxplo s o s6536295 (n = 197) and s10020166 (n = 225), espec i ely. Da a a e ep esen ed as mean ±SEM. *p < 0.05, Wilcoxon signed- ank es . Cell Repo s 31, 107548, Ap il 21, 2020 3 (legend on nex page) 4Cell Repo s 31, 107548, Ap il 21, 2020 o he s imuli, we es ed o associa ions among SNPs nea SETD7 and a ia ion in he magni ude o cy okine esponses o indi iduals ained wi h BCG and oxidized low-densi y lipop o- ein (oxLDL). We iden i ied nume ous SNPs sugges i ely associ- a ed (p < 9.99 310 3 ) wi h adap i e changes in p o-in lamma- o y cy okine p oduc ion mapped wi hin 250 kb o SETD7. Va ian s s795971 (loca ed app oxima ely 250 kb ups eam o SETD7 [p = 0.007]) and s6816973 (loca ed app oxima ely 160 kb ups eam o SETD7 [p < 0.003]) we e associa ed wi h he po en ia ion o TNFaand IL-6 p oduc ion, espec i ely, upon in- duc ion o ained immuni y by BCG (Figu e 1E). In addi ion, we iden i ied ha a ian s s6536295 (loca ed app oxima ely 3 kb ups eam o SETD7 [p < 0.007]) and s10020166 (loca ed app oxima ely 15 kb ups eam o SETD7 [p = 0.01]) we e associ- a ed wi h he p oduc ion o TNFaand IL-6, espec i ely, upon in- duc ion o ained immuni y by oxLDL (Figu e 1F). Pha macological Inhibi ion o Se 7 A enua es b-Glucan-Induced T ained Immuni y In Vi o Recen s udies demons a ed speci ic inhibi ion o Se 7 ac i i y by cyp ohep adine (CPH) in human b eas cance cells (Take- mo o e al., 2016). To es whe he CPH simila ly inhibi s Se 7 in p ima y cells, we measu ed Rpl29K5me2 le els o mac o- phages ained wi h b-glucan and obse ed a s ong educ ion o his pos ansla ional modi ica ion ollowing 24-h incuba ion wi h 100 mM CPH (Figu e 2A). To cha ac e ize he e ec s o CPH on ained immuni y, we conduc ed in i o aining expe i- men s on monocy es exposed o a ange o CPH concen a ions (Figu e 2B). When co-incuba ed wi h b-glucan o only he i s 24 h o he aining p o ocol, CPH dose-dependen ly a enua ed he heigh ened esponsi eness o ained cells o es imula ion wi h LPS (Figu es 2C and 2D). This e ec was mos p onounced a he highes concen a ion o CPH es ed (100 mM); howe e , a signi ican educ ion in TNFap oduc ion was obse ed a e co- incuba ion wi h 25 mM CPH. Simila inhibi ion o b-glucan-induced ained immuni y by CPH (100 mM) was obse ed when he cells we e es imula ed wi h he Toll-like ecep o 2 agonis Pam3Cys (10 mg/mL) (Fig- u e 2E). Exposu e o his concen a ion o CPH did no al e he mRNA exp ession o SETD7 (measu ed on day 6, p io o es im- ula ion), indica ing ha he inhibi o y e ec on ained immuni y occu ed a he le el o Se 7 ac i i y (Figu e 2F, open ba ep e- sen s DMSO ehicle con ols). Simila ly, RPL29 mRNA exp es- sion was una ec ed by 100 mM CPH (Figu e 2G, open ba ep e- sen s DMSO ehicle con ols). The a enua ing e ec o CPH on cy okine p oduc ion was no due o cy o oxici y o he com- pound (Figu e 2H) o induc ion o apop osis (Figu e 2I). We excluded he possibili y ha CPH inhibi s ained immuni y ia i s an ihis amine p ope ies by pe o ming expe imen s using an al e na i e an ihis amine inhibi o , diphenhyd amide, which did no al e ained immuni y induced by b-glucan (Figu e S1). To alida e he obse a ion o Se 7-dependen egula ion o cy okine p oduc ion, we also es ed he Se 7 inhibi o sine ungin (Sasaki e al., 2016). Sine ungin dose-dependen ly inhibi ed he heigh ened p oduc ion o TNFaand IL-6 by ained cells ollowing es imula ion wi h LPS (Figu es 2J and 2K). To es he b oade ole o Se 7 as a key egula o o ained immuni y, we assessed he e ec s o CPH on ained cy okine p oduc ion by o he compounds p e iously shown o induce ained immu- ni y. Indeed, incuba ion wi h 100 mM CPH also inhibi ed he in- duc ion o ained immuni y by BCG (Figu e 2L). Simila ly, he dec in-1 ligand lamina in was p e iously demons a ed o induce ained immuni y (Pe i e al., 2019). He e, we show ha he augmen ed TNFap oduc ion exhibi ed by mac ophages ained wi h lamina in was inhibi ed by CPH. A simila end was obse ed o IL-6 p oduc ion, wi h no di e ence obse ed be- ween cells ha we e incuba ed wi h lamina in and CPH, o lami- na in alone (Figu e 2M). To unde s and he mechanism o Se 7-dependen cy okine p oduc ion in ained immuni y, we assessed he TNF and IL6 Figu e 2. Pha macological Inhibi ion o Se 7 Dose-Dependen ly A enua es he P o-in lamma o y Cy okine Response o T ained Immuni y In Vi o (A) Wes e n blo analysis o b-glucan- ained mac ophages incuba ed wi h cyp ohep adine (CPH) o 24 h. HSP90 was used as a loading con ol. (B) G aphical o e iew o in i o aining me hods. Adhe en monocy es (Mo) we e s imula ed wi h b-glucan o RPMI cul u e medium o 24 h in he p esence o CPH o DMSO ehicle con ol, allowed o di e en ia e o mac ophages (M4), and es imula ed o 24 h wi h LPS, Pam3Cys, o RPMI on day 6. (C and D) P oduc ion o (C) TNFaand (D) IL-6 by b-glucan- ained mac ophages incuba ed wi h CPH o 50-me hyl hioadenosine (MTA) o he i s 24 h o in i o aining and es imula ed wi h LPS (n = 7 heal hy olun ee s). (E) P oduc ion o TNFaby b-glucan- ained mac ophages incuba ed wi h CPH o he i s 24 h o in i o aining and es imula ed wi h Pam3Cys (n = 3 heal hy olun ee s). (F) Exp ession o SETD7 mRNA on day 6 by cells ained wi h b-glucan in he p esence o 100 mM CPH (n = 6 heal hy olun ee s; open ba ep esen s DMSO ehicle con ol). (G) Exp ession o RPL29 mRNA a 24 h by cells ained wi h b-glucan in he p esence o 100 mM CPH (n = 3 heal hy olun ee s; open ba ep esen s DMSO ehicle con ol). (H) Analysis o lac a e dehyd ogenase (LDH) as a measu e o cy o oxici y in cells incuba ed wi h 100 mM CPH o 24 h (n = 3 heal hy olun ee s). (I) Analysis o iabili y and apop osis wi h Annexin V and PI s aining in cells incuba ed wi h 100 mM CPH e sus DMSO ehicle con ols o 24 h (n = 3 heal hy olun ee s). Fold change di e ence be ween CPH and ehicle con ols. (J and K) P oduc ion o (J) TNFaand (K) IL-6 by b-glucan- ained mac ophages incuba ed wi h sine ungin o he i s 24 h o in i o aining and es imula ed wi h LPS (n = 6 heal hy olun ee s). (L) P oduc ion o TNFaand IL-6 by bacillus Calme e-Gue ´ in (BCG)- ained mac ophages incuba ed wi h 100 mM CPH o he i s 24 h o in i o aining and es imula ed wi h LPS (n = 6 heal hy olun ee s). (M) P oduc ion o TNFaand IL-6 by lamina in- ained mac ophages incuba ed wi h 100 mM CPH o he i s 24 h o in i o aining and es imula ed wi h LPS (n = 6 heal hy olun ee s). Da a a e ep esen ed as mean ±SEM. *p < 0.05, Wilcoxon signed- ank es o es whe e app op ia e. See also Figu es S1 and S2. Cell Repo s 31, 107548, Ap il 21, 2020 5 p omo e s by ch oma in immunop ecipi a ion (ChIP) on day 6. We did no obse e signi ican inc eases in H3K4me1 en ich- men a ei he p omo e in cells ained wi h b-glucan. A simila pa e n o H3K4me1 en ichmen was obse ed o cells ained in he p esence o 100 mM CPH (Figu e S2), sugges ing ha Se 7 does no di ec ly egula e TNF and IL6 exp ession ia p o- mo e his one me hyla ion. Toge he , hese indings iden i y an impo an ole o Se 7 in ained immuni y in i o. Se 7 Regula es T ained Immuni y In Vi o Using mu ine models, we and o he s (Cheng e al., 2014; Ga cia- Val anen e al., 2017) ha e desc ibed he speci ic con ibu ion o b-glucan o he ac i a ion o ained immuni y in i o. Wild- ype mice ha ecei ed b-glucan injec ions exhibi ed enhanced cy o- kine p oduc ion by inna e immune cells in esponse o a second- a y challenge o in ec ion (A s e al., 2016a). We adop ed a simila app oach o es he ole o Se 7 in ained immuni y in i o. We gene a ed a Se 7 cons i u i e knockou (Se d7 KO) mouse model by he dele ion o Se d7 exon 2 egion (unpub- lished da a). Dele ion o exon 2, which encodes he i s MORN (memb ane occupa ion and ecogni ion nexus) epea , esul s in a ameshi and inac i a ion o Se 7. Wes e n blo and gene exp ession analyses con i med ha Se 7 was absen in bone ma ow (BM) o he homozygous Se d7 KO mice (Figu e 3A). Wild- ype and Se d7 KO mice we e sys emically adminis e ed a single in ape i oneal 1-mg dose o b-glucan as desc ibed p e- iously (Cheng e al., 2014). Con ol mice we e injec ed wi h endo oxin- ee phospha e-bu e ed saline (PBS). Fi e days a e b-glucan adminis a ion, he mice we e challenged wi h in ape - i oneal injec ions o 10 mg o LPS, and a e 3 h he se um le els o cy okines we e quan i ied (Figu e 3B). Con as ing he augmen ed p o-in lamma o y cy okine p oduc ion in wild- ype mice adminis e ed b-glucan, mice lacking unc ional Se 7 we e unable o moun ained immuni y agains endo oxin challenge wi h ega d o TNFaand IL-1bp oduc ion. Howe e , he e ec s o Se 7 dele ion on he ained p oduc ion o IL-6 in i o a e less clea , because IL-6 p oduc ion was al eady a high le els in wild- ype mice ha ecei ed PBS (Figu e 3C). The modula ion o myeloid p ogeni o s is an in eg al compo- nen o ained immuni y (Ch is e al., 2018; Kau mann e al., 2018; Mi oulis e al., 2018), which can explain he sus ained ac i- a ion o he inna e immune sys em beyond he sho li e span o ci cula ing myeloid cells. A ecen s udy showed ha adminis a- ion o b-glucan o mice esul ed in he expansion and pola iza- ion o hema opoie ic s em and p ogeni o cells (HSPCs) owa d myelopoiesis, which was associa ed wi h ele a ed signaling by inna e immune media o s such as IL-1band g anulocy e-mac o- phage colony-s imula ing ac o (GM-CSF), as well as changes in lipid and glucose me abolism (Mi oulis e al., 2018). To in es i- ga e he ole o Se 7 in hese adap a ions, we analyzed Se d7 mRNA in he BM o wild- ype mice and obse ed a end o in- c ease exp ession in mice adminis e ed b-glucan (Figu e 3D). We eplica ed key indings o he p e ious s udy (Mi oulis e al., 2018), albei in whole bone ma ow, and ound ha b-glucan-dependen ansc ip ional induc ion o Cs 2 (GM- CSF) and Il1b was signi ican ly educed in Se d7 KO mice. We also obse ed a educ ion in he exp ession o he su oga e ma ke o HSPCs Cd34 in Se 7 null mice; howe e , he exp es- sion o his gene emained ele a ed ela i e o Se 7-null mice ha ecei ed PBS injec ions (Figu e 3E). These da a demons a e ha Se 7 egula es he in i o p o-in lamma o y cy okine esponse o induc ion o ained immuni y by b-glucan and indi- ca e an impo an ole in hema opoie ic adap a ions ha suppo he sus ained pheno ype. Se 7 Regula es Key Me abolic Changes in Mac ophages T ained wi h b-Glucan T ained immuni y induced by b-glucan o BCG is cha ac e ized by me abolic ep og amming including inc eased glycolysis and in acellula accumula ion o uma a e and me alona e (A s e al., 2016a; Bekke ing e al., 2018). To explo e he po en ial ole o Se 7 in he hallma k glycoly ic me abolism o ained im- muni y, we measu ed ex acellula lac a e le els in day 6 mac o- phages ained wi h b-glucan. We obse ed a signi ican inc ease in hese le els in b-glucan- ained cells ha was abolished by co- incuba ion wi h 100 mM CPH o he i s 24 h o in i o aining (Figu e 4A). In addi ion o changes in glycolysis, p e ious s udies ound ha b-glucan aining is accompanied by he ep ession o OXPHOS. In con as o hose indings (Cheng e al., 2014), we obse ed inc eased oxygen consump ion a day 6 by cells ained wi h 1 mg/mL b-glucan (Figu e 4B). By pe o ming pa allel espi- ome y expe imen s wi h he Seaho se XF Ex acellula Flux Analyze and he Oxyg aph-2k om O obo os ( he ins umen used o measu ing oxygen consump ion by Cheng e al., 2014), we ound ha he s imula o y dose o b-glucan can explain he disc epancy be ween he cu en (inc eased oxygen con- sump ion) and p e iously epo ed indings (dec eased oxygen consump ion) (Cheng e al., 2014). Speci ically, we obse ed, us- ing he Oxyg aph-2k, aining wi h 1 mg/mL b-glucan s imula ed oxygen consump ion measu ed on day 6. On he o he hand, he s imula o y dose o 10 mg/mL desc ibed by Cheng e al. led o an o e all educ ion o oxygen consump ion by ained mac o- phages (Figu es S3A and S3B). To con i m hese obse a ions and o ule ou dono -speci ic a ia ion as he cause, we ained cells om he same se o dono s wi h 1 mg/mL b-glucan o 10 mg/mL b-glucan and analyzed hem in pa allel using he Sea- ho se sys em. While bo h s imula o y doses showed a end o in- c ease he ex acellula acidi ica ion a e, aining wi h 1 mg/mL b-glucan inc eased oxygen consump ion, whe eas aining wi h 10 mg/mL b-glucan educed oxygen consump ion o each dono (Figu es S3C and S3D). Nex , we sough o de e mine whe he Se 7 was mechanis ically in ol ed in he up egula ion o OX- PHOS by cells ained wi h b-glucan. Pa alleling i s a enua ing e ec on cy okine p oduc ion, CPH blun ed he inc ease in oxy- gen consump ion induced by b-glucan (Figu e 4B; open ba s ep esen DMSO ehicle con ols). To unde s and he signi icance o his change in oxygen con- sump ion o he induc ion o ained immuni y, we in es iga ed he e ec o gene ic a ia ion on indi idual esponses o b-glucan. D awing om ou gene ic s udy o PBMCs isola ed om 267 heal hy olun ee s (coho 1; 300BCG), we es ed o associa ions among common SNPs (mino allele equency >5%) and a ia ion in he magni ude o b-glucan- ained TNFa and IL-6 esponses o indi idual subjec s. Al hough genome- wide signi ican cy okine quan i a i e ai loci (cQTLs) we e no 6Cell Repo s 31, 107548, Ap il 21, 2020 obse ed, we iden i ied nume ous SNPs sugges i ely associ- a ed (p < 9.99 310 3 ) wi h adap i e changes in p o-in lamma- o y cy okine p oduc ion mapped wi hin 250 kb o genes ela ed o OXPHOS as well as genes encoding key ica boxylic acid (TCA) cycle enzymes. Va ia ion in genes encoding isoci a e de- hyd ogenase enzymes was associa ed wi h he po en ia ion o TNFap oduc ion upon aining wi h b-glucan. Simila ly, a ia ion in genes ha encode subuni s o he succina e dehyd ogenase Figu e 3. Se 7 Regula es T ained Immuni y Induced by b-Glucan In Vi o (A) Rep esen a i e wes e n blo o Se 7 p o ein exp ession in he bone ma ow o wild- ype (WT) and Se d7 KO mice. b-Ac in was used as loading con ol. Exp ession o Se d7 mRNA in he bone ma ow o WT and Se d7 KO mice (n = 7 mice pe g oup). (B) Schema ic o e iew o in i o induc ion o ained immuni y by b-glucan. (C) Plasma le els o TNFa, IL-6, and IL-1bin WT and Se d7 KO mice ained wi h PBS o b-glucan on day 1 and adminis e ed LPS on day 6 (n = 6–9 mice pe g oup). (D) Day 6 analysis o Se d7 mRNA exp ession in he bone ma ow o WT mice adminis e ed PBS o b-glucan (n = 7 mice pe g oup). (E) Bone ma ow mRNA exp ession o Cs 2,Il1b, and Cd34 in WT and Se d7 KO mice ained wi h PBS o b-glucan on day 1 and adminis e ed LPS on day 6 (n = 6–7 mice pe g oup). Da a a e ep esen ed as mean ±SEM. *p < 0.05, **p < 0.01, Mann-Whi ney es . Cell Repo s 31, 107548, Ap il 21, 2020 7 (legend on nex page) 8Cell Repo s 31, 107548, Ap il 21, 2020 S.F., J.O., K.H., J.H. .P., L.H., M.P.N., L.C.J.d.B., V.A.C.M.K., S.J.C.F.M.M., V.P.M., J.D.-A., M.O., E.P.B., and W.J.H.K.; W i ing—O iginal D a , S.T.K.; W i ing—Re iew and Edi ing, S.T.K., L.G., C.D.C.C. .d.H., J.C.d.S., S.F., J.O., J.H. .P., M.P.N., V.M., L.C.J.d.B., V.A.C.M.K., S.J.C.F.M.M., V.P.M., J.D.-A., M.O., E.P.B., W.J.H.K., L.A.B.J., M.G.N., and N.P.R.; Funding Acqui- si ion, L.A.B.J., M.G.N., and N.P.R.; Supe ision, J.O., M.M., A.E.-O., L.A.B.J., M.G.N., and N.P.R. DECLARATION OF INTERESTS W.J.H.K. is a scien i ic ad iso o Khond ion (Nijmegen, he Ne he lands) and o Fo i y The apeu ics. These subjec ma e expe s had no in ol emen in he da a collec ion, analysis and in e p e a ion, w i ing o he manusc ip , and he decision o submi he manusc ip o publica ion. 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Rep. 6, 21718. 16 Cell Repo s 31, 107548, Ap il 21, 2020 STAR+METHODS KEY RESOURCES TABLE REAGENT o RESOURCE SOURCE IDENTIFIER An ibodies Rabbi monoclonal an i-mono-me hyl-His one H3 (Lys4) Cell Signaling Technology Ca #5326; RRID: AB_10695148 Rabbi monoclonal an i-di-me hyl-Rpl29 (Lys5) Cell Signaling Technology Ca #19495; RRID: AB_2798819 Rabbi polyclonal an i-Se 7 Cell Signaling Technology Ca #2813; RRID: AB_823636 Rabbi polyclonal an i-HSP90 Cell Signaling Technology Ca #4874; RRID: AB_2121214 Rabbi polyclonal an i-b-ac in Sigma-Ald ich Ca #A2066; RRID: AB_476693 Mouse monoclonal an i-b-ac in Cell Signaling Technology Ca #3700; RRID: AB_2242334 swine-an i- abbi Ig polyclonal seconda y HRP an ibody Dako Ca #P0217; RRID: AB_2728719 Chemicals, Pep ides, and Recombinan P o eins b-glucan (b1,3-(D)-glucan P o esso Da id Williams, College o Medicine, Johnson Ci y, USA N/A Lipopolysaccha ide Sigma-Ald ich Ca #L2880 F om E.coli se o ype 055:B5 Pe coll Sigma-Ald ich Ca #P1644 Ficoll-Paque GE Heal hca e Ca #17-1440-03 Roswell Pa k Memo ial Ins i u e medium (RPMI) In i ogen Ca #22406031 iSc ip e e se ansc ip ase Bio-Rad Ca #1708840 TRIzol eagen Li e Technologies Ca #15596018 SYBR G een Applied Biosciences Ca #4368708 16% Fo maldehyde Fishe Scien i ic Ca #28908 Cyp ohep adine Selleckchem Ca #S2044 Sine ungin Sigma-Ald ich Ca #S8559 Bacillus Calme e–Gue ´ in accine S a ens Se um Ins i u , Copenhagen, Denma k N/A Lamina in Sigma-Ald ich Ca #L9634 Diphenhyd amide hyd ochlo ide Sigma-Ald ich Ca #D3630 C i ical Comme cial Assays Pie ce BCA p o ein assay ki The moFishe Scien i ic Ca #23225 Human TNFaDuoSe ELISA R&D sys ems Ca #DY210 Human IL-6 DuoSe ELISA R&D sys ems Ca #DY206 Mouse TNFaQuan ikine ELISA R&D sys ems Ca #MTA00B Mouse IL-6 Quan ikine ELISA R&D sys ems Ca #M6000B Mouse IL-1bQuan ikine ELISA R&D sys ems Ca #MLB00C Lac a e Fluo ome ic Assay ki Bio ision Ca #K607 MinElu e PCR pu i ica ion column QIAGEN Ca #28006 iSc ip cDNA syn hesis ki Bio-Rad Ca #1708891 Cy ox 96 assay P omega Ca #G1780 Succina e colo ime ic assay ki Sigma-Ald ich Ca #MAK184 Fuma a e colo ime ic assay ki Sigma-Ald ich Ca #MAK060 Mala e colo ime ic assay ki Sigma-Ald ich Ca #MAK067 Oxaloace a e colo ime ic assay ki Sigma-Ald ich Ca #MAK070 Ci a e colo ime ic assay ki Sigma-Ald ich Ca #MAK057 Expe imen al Models: O ganisms/S ains 300BCG coho (Human Func ional Genomics P ojec ) N/A h ps://www.human unc ionalgenomics.o g 200FG coho (Human Func ional Genomics P ojec ) N/A h ps://www.human unc ionalgenomics.o g (Con inued on nex page) Cell Repo s 31, 107548, Ap il 21, 2020 e1 RESOURCE AVAILABILITY Lead Con ac Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he Lead Con ac , Niels P. Riksen ([email p o ec ed]). Ma e ials A ailabili y This s udy did no gene a e new unique eagen s. Da a and Code A ailabili y This s udy did no gene a e any unique da ase s o code. EXPERIMENTAL MODEL AND SUBJECT DETAILS Human subjec s Wi h ega d o he in i o s udies, bu y coa s om male and emale heal hy dono s we e ob ained a e w i en in o med consen (Sanquin blood bank, Nijmegen, he Ne he lands). Cells we e isola ed and expe imen s conduc ed on he same day. Human coho s The 300BCG coho consis s o 267 heal hy males and emales o Wes e n Eu opean ances y. The second coho consis s o 119 heal hy indi iduals o Wes e n Eu opean ances y om he 200 Func ional Genomics coho (2011/399) o he Human Func ional Genomics P ojec . The 300BCG coho and 200FG coho s udies we e app o ed by he local e hics commi ee (CMO egio A nhem- Nijmegen, numbe NL58553.091.16 and numbe 2011-399, espec i ely). Inclusion o olun ee s and expe imen s we e conduc ed acco ding o he p inciples exp essed in he Decla a ion o Helsinki. All olun ee s ga e w i en in o med consen be o e any ma e ial was aken. Mice The Se d7 knockou (KO) mice we e gene a ed by genOway (Lyon, F ance). The Se d7 a ge ing ec o was designed wi hin he i s MORN domain o exon 2 con aining wo loxP si es (unpublished da a). Inse ion o loxP si es was in oduced 1.6kb ups eam o exon 2 by he in eg a ion o a loxP- lanked neomycine casse e. A a ge ing ec o con aining loxP si es lanking exon 2 o he Se d7 gene was in eg a ed by homologous ecombina ion in mouse emb yonic s em cells. Recombinan clones we e injec ed in o mouse C57BL/6J s ain blas ocys s and implan ed in o pseudop egnan emales. Once he cons uc was in eg a ed in o C57BL/6J back- g ound mice, i was c ossed wi h a CMV p omo e -d i en C e ecombinase mouse o c ea e a cons i u i e Se d7 KO mouse. Wild- ype and Se d7 KO mice housed unde speci ic pa hogen- ee condi ions we e used a he age o 9-11 weeks. Food and wa e was p o ided ad libi um. All animal s udies we e app o ed by he Al ed Medical Resea ch and Educa ion P ecinc (AMREP) Animal E hics Commi ee unde guidelines laid down by he Na ional Heal h and Medical Resea ch Council (NHMRC) o Aus alia. METHOD DETAILS Cells and eagen s Human pe iphe al blood mononuclea cells (PBMCs) we e isola ed om heal hy olun ee s by densi y-g adien cen i uga ion o e Ficoll-Paque (GE Heal hca e). Pe coll isola ion o monocy es was pe o med as p e iously desc ibed (A s e al., 2016a). Cells we e cul u ed in RPMI 1640 Du ch-modi ied cul u e medium (RPMI medium, In i ogen) supplemen ed wi h 10 mg/mL gen amicin (Cen a o m), 2 mM Glu amax (In i ogen), 1 mM py u a e (In i ogen), and 10% pooled human se um. S imuli and inhibi o s used we e Esche ichia coli lipopolysaccha ide (LPS; se o ype 055:B5, Sigma-Ald ich, 10 ng/mL), and Pam3Cys (EMC mic ocollec ions, Con inued REAGENT o RESOURCE SOURCE IDENTIFIER Se d7 knockou mouse P o esso Assam El-Os a, Monash Uni e si y, Melbou ne N/A Oligonucleo ides See Table S1 This pape N/A So wa e and Algo i hms G aphPad P ism 8.12 G aphpad so wa e h ps://www.g aphpad.com R s a is ical p og amming N/A RRID:SCR_001905 e2 Cell Repo s 31, 107548, Ap il 21, 2020 L2000, 10 mg/mL), Lamina in (Sigma), BCG (S a ens Se um Ins i u , Copenhagen, Denma k), cyp ohep adine (Selleckchem), sine un- gin (Sigma), and diphenhyd amide (Sigma). In i o aining and pha macological inhibi ion b-1,3-(D)-glucan (b-glucan) was kindly p o ided by P o esso Da id Williams (College o Medicine, Johnson Ci y, USA). Fo isola ion o cell wall b-glucans C. albicans was cul i a ed in 25 mL o YPD (1% yeas ex ac , 2% dex ose, 2% pep one) o 48 hou s a 30C. The cells we e ha es ed by cen i uga ion a 5,000x g o 5 minu es and pelle washed once wi h dH 2 O. The washed cell pelle s we e hen ozen a 20C o e nigh . P io o ex ac ing he cell wall b-glucans, he cell pelle s we e subjec ed o epea ed eeze- haw cycles (3X) o lyse he cells. Cell pelle s we e hen ex ac ed wi h a base/acid isola ion app oach. The supe na an con ained he wa e soluble mannans. Glucans a e wa e insoluble and we e ha es ed by cen i uga ion and washing in dH 2 O p io o lyophiliza ion. The s uc u e and pu i y o he b-glucans was de e mined by solu ion, high ield one and wo-dimensional Nuclea Magne ic Resonance Spec oscopy (1 and 2-D NMR). Adhe en monocy es we e ained as desc ibed p e iously (Bekke ing e al., 2014). Cells we e incuba ed wi h b-glucan (1 mg/mL), Lamina in (1 mg/mL), o BCG (5 mg/mL) o 24 hou s, washed wi h wa m phospha e bu e ed saline (PBS) and incuba ed in no mal cul u e medium a 37C, 5% CO 2 . Fo pha macological inhibi ion expe imen s, cells we e p e-incuba ed wi h cyp ohep adine (25- 100 mM), sine ungin (1, 10, 50, and 100 mg/mL), o diphenhyd amide (10, 50, 100 mg/mlL), o 1 hou p io o s imula ion. Following 5 days in cul u e, cells we e es imula ed wi h medium alone, 10 ng/mL LPS, o 10 mg/mL Pam3Cys o 24 hou s a 37C, 5% CO 2 . Cy okine p oduc ion was measu ed in supe na an s by enzyme-linked immunoso ben assay (ELISA) acco ding o he manu- ac u e ’s ins uc ions (R&D Sys ems). Cy okine measu emen Cy okine p oduc ion in supe na an s and plasmas was de e mined using comme cial enzyme-linked immunoso ben assay ki s o TNFa, IL-6, IL-1b(R&D Sys ems, MN, USA), IL-10 (Sanquin) acco ding o he ins uc ions o he manu ac u e s. Annexin V/PI s aining and lac a e dehyd ogenase (LDH) measu emen s o cell iabili y Apop osis and cell iabili y o monocy es a e 24h exposu e o 100 mM CPH o ehicle con ol was e alua ed wi h Annexin V-FITC (Bio ision) and P opidium Iodide ECD (Bio ision) luo escence wi h cy oFLEX low cy ome e (Beckman Coul e ) and analyzed wi h Kaluza 2.1 (Beckman Coul e ). Analysis o LDH as a measu e o cy o oxici y in cells incuba ed wi h CPH o 24 hou s was assessed in he supe na an s by using a Cy o ox 96 ki (P omega). Quan i a i e RT-PCR To al RNA was isola ed om human p ima y mac ophages and o al bone ma ow o mice using TRIzol eagen acco ding o he man- u ac u e ’s ins uc ions. 0.5-1 mg o o al RNA was used o syn hesize cDNA wi h he Supe Sc ip III Fi s -S and Syn hesis Sys em (The mo Fishe Scien i ic) acco ding o he manu ac u e ’s p o ocol. Quan i a i e RT-PCR was pe o med using an Applied Biosci- ences S epOne PLUS qRT-PCR machine using SYBR G een (In i ogen). All eac ions we e pe o med o a leas 6 biological ep- lica es and he alues exp essed as old inc ease in mRNA le els ela i e o hose in non- ained cells. 18s (human) o H3 3a (mouse) was used as a housekeeping gene. qRT-PCR p ime s a e lis ed in Table S1. Wes e n blo analysis o p ima y human cells Fo p o ein exp ession analysis o p ima y human cells, app oxima ely 1 310 6 monocy es exposed o 24 hou s o 100 mM CPH and 1310 6 mac ophages we e lysed wi h 100 mL o lysis bu e (1M T is pH 7.4), 5M NaCl, 0.5M EDTA, 10% NP-40, 0.5M NaF, 2.5% sodium deoxychola e, PhosSTOP (Roche), cOmple e (Roche)) p io o s imula ion on day 6. The homogena e was ozen, hen hawed and cen i uged a 4C o 10 min a 15,000 x g, and he supe na an was aken o analysis. The wes e n blo was pe o med using a T ans Tu bo Blo Sys em (Bio-Rad) acco ding o he manu ac u e ’s ins uc ions. P o ein was loaded and sepa a ed on SDS- PAGE using 4%–15% g adien p ecas gels and ans e ed o ni ocellulose memb anes using he semi-d y me hod (Bio-Rad). Rab- bi polyclonal p ima y an ibodies we e used o bo h Se 7 (1:1000, 2813, Cell Signaling Technology) and Rpl29k5me2 (1:1000, 19495, Cell Signaling Techonolgy. Swine-an i- abbi polyclonal seconda y HRP an ibody (1:5000, P0217, Dako) was used o de ec Se 7 and Rpl29k5me2 p o ein exp essions. b-ac in was de ec ed on he blo s using abbi polyclonal p ima y an ibody (1:1000, A2066, Sigma- Ald ich) and swine-an i- abbi polyclonal seconda y HRP an ibody (1:5000, P0217, Dako). HSP90 was de ec ed on he blo s using abbi polyclonal p ima y an ibody (1:1000, 4874, Cell Signaling Technology) and swine-an i- abbi polyclonal seconda y HRP an i- body (1:5000, P0217, Dako). Blo s we e de eloped wi h ECL (GE Heal hca e) acco ding o he manu ac u e ’s ins uc ions. Mouse expe imen s Fo he in i o s udy o ained immuni y, mice we e injec ed in ape i oneally wi h 1 mg o b-glucan in 200 mL o endo oxin- ee phos- pha e-bu e ed saline (PBS). In ape i oneal injec ions o PBS we e pe o med as con ol. Fi e days a e b-glucan adminis a ion, he mice we e injec ed in ape i oneally wi h 10 mg o LPS om E. coli 055:B5 (Sigma) as a seconda y challenge. Mice we e eu hanized a 3 hou s a e he LPS challenge. Cell Repo s 31, 107548, Ap il 21, 2020 e3 To isola e bone ma ow cells (BMCs), mouse emu s and ibias we e collec ed, immed and lushed wi h DPBS (GIBCO) using a 20 mL sy inge wi h a 25 gauge needle o elease BMCs. Bone ma ow suspensions we e gen ly ha es ed on 40 mm nylon mesh s aine (Falcon) in 50 mL conical ubes. A e cen i uga ion (5 min, 350 x g,4 C), he cells we e suspended wi h RBC lysis bu e (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1mM EDTA) o emo e e y h ocy es. P o ein analyses we e pe o med as desc ibed p e iously (Okabe e al., 2012). B ie ly, app oxima ely 5 310 6 BMCs we e lysed wi h 250 mL o bu e C (20 mM HEPES-KOH (pH 7.5), 25% Glyce ol, 520 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1mM DTT, 0.5 mM PMSF, 0.2% NP-40 and p o einase inhibi o cock ail) o 15 min a 4C, hen cen i uged o 15 min a 15,000 x g,4 C. The supe na an was collec ed o analysis. P ima y an ibodies we e used o bo h Se 7 (1:2,000, 2813, Cell Signaling Technology) and b-ac in (1:10,000, 3700, Cell Signaling Technology). IRDye 800CW Donkey an i-mouse IgG and IRDye 680RD Donkey an i- abbi IgG seconda y an ibodies (1:10,000 each, LI-COR) we e used o de ec Se 7 and b-ac in p o ein signals simul aneously. Me abolic analysis App oxima ely 1 310 7 monocy es we e ained wi h b-glucan (1 mg/mL) in 10 cm Pe i dishes (G eine ) in 10 mL medium olumes o 24 hou s, washed wi h wa m PBS and incuba ed in no mal cul u e medium a 37C, 5% CO 2 . Following 5 days in cul u e, cells we e de ached wi h e sene solu ion (The moFishe Scien i ic) and 1 310 5 cells we e pla ed o o e nigh -calib a ed ca idges in assay medium (RPMI wi h 0.6 mM glu amine, 5 mM glucose and 1 mM py u a e [pH adjus ed o 7.4]) and incuba ed o 1 hou in a non-CO 2 - co ec ed incuba o a 37C. Oxygen consump ion a e (OCR) was measu ed using a Cell Mi o S ess Ki ( o OCR) o a glycolysis s ess es ki in an XFp Analyze (Seaho se Bioscience), wi h inal concen a ions o 1 mM oligomycin, 1 mM FCCP, and 0.5 mM o e- none/an imycin A. Oxygen consump ion measu emen Cul u e medium was collec ed om cells ea ed wi h ei he RPMI o b-glucan (1 mg/mL o 10 mg/mL). A e s imula ion, he cells we e ypsinized, washed, and esuspended in he collec ed cul u e medium. Cell suspensions con aining 1 310 6 cells we e hen used o cellula O 2 consump ion analysis. Oxygen consump ion was measu ed a 37C using pola og aphic oxygen senso s in a wo-cham- be Oxyg aph (OROBOROS Ins umen s, Innsb uck, Aus ia). Da Lab so wa e (O obo os) was used o da a acquisi ion (2 s ime in- e al) and analysis (Gnaige , 2001). Fi s , basal espi a ion (baseline oxygen consump ion) was measu ed. Nex , leak espi a ion was de e mined by addi ion o 2.5 mM o he speci ic complex V inhibi o oligomycin A (OLI). Then, maximal elec on anspo chain com- plex (ETC) capaci y (maximum oxygen consump ion) was quan i ied by applying inc easing concen a ions o he mi ochond ial un- couple FCCP (0.25 o 20 mM inal maximal concen a ion). Finally, minimal (non-mi ochond ial) espi a ion was assessed by addi ion o he speci ic complex I inhibi o o enone (ROT; 100 nM) and he complex III inhibi o an imycin A (AA; 2.5 mM). Me aboli e measu emen s Cells we e ained wi h b-glucan (1 mg/mL) wi h and wi hou CPH (100 mM) as desc ibed abo e, using DMSO as a ehicle con ol. Me aboli e concen a ions measu ed om a leas 1 310 6 ained monocy es o succina e, uma a e, mala e, oxaloace a e, and ci - a e we e de e mined using a comme cial colo ime ic assay ki (Sigma) acco ding o he manu ac u e ’s ins uc ions. Gene ic analysis We conduc ed in i o b-glucan, BCG and oxLDL aining o adhe en PBMCs om 267 heal hy indi iduals o Wes e n Eu opean ances y om he 300BCG coho (NL58553.091.16). DNA samples o hese indi iduals we e geno yped using he comme cially a ailable SNP chip, In inium Global Sc eening A ay MD 1.0 om Illumina. Geno ype in o ma ion on app oxima ely 4 million sin- gle-nucleo ide polymo phisms (SNPs) was ob ained upon impu a ion (MAF > 5% and R 2 > 0.3 o impu a ion quali y). Fi s , aw cy o- kine le els we e log- ans o med and he a io be ween ained and non- ained cy okine le els we e aken as he change o cy okine le els. The cy okine changes we e mapped o geno ype da a using a linea eg ession model wi h age and sex as co a ia es. Gene ic ou lie s (n = 15) and samples s imula ed wi h low b-glucan (< 1 mg/mL) we e emo ed be o e QTL mapping. We also conduc ed in i o b-glucan aining o adhe en PBMCs in a second coho o 119 heal hy indi iduals o Wes e n Eu opean ances y om he 200 Func ional Genomics coho (2011/399) o he Human Func ional Genomics P ojec (www. human unc ionalgenomics.o g). Geno ype in o ma ion on app oxima ely 4 million single-nucleo ide polymo phisms (SNPs) was ob- ained using Illumina HumanOmniExp essExome SNP chip upon impu a ion. Only SNPs wi h a mino allele equency o R5% ha passed s anda d quali y il e s we e included in he analysis. Raw cy okine le els we e log- ans o med and he a io be ween ained and non- ained cy okine le els was used o quan i y he ained immuni y esponse. They we e subsequen ly mapped o geno ype da a using a linea eg ession model wi h age and sex as co- a ia es (Li e al., 2016). ChIP-seq and ChIA-PET analysis This s udy makes use o H3K4me1 ChIP-seq da ase s gene a ed by he Bluep in Conso ium (Adams e al., 2012). This s udy makes use o ChIA-PET da a (accession numbe GSM970213). In he g aphical display o he ChIA-PET da a, he pai ed end ags (PETs) o ch omosomal in e ac ions a e ep esen ed by wo blocks o each end o he con ac , connec ed by a ho izon al line. The numbe o PETs in a clus e e lec s he s eng h o he ch omosomal in e ac ion. The p e-p ocessed da ase s we e isualized using he UCSC genome b owse wi h he GRCh37/hg19 assembly (Ken e al., 2002). Enhance s we e iden i ied om he GeneHance da abase o e4 Cell Repo s 31, 107548, Ap il 21, 2020 human egula o y elemen s (Fishile ich e al., 2017). Enhance s wi h he highes anno a ion-de i ed con idence sco e we e selec ed. The s udy makes use o Hi-C da a om he K562 cell line (accession GSE63525). Hi-C maps we e gene a ed a 5kb esolu ion using he 3D genome b owse wi h he GRCh37/hg19 assembly (Wang e al., 2018). Ch oma in immunop ecipi a ion T ained monocy es on day 6 we e c oss-linked in me hanol ee 1% o maldehyde, ollowed by sonica ion and immunop ecipi a ion using an ibodies agains H3K4me1 (Cell Signaling Technology). Immunop ecipi a ed ch oma in was p ocessed u he o qRT-PCR analysis using he MiniElu e DNA pu i ica ion ki (QIAGEN). P ime s used in he eac ion a e lis ed in Table S1. Samples we e analyzed wi h a compa a i e C me hod on he S epOne PLUS qPCR machine (Applied Biosys ems) using SYBR g een (In i ogen) in acco - dance wi h he manu ac u e ’s ins uc ions. QUANTIFICATION AND STATISTICAL ANALYSIS S a is ical pa ame e s including he exac alue o n, he de ini ion o cen e , dispe sion and p ecision measu es (mean ±SEM), and s a is ical signi icance a e epo ed in he igu es and igu e legends. S a is ical analysis was pe o med using G aphPad P ism 8.12 (G aphPad Inc.). Analysis o human qPCR, ELISA and cellula assays was pe o med using Wilcoxon signed- ank es , es o non- pa ame ic Mann-Whi ney es s, as app op ia e. Analysis o mouse da a used Mann-Whi ney es s o compa isons be ween g oups. R-package Ma ix-eQTL was used o cy okine QTL mapping. A p alue < 0.05 (*) was conside ed s a is ically signi ican , (**) p < 0.01. Da a a e shown as mean ±SEM. Cell Repo s 31, 107548, Ap il 21, 2020 e5