Hyperglycemia Induces Trained Immunity hauv Macrophages Thiab Lawv Precursors Thiab Txhawb Atherosclerosis

POB ZEB:

Kev pheej hmoo mob plawv hauv ntshav qab zib tseem nce siab txawm tias muaj kev kho mob ntshav qab zib. Peb xav tias hyperglycemia ua rau muaj kev tiv thaiv kev tiv thaiv hauv macrophages, txhawb cov yam ntxwv tsis tu ncua proatherogenic.

Tsis tas li ntawd, nyob rau hauv ntau yam ntaub ntawv, peb kuj tau kawm tias ib tsob nroj hu ua Cistanche muaj txiaj ntsig zoo rau kev txhim kho kev tiv thaiv. Cistanche yog tshuaj suav tshuaj suav nrog ntau yam kev tiv thaiv kab mob. Kev tso tawm ntawm yam, txhim kho kev ua haujlwm ntawm lub cev tiv thaiv kab mob, thiab lwm txoj hauv kev los txhim kho kev tiv thaiv ntawm tib neeg lub cev. Yog li ntawd, hauv lub neej niaj hnub, tib neeg tuaj yeem txhim kho lawv txoj kev tiv thaiv kab mob los ntawm kev noj Cistanche deserticola nyob rau hauv ib qho me me, tiv thaiv thiab kho ntau yam kab mob.

Nyem cistanche deserticola ntxiv khoom

Txoj kev:

Cov pob txha pob txha-los ntawm macrophages los ntawm kev tswj cov nas thiab nas uas muaj ntshav qab zib tau loj hlob hauv cov piam thaj hauv lub cev (5 mmol / L) thiab raug RNA sequencing (n=6), kev soj ntsuam rau transposase nkag tau chromatin sequencing (n=6) ), thiab chromatin immunoprecipitation sequencing (n=6) rau kev txiav txim siab ntawm hyperglycemia-vim kev tiv thaiv kab mob. Kev hloov cov pob txha pob txha los ntawm nas nrog (n=9) lossis tsis muaj (n=6) ntshav qab zib rau hauv (normoglycemic) Ldlr −/− nas tau siv los ntsuas nws qhov tseem ceeb hauv vivo. Cov ntaub ntawv pov thawj ntawm hyperglycemia-vim kev tiv thaiv kev tiv thaiv tau raug nrhiav hauv tib neeg peripheral ntshav mononuclear hlwb los ntawm cov neeg mob ntshav qab zib (n=8) ​​piv nrog cov kev tswj hwm (n=16) thiab hauv tib neeg atherosclerotic plaque macrophages excised los ntawm laser ntes microdissection. .

TSEEM CEEB:

Nyob rau hauv macrophages, siab extracellular qabzib txhawb proinflammatory gene qhia thiab proatherogenic functional yam ntxwv los ntawm glycolysis-dependent mechanisms. Cov pob txha pob txha-los ntawm macrophages los ntawm cov nas mob ntshav qab zib tau khaws cov yam ntxwv no, txawm tias thaum coj los ntawm cov piam thaj hauv lub cev, qhia txog kev tiv thaiv kev tiv thaiv hyperglycemia. Kev hloov cov pob txha pob txha los ntawm cov nas mob ntshav qab zib mus rau hauv (normoglycemic) Ldlr −/- nas nce aortic cag atherosclerosis, paub meej tias muaj kab mob cuam tshuam thiab tsis tu ncua ntawm kev kawm hauv lub cev tiv thaiv kab mob. Kev sib xyaw ua ke rau transposase-accessible chromatin, chromatin immunoprecipitation, thiab RNA sequencing analyses ntawm hematopoietic qia hlwb thiab cov pob txha pob txha-derived macrophages tau qhia txog cov txiaj ntsig ntawm kev mob ntshav qab zib. Tus qauv ntawm qhib chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Ib yam li ntawd, transcriptomes ntawm atherosclerotic plaque macrophages thiab peripheral leukocytes nyob rau hauv cov neeg mob uas muaj hom 2 mob ntshav qab zib tau enriched rau Runx1 lub hom phiaj, zoo ib yam li lub luag hauj lwm nyob rau hauv tib neeg kab mob. Pharmacological inhibition ntawm Runx1 hauv vitro inhibited qhov kev cob qhia phenotype.

Cov ntsiab lus:

Hyperglycemia-vim kev tiv thaiv kev tiv thaiv kab mob tuaj yeem piav qhia vim li cas lub hom phiaj siab dua cov piam thaj tsis zoo hauv kev txo qis kev pheej hmoo ntawm macrovascular hauv ntshav qab zib thiab qhia lub hom phiaj tshiab rau kev tiv thaiv kab mob thiab kho.

Lo lus tseem ceeb:

Mob ntshav qab zib mellitus ◼ epigenetics ◼ qabzib ◼ mob ◼ macrophages

Ntshav qab zib yog txuam nrog kev pheej hmoo siab ntawm atherosclerosis thiab nws cov teeb meem, suav nrog myocardial infarction.1 Hyperglycemia yog ib qho tseem ceeb hauv ob hom ntshav qab zib hom 1 thiab hom 2, thiab kev kho mob tau tsom mus rau kev txo cov ntshav qabzib ntau. Kev kho mob ntshav qab zib kom qis yog qhov ua tau zoo hauv kev txo qis vascular kev pheej hmoo ntawm cov neeg mob ntshav qab zib hom 1, tab sis cov txiaj ntsig tau ncua sijhawm ntau xyoo 2 thiab kev cuam tshuam ib ntus los ntawm kev tswj cov ntshav qabzib.3 Hauv hom 2 mob ntshav qab zib, kev txo cov piam thaj tau pom tias tsis muaj txiaj ntsig lossis Cov txiaj ntsig me me ntawm cov txiaj ntsig ntawm atherosclerosis-txog vascular tshwm sim xws li mob myocardial infarction.4-6 Kev pheej hmoo pheej hmoo ntawm cov kab mob plawv, txawm tias tom qab cov piam thaj txo qis, tau raug hu ua cov txiaj ntsig qub txeeg qub teg lossis kev nco qab metabolic, 4,7 tab sis cov txheej txheem hauv qab tseem tsis meej.

Atherosclerosis yog ib hom kab mob inflammatory uas tshwm sim los ntawm kev tso tawm thiab khaws cia ntawm cov lipoproteins hloov pauv thiab kev sib sau ntawm cov tshuaj tiv thaiv kab mob hauv cov phab ntsa ntawm cov hlab ntsha loj. Macrophages yog qhov tseem ceeb hauv txhua theem ntawm atherosclerosis thiab yog dav suav hais tias yog cov hom phiaj kho mob.8 Lawv yog cov heterogeneous thiab functionally teb rau microenvironmental cues. Macrophages tau raug subtyped rau hauv kev tawm tsam phenotypes, uas yog proinflammatory (M1), nyob rau hauv teb rau tus xov tooj-zoo li receptor ligand lipopolysaccharide (LPS) thiab interferon- (IFN), los yog cov ntaub so ntswg reparative (M2), piv txwv li, teb rau interleukin (IL. ) -4, txawm hais tias nyob rau hauv vivo lawv lub luag haujlwm ua haujlwm yog qhov nyuaj dua.9

Hyperglycemia exacerbates atherosclerosis kev loj hlob thiab retards plaque regression, 10 nrog nce kev qhia ntawm proinflammatory genes thiab tsis kam mus induction ntawm M2-sociated gene expression.10,11 Nyob rau hauv monocytes thiab macrophages, cellular zog txoj kev yog tswj los ntawm ib tug interplay ntawm cell- intrinsic thiab ib puag ncig stimuli, uas, nyob rau hauv lem, markedly cuam tshuam rau lub cev tiv thaiv kev ua hauj lwm thiab noob qhia.12 Nyob rau hauv teb rau IL-4, M2 macrophages feem ntau metabolize fatty acids thiab cia siab rau oxidative phosphorylation.13 Hauv qhov sib piv, M1 macrophages xav tau cov piam thaj thiab hloov mus rau aerobic glycolysis, zoo ib yam li Warburg cov nyhuv hauv kev mob qog noj ntshav.14,15 Cov kev hloov pauv hauv metabolic no tshwm sim vim qhov tshwm sim ntawm cytokine stimulation tab sis kuj tuaj yeem txiav txim siab ua haujlwm ntawm macrophage.16 Tsis tas li ntawd, cov ntaub ntawv tsis ntev los no qhia tau hais tias kev hloov pauv hauv monocyte thiab macrophage metabolism. , suav nrog aerobic glycolysis lossis ua kom txoj hauv kev mevalonate, 17,18 ua rau lub sijhawm ntev ntawm lub cev tiv thaiv kab mob hauv lub cev (hu ua kev tiv thaiv kab mob) los ntawm kev hloov pauv ntawm epigenetic thiab cov qauv chromatin hloov. ntawm lawv cov pob txha pob txha (BM) progenitors.20

Peb xav tias kev cob qhia kev tiv thaiv kab mob hauv cov lus teb rau cov ntshav qab zib kom siab rau cov ntshav qab zib hyperglycemic nco txog atherosclerosis. Yog li, peb tau nrhiav kev txiav txim siab seb puas muaj tus kab mob hyperglycemia-vim cov kab mob cuam tshuam txog kev hloov pauv hauv monocyte thiab macrophage muaj nuj nqi thiab seb cov kev hloov pauv no puas muaj nyob tom qab kho cov piam thaj ib txwm, hais txog qhov tseem ceeb reprogramming. Peb ua ke cov kev tshawb fawb ntawm cellular muaj nuj nqi, metabolomics, transcriptomics, thiab epigenomics los txheeb xyuas seb hyperglycemia hloov pauv cov metabolism hauv kev hloov pauv mus ntev li cas los ntawm kev hloov pauv epigenetic. Peb tshawb nrhiav seb cov kev hloov no yuav tshwm sim li cas ntawm qib hematopoietic qia cell (HSC) thiab hauv cov macrophages sib txawv. Kev ua haujlwm tseem ceeb yog kuaj los ntawm BM hloov pauv hauv tus qauv nas ntawm atherosclerosis, thiab cov kev tshawb pom tau lees paub los ntawm kev nug ntawm macrophages muab rho tawm los ntawm tib neeg cov ntshav peripheral thiab atherosclerotic plaques los ntawm laser capture microdissection (LCM). Peb qhov kev tshawb pom yuav piav qhia txog qhov tsis kam ntawm cov teeb meem macrovascular ntawm ntshav qab zib rau cov kev kho mob ntshav qab zib kom qis.

Txoj kev

Cov ntaub ntawv pov thawj

Cov ntaub ntawv tsim tawm hauv txoj kev tshawb no tau muab tso rau hauv National Center for Biotechnology Information Gene Expression Omnibus thiab nkag mus tau los ntawm tus lej nkag GSE176068.

Tsiaj txhu

Murine BM transplantation experiments were performed at The Jackson Laboratory (Bar Harbor, ME) in vivo facility by the Institutional Animal Care and Use Committee regulations (Association for Assessment and Accreditation of Laboratory Animal Care accredited facility, project reference 70195 IVAT). All other animal protocols were conducted by the UK Home Office under the guidance of the operation of the Animals (Scientific Procedures) Act 1986, complying with all ethical regulations and the institutional review board guidelines (project license 30/3374; January 27, 2016). Wild-type C57BL/6J (12–14 weeks old) were randomly assigned to control or diabetic experimental groups. Diabetes was induced through an intraperitoneal injection of streptozotocin at a low dose range (42–45 mg·kg−1·/d−1) over 5 consecutive days. After 6 weeks after the final streptozotocin injection, diabetic mice with nonfasted blood glucose levels >13.9 mmol / L yog euthanized, thiab cov ntaub so ntswg tau sau.

Kab Kab

Tag nrho cov kab ntawm tes tau loj hlob ntawm 37 degree thiab 5 feem pua ​​​​CO2. L929 hlwb tau loj hlob hauv Roswell Park Memorial Institute nruab nrab ntxiv nrog 10 feem pua ​​​​fetal bovine serum (FBS), 2 feem pua ​​penicillin-streptomycin, thiab 2 mmol / L glutamine. L929 hlwb tau loj hlob mus rau tag nrho cov confluence rau 7 mus rau 8 hnub, thiab cov conditioned media raug tshem tawm, muab faib ua aliquots, thiab khaws cia ntawm −20 degree kom txog thaum xav tau. Cov kab mob ntawm daim tawv nqaij los ntawm nas endothelial tau coj mus rau hauv 25 mmol / L qabzib DMEM nrog 10% FBS, 2% penicillin-streptomycin, thiab 2 mmol / L glutamine.

BM-Drived Macrophages

BM-derived macrophages (BMDMs) siv nyob rau hauv txoj kev tshawb no tau tsim los ntawm kev tshem tawm BM los ntawm murine femurs thiab tibias thiab plating ntawm cov nqaij mos kab lis kev cai-kho Petri cov tais diav ua tiav 5 mmol / L qabzib DMEM (ntxiv nrog 10 feem pua ​​FBS, 2 feem pua ​​penicillin. -streptomycin, thiab 2 mmol / L glutamine) ntxiv rau 20 feem pua ​​​​L929 cell-conditioned media rau kev sib txawv. Tom qab sib txawv rau 7 hnub, BMDMs tau raug xaiv zoo, plated ntawm qhov ceev ntawm 2 × 105 hlwb / mL, thiab ua kom tiav 5 mmol / L qabzib DMEM (tshwj tsis yog hais tias lwm yam) nrog rau 10 ng / mL recombinant murine interferon- (IFN. - ; R&D Systems, UK) ntxiv rau 100 ng/mL LPS (Sigma-Aldrich, UK), 10 ng/mL recombinant nas IL-4 (R&D Systems, UK), lossis kev tshaj tawm-tsuas yog tswj rau 16 teev ua ntej siv . BMDMs kuj tau txais kev coj noj coj ua nyob rau hauv ntau yam glycemic tej yam kev mob (5 mmol / L qabzib DMEM nrog ntxiv qabzib kom ua tiav qhov kawg ntawm 11 lossis 20 mmol / L qabzib) lossis kev tswj osmotic (mannitol; 5 mmol / L glucose DMEM) ntxiv rau 15 mmol / L mannitol). Cov kev tshawb fawb metabolic tau ua thaum tsis muaj lossis muaj 10 mmol / L dichloroacetates (Sigma-Aldrich, UK) lossis 1 mmol / L 2-deoxy-d-glucose (Sigma-Aldrich, UK).

Kev rho tawm HSC

HSCs raug cais nrog MagniSort Mouse Hematopoietic Lineage Depletion Kit (ThermoFisher Scientific, UK) los ntawm kev yaug BM los ntawm murine femurs thiab tibias thiab incubating nrog cov cim kom tshem tawm cov kab mob (CD3, CD4, B220, Ter119, thiab Gr1) thiab ntxiv rau. HSCs. HSCs tau coj mus kuaj hauv 5 mL fluorescence-activated cell sorting tubes ua tiav 5 mmol / L DMEM (los yog 20 mmol / L qabzib lossis mannitol rau kev qhia tawm hom tsiaj qus) nrog ntxiv ntawm 100 ng / mL LPS ntxiv rau 10 ng / mL IFN - los yog 10 ng/mL recombinant nas IL-1 (R&D Systems, UK) rau 16 teev rau Western blot tsom los yog 2 teev rau kev ntsuam xyuas rau transposase accessible chromatin sequencing (ATAC-seq) tsom xam.

BMDM Hauv Vitro Memory Model

Mouse BM tau muab faib ua BMDMs raws li tau piav qhia saum toj no hauv 5 lossis 20 mmol / L qabzib rau 1, 2, 4, lossis 7 hnub. BMDMs tau raug xaiv zoo los ntawm kev ua kom adhesion, rov ua kom tiav 5 mmol / L DMEM, thiab sab laug rau incubate rau 48 teev kom tso cai rau lub sijhawm ntawm cov piam thaj normalization. BMDMs tau raug txhawb kom siv nrog 10 ng/mL IFN- ntxiv rau 100 ng/mL LPS lossis tswj kev tshaj tawm rau 16 teev thaum tsis muaj lossis muaj 0.25, 0.5, 1, 5, lossis 10 µmol/L Ro 5-3335 (Merck Millipore, UK) ua ntej siv rau kev tshuaj ntsuam gene.

Mouse Monocyte Isolation

Murine monocytes raug cais tawm ntawm cov ntshav peripheral nrog EasySep Mouse Monocyte Isolation Kit (Stemcell Technologies, Canada) raws li cov chaw tsim khoom raws tu qauv. Cells tau siv tam sim ntawd rau kev soj ntsuam adhesion.

Seahorse XFe 96 Real-Time ATP Rate Assay Kit

Extracellular flux tsom xam tau ua nrog XFp RealTime ATP Rate Assay cov khoom siv ntawm BMDMs (8 × 104 hlwb rau ib qhov dej; 5 replicate qhov dej rau ib tus tsiaj thiab mob), raws li cov chaw tsim khoom cov lus qhia. Peb lub hauv paus kev noj cov pa oxygen thiab ntsuas qhov ntsuas ntawm cov cellular acidification tau ua ntej qhov sib txawv ntawm cov piam thaj (5 lossis 20 mmol / L) lossis tswj (mannitol; qhov chaw txhaj tshuaj A) qhov nruab nrab tau txhaj. Tom qab kev ntsuas 7 ntxiv, cov tshuaj oligomycin (2 µmol / L; txhaj tshuaj chaw nres nkoj B) thiab ua ke antimycin A (0.5 µmol / L) thiab rotenone (0.5 µmol / L; qhov chaw txhaj tshuaj C), los ntawm XFp Real-Time ATP Rate Assay cov khoom siv, tau txhaj tshuaj nrog 3 qhov kev ntsuas tom qab. Tus nqi ntawm ATP ua los ntawm glycolysis thiab ATP tus nqi ntsuas (mitoATP tus nqi ntau lawm / glycolytic ATP ntau lawm tus nqi) tau txiav txim siab nrog Wave (version 2.6, Agilent).

Static Adhesion Assay

Cov tawv nqaij-derived nas endothelial hlwb tau loj hlob mus rau confluence ntawm iav npog hauv 12- cov phiaj zoo thiab qhib los ntawm 10 ng/mL recombinant nas qog necrosis factor- (R&D Systems, UK) rau 16 teev. Unstimulated los yog IFN- ntxiv rau LPS-stimulated peripheral ntshav monocytes (los yog BMDMs los ntawm streptozotocin mob ntshav qab zib nas kab lis kev cai hauv 5 mmol / L qabzib) tau fluorescently sau ntawv ntsuab nrog PKH67 fluorescent cell linker cov khoom (Sigma-Aldrich, UK) raws li cov chaw tsim khoom cov lus qhia. Labeled hlwb raug muab ntxiv rau cov hlwb endothelial ntawm qhov ntom ntawm 5 × 104 cell ib daim npog rau 1 teev ua ntej cov xov xwm thiab cov hlwb tsis sib txuas tau raug tshem tawm, ntxuav nrog phosphate-buffered saline (PBS), thiab tsau hauv 4 feem pua ​​​​ntawm paraformaldehyde. Plaub daim duab ib daim npog tau txais; tus naj npawb ntawm adhered, sau lub hlwb raug suav; thiab cov dluab ib coverlip tau nruab nrab. Cov duab pom tau sib npaug li qhov ci ntawm ImageJ.

Foam Cell Assay

Unstimulated los yog stimulated (LPS plus IFN- ) BMDMs tau loj hlob ntawm iav npog hauv 12- cov phiaj zoo thiab incubated nrog 25 µg/mL DiI-acetylated low-density lipoprotein (LDL; Bioquote, US) rau 48 teev. Media thiab seem DiI-acetylated LDL raug tshem tawm, thiab cov hlwb raug ntxuav 3 zaug nrog PBS thiab kho hauv 4 feem pua ​​paraformaldehyde. Cov lipids tau stained nrog Roj Liab O, thiab cov ntawv npog tau DAPI mounted. Cov duab theem sib piv ntawm Cov Roj Liab O staining thiab fluorescent dluab ntawm lub nuclei raug coj mus (4 dluab ib daim npog) thiab siv los ntsuas cov lipids los yog pes tsawg lub hlwb, raws li, nrog ImageJ software. Lipid cheeb tsam ntawm ib daim duab yog tom qab ntawd normalized rau cov cell ntawm ib daim duab, thiab cov txiaj ntsig tau nthuav tawm raws li kev hloov pauv ntau dua 5 mmol / L glucose control samples (rau BMDM glucose thwmsim) thiab tswj cov qauv (rau cov kev sim ntshav qab zib BMDM). Cov duab pom tau zoo ib yam li qhov ci ntsa iab.

Diabetic BM Transplantation Experiment

BM transplantation experiments were performed at The Jackson Laboratory. Diabetes was induced in CD68–green fluorescent protein (GFP) transgenic mice as previously described for 4 weeks. Diabetic CD68-GFP mice with nonfasted blood glucose levels >13.9 mmol/L thiab tswj CD68-GFP nas ntawm tib lub hnub nyoog tau siv los ua BM pub. BM tau sau los ntawm cov nas pub dawb, thiab 5 × 106 cov hlwb siv tau raug txhaj tshuaj rau hauv cov kab mob tuag taus (2 × 500 rads) LdLr−/− tus neeg txais B6.129S7- Ldlr-tm1Her/J nas (JAX Tshuag No. 002207 ). Tom qab engraftment, tag nrho cov neeg tau txais cov tsiaj tau muab tso rau hauv Western noj zaub mov (Open Source Diets D12079B; 1 feem pua ​​​​ rog, 40 feem pua ​​​​kcal) rau 12 lub lis piam. Tag nrho cov ntshav tau sau rau ntawm 4, 8, thiab 12 lub lis piam tom qab engraftment rau flow cytometric tsom xam ntawm peripheral ntshav mononuclear hlwb (PBMCs) thiab saib xyuas cov qib engraftment thiab ntsuam xyuas leukocyte cov pejxeem. Engraftment, soj ntsuam txhua 4 lub lis piam tom qab engraftment, tau soj ntsuam los ntawm staining hlwb nrog antiCD45 thiab ntsuam xyuas cov txheeb ze ntawm CD45 ntxiv rau GFP ntxiv hlwb ntawm tag nrho cov CD45 ntxiv cov pejxeem. Thaum 12 lub lis piam, nas tau euthanized, tag nrho cov ntshav tau sau, thiab sera tau ua tiav rau triglycerides, nonesterified fatty acids, LDL, thiab high-density lipoprotein cholesterol ntsuas. Cov nas tau perfused nrog PBS, ua raws li perfusion thiab kho nrog 4 feem pua ​​​​ntawm paraformaldehyde thaum hmo ntuj. Lub plawv thiab aorta tau raug txiav thiab muab tso rau hauv 70 feem pua ​​​​ethanol kom txog thaum ua tiav.

Aortic Root Plaque Analysis thiab Immunofluorescence

Txhawm rau kom muaj nuj nqis ntawm cov quav hniav, paraformaldehyde-taw aortic cov hauv paus hniav tau muab tso rau hauv qhov pom kev txiav nruab nrab thiab muab faib rau ntawm lub dav hlau sib npaug mus rau atria. Txhawm rau kom muaj nuj nqis ntawm cov quav hniav, cov plaque lipid cov ntsiab lus, 21 thiab cov ntsiab lus collagen, 5 ntu, sib npaug ntawm cov hauv paus aortic, yog Masson-Goldner trichrome stained (Merck, UK). Luv luv, ntu tau kho hauv Bouin tov ntawm chav tsev kub rau 1 teev, ntxuav hauv dej rau 5 feeb, thiab tom qab ntawd muab tso rau hauv Masson-Goldner staining kit protocol los ntawm qhov thib ob 70 feem pua ​​​​ethanol incubation raws li cov chaw tsim khoom. raws tu qauv sij hawm. Xylene-ntub slides yog ces mounted nrog Neo-Mount thiab kaw nrog iav npog. Cov yam ntxwv ntawm cov quav hniav tau txheeb xyuas ntawm Image Pro Plus software version 6.0 (Media Cybernetics, Silverspring, MD), thiab cov ntaub ntawv tau tshwm sim raws li qhov nruab nrab ntawm txhua ntu.

Immunofluorescence tau ua tiav ntawm 6 ntu uas nyob ib sab. Cov seem tau stained rau cov ntsiab lus macrophage nrog GFP (luv anti-GFP, A6455, Invitrogen) lossis macrophage marker galectin -3 (tshis anti-galectin 3, AF1197, R&D Systems), rau cov leeg nqaij leeg cov ntsiab lus nrog -actin ( luav anti-smooth nqaij actin, ab32575, Abcam PLC), thiab rau cov cim epigenetic H3K4me3 (luav polyclonal rau histone H3 [trimethyl K4], Ab8580, Abcam PLC) thiab H3K27ac (luv polyclonal rau histone H3 [acetyl K27, Abstract], Abcam PLC). Rau immunofluorescence staining, ntu tau rov ua dej nrog PBS rau 5 feeb ntawm chav tsev kub thiab thaiv rau 1 teev nrog DAKO thaiv tsis pub (Agilent, UK). Thawj cov tshuaj tiv thaiv tau diluted rau 1 µg / mL hauv DAKO thiab ntxiv rau cov ntu thaum hmo ntuj ntawm 4 degree. Cov tshuaj tiv thaiv thib ob tau diluted 1:200 hauv DAKO thiab ntxiv rau 1 teev nyob rau hauv chav tsev kub (loj tiv thaiv luav [A488], A21206, Lub Neej Technologies; nees luav antigoat [A594], A11058, ThermoFisher Scientific). Cov ntu tau kawg DAPI mounted (glycerol mounting nruab nrab nrog DAPI thiab DABCO, Abcam, UK). Cov duab fluorescent tau txheeb xyuas nrog Image J software.

Gene Expression

Tag nrho RNA raug cais nrog RNeasy Plus Mini cov khoom siv (Qiagen, UK) thiab suav nrog Nanodrop 2000 UV-pom spectrophotometer. cDNA synthesis tau ua nrog 0.2 mus rau 2 µg RNA nyob rau hauv ib tug polymerase saw cov tshuaj tiv thaiv (PCR) nrog QuantTect reverse transcription kit (Qiagen, UK), raws li cov chaw tsim tshuaj paus cov lus qhia. PCR quantitative real-time tau ua tiav ntawm 5 ng/µL cDNA nrog Taqman sojntsuam tshwj xeeb rau B2m (Mm00437762), Il-6 (Mm00446190), Il-1 (Mm00462531_ m1), iNos (Mm00440502), Ym1 (Mm00657009), thiab Fizz1 (Mm00556208). Quantitative PCR tau ua nrog TaqMan universal master mix II nrog UNG ntawm QuantStudioTM 7 Flex Real-Time PCR System (ThermoFisher). Tag nrho cov ntaub ntawv PCR tau ua kom zoo rau cov neeg saib xyuas hauv tsev gene qhia (B2m) thiab tshuaj xyuas los ntawm 2−ΔCT txoj kev los xam cov txheeb ze mRNA qhia.

Western Blot Analysis

Cells raug ntxuav hauv dej txias PBS thiab lysed hauv radioimmunoprecipitation assay lysis buffer (150 mmol/L NaCl, 1.0 feem pua ​​IGEPAL CA-630, 0.5 feem pua ​​sodium deoxycholate, 0.1 feem pua ​​SDS, 50 mmol/L Tris, pH 8.0) supplemented nrog protease thiab phosphatase inhibitors. Protein concentration tau txiav txim siab los ntawm bicinchoninic acid assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, UK) raws li kev soj ntsuam cov lus qhia. Qhov sib npaug ntawm cov protein tau thauj khoom thiab khiav ntawm SDS-PAGE gel (4 feem pua ​​​​rau 12 feem pua ​​​​Bis-Tris Gel, Life Technologies, UK) cais. Cov proteins tau raug xa mus rau ib daim nyias nyias ntawm nitrocellulose (Bio-Rad, UK) thiab soj ntsuam nrog cov tshuaj tiv thaiv tshwj xeeb raws li qhia nrog -actin (nas anti- -actin [15G5A11/E2], Invitrogen) ua tus saib xyuas hauv tsev thiab tswj kev thauj khoom. Membranes tau tsim (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, UK) thiab kuaj pom los ntawm chemiluminescence ntawm Biorad Image Station.

Tib neeg Samples thiab LCM Isolation ntawm Plaque Macrophages

Txhua qhov kev tshawb fawb soj ntsuam tau ua los ntawm Kev Tshaj Tawm ntawm Helsinki, thiab cov qauv raug khaws cia raws li UK Human Tissue Act (2004). Txoj kev tshawb no tau pom zoo los ntawm pawg saib xyuas kev coj ncaj ncees, thiab txhua yam kev kawm tau txais kev pom zoo sau tseg. Cov neeg mob tos carotid endarterectomy tau raug xaiv, thiab carotid plaques tau sau tshiab thaum lub sijhawm phais (tus neeg mob cov yam ntxwv tau sau tseg hauv Excel File I hauv Cov Ntaub Ntawv Ntxiv). Cog carotid plaques raug ntxuav hauv cov dej khov-txias tsis muaj PBS, snap-krozen hauv qhov nruab nrab txiav, thiab khaws cia ntawm -80 degree kom txog thaum ua tiav.

LCM tau ua tiav nrog PALM Microbeam LCM system (Carl Zeiss GmbH, Lub teb chaws Yelemees) siv qhov hloov kho "qhia ntawv swb" mus kom ze.22,23 Carotid plaque kuaj tau tshiab tshiab ntawm 15 µm. Cov ntawv qhia slides muaj 3 ntu sib law liag: Thawj ntu tau stained nrog Masson-Goldner staining kit raws tu qauv los pab rau kev txiav txim siab ntawm microanatomic qhov chaw, thiab 2 tom ntej no tau txhaj tshuaj tiv thaiv kab mob rau ob qho tib si macrophages (nas antihuman CD68 [clone KP{{7}) }], Dako, Cambridge UK) lossis cov leeg nqaij du (nas antihuman -actin [clone 1A4], Dako, Cambridge UK). Cov ntu tom ntej (txog 15) raug txiav mus rau LCM membrane slides (ThermoFisher Scientific, UK) thiab cresyl violet stained npaj rau kev ua tam sim. Macrophage cell pawg tau txheeb xyuas manually raws li cov lus qhia-slide staining siv PALM RoboSoftware (version 4.5, Carl Zeiss GmbH, Lub teb chaws Yelemees). Tag nrho qhov sib xyaw ua ke ntawm thaj tsam ntawm 3 thiab 5 × 106 µm2 tau laser-captured ntawm Zeiss PALM AdhesiveCap (Carl Zeiss GmbH, Lub Tebchaws Yelemees). Cells tau lysed tam sim ntawd, snap-khov rau ntawm cov dej khov qhuav, thiab khaws cia ntawm −80 degree kom txog thaum RNA tau muab rho tawm nrog RNEasy micro cov khoom (Qiagen, UK), raws li cov chaw tsim khoom raws tu qauv.

Tib neeg Plaque Macrophage Microarray

Isolated plaque macrophage RNA concentration was determined with the Agilent RNA 6000 Pico LabChip on the Agilent Bioanalyzer 2100 (Agilent Technologies, US). Samples with an RNA integrity number >5 tau coj mus rau RNA amplification, biotinylation, thiab gene qhia microarray tsom xam. Cov qauv no tau xa mus rau Cambridge Genomic Services (Department of Pathology, University of Cambridge, UK) rau kev ua haujlwm ntxiv. Amplification thiab biotinylation tau ua nrog Ovation Pico WTA V2 cov khoom siv (Nugen Technologies Inc, US), thiab tom qab ntawd hybridization tau sib txawv hauv qhov kev txiav txim siab ntawm 2 Illumina Human HT12 v4.0 BeadChips (Illumina, US) kom txo qis batch / chip variation. Illumina BeadArrays tau ua tiav nrog Bioconductor hlaws array pob (version 2.24.0)24,25 siv cov exponential normalization. Cov txheej txheem sojntsuam tau tawg rau ib lub noob raws li qhov siab tshaj plaws interquartile ntau yam kom tsis txhob muaj qhov tseem ceeb tab sis ntes qhov sib txawv ntawm txhua qhov qauv. Unannotated thiab tswj cov kev sojntsuam tau raug tshem tawm los ntawm kev tshuaj xyuas ntxiv. Differential qhia (DE) ntawm cov npe kawg ntawm 21036 noob tau soj ntsuam nrog Bioconductor limma pob (version 3.30.13)26 los sib piv cov ntshav qab zib nrog kev tswj hwm, kho cov tsos mob / asymptomatic. Muab qhov muaj ntawm cov ntaub ntawv orthogonal cov ntaub ntawv thiab 8 cov qauv rau ib pawg, peb siv tus nqi hloov kho tus nqi ntawm P.<0.2 with no fold change cutoff to select genes to pursue.

Tib neeg Monocyte Isolation

Cov ntshav peripheral venous (15–20 mL) tau txais los ntawm cov neeg pub noj qab haus huv hauv K2-EDTA-coated hlab (BD Vacutainer, New Jersey) thiab ua tiav hauv 1 teev. Plasma thiab mononuclear cell-enriched feem yog tau los ntawm centrifuging tag nrho cov ntshav ntawm Accuspin Histopaque-1077 kab (Sigma-Aldrich, US) raws li cov chaw tsim khoom cov lus qhia. Lub mononuclear cell-enriched feem yog coj mus rau pem hauv ntej rau monocyte kev rho tawm nrog cov khoom siv tsis zoo xaiv hlaws cais (EasySep human monocyte enrichment kit yam tsis muaj CD16 depletion, StemCell Technologies, Canada), ua raws li cov chaw tsim khoom tau piav qhia.

Tib neeg Peripheral Ntshav Mononuclear Cell Isolation thiab Stimulation

Cov neeg mob ntshav qab zib thiab cov neeg mob tswj tau raug xaiv los rau kev tshawb fawb soj ntsuam tom qab kev pom zoo hauv zos thiab cheeb tsam kev coj ncaj ncees ntawm Karolinska Institute, Stockholm (2011/1002-31/1 thiab 2009/1881-31/1). PBMCs raug cais tawm los ntawm cov qauv ntshav peripheral sau tom qab kev yoo mov thaum hmo ntuj nrog SepMate (StemCell Technologies) thiab Lymphoprep (StemCell Technologies) raws li cov chaw tsim khoom cov lus qhia. PBMCs cais tau khov thiab khaws cia hauv 90 feem pua ​​FBS thiab 10 feem pua ​​dimethyl sulfoxide.

Khaws PBMCs tau thawed thiab ntxuav hauv PBS los ntawm centrifugation (500g rau 10 feeb), cov hlwb siv tau raug suav thiab plated rau hauv ib lub qhov dej ntawm 12-cov ntaub so ntswg kab lis kev cai hauv lub cev (tsawg) qabzib DMEM, 10 feem pua ​​FBS, 2 mmol / L l-glutamine, thiab 1% penicillin thiab streptomycin. PBMCs tau so thaum hmo ntuj thiab kho nrog PBS-tsheb lossis 100 ng / mL LPS thiab 10 ng / mL ntawm recombinant IFN- (R&D Systems) rau 6 teev. Tom qab stimulation, PBMCs tau lysed hauv RLT (QIAGEN), snap-khov, thiab khaws cia ntawm -80 degree . RNA raug cais tawm thiab raug rau RNA-sequencing (RNAseq) raws li tau piav qhia.

Metabolomics thiab Metabolite Measurements

Cov tib neeg monocytes raug cais tawm ntawm qhov ceev ntawm 3.5 × 105 lub hlwb rau ib tus qauv ua tiav 5 mmol / L qabzib DMEM, cov xov xwm ntxiv nrog cov piam thaj kom ua rau qhov kawg ntawm 20 mmol / L qabzib. , lossis media nrog 15 mmol / L mannitol hauv 5 mL fluorescence-activated cell sorting tubes rau 12 teev. Cell lysates tau npaj los ntawm kev rho tawm nrog dej khov-txias methanol rau 20 feeb ntawm cov dej khov qhuav thiab snap-khov hauv cov kua nitrogen. Nyob rau hnub ntawm kev tshuaj ntsuam, cov qauv tau vortexed thiab ces centrifuged ntawm 10, 000g rau 10 feeb ntawm 4 degree . Ib qho piv txwv tswj tau zoo tau npaj los ntawm kev sib xyaw 60 µL txhua tus qauv rho tawm thiab tshuaj xyuas ib ntus thoob plaws txhua qhov kev tshuaj ntsuam xyuas kom paub meej tias txoj kev ua tau zoo. Cov qauv raug lim nrog 0. Cov kev soj ntsuam tsis yog hom phiaj metabolomics tau ua raws li tau piav qhia yav dhau los.27 Luv luv, cov qauv tau txheeb xyuas ntawm Thermo Ultimate 3000 ultrahigh kev ua tau zoo ua kua chromatography txuas nrog Q-Exactive Orbitrap siv polarity switching (positive-negative) kev lag luam ntawm kev daws teeb meem loj ntawm 7000 tag nrho dav ntawm ib nrab siab tshaj plaws. Hydrophilic compounds tau chromatographically sib cais ntawm Merck Sequant ZIC-HILIC kem (150 × 4.6 mm, 5-µm particle size) thiab hydrophobic compounds siv lub Thermo Accucore aQ RP C18 kem (150 × 2.1 mm, 2.{{38 }}µm particle loj). Metabolites tau txheeb xyuas siv cov ntaub ntawv hauv tsev.27 Metabolite Set Enrichment thiab Metabolic Pathway Analysis tau ua rau txhua qhov kev ntsuas metabolite siv MetaboAnalyst (version 4.0).

Rau BMDM thiab HSC cov kev ntsuas metabolite, cov hlwb tau ua raws li tau piav qhia yav dhau los ntawm qhov ceev ntawm 5 × 105 ib qhov dej, thiab kab lis kev cai supernatants tau khiav ntawm ABX Pentra 400 (Horiba) los ntsuas cov piam thaj (ABX Pentra Glucose PAP CP, Horiba, UK) thiab lactate (ABX Pentra Lactic Acid) qib. Kev ntsuas tau normalized rau cov piam thaj thiab lactate concentrations tam sim no nyob rau hauv kev tswj tag nrho cell kab lis kev cai xov xwm uas tau incubated nyob rau hauv tib yam mob tab sis tsis nyob rau hauv lub xub ntiag ntawm cell. BMDM cell lysates tau siv los ntsuas cov qib succinate (Succinate Assay Kit, Abcam, UK) raws li cov chaw tsim khoom cov lus qhia.

ATAC-seq Analysis

Control or diabetic isolated HSCs or BMDMs, unstimulated or stimulated with IL-1β or LPS+IFN-γ for 2 hours, respectively, in 5 mmol/L glucose DMEM were used for ATAC-seq analysis according to a previously described protocol.28 Briefly, nuclei from 50000 HSCs or 75000 BMDMs per replicate were isolated, lysed on ice, and immediately put through transposition reaction using Tn5 transposase and TD buffer (Illumina, US) for 30 minutes at 37°C. Library amplification (NEBNext High Fidelity 2× PCR master mix, New England Biolabs, US) was followed by SPRI size selection to exclude fragments >1200 Ib. DNA concentrations tau ntsuas nrog Qubit fluorometer (Lub Neej Technologies), thiab lub tsev qiv ntawv loj faib tau soj ntsuam nrog Bioanalyser DNA HighSensitivity Chip (Agilent, UK). Lub tsev qiv ntawv amplification tau ua nrog kev cai Nextera primers.28 Cov tsev qiv ntawv tau ua raws los ntawm Biomedical Sequencing Facility ntawm CeMM siv lub platform Illumina HiSeq 3000/4000 thiab 50-bp kev teeb tsa ib zaug nyeem.

Kev nyeem tau raug tswj kom ua tau zoo thiab txiav tawm ntawm cov khoom txuas ntxiv. Tom qab ntawv nyeem tau ua raws li nas genome (Mm10) nrog Bowtie (version 2.2.6) hauv "rhiab hauv zos" hom. Aligned nyeem tau lim kom tshem tawm qhov tsis zoo qhov sib tw (mapping zoo Tsawg dua lossis sib npaug li 10) lossis nyeem daim ntawv qhia txog mitochondrial sequences. Alignment pib txoj haujlwm tau hloov pauv hauv txoj kev paub txog (4 bp, −5bp, 28) ua ntej kev hu xov tooj nrog MACS2 (version 2.1.129). Cov cheeb tsam sib txawv tau txheeb xyuas nrog DiffBind (version 2.4.830) nrog qhov siab tshaj plaws nruab nrab (summits=100) thiab edgeR.31

Txoj kev tsom xam tom qab ntawd tau ua tiav ntawm qhov siab tshaj plaws hauv cov qauv kuaj ntshav qab zib (tus lej nrhiav pom tsis tseeb [FDR] Tsawg dua lossis sib npaug rau {{0}}.1) los ntawm kev nkag mus rau qhov chaw chromosome ncov rau hauv Genomic Regions Enrichment of Annotations Tool ( version 3. motifs E<0.05; minimum width, 6; minimum number of sites, 3).

Runt-Related Transcription Factor 1 Target Identification

MEME-ChIP ntawm ATAC-seq cheeb tsam tau txheeb xyuas Runt-related transcription factor 1 (RUNX1) ua tus tswj hwm muaj peev xwm hauv ob qho tib si IL-1 thiab HSCs tsis muaj ntshav qab zib. MEME GOMo (version 4.12. Ntxiv rau Gene Ontology enrichment tsom xam, GOMo rov qab cov npe ntawm cov noob txheeb xyuas raws li lub hom phiaj. Cov hits no tau txwv rau cov neeg txhawb nqa kev ruaj ntseg khi rau ob leeg nas thiab tib neeg nrog qhov muaj txiaj ntsig P.<0.05 or 0.01. Mouse Genome Informatics identifiers were converted to Mouse Ensembl identifications on the Mouse Genome Informatics website and then to Human Ensembl identification and gene symbol using the Ensembl BioMart interface. For values of P<0.05, 1300 target genes were converted, of which 1251 were represented on the arrays; for values of P<0.01, there were 355 converted and 347 on the arrays. The overrepresentation of RUNX1 target genes among the DE genes was assessed by the Fisher exact test as implemented in R. The heatmap package (version 1.0.8) was used to generate the heat maps, with row-normalized log2 values shown for DE genes. Both genes and samples were clustered by Pearson correlation.

RNA-seq Analysis

Tswj lossis mob ntshav qab zib BMDMs (n=6), kab lis kev cai hauv 5 mmol / L thiab txhawb nqa nrog LPS ntxiv rau IFN- rau 6 teev lossis tsis muaj zog tswj hlwb, tau siv rau kev tshuaj xyuas RNA-seq. Luv luv, RNA raug cais nrog mirVana mRNA / miRNA Isolation Kit (ThermoFisher Scientific, UK), raws li cov chaw tsim khoom cov lus qhia. Tus nqi ntawm tag nrho RNA tau suav nrog Qubit Fluorometric Quantitation system (Life Technologies), thiab tus lej RNA kev ncaj ncees tau txiav txim siab nrog Kev Tshawb Fawb Automated Electrophoresis System (Bio-Rad). Cov tsev qiv ntawv RNA-seq tau npaj nrog TruSeq Stranded mRNA LT qauv npaj cov khoom siv (Illumina, UK) siv ob qho tib si Sciclone thiab Zephyr kua tuav neeg hlau (PerkinElmer, UK). Cov tsev qiv ntawv ntau tau suav nrog Qubit Fluorometric Quantitation system (Life Technologies, UK), thiab qhov loj me tau raug soj ntsuam nrog Experion Automated Electrophoresis System (Bio-Rad, US).

Rau sequencing, cov qauv tau diluted thiab sib sau ua ke hauv qhov sib npaug thiab sib npaug ntawm Illumina HiSeq 3000/4000 cov cuab yeej hauv 50-bp kev teeb tsa ib zaug nyeem los ntawm Biomedical Sequencing Facility ntawm CeMM. Lub hauv paus hu tau muab los ntawm Illumina Real-Time Analysis software tau tom qab hloov mus rau hauv binary alignment daim ntawv qhia hom (Illumina2bam) ua ntej demultiplexing (BamIndexDecoder) rau hauv tus kheej, cov qauv tshwj xeeb binary alignment daim ntawv qhia cov ntaub ntawv ntawm Illumina2bam cov cuab yeej (1.17.3). Cov tsev qiv ntawv tau npaj thiab ua tiav los ntawm Biomedical Sequencing Facility ntawm CeMM.

Cov ntawv nyeem tau ua raws li nas genome (GRCm38 / mm10) siv STAR (version 2.5.3a34) hauv cov noob suav hom (quantMode GeneCounts) nrog Gencode (version M16) cov lus piav qhia (los ntawm qhov mega transcript Gm20388 raug tshem tawm kom tsis txhob sib tshooj gene). Cov suav raug thauj mus rau R (version 3.3.3) thiab tshuaj xyuas nrog edgeR (version 3.16.535) tom qab tshem tawm cov ntawv sau nrog<0.5 mapped reads per million sequenced in all samples (13568 transcripts retained). Genes with adjusted values of P<0.05 and fold change >1.5 tau suav tias yog DE. DE cov lus thiab cov npe noob tau suav nrog hauv Excel File II hauv Cov Ntaub Ntawv Ntxiv (unstimulated) thiab Excel File III hauv Cov Ntaub Ntawv Ntxiv (LPS ntxiv rau IFN- stimulated). Lub edgeR muaj nuj nqi cpm tau siv los teev-normalize suav (Excel File IV nyob rau hauv cov ntaub ntawv ntxiv) rau cov tshav kub maps thiab lwm qhov chaw. Daim ntawv qhia tshav kub tau tsim nrog R pob heatmap (version 1.0.836). Overpresentation ntawm RUNX1 lub hom phiaj genes tsom xam ntawm DE noob hauv ntshav qab zib piv nrog kev tswj LPS ntxiv rau IFN- cov qauv tau ua raws li tau piav qhia yav dhau los rau tib neeg cov ntaub ntawv.

Chip Sequence

Tag nrho ntawm 1 × 106 hlwb / mL tau kho nrog 1 feem pua ​​​​ formaldehyde ntawm chav tsev kub rau 10 feeb, ntxuav nrog PBS, thiab khaws cia ntawm −80 degree. Cells raug sheared nrog Chromatin EasyShear Kit-Low SDS (Diagenode, Belgium) thiab sonicated nrog Bioruptor Pico sonication ntaus ntawv (catalog No. B01080010, Diagenode, Belgium) raws li cov chaw tsim khoom pom zoo. MAGnify Chromatin Immunoprecipitation System (Thermo Scientific) tau siv los xaiv chromatin fragments siv anti-histone H3 (trimethyl K4; ab8580) los yog anti-histone H3 (acetyl K27; ab4729). Cov tsev qiv ntawv Sequencing tau npaj nrog NEBNext Ultra II DNA Library Prep Kit raws li cov chaw tsim khoom cov lus qhia. Nyob rau hauv luv luv, purified Chip los yog input DNA tau kawg-kho thiab A-tailed ua raws li adapter ligation. Lub adaptor-ligated DNA tau loj xaiv nrog SPRI Ampure XP hlaws dai (Agencourt) thiab ntxiv nrog Illumina-tshaj li cov primers. Cov tsev qiv ntawv zaum kawg tau ua kom huv nrog SPRI Ampure XP hlaws dai kom tshem tawm cov hloov pauv dimers thiab ua raws los ntawm Biomedical Sequencing Facility ntawm CeMM siv Illumina HiSeq 3000/4000 platform thiab 50-bp ib leeg-nyeem teeb tsa.

Bioinformatics

Txhawm rau tshuaj xyuas kev sib raug zoo ntawm chromatin qhib thiab cov noob qhia hauv BMDMs tsis muaj zog, ntshav qab zib tiv thaiv ATAC-seq peaks (log2 fold hloov) thiab mRNA qib ntawm cov noob sib thooj (log2 fold hloov) tau muab piv los ntawm Pearson linear correlation.

Nyeem los ntawm ATAC-seq, H3K4me3 Chip-sequencing (ChIPseq), thiab H3K27ac Chip-seq thwmsim tau pom nyob rau hauv cov qauv neeg sawv cev siv Integrative Genome Browser, nrog cov kev ntsuas qhov siab ntawm ib qho kev sim thiab hom ntawm tes los suav rau qhov sib txawv hauv kev nyeem qhov tob.

Kev txheeb cais

Tag nrho cov ntaub ntawv tsis raug txheeb xyuas txheeb xyuas tau ua hauv GraphPad Prism software. Cov ntaub ntawv raug txheeb xyuas rau kev faib tawm ib txwm los ntawm Shapiro-Wilk normality test. Kev sib piv ntawm 2 pab pawg tau suav los ntawm 2-tailed Student t-tests or Mann-Whitney U tests for a normally or nonnormally distribution data, ntsig txog. Kev sib piv ntawm ntau pawg tau suav los ntawm 1- txoj kev lossis 2- txoj kev ANOVA tsom xam nrog Bonferroni post hoc kho. Cov ntaub ntawv raug tshaj tawm raws li txhais tau tias ± SD rau ib txwm faib cov ntaub ntawv lossis nruab nrab ± interquartile ntau yam rau cov ntaub ntawv tsis raug faib tawm. Ob peb replicates thiab lwm yam ntaub ntawv txheeb cais muaj nyob rau hauv daim duab legends. Rau ob qho tib si hauv vivo thiab hauv vitro kev sim, n yog tus naj npawb ntawm cov tsiaj nyeg lossis tus neeg noj qab haus huv pab dawb lossis cov neeg mob. Fold hloov yog xam raws li qhov nruab nrab qhov ntsuas tus nqi faib los ntawm qhov nruab nrab tswj tus nqi.

TSEEM CEEB

Mob ntshav qab zib hyperglycemia hloov pauv Cellular Metabolism, Tsav Proinflammatory Gene Qhia thiab Kev Ua Haujlwm

Aerobic glycolysis tsub kom proinflammatory (M1) noob qhia, 37 thiab hyperglycemia kuj txhawb M1- txuam gene expression.10,11 Yog li ntawd, peb xav tias siab extracellular qabzib drives nce glycolysis, ua rau txhim kho polarization ntawm macrophages mus rau ib tug M1 phenotype. .

Kev tshuaj xyuas metabolome ntawm thawj tib neeg monocytes tau qhia tias muaj cov piam thaj ntau ntxiv (20 mmol / L) hloov pauv cov metabolic profile piv nrog osmotically matched physiological qabzib (5 mmol / L). Txoj kev tsom xam (Daim duab 1A) qhia tias cov piam thaj siab ua rau muaj kev hloov pauv loj hauv lub voj voog tricarboxylic acid (FDR, 0.002) thiab pyruvate metabolism (FDR, 0.005), qhia los ntawm kev nce succinate, malate ( Daim duab 1B), lactate, thiab pyruvate (Daim duab 1C) qib, raws li, nrog metabolite teeb enrichment tsom xam (Daim duab 1D) qhia txog cov nyhuv Warburg (FDR, 0.04).

Daim duab 1. Cov qabzib siab hloov cellular metabolism. Kev ntsuam xyuas Metabolic pathway. Kev ntsuas tus kheej rau (B) succinate, malate, (C) lactate thiab pyruvate thiab (D) metabolite teem enrichment tsom xam (Metaboanalyst) rau tib neeg monocyte cell lysate metabolomics kuaj (n=5). Extracellular flux tsom xam ntawm nas pob txha hlwb-derived macrophages (Seahorse bioanalyzer; n=6 ib tug tsiaj) ntsuas (E) extracellular acidification tus nqi (ECAR) thiab (F) oxygen noj tus nqi (OCR) nyob rau hauv teb rau ntau yam extracellular qabzib, osmotic tswj (mannitol), thiab kev txhaj tshuaj metabolic inhibitor. Cov piam thaj ntau (G) nce glycolytic ATP (glycoATP) ntau lawm thiab (H) txo qis ATP tus nqi Performance index (mitochondrial ATP ntau lawm tus nqi / glycoATP ntau lawm), qhia txog kev hloov mus rau ntau glycolytic phenotype. Cov ntaub ntawv tau txheeb xyuas los ntawm (B thiab C) 2- txoj kev ANOVA lossis (E–H) 1- txoj kev ANOVA ntxiv rau Bonferroni post hoc xeem. Tag nrho cov ntaub ntawv qhia yog txhais tau tias ± SD. AU qhia arbitrary units; FDR, tus nqi nrhiav tsis tseeb; thiab TCA, tricarboxylic acid. *P<0.05; **P<0.01; ***P<0.001.

Ib qho ntxiv glycolytic (thiab tsawg dua oxidative) phenotype hauv cov lus teb rau cov piam thaj ntau (20 mmol / L) tau lees paub hauv nas BMDMs, uas pom tias muaj kev nce ntxiv hauv cov kua qaub ntxiv, nce glycolytic ATP ntau lawm (2-fold; P<0.01), decreased ATP rate index (by 56%; P<0.05; Figure 1E–1H), and increased glucose consumption and lactate production (P<0.001; Figure Ia and Ib in the Data Supplement). Cultured BMDMs also increased succinate levels in response to high extracellular glucose in both unstimulated (2.5-fold, P<0.05) and M1-stimulated states (1.9-fold; P<0.001; Figure Ic in the Data Supplement).

Qhov kev nce qib ntawm glycolytic no tau tiv thaiv los ntawm inhibitor ntawm glycolysis dichloroacetate (Daim duab Ia thiab Ib hauv Cov Ntaub Ntawv Ntxiv).

Hauv BMDMs, cov qabzib siab ob leeg txhawb nqa proinflammatory M1-sociated Il-6 qhia (2.7-fold; P<0.001; Figure 2A) and suppressed M2-associated Ym1 (70%; P<0.001) and Fizz1 (42%; P<0.001) expression (Figures 2C and 2D). These bidirectional changes in gene expression were restored in the presence of dichloro-acetate (Figure 2A–2D) and 2-deoxy-d-glucose (Figure IIa–IId in the Data Supplement), together indicating that the proinflammatory effects of high extracellular glucose were mediated through changes in glycolysis and were not merely the result of nonspecific changes to overall metabolic activity.

Daim duab 2. Cov qabzib siab hloov cov macrophage noob qhia thiab ua haujlwm. M1 (lipopolysaccharide [LPS] ntxiv rau interferon- [IFN-] stimulated) pob txha hlwb-derived macrophage (BMDM) (A) Il-6 thiab (B) yog cov noob qhia nyob rau hauv muaj lossis tsis muaj dichloroacetate (DCA). M2 (interleukin-4 stimulated) BMDM (C) Ym1 thiab (D) Fizz1 noob qhia. A txog D, Quantitative polymerase saw cov tshuaj tiv thaiv cov ntaub ntawv yog normalized rau B2m qhia; n=7 mus rau 10. E, Cov duab quantification ntawm nas monocyte adhesion rau endothelial hlwb (n=5). F, Cov duab kom muaj nuj nqis ntawm acetylated low-density lipoprotein uptake los ntawm BMDMs, normalized los ntawm xov tooj ntawm tes ib daim duab. Tus neeg sawv cev (G) cov duab zoo li qub ntawm cov fluorescently PKH67-labeled nas monocytes rau endothelial hlwb thiab (H) Roj Liab O (ORO)– thiab DAPI-stained foam cell dluab. Tag nrho cov ntaub ntawv (A–H) yog qhia raws li txhais tau tias ± SD; (A–D) 1- txoj kev ANOVA lossis (E thiab F) 2- txoj kev ANOVA nrog Bonferroni post hoc tsom xam. Txhua lub ntsiab lus sawv cev rau ib tus tsiaj (nruab nrab ntawm 4 cov duab rau static adhesion thiab foam cell tsim). AU qhia arbitrary units; NS tsis tseem ceeb. *P<0.05; **P<0.01; ***P<0.001.

Muab nws cov teebmeem ntawm kev txhawb kev mob, peb mam li kuaj seb puas muaj cov piam thaj ntau ntxiv hloov pauv monocyte thiab macrophage ua haujlwm cuam tshuam rau atherogenesis.38,39 Cov piam thaj ntau ntxiv rau nas monocyte adhesion rau cov hlwb endothelial (2.{6-fold; P<0.001; Figure 2E and 2G) and promoted BMDM uptake of modified LDL to form foam cells (4.1-fold [and 2.7-fold when M1 stimulated]; P<0.001 for both; Figure 2F and 2H). The effects on monocyte adhesion were negated entirely by inhibition of glycolysis with dichloroacetate (P<0.001; Figure 2E and 2G) and 2-deoxy-glucose (P<0.001; Figure IIe in the Data Supplement), whereas foam cell formation, was decreased by 30% (and by 42% when M1-stimulated; P<0.001 for both; Figure 2F and 2H).

BM-Drived Cells Los Ntawm Mob Ntshav Qab Zib Mice Qhia Txog Hyperglycemia-Induced Trained Immunity

Peb tom ntej no siv tus qauv nas ntshav qab zib los kuaj seb puas yog hyperglycemia-vim tsis tu ncua kev hloov pauv hauv macrophages, txawm tias tom qab cov piam thaj normalization. Peb tau txais BM los ntawm cov nas uas tsis yog mob ntshav qab zib, muaj hnub nyoog-match nrog cov nas thiab los ntawm cov nas uas muaj ntshav qab zib mellitus uas yog hyperglycemia tau raug ntxias los ntawm streptozotocin thiab khaws cia rau 6 lub lis piam hauv vivo (Daim duab IIIa-IIId hauv Cov Ntaub Ntawv Ntxiv). Cells tau sib txawv rau hauv BMDMs nyob rau hauv physiological qabzib (5 mmol / L; Daim duab 3A). Tom qab kev txhawb nqa, BMDMs ntawm cov kab mob ntshav qab zib nas tuaj yeem nthuav tawm txhim kho M1-sociated Il-6 gene qhia (4.1-fold; P<0.0001; Figure 3B) and, with IL-4 treatment, decreased M2-associated Ym1 expression (by 75%; P=0.0003) and Fizz1 (by 70%; P=0.0326; Figure 3D and 3E). RNA-seq analysis of control and diabetic BMDMs revealed clear delineation based on gene expression profiles, which was further accentuated by LPS and IFN-γ stimulation (Figure 3F). Indeed, when expression levels of a panel of 39 previously identified M1- and M2-associated genes were examined,9 BMDMs from control and diabetic origin showed clear segregation after LPS+IFN-γ stimulation (Figure 3G).

Daim duab 3. Cov pob txha pob txha (BM)-derived macrophages (BMDMs) los ntawm cov nas mob ntshav qab zib manifest hyperglycemic nco hauv gene qhia. A, Schematic ntawm ntshav qab zib thiab tswj BMDM raws tu qauv (n=10–12). M1 gene expression analysis of (B) Il-6 (P<0.0001) and (C) is. M2 (D) Ym1 (P=0.0003) and (E) Fizz1 (P=0.0326) gene expression. B through E, All quantitative polymerase chain reaction data are normalized to B2m expression and analyzed by the unpaired t-test. F, Principal component (PC) analysis of all control and diabetic BMDM RNA sequencing (RNA-seq) transcript reads (n=6); percent indicates sample variability. G, Unsupervised hierarchical clustering shows marked segregation of 39 previously identified M1 and M2 macrophage marker genes in lipopolysaccharide (LPS)+interferon-γ (IFN-γ)–stimulated BMDMs from diabetic or control origin (RNA-seq analysis). IL indicates interleukin.

Tsis tas li ntawd, muaj zog adhesion rau activated endothelium (los ntawm 6.3-fold; P<0.05; and by 9.4-fold when LPS-stimulated; P<0.001; Figure 4A and 4C) and enhanced modified LDL uptake and foam cell formation (LPS stimulated, 1.6-fold; P<0.01; Figure 4B and 4D). This array of persistently heightened responses was indicative of hyperglycemia-induced trained immunity.

Daim duab 4. Cov pob txha pob txha-derived macrophages (BMDMs) los ntawm cov nas mob ntshav qab zib qhia cov haujlwm hloov pauv txawm tias cov piam thaj normalization. BMDMs los ntawm cov nas mob ntshav qab zib tau sib txawv hauv 5 mmol / L (n=5-8). A, Cov duab kom muaj nuj nqis ntawm BMDM adhesion rau endothelial hlwb (n=8). B, Cov duab kom muaj nuj nqis ntawm acetylated low-density lipoprotein uptake los ntawm BMDMs, normalized los ntawm xov tooj ntawm tes ib daim duab. Tus neeg sawv cev (C) cov duab zoo li qub ntawm fluorescently (PKH67) sau BMDMs rau cov hlwb endothelial thiab (D) Roj Liab O (ORO)- thiab DAPI-stained foam cell dluab. Tag nrho cov ntaub ntawv yog qhia raws li qhov txhais tau tias ± SD, thiab txhua lub ntsiab lus sawv cev rau ib tus tsiaj (nruab nrab ntawm 4 dluab rau static adhesion thiab foam cell tsim). Cov ntaub ntawv tau txheeb xyuas los ntawm 2- txoj kev ANOVA nrog Bonferroni post hoc xeem. IFN- qhia tias interferon-; thiab LPS, lipopolysaccharide. *P<0.05; **P<0.01; ***P<0.001.

Hyperglycemia-Induced Trained Immunity Drives Atherosclerosis

Yog li, txhawm rau ntsuas qhov tseem ceeb ntawm kev ua haujlwm ntawm hyperglycemia-vim kev tiv thaiv kev tiv thaiv kab mob hauv atherosclerosis, peb hloov BM los ntawm kev tswj hwm normoglycemic lossis streptozotocin-induced ntshav qab zib cov nas (cov ntshav qabzib, 33.9 ± 6.9 mmol / L) rau hauv atherosclerosis-L-procemic normoglycemic. - cov nas tau txais (Daim duab 5A). Hauv txhua kis, cov nas pub dawb tau hloov pauv rau GFP raws li kev tswj hwm ntawm tib neeg CD68 txhawb nqa.40 Kev siv tau zoo thiab sib npaug tau pom los ntawm kev khiav cytometric tsom ntawm PBMCs, lees paub tias muaj CD45 ntxiv rau GFP ntxiv cov hlwb zoo rau CD11b lossis Ly6C ( Daim duab IVa thiab IVb hauv Cov Ntaub Ntawv Ntxiv). Tom qab 12 lub lis piam ntawm kev noj zaub mov sab hnub poob, tsis muaj qhov sib txawv ntawm cov ntshav lipid ntsuas (Daim duab IVc thiab IVd hauv Cov Ntaub Ntawv Ntxiv) lossis leukocyte subsets raws li kev soj ntsuam los ntawm kev ntws cytometry (Daim duab IVe hauv Cov Ntaub Ntawv Ntxiv).

Cov nas uas tau txais cov ntshav qab zib BM pom tau tias muaj cov kab mob atherosclerosis, feem ntau muaj ntau tshaj qhov hnyav tshaj plaws atherosclerosis hauv cov neeg tsis muaj ntshav qab zib (qhov nruab nrab ntawm cov quav hniav, 44461 µm2 [interquartile range, 18368–59597 µm2] [501µm27] 70µm2. ; Mann-Whitney, P=0.036). Cov nas no kuj tau pom qhov nce hauv cov ntsiab lus macrophage ntawm cov plaques (P=0.052; Daim duab V hauv Cov Ntaub Ntawv Ntxiv), lipid-nplua nuj necrotic core cheeb tsam (2.8-fold, P{{18} }}.0076; Daim duab VIa hauv Cov Ntaub Ntawv Ntxiv), thiab feem ntau cov lipid-nplua nuj necrotic core (2.4-fold, P=0.046; Daim duab VIb hauv Cov Ntaub Ntawv Ntxiv), tab sis tsis muaj Qhov sib txawv ntawm cov ntsiab lus collagen (plaque feem pua; Daim duab VIc thiab VId hauv Cov Ntaub Ntawv Ntxiv) lossis cov leeg nqaij du (plaque feem pua; Daim duab VIe thiab VIf hauv Cov Ntaub Ntawv Ntxiv).

Hyperglycemia-Induced Trained Immunity nyob rau hauv HSCs yog tsav los ntawm kev hloov pauv hauv metabolism ua rau Epigenetic Reprogramming

Muab qhov ua qauv qhia tias BM los ntawm cov nas hyperglycemic txhawb nqa atherosclerosis, nrog rau cov kab mob proinflammatory gene qhia thiab cov lus teb ua haujlwm tau pom hauv BMDM hauv vitro, peb tau xav tias qhov kev nco no yog qhov tshwm sim ntawm kev kawm tiv thaiv kab mob hauv BM-derived hlwb. Kev cob qhia kev tiv thaiv kab mob tau cuam tshuam nrog kev hloov pauv hauv cov cellular metabolism uas ua rau lub sijhawm ntev, chromatin-hloov epigenetic cim xws li H3K4me3 thiab H3K27ac.17,18,41 Raws li qhov no, peb pom tias HSC (lineage negative: tshem tawm cov laus T hlwb. , B hlwb, lub hlwb tua neeg, cov hlwb dendritic, monocytes, granulocytes, erythroid cells: CD3-, CD4-, B220-, Ter119, Gr1- kab lis kev cai nyob rau hauv cov kab mob siab extracellular qabzib kuj pom muaj zog cov piam thaj thiab lactate ntau lawm, qhia dysregulated glycolysis. (P<0.001; Figure VIIa–VIId in the Data Supplement), and both H3K4me3 and H3K27ac modifications were increased (Figure VIIIa–VIIIf in the Data Supplement). In the case of H3K4me3 (but not H3K27Ac), this effect was negated with dichloroacetate inhibition (Figure VIIIa, VIIIc, and VIIId in the Data Supplement). These chromatin marks were also consistently increased in HSCs from diabetic mice compared with control HSCs and remained heightened after differentiation into BMDMs using physiological glucose levels (5 mmol/L; Figure VIIIb, VIIIe, and VIIIf in the Data Supplement).

Hauv aortic cag plaques, qhov kev faib ua feem ntawm macrophage-txuas nrog nuclei stained zoo rau H3K4me3 yog siab dua hauv cov nas uas tau txais BM los ntawm cov neeg mob ntshav qab zib (1.8-fold, P<0.05) compared with control donors (Figure 5E and 5F), which is in line with the corresponding analysis of BMDM in vitro experiments (Figure VIIIb and VIIIe in the Data Supplement). These data point to a direct causal link between increased glycolytic rate and the development of hyperglycemia-induced trained immunity. In addition to metabolic mediators,17,18 recent work has implicated the inflammatory cytokine IL-1β in the development of trained immunity.42 This is particularly relevant because IL-1β has been identified in diabetes43 as an important driver of atherosclerosis and in obesity in which adipose tissue macrophage-produced IL-1β induces the proliferation of BM progenitors.44,45 Therefore, we hypothesized that IL-1β and hyperglycemia may work synergistically to induce atherosclerosis-relevant epigenetic changes that alter chromatin structure. To interrogate chromatin accessibility, we performed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) on HSCs from diabetic or control mice, both with and without IL-1β stimulation28 (sample processing summarized in Excel File V in the Data Supplement). HSCs from a diabetic origin showed differential chromatin profiles (summarized in Excel File VI in the Data Supplement) in response to IL-1β exposure, with 530 differential peaks (FDR ≤0.1) of which 50% exhibited increased accessibility in diabetic cells. 

Daim duab 5. Mob ntshav qab zib pob txha (BM) ua rau atherosclerosis txawm tias ntev npaum li cas cov piam thaj normalization. A, Schematic ntawm BM hloov pauv kev sim. B, Flow cytometric tsom xam ntawm peripheral leukocyte cov neeg nyob hauv cov nas tom qab 12 lub lis piam ntawm kev hloov pob txha pob txha. C, Tus neeg sawv cev aortic hauv paus duab los ntawm Ldlr−/− nas uas tau txais cov neeg mob ntshav qab zib lossis tswj cov neeg pub dawb CD68- GFP BM. Masson Trichrome stained (sab laug) thiab immunofluorescence staining rau BM transplant-derived macrophages (ntsuab qhia ntsuab fluorescent protein [GFP]) los yog tag nrho cov macrophages (liab qhia galectin-3 [GAL3]). Duab kom muaj nuj nqis ntawm (D) plaque ntim (P=0.036). Cov ntaub ntawv qhia tias txhais tau tias ± SD, txheeb xyuas los ntawm 1- txoj kev ANOVA nrog Bonferroni post hoc test (B), lossis nruab nrab ± interquartile ntau yam, txheeb xyuas los ntawm Mann-Whitney U test (D); tswj n=6, mob ntshav qab zib n{10}}. E, Tus sawv cev aortic hauv paus duab los ntawm Ldlr-/- nas uas tau txais cov ntshav qab zib lossis tswj BM hloov pauv. F, Quantification ntawm feem pua ​​ntawm nuclei stained zoo rau H3K4me3 los yog H3K27ac nyob rau hauv lub GAL3 macrophage zoo cheeb tsam. Txhua cov ntaub ntawv taw qhia sawv cev rau ib tus tsiaj (nruab nrab, 6 ntu; n{18}} txog 10). Qhia raws li txhais tau tias ± SD. Ib txoj kev ANOVA tsom xam. BMDM qhia BM-derived macrophages; HSC, hematopoietic qia cell; IFN-, interferon-; thiab LPS, lipopolysaccharide. *P<0.05.

Txhawm rau tshawb nrhiav qhov cuam tshuam txog kev lom neeg ntawm cov cheeb tsam uas tsis yog coding genomic (nrog rau qhov tseem ceeb ntawm kev ua haujlwm cis-kev tswj hwm), cov cheeb tsam ntawm chromatin nrog kev nkag mus tau zoo hauv cov kab mob ntshav qab zib tau raug xa mus rau 2 cov noob ze tshaj (hauv 10{{ 11}}0 kb) thiab soj ntsuam nrog Genomic Regions Enrichment of Annotations Tool.33 Txoj kev tsom xam no tau nthuav tawm ntau txoj hauv kev mob ntsig txog kev mob, suav nrog kev ua kom leukocyte (3.3-fold; FDR, 0.0005 ), thiab cov noob muaj feem xyuam nrog LPS kam rau hauv macrophages (2.7-fold; FDR, 0.0009). Txhawm rau kom nkag siab seb cov piam thaj siab hauv kev sib cais puas tuaj yeem ua rau muaj kev hloov pauv chromatin, peb siv ATAC-seq los saib HSCs los ntawm kev tswj hwm thiab cov nas mob ntshav qab zib thaum tsis muaj IL-1 stimulation. Unstimulated HSCs tso tawm 357 qhov sib txawv (FDR Tsawg dua lossis sib npaug rau 0.2), ntawm qhov uas 51 feem pua ​​​​tau muaj kev nkag tau zoo hauv cov hlwb los ntawm cov kab mob ntshav qab zib piv nrog kev tswj hwm, nrog rau feem ntau hauv cheeb tsam intronic (48%) lossis intergenic (21%).

Muab cov kev tshawb pom no hauv HSCs, peb txuas ntxiv mus kuaj chromatin nkag tau thiab cov noob qhia hauv lawv cov derivative BMDMs. Peb tau ua ATAC-seq thiab RNA-seq ntawm BMDMs los ntawm kev tswj hwm thiab cov nas mob ntshav qab zib nyob rau hauv cov xwm txheej uas tsis muaj zog lossis tsis muaj zog (LPS ntxiv rau IFN-) cov xwm txheej. BMDMs los ntawm cov nas mob ntshav qab zib tau pom qhov sib txawv ntawm chromatin nkag mus rau hauv cov xwm txheej tsis muaj zog, nrog rau 1047 thaj chaw sib txawv (FDR<0.05; Figure 6A) but only 40 differentially open regions (FDR <0.05) after stimulation (Figure 6B). In contrast, unstimulated BMDMs from diabetic mice differentially express 632 genes (324 upregulated expression and 308 downregulated expression; Excel File II in the Data Supplement) compared with unstimulated control BMDMs, but this increases to 1348 genes (802 increased and 546 decreased; Excel File III in the Data Supplement) on stimulation (FDR <0.05; fold change >1.5). Ua ke, ATAC-seq thiab RNAseq cov ntaub ntawv qhia tias cov ntshav qab zib ua rau cov macrophages rau qhov ua rau cov lus teb tsis txaus ntseeg rau cov tshuaj tiv thaiv.

Daim duab 6. Ntshav qab zib hloov cov qib H3K4me3 thiab H3K27ac thiab chromatin nkag tau. MA zaj duab xis ntawm tag nrho cov kev ntsuam xyuas rau transposase nkag tau chromatin sequencing chromatin nyeem nyob rau hauv (A) unstimulated los yog (B) stimulated pob txha pob txha-derived macrophages (BMDMs), qhov twg peaks nrog sib txawv nkag tau (tus nqi nrhiav tsis tseeb<0.05, fold change >1.5) tau qhia hauv liab (1047 unstimulated, 40 stimulated, n=6). C, Cov cheeb tsam sib txawv ntawm H3K4me3 thiab H3K27ac ntsuas los ntawm chromatin immunoprecipitation sequencing hauv hematopoietic stem cells (HSCs) thiab BMDMs los ntawm kev tswj hwm cov nas ntshav qab zib (n=6 ib pab pawg). D, Histogram ntawm qhov nruab nrab qhov kev pab them nqi ntawm H3K27ac thiab K3K4me3 hauv HSCs, unstimulated BMDMs, thiab proinflammatory stimulated BMDMs (lipopolysaccharide [LPS] ntxiv rau interferon- [IFN]) ntawm transcription pib qhov chaw ± 1 kb ntawm protein-coding noob ze rau thaj tsam ntawm qhov nce. nkag mus tau rau hauv unstimulated BMDMs (vs tswj; n=405) thiab piv nrog rau theem tom ntej hauv cheeb tsam uas tsis tau xaiv.

We further undertook an unbiased examination of the relationship between differential gene expression and altered chromatin accessibility in the diabetic BMDMs. We annotated the nearest or overlapping gene for each region of differentially accessible chromatin in BMDMs (M0 state) from diabetic mice versus controls. For genes within this set that were also DE (cutoff FDR, 0.1; log fold change >0.5 rau txhua tus), peb piv qhov hloov pauv rau qhov sib txawv ATAC-seq ncov piv rau qhov hloov pauv rau DE. Kev tshuaj xyuas raws li qhov sib txawv tsis tu ncua, muaj kev sib raug zoo ntawm kev hloov pauv hauv chromatin thiab kev qhia ntawm cov noob caj noob ces (R=0.65; P<1×10−6).

Furthermore, to ascertain the nature of diabetes-induced chromatin modifications, we undertook ChIPseq in HSCs and BMDMs from control versus diabetic mice (n=6 per group). Diabetic HSCs and BMDMs were distinguishable from control samples based on the quantification of both H3K27ac and H3K4me3 histone modifications (Figure 6C), and we confirmed these results by Western blot analysis (Figure VIII in the Data Supplement). To integrate the ChIP-seq and ATACseq data, we examined the distribution of H3K4me3 and H3K27ac histone modifications in the regions of open chromatin that were identified by ATACseq in unstimulated diabetic BMDMs. A majority of these regions were identified as enhancers, located >1 kb los ntawm annotated transcription pib qhov chaw. Peb tsom mus rau cov noob nyob ze rau cov kev txhim kho no vim tias lawv thaj chaw txhawb nqa cov noob (cov chaw pib hloov pauv ± 1 kb) sawv cev rau cov hom phiaj feem ntau yuav tswj hwm. ATAC-seq tau txheeb xyuas 405 thaj tsam ntawm qhov sib txawv qhib chromatin (P<0.05) in BMDMs derived from diabetic mice versus control mice under basal conditions. In the HSCs from diabetic mice, there was a marked increase in promoter-associated H3K27ac compared with control HSCs (Figure 6D). 

Daim duab 6. Ntshav qab zib hloov cov qib H3K4me3 thiab H3K27ac thiab chromatin nkag tau. MA zaj duab xis ntawm tag nrho cov kev ntsuam xyuas rau transposase nkag tau chromatin sequencing chromatin nyeem nyob rau hauv (A) unstimulated los yog (B) stimulated pob txha pob txha-derived macrophages (BMDMs), qhov twg peaks nrog sib txawv nkag tau (tus nqi nrhiav tsis tseeb<0.05, fold change >1.5) tau qhia hauv liab (1047 unstimulated, 40 stimulated, n=6). C, Cov cheeb tsam sib txawv ntawm H3K4me3 thiab H3K27ac ntsuas los ntawm chromatin immunoprecipitation sequencing hauv hematopoietic stem cells (HSCs) thiab BMDMs los ntawm kev tswj hwm cov nas ntshav qab zib (n=6 ib pab pawg). D, Histogram ntawm qhov nruab nrab qhov kev pab them nqi ntawm H3K27ac thiab K3K4me3 hauv HSCs, unstimulated BMDMs, thiab proinflammatory stimulated BMDMs (lipopolysaccharide [LPS] ntxiv rau interferon- [IFN]) ntawm transcription pib qhov chaw ± 1 kb ntawm protein-coding noob ze rau thaj tsam ntawm qhov nce. nkag mus tau rau hauv unstimulated BMDMs (vs tswj; n=405) thiab piv nrog rau theem tom ntej hauv cheeb tsam uas tsis tau xaiv.

Cov kev sib txawv no tau txo qis hauv BMDMs (Daim duab 6D), ib feem vim yog nce H3K27ac hauv cov hlwb tsis muaj ntshav qab zib tom qab sib txawv. Txawm li cas los xij, lub cim H3K4me3 tau nce ntxiv ntawm cov neeg txhawb nqa hauv HSC, nrog rau kev txheeb xyuas qhov sib txawv ntawm qhov sib txawv ntawm cov macrophages, suav nrog tom qab cytokine stimulation (Daim duab 6D). Cov qauv no ntawm kev hloov H3K27ac thiab tsis tu ncua H3K4me3 cov qhab nia zoo ib yam nrog peb qhov kev tshawb pom hauv ob qho tib si kab lis kev cai (Daim duab VIII hauv Cov Ntaub Ntawv Ntxiv) thiab immunohistochemistry hauv nas plaque macrophages (Daim duab 5E thiab 5F).

Cov cheeb tsam ntawm qhov sib txawv qhib chromatin txheeb xyuas los ntawm ATAC-seq tau cuam tshuam nrog kev hloov pauv ntawm cov noob caj noob ces. Tsis tas li ntawd, qhov sib txawv ntawm qhov sib txawv ntawm chromatin nkag tau (mob ntshav qab zib tiv thaiv kev tswj hwm) thiab kev sib piv ncaj qha nrog kev hloov pauv hauv mRNA qhia qib ntawm cov gene proximate tau sib raug zoo heev (R=0.5; P=6.2e − 15; Fig 7A). Txhawm rau tshuaj xyuas cov teebmeem no ntawm qhov cuam tshuam genomic loci, peb ua ke ATAC-seq, H3K4me3 Chip-seq, thiab H3K27ac Chip-seq cov ntaub ntawv ntawm cov neeg txhawb nqa ntawm 2 cov noob yav dhau los cuam tshuam hauv glycolysis-induced kev tiv thaiv kev tiv thaiv thiab nrog cov khoom cuam tshuam rau kev tsav tsheb o (Il -6) thiab qabzib metabolism (Hk1). Cov noob no tau nthuav tawm thaj chaw ntawm cov chromatin nkag tau ntawm qhov chaw pib sau ntawv, nrog rau H3K4me3 thiab H3K27ac peaks ntawm cov genomic loci (Daim duab 7B).

Txhawm rau kom nkag siab txog qhov twg cov ntsiab lus hloov pauv tuaj yeem cuam tshuam raws li tus neeg nruab nrab ntawm cov qauv no ntawm hyperglycemia-vim kev tiv thaiv kev tiv thaiv, Motif Enrichment Analysis33 tau ua nyob rau thaj tsam ntawm chromatin nrog kev nkag mus tau zoo dua hauv cov mob ntshav qab zib. Qhov tseem ceeb ntawm kev hloov pauv cov ntsiab lus tseem ceeb tshwj xeeb suav nrog RUNX1 (FDR, 0.0001), uas yav dhau los tau cuam tshuam nrog kev nco txog kev tiv thaiv kab mob, 46 nrog rau PU.1 (FDR, 0.04) thiab CCCTC-binding yam (FDR, 1.8 × 10−7; Daim duab 8A). Tau cuam tshuam qhov kev hloov pauv ntawm RUNX147 raws li kev nkag mus tau ntawm kev sib txuas motifs, peb tau tshawb xyuas seb RUNX1 cov hom phiaj cov noob tau muaj ntau dhau ntawm cov noob DE hauv LPS ntxiv rau IFN- -stimulated BMDMs. Qhov kev tshuaj ntsuam no tau txheeb xyuas 95 ntawm 1348 DE noob raws li RUNX1 lub hom phiaj (P<0.05; odds ratio, 1.3; RUNX1 binding site P<0.05; Figure 8B and full differential gene list with RUNX1 target genes marked in Excel File VII in the Data Supplement), confirming its relevance in these disease-modifying cells. To test for RUNX1 involvement in human disease, we interrogated the gene expression of macrophages obtained from carotid atherosclerotic plaques of patients with or without diabetes (Excel File I in the Data Supplement). LCM was used to isolate macrophages from human carotid atherosclerosis plaques in patients undergoing clinically driven endarterectomy. Figure 8C shows the DE genes between diabetic and nondiabetic plaque macrophages (FDR <0.2) and highlights in red the RUNX1-associated genes (further detailed in Figure IX in the Data Supplement). RUNX1 target genes were significantly overrepresented among the DE genes, even more so when target binding sites were more stringently restricted: At a binding site P<0.05, 12 of 106 DE genes were among the 1251 RUNX1 targets (P=0.03; odds ratio, 2.0). These target genes include diabetes-induced DE of atherosclerosis-relevant genes such as Traf3ip3 (log fold change, 0.56; FDR, 0.02),48 Crnkl1 (log fold change, 0.63; FDR, 0.04),49 and Pla2g5 (log fold change, 0.16; FDR, 0.1).50

Txhawm rau txhawm rau txheeb xyuas seb tus tib neeg cov quav hniav macrophage ua haujlwm tau zoo npaum li cas ntawm cov nas macrophages, peb muab piv nrog cov txheej txheem ntawm 39 M1 thiab M2 cov noob nrog cov lus qhia uas tau ntsuas hauv BMDMs los ntawm cov ntshav qab zib tiv thaiv cov nas (Daim duab 3G). Cov no tau sau tseg los ntawm qhov zoo lossis tsis zoo log hloov pauv (mob ntshav qab zib tiv thaiv kev tswj hwm) thiab piv nrog cov kev hloov pauv sib npaug hauv tib neeg cov quav hniav macrophages ntawm cov neeg mob ntshav qab zib hom 2 (piv txwv). Ib qho kev sim Fisher tseeb qhia tau tias muaj kev sib raug zoo ntawm tus nas BMDM thiab tib neeg cov quav quav macrophage cov ntaub ntawv teev rau cov genes upregulated lossis downregulated nyob rau hauv cov ntshav qab zib tiv thaiv cov ntsiab lus (P=0.0155; odds ratio, 6.83).

Having shown a hyperglycemia-induced trained immunity phenotype in mouse HSCs and BMDMs and confirmed shared transcriptional features with macrophages from human plaques, we hypothesized that circulating human blood cells from patients with diabetes might also show evidence of priming. We obtained PBMCs from patients with type 2 diabetes and control subjects without diabetes matched for age and body mass index (Figure X in the Data Supplement). After isolation, PBMCs were restored to normal glucose conditions (5 mmol/L) in tissue culture for 24 hours. Comparing transcriptomes in basal conditions and after stimulation with LPS+IFN-γ, we found that the cells derived from patients with diabetes showed DE of >3000 cov noob piv nrog cov kev tswj tsis muaj ntshav qab zib. Gene teem enrichment tsom xam ntawm altered noob pom zoo enrichment nyob rau hauv stimulated mob ntshav qab zib PBMCs (piv txwv li) rau Hallmark txoj kev, nrog rau cov inflammatory teb (P=1.70 × 10−5) thiab IL-6 JAK STAT3 signaling ( P=0.0221), thiab Reactome pathways, suav nrog cov pa oxygen reactive thiab reactive nitrogen hom kev tsim tawm (P=0.0049), IL- /IL- signaling (P=0.0110) , thiab Tus Xov Tooj Zoo li 1/2 cascade (P=0.0033). Cov txheej txheem no qhia tau hais tias cov cell peripheral no kuj pom cov yam ntxwv zoo ib yam li cov uas peb tau tshaj tawm hauv BMDMs los ntawm cov nas mob ntshav qab zib (Daim duab 3G).

Thaum kawg, txhawm rau tshawb fawb ntxiv txog qhov ua tau ua rau RUNX1 hauv cov lus teb ntawm hyperglycemia-vim kev tiv thaiv kab mob, BMDMs uas yav dhau los tau sib txawv hauv lub cev lossis cov qabzib siab tau rov qab los rau 5 mmol / L qabzib thiab tom qab ntawd ua rau tsis muaj lossis muaj cov tshuaj RUNX. 1- tshwj xeeb inhibitor Ro5-333551 (Daim duab 8D). Cov cell sib txawv hauv cov piam thaj siab tau nthuav tawm ntau ntxiv ntawm Il-6 (2-fold, P<0.001) and Il-1β (2.1-fold, P<0.001), despite normalization of glucose levels (Figure 8E and 8F and Figure XIb and XIc n the Data Supplement). Ro5-3335 normalized heightened Il-6 and Il-1β expression in a dose-dependent manner (Figure 8E and 8F), with the starting effective dose (0.5 µmol/L) being equal to the Ro5-3335 IC50 value.52

Daim duab 8. Lub luag haujlwm rau RUNX1 hyperglycemia-induced cob qhia kev tiv thaiv. Sab saum toj 3 qhov kev hloov pauv tau pom nyob rau hauv ib qho kev ntsuam xyuas rau transposase accessible chromatin sequencing (ATAC-seq) peaks differentially enriched (A) unstimulated diabetic HSC (n=3) piv nrog unstimulated tswj hematopoietic qia hlwb (HSCs; n{{6 }}). Unsupervised hierarchical pawg ntawm kev sib txawv ntawm cov noob los ntawm RNA sequencing (RNA-seq) tsom xam ntawm (B) ntshav qab zib (n=6) ​​lossis tswj (n=6) pob txha pob txha (BM)-derived macrophages (BMDMs) stimulated nrog lipopolysaccharide (LPS) ntxiv rau interferon- (IFN-). Unsupervised hierarchical pawg ntawm qhov sib txawv ntawm cov noob los ntawm (C) ntshav qab zib (n=8) lossis tswj (n{13}}) laser ntes microdissected tib neeg carotid plaque macrophage kuaj. B thiab C, Runt-related transcription factor 1 (Runx1) target genes (RUNX1) (P<0.05 stringency) are highlighted in red to the left of the heat map. Wild-type (WT) BM was differentiated into BMDMs in physiological (5 mmol/L) or high (20 mmol/L) glucose, rested for 2 days in 5 mmol/L glucose, and then stimulated in 5 mmol/L glucose±varying concentrations of Ro5-3335, (D) as illustrated. M1 gene expression of (E) Il-6 and (F) Il-1β assessed by quantitative polymerase chain reaction; data normalized to B2m expression. Data displayed as mean±SD, 2-way ANOVA with Bonferroni post hoc. Sample n=5 individual animals. FC indicates fold change. *P<0.05; ***P<0.001.

Kev sib tham

Feem ntau ntawm cov teeb meem hauv plawv tseem muaj nyob hauv cov neeg mob ntshav qab zib txawm tias muaj kev tswj hwm cov piam thaj ntau.52 Ntawm no, peb muab cov pov thawj tias hyperglycemia ua rau muaj kev tiv thaiv kev tiv thaiv hauv HSCs thiab macrophages thiab qhov no ua rau muaj kev cuam tshuam atherosclerosis. Peb pom tias hyperglycemia ua rau muaj kev hloov pauv tsis tu ncua hauv BM HSCs los ntawm kev tsav tsheb aerobic glycolysis, ua rau cov qauv tshwj xeeb ntawm chromatin nkag tau, nrog cov pov thawj rau lub xeev proinflammatory priming uas pheej mus rau qhov sib txawv macrophages. Peb qhov kev tshawb pom muaj feem cuam tshuam txog kev hloov pauv, tshwj xeeb yog RUNX1, uas yog tus neeg nruab nrab ntawm kev tiv thaiv kev tiv thaiv. Pharmacological inhibition ntawm RUNX1 tshem tawm cov kev tshwm sim ntawm hyperglycemia-vim kev cob qhia kev tiv thaiv hauv vitro.

Kev hloov pauv hauv cellular metabolism tuaj yeem ua rau muaj kev tiv thaiv kev tiv thaiv, ib lo lus uas qhia txog kev tiv thaiv kab mob hauv lub cev uas tuaj yeem txhim kho tom qab luv luv stimulation nrog cov khoom siv microbial lossis endogenous atherogenic tshuaj, suav nrog oxidized LDL thiab lipoprotein (a).53 Piv txwv li, kev cob qhia kev tiv thaiv kab mob los ntawm -glucan , lub cell-phab ntsa tivthaiv ntawm Candida albicans, yog kho los ntawm kev ua kom lub dectin -1–Akt–mTOR–HIF-1 txoj kev, uas ua rau hloov ntawm oxidative phosphorylation mus rau aerobic glycolysis (Warburg nyhuv). 18 Tseeb, ntau cov metabolites los ntawm glycolysis thiab lub voj voog tricarboxylic acid ua cofactors rau histone-hloov enzymes, uas nyob rau hauv lem txiav txim siab chromatin accessibility; Piv txwv li, histone acetyltransferase thiab deacetylases xav tau acetyl-CoA thiab NAD ntxiv, whereas demethylase xws li Tet thiab JmjC siv -ketoglutarate ua cofactor.19,54 Tsis ntev los no, cov kev cai metabolic ntawm macrophage inflammatory gene qhia tau ncaj qha mus rau kev hloov kho histone. acetylation muab tau los ntawm kev nce qib lactate.55

Cov kev sib txuas ncaj qha ntawm cov piam thaj metabolism thiab kev hloov pauv ntawm epigenetic, ua ke nrog cov kev soj ntsuam qhia pom tias hyperglycemic nco hauv cov neeg mob ntshav qab zib, coj peb mus rau qhov kev xav tias hyperglycemia nws tus kheej tuaj yeem ua rau muaj kev hloov pauv hauv cellular metabolism, ua rau lub cev tsis muaj zog hauv cov ntshav qab zib. Hauv cov ntaub ntawv ntawm -glucan, nce aerobic glycolysis hauv macrophages ua rau muaj zog histone 3 Lys4 trimethylation (H3K4me3) thiab histone 3 Lys27 acetylation (H3K27ac). Ob qho ntawm cov kev hloov kho chromatin no tau nce ntxiv hauv cov ntsiab lus ntawm cov piam thaj-tsav glycolysis siab. Raws li tau pom ntawm no thiab hauv cov kev tshawb fawb yav dhau los, muaj kev txhawb nqa ntawm H3K4me3 ntawm kev txhawb nqa ntawm cov noob muaj feem xyuam rau kev mob (xws li, Il-6) thiab cov piam thaj metabolism (piv txwv li, Hk1).16,17 Lub voj voog tricarboxylic acid intermediates succinate thiab fumarate tau tshaj tawm los ua antagonists ntawm histone thiab DNA demethylase, 54 thiab ua raws li qhov no, peb kuj pom tias tricarboxylic acid voj voog metabolites succinate thiab malate tau nce hauv cov hlwb hyperglycemic.

Metropolis et al20 tau qhia tias, nyob rau hauv cov ntsiab lus ntawm -glucan-induced innate tiv thaiv kab mob hauv myeloid cell progenitors, cytokines tuaj yeem kho cov kev tiv thaiv kab mob. Tshwj xeeb, inhibition ntawm IL-1 nrog receptor antagonist anakinra inhibited -glucan-induced kawm tiv thaiv kab mob. Kev noj zaub mov sab hnub poob kuj tau pom tias ua rau muaj kev cuam tshuam ntawm epigenetic reprogramming nyob rau hauv BM progenitor hlwb, ua rau ib tug inflammasome-mediated cob qhia kev tiv thaiv kab mob, implicating IL-1 signaling pathways nyob rau hauv kev kawm tiv thaiv kab mob.56 Muab hais tias IL-1 mediates ntau pathological txheej txheem hauv ntshav qab zib, 44 peb tau tshuaj xyuas seb IL-1 puas yuav txhawb nqa cov txiaj ntsig ntawm cov piam thaj siab hauv kev txhim kho kev tiv thaiv kab mob. Txawm hais tias mob ntshav qab zib ib leeg tau pom muaj txiaj ntsig ntawm chromatin nkag tau (nrog kev tshuaj ntsuam motif cuam tshuam nrog PU.1, RUNX1, thiab CCCTCbinding factor), cov teebmeem ntawm IL-1 , nyob rau hauv cov ntsiab lus ntawm cov hlwb uas tau tsim los ntawm cov ntshav qab zib, tau hais ntau dua ( thiab cuam tshuam ERG, ZNF263 [zinc ntiv tes protein 263], thiab RUNX1). Qhov no qhia txog kev sib raug zoo hierarchical uas macrophages tau primed los ntawm hyperglycemia tab sis tshwm sim ntau qhov kev hloov pauv hauv cov lus teb rau qhov kev txhawb nqa thib ob.43 Tseeb tiag, nws tau lees paub zoo tias cov kev hloov pauv hauv chromatin xeev ntawm cov neeg txhawb nqa tuaj yeem cuam tshuam kev sib cuam tshuam ntawm kev sib txawv ntawm macrophage-activating stimuli. Piv txwv li, txawm hais tias IFN- tsis tuaj yeem qhib ntau LPS-inducible genes, nws tuaj yeem ua rau cov tshuaj histone acetylation thiab chromatin hloov kho ntawm lawv cov neeg txhawb nqa, yog li priming lub cell rau yav tom ntej ua kom teb rau LPS.57.

Motif tsom rau ntawm qhov chaw qhib chromatin tau pom muaj kev txhawb nqa hauv RUNX1 qhov chaw sib khi. Los ntawm kev pom kev ua haujlwm, RUNX1 yuav tsum muaj rau tiam thiab kev saib xyuas ntawm HSCs thiab kev sib txawv ntawm ntau hom kab. -1) receptor, uas yog qhov tseem ceeb rau kev ciaj sia, kev sib txawv, thiab nthuav dav ntawm macrophages.59 Ntawm qhov cuam tshuam rau kev ua haujlwm nco tau zoo uas peb tau pom hauv monocyte adhesion, RUNX1 kuj yog ib qho tseem ceeb ntawm kev sib cuam tshuam ntawm monocyte-endothelium los ntawm Kev tswj hwm ntawm lymphocyte muaj nuj nqi-koom nrog antigen 1/CD11a.31 Thaum kawg, Tus Xov Tooj-zoo li receptor 4-mediated o thiab RUNX1 overexpression nce qhov tsim ntawm Tus Hu-zoo li receptor 4-induced IL-6 thiab IL-1 los ntawm RUNX1 khi rau lub nuclear factor-κB subunit p50, synergizing raws li ib tug transcriptional coactivator.60 Coj ua ke, muaj pov thawj rau muaj zog convergence ntawm RUNX1 muaj nuj nqi nyob rau hauv macrophages mus rau regulating txheej txheem uas tseem ceeb nyob rau hauv atherogenesis.

Peb, yog li ntawd, cross-referenced genes under putative control of RUNX1 with the transcriptional profiles of cells derived from nas BMDMs and in macrophages from a related human human in vivo setting. Tib neeg atherosclerotic plaque macrophages thiab BMDMs, thaum muaj ntshav qab zib, pom DE ntawm RUNX1- tswj cov noob, suav nrog ntau yam yav dhau los txuas rau o thiab atherosclerosis.49,50 Peb txoj haujlwm txuas ntxiv ntawm Alrdahe thiab cov npoj yaig, 61 uas nyuam qhuav pom. uas cov neeg mob ntshav qab zib tau los ntawm cov macrophages kab lis kev cai rau 6 hnub hauv vitro tswj cov proinflammatory priming thiab hyperpolarize rau proinflammatory phenotype thaum stimulated nrog LPS thiab IFN- lossis qog necrosis factor-.

Interestingly, qee qhov RUNX1 lub hom phiaj cov noob tau nthuav tawm ntau ntxiv hauv cov quav hniav los ntawm cov neeg mob ntshav qab zib, xws li CRNKL1, thiab PLA2G5, thaum lwm tus, xws li ZNF32 (zinc ntiv tes protein 32) thiab NUP214 (nucleoporin 214), qhia qis dua hauv cov ntshav qab zib thiab tsis muaj ntshav qab zib. plaque macrophages. Txawm hais tias qhov no tej zaum yuav zoo li tsis sib haum, RUNX1 tau ua pov thawj rau ob qho tib si txhawb cov noob qhia thiab ua raws li kev hloov pauv hloov pauv.62 Qhov no tshwm sim feem ntau los ntawm kev nrhiav histone deacetylases xws li HDAC1 thiab HDAC663 thiab histone methyltransferases.62 Ntxiv mus, kev sib cuam tshuam ntawm lub cev ntawm RUNX1. nrog chromatin tuaj yeem pab txhawb rau tus kheej ntawm cell progeny. Piv txwv li, kev cuam tshuam ntawm RUNX1 kev sib cuam tshuam thaum lub sij hawm mitosis ua rau epithelial-to-mesenchymal transition.64 Pharmacological inhibition ntawm RUNX1 nrog Ro5-3335 normalized cov hloov inflammatory gene qhia cuam tshuam nrog hauv vitro hyperglycemia-vim kev tiv thaiv kev tiv thaiv. Lub hom phiaj kev cuam tshuam tawm tsam RUNX1 muab pov thawj ntxiv ntawm kev koom tes ntawm RUNX1 hauv cov kev cuam tshuam hauv qab ntawm hyperglycemia-induced kawm tiv thaiv kab mob hauv macrophages.

Induction ntawm hyperglycemia-vim kev cob qhia kev tiv thaiv kab mob hauv peripheral macrophages ib leeg tuaj yeem ua rau muaj kab mob loj zuj zus, tab sis muab qhov cuam tshuam ntev ntawm kev tiv thaiv kev tiv thaiv thiab lub neej luv luv ntawm peripheral myeloid cells, peb xav tias kev tiv thaiv kev tiv thaiv kuj tseem yuav raug ntxias. nyob rau theem ntawm HSCs, raws li tsis ntev los no tau qhia rau -glucan thiab Western-type diet.20 Txawm hais tias yav dhau los cov kev tshawb fawb muaj feem ntau cuam tshuam txog kev hloov pauv hauv kev tiv thaiv kab mob rau hauv vitro phenotype (xws li, cytokine ntau lawm), peb qhia tau hais tias kev tiv thaiv kab mob ncaj qha li cas. txhawb kev loj hlob ntawm atherosclerosis, ib qho tseem ceeb pathology nrog ib qho tseem ceeb inflammatory tivthaiv. Kev hloov pauv BM los ntawm cov nas mob ntshav qab zib mus rau cov nas normoglycemic atherosclerosis-nrhiav cov nas tau ua rau muaj kev cuam tshuam ntawm qhov kev mob tshwm sim thiab tau cuam tshuam nrog cov lipid-nplua nuj necrotic core, zoo ib yam nrog rau kev txhoj puab heev ntawm atherosclerosis pom hauv ntshav qab zib hauv tib neeg.65

Txoj kev tshawb no yog tsim los ntawm kev kuaj xyuas cov teebmeem ntawm cov piam thaj / hyperglycemia. Txhawm rau tso cai rau peb kom pom qhov cuam tshuam ntev ntawm hyperglycemia, cov yam ntxwv tseem ceeb ntawm cov ntshav qab zib, peb siv cov qauv streptozotocin nas cov ntshav qab zib ntau dua li hom 2 mob ntshav qab zib nas tus qauv, uas yuav muaj ntau yam nuanced nta ntawm tus mob ntawd tab sis yuav ua rau muaj kev cuam tshuam zoo. kev tshawb fawb txog cov teebmeem ntawm cov piam thaj. Muaj qhov sib txawv tseem ceeb ntawm hom 1 thiab hom 2 mob ntshav qab zib. Txawm li cas los xij, hyperglycemia yog qhov tshwm sim rau lawv qhov kev kuaj mob, ua rau muaj kev pheej hmoo ntawm cov hlab plawv hauv txhua qhov, thiab yog lub hom phiaj tseem ceeb ntawm kev kho mob thiab saib xyuas ob hom ntshav qab zib tau ntau xyoo. Peb cia siab tias qhov tshiab tau txheeb xyuas qhov kev tiv thaiv hyperglycemia-vim kev tiv thaiv kev tiv thaiv tau tshaj tawm ntawm no yuav raug piav qhia yav tom ntej kom coj mus rau hauv tus account tshwj xeeb kev hloov kho ntawm ntau lwm yam muaj feem cuam tshuam (xws li kev noj qab haus huv, 66 qauv ntawm cov piam thaj nce, 67 concomitant hyperlipidemia57).

Peb qhov kev tshawb pom muaj qhov cuam tshuam tseem ceeb rau kev tswj hwm cov teeb meem atherosclerotic ntawm ntshav qab zib. Qhov tseem ceeb tshaj plaws, cov yam ntxwv proatherogenic ntawm cov hlwb myeloid thiab BM precursors tseem nyob tom qab normalization ntawm cov ntshav qabzib. Qhov no tuaj yeem pab piav qhia txog qhov tsis muaj txiaj ntsig ntawm cov kev kho mob ib txwm muaj thiab ua rau muaj kev nyuaj siab rau txoj hauv kev los tswj kev pheej hmoo ntawm vascular thiab teeb meem ntshav qab zib. Kev txheeb xyuas cov neeg tsav tsheb metabolic thiab kev hloov kho epigenetic tshwj xeeb kuj pom zoo rau cov hom phiaj kho mob tshiab.

NYEEM NTXIV

Tau txais Lub Ob Hlis 27, 2020; tau txais Lub Rau Hli 23, 2021.

Kev koom tes

Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, UK (LE, NA, ATB, JB, JTC, MA, KZ, RA, TJC, MJC, KMC, RC, RPC). CeMM Research Center for Molecular Medicine ntawm Austrian Academy of Sciences, Vienna, Austria (TK, AFR, CB). Division of Physiological Chemistry II, Department of Medical Biochemistry thiab Biophysics, Karolinska Institutet, Stockholm, Sweden (HG-A., CEW). Department of Respiratory Medicine and Allergy (HG-A., CEW) and Department of Medicine (H7) (JL, MR), Karolinska University Hospital, Stockholm, Sweden. Kennedy Institute of Rheumatology, University of Oxford, UK (ALC, TEK, IAU). Lub koom haum ntawm Artificial Intelligence thiab kev txiav txim siab txhawb nqa, Center for Medical Statistics, Informatics, thiab Intelligent Systems, Medical University of Vienna, Austria (CB). Bioinfo, Plantagenet, ON, Canada (MEL). Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Lub Netherlands (NPR., MGN). Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Lub teb chaws Yelemees (MGN).

Kev lees paub

Cov kws sau ntawv ua tsaug rau Lisa Heather, Maria da Luz Sousa Fiahlo, thiab Thomas Milne rau lawv txoj kev pab. RPC, NA, thiab LE tau tsim txoj kev kawm. LE tau ua txhua yam hauv vitro kev sim, kev tshuaj ntsuam gene qhia, thiab kev tiv thaiv kab mob tau piav qhia. MA tau pab txhawb rau cov kev tshawb fawb hauv tib neeg cov qauv. CEW thiab HG-A. Nws tau ua tiav cov txheej txheem metabolomics loj. JTC tau ua tib neeg cov quav hniav macrophage LCM ua haujlwm thiab npaj array. LE tau ua Seahorse Bioanalyser thwmsim nyob rau hauv kev taw qhia ntawm JB thiab kev saib xyuas ntawm MJCALC thiab ua kev txheeb xyuas ntawm tes. LE npaj cov qauv RNA-seq. LE tau npaj ATAC-seq cov qauv raws li TEK cov lus qhia thiab IAU kev saib xyuas. NA tau ua txhua yam kev npaj ntawm tes ntawm ChiP. Sequence tau ua los ntawm TK thiab AFR nyob rau hauv CB tus saib xyuas. MEL thiab ATB tau khiav cov ntaub ntawv sib txuas ua ke, normalization, thiab kev tswj kom zoo, nrog LE ua cov kev soj ntsuam hauv qab. MEL thiab ATB ua array thiab sequencing cov ntaub ntawv tsom xam. KZ, RA, thiab RC coj kev tshawb nrhiav tsiaj. LE, NA, TJC, NPR, thiab MGN tau koom nrog kev sim tsim thiab ua tiav. RPC, LE, NA, thiab MEL npaj cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo qhov kev xa tawm kawg ntawm cov ntawv sau.

Cov peev txheej nyiaj txiag

Txoj haujlwm no tau txais kev txhawb nqa los ntawm British Heart Foundation Center of Research Excellence Oxford (RE/13/1/30181), British Heart Foundation Project Grant (Drs Akbar thiab Choudhury: PG/18/53/33895), National Institute for Health Research Oxford Biomedical Research Center, Tripartite Immunometabolism Consortium–Novo Nordisk Foundation (grant NNF15CC0018486; Drs Choudhury, Udalova, Rydén, Channon, thiab Wheelock), Metabolite-Related Inflammation in Diabetes-Spectrum Diseases (Norvo AD-Spectrum Diseases) , nyiaj pab 0064142; Drs Choudhury, Udalova, Rydén, thiab Channon), thiab ib xyoo 4-xyoo British Heart Foundation Ph.D. kawm ntawv thiab Doris Field Trust (Dr. Edgar). Pawg High-Throughput Genomics ntawm Wellcome Trust Center rau Human Genetics tau txais nyiaj los ntawm Wellcome Trust kev pab nyiaj siv 090532/Z/09/Z thiab MRC Hub muab G0900747 91070. Biomedical Sequencing Facility ntawm CeMM Research Center for Molecular Medicine ntawm Austrian Academy of Sciences hauv Vienna Austria. Drs Riksen thiab Netea tau txais nyiaj los ntawm European Union's Horizon 2020 kev tshawb fawb thiab kev tsim kho tshiab raws li kev pom zoo pub dawb 667837. Dr. Netea tau txais kev txhawb nqa los ntawm ERC Consolidator Grant (No. 310372) thiab Netherlands Lub Koom Haum rau Kev Tshawb Fawb Txog Kev Tshawb Fawb.

Qhia tawm

Tsis muaj.

Cov khoom siv ntxiv

Cov Ntaub Ntawv Ntxiv Figures I-XI

Cov ntaub ntawv ntxiv Excel Files I-VII

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Laurence Edgar, DPhil*; Naveed Akbar, Ph.D.*; Adas T. Braithwaite, Ph.D.; Thomas Krausgruber, Ph.D. Héctor Gallart-Ayala, Ph.D.; Jade Bailey, DPhil; Alastair L. Corbin, DPhil; Tariq E. Khoyratty, DPhil; Joshua T. Chai, DPhil; Mohammad Alkhalil, DPhil; André F. Rendeiro, Ph.D.; Klemen Ziberna, DPhil; Ritu Arya, MRes; Thomas J. Cahill, DPhil; Christoph Bock, Ph.D.; Jurga Laurencikiene, Ph.D. Mark J. Crabtree, Ph.D.; Madeleine E. Lemieux, Ph.D.; Niels P. Riksen, MD, Ph.D.; Mihai G. Netea, MD, Ph.D.; Craig E. Wheelock, Ph.D.; Keith M. Channon, MD; Mikael Rydén, MD, Ph.D.; Irina A. Udalova, Ph.D.; Ricardo Carnicer, Ph.D. Robin P. Choudhury, DM.

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