Rapamycin Hloov Kho Mitochondrial Dynamics hauv Dendritic Cells kom txo qis raum Ischemic Reperfusion Injury
Feb 22, 2022
Abstract:Dendritic cells (DCs) yog cov tshuaj tiv thaiv kab mob tshwj xeeb uas tuaj yeem txuas lub cev thiab hloov lub cev tiv thaiv kab mob thiab Immunometabolism cuam tshuam rau lawv cov phenotype. Rapamycin yog ib qho tshuaj macrolide uas muaj cov tshuaj tiv thaiv kab mob thiab siv los tiv thaiv kev poob ntawm kev hloov hauv lub raum. Txoj kev tshawb fawb tam sim no tau soj ntsuam cov peev xwm kho mob ntawm ex-vivo rapamycin kho DCs los tiv thaivlub raumnyob rau hauv ib tug nas qauv ntawm mobmob raum (AKI). Rau kev kho mob rapamycin ib leeg (S) (Rapa-S-DC), Veh-DCs tau kho nrog rapamycin (10 ng / mL) rau 1 teev ua ntej LPS. Hauv qhov sib piv, rapamycin ntau (M) kev kho mob (Rapa-M-DC) tau raug rau 3 qhov kev kho mob dhau 7 hnub. Tsuas yog ntau yam ex-vivo rapamycin kev kho mob ntawm DCs induced ib tug pheej reprogramming ntawm mitochondrial metabolism. Cov DCs no tau 18-fold mitochondria ntau dua, yuav luag 4-fold cov pa oxygen ntau dua, thiab tsim ATP ntau dua piv rau Veh-DCs (Veh kho DCs). Txoj kev tsom xam pom IL10 teeb liab ua txoj hauv kev tseem ceeb rau kev hloov pauv ntawm immunophenotype tom qab kev kho mob Rapamycin piv rau lub tsheb nrog qis qis cytokines Tnfa, Il1b, thiab Il6, thaum cov neeg tswj hwm ntawm cov ntsiab lus mitochondrial Pgc1a, Tfam, thiab Ho1 tseem nce siab. Qhov tseem ceeb, kev hloov pauv ntawm rapamycin-kho DCs rau WT cov neeg tau txais 24 teev ua ntej ob tograumischemia tseem ceeb tiv thaiv covlub raumlos ntawm kev raug mob nrog qhov tseem ceeb 3-fold txhim kho hauvlub raum ua haujlwm. Thaum kawg, cov dej ntawm DCs uas muaj cov lej mitochondria ntau dua (kho ex-vivo nrog kev noj qab haus huv cais mitochondria (10 µg / mL) ib hnub ua ntej) kuj tau tiv thaiv qee qhovlub raumlos ntawm IRI. Cov kev tshawb fawb no qhia tau hais tias ua ntej emptive infusion ntawm ex-vivo reprogrammed DCs uas muaj cov ntsiab lus mitochondria siab dua muaj peev xwm kho tau los ua kom muaj kev tiv thaiv kab mob phenotype los tiv thaivlub raumlos ntawm kev raug mob.
Ntsiab lus:dendritic cell; rapamycin; mitochondria; mob raum mob; ischemic reperfusion raug mob
Taw qhiaLub pathobiology ntawm mob hnyavmob raumyog multifactorial thiab koom nrog intricate kev sib cuam tshuam ntawmlub raumparenchymal hlwb thiab lub cev tiv thaiv kab mob [1,2]. Hauvraum,Cov pa dendritic cells (DCs) yog cov kab mob loj hauv lub cev tiv thaiv kab mob thiab pom muaj kev sib raug zoo hauv qhov chaw sib cuam tshuam thoob plaws hauv lub cev.raum[3–5]. DCs yog ib qho ntawm cov xov tooj tseem ceeb uas koom nrog hauv kev tiv thaiv kab mob hauv lub cev thiab yog qhov tseem ceeb hauv kev muab kev tiv thaiv kab mob. Peb cov ntawv tshaj tawm yav dhau los siv cov pob txha pob txha muab los ntawm DCs tau pom tias DCs kev kho mob tuaj yeem tswj hwm ntau yam kev tiv thaiv kab mob hauv lub cev thiab hloov kho lub cev tiv thaiv kab mob cuam tshuam nrog AKI [6,7].
DCs muaj nyob rau hauv tag nrho cov ntaub so ntswg, nrog ib tug subset ntawm DCs (CD11c ntxiv rau DCs) nyob rau hauv lublub raumparenchyma. Txawm li cas los xij, cov CD11c ntxiv rau DCs kuj tseem ua lub luag haujlwm ua rau mob hnyav zuj zusmob raumtom qab ischemia-reperfusion raug mob (IRI) [8]. IRI yog txhais tau tias yog kev raug mob los ntawm kev rov qab muab cov ntshav oxygenated rau hauv lub cev tom qab lub sij hawm ntawm ischemia thiab feem ntau pom hauv myocardial infarction, mob stroke, thiab hloov khoom nruab nrog cev. Hauv kev teb rau IRI, CD11c ntxiv rau DCs suav nrog feem ntau ntawm cov cell inflammatory infifiltrate hauvlub raumntaub so ntswg.
DCs muaj peev xwm paub txog cov kab mob sib txuas nrog cov qauv molecular (PAMPs) los ntawm ntau yam qauv lees paub receptors nyob rau saum npoo ntawm DCs xws li tolllike receptors (TLRs) thiab NOD-zoo li receptors. Kev khi ntawm cov qauv lees paub receptors (PRRs) rau lawv cov PAMPs tshwj xeeb ua rau muaj kev ua kom muaj ntau txoj hauv kev, uas suav nrog cov lus teb proinflammation, antimicrobial peptide synthesis, thiab induction ntawm lymphocyte mediated adaptive immunity [9]. Cov kev tshawb fawb tseem ceeb tau mob siab rau kev kawm txog kev hloov pauv hauv cellular bioenergetics ntawm DCs thaum ua haujlwm los ntawm PAMPs. Nws tau raug tsim los ua kom DCs nrog lipopolysaccharide (LPS; PAMP txawv rau cov kab mob gramnegative), los ntawm kev khi rau nws cov receptor tshwj xeeb TLR4 ua rau muaj kev hloov pauv hauv metabolic los ntawm kev ua tau zoo oxidative phosphorylation rau cov txheej txheem zoo ib yam li Warburg metabolism, lossis aerobic glycolysis. . Warburg metabolism yog tus cwj pwm los ntawm kev hloov pauv pyruvate tsim los ntawm glycolysis ntawm kev hloov pauv mus rau lactate rau lub zog tsim khoom, thiab deb ntawm kev thauj mus rau hauv mitochondria kom dhau mus rau catabolism ntawm TCA lub voj voog, txawm tias muaj cov pa txaus txaus. Ib qho kev piav qhia rau qhov tshwm sim no suav nrog ib qho ntawm ntau qhov kev cuam tshuam ntawm TLR4 ua kom muaj zog, uas yog kev ua kom muaj zog ntawm serine / threonine kinase lub hom phiaj ntawm cov tsiaj txhu ntawm rapamycin (mTOR). mTOR inhibition los ntawm rapamycin khaws cia mitochondrial muaj nuj nqi nyob rau hauv activated DCs los ntawm downregulating iNOS, nrog rapamycin-kho DCs stimulated nyob rau hauv lub xub ntiag ntawm LPS exhibiting txo qib ntawm aerobic glycolysis [10]. mTORC1 inhibitors zoo li rapamycin tam sim no siv rau hauv kev hloov pauv hauv nruab nrog cev los ua lwm txoj hauv kev rau calcineurin inhibitors kom tsis txhob mob ntev.lub raumallograft puas tsuaj raws li nws kuj muaj peev xwm nthuav dav ntawm CD4 ntxiv rau CD25 ntxiv rau kev tswj T-cells [11,12]. Txawm li cas los xij, kev siv rapamycin hauv kev hloov pauv tuaj yeem ua rau muaj teeb meem tshwm sim uas suav nrog peripheral edema, creatinine nce, thiab thrombocytopenia. Cov kev tshawb fawb yav dhau los tau pom tias cov kev tsis zoo ntawm hypoxic stimuli yog txhim kho los ntawm LPS raug thiab attenuated los ntawm rapamycin [8,13].
Lub hom phiaj yog los tsim txoj hauv kev los siv FDA pom zoo cov tshuaj qib tshuaj xws li Rapamycin los txhawb kev tswj hwm DCs uas tuaj yeem tswj hwm qhov tsis xav tau hauv lub cev thiab hloov lub cev tiv thaiv kab mob. Lub hom phiaj ntawm txoj kev tshawb no yog los txiav txim siab txog qhov muaj peev xwm tiv thaiv cov txheej txheem ntawm rapamycin stimulated BMDCs hauv tus qauv nas preclinical ntawmraumIRI. Kev kho mob ntawm BMDCs ex-vivo nrog rapamycin zam kev cuam tshuam tsis zoo ntawm lub hom phiaj thiab cov teeb meem o uas cuam tshuam nrog kev txhaj tshuaj. Cov koob tshuaj zoo tshaj plaws thiab lub sijhawm ntawm cov tshuaj pharmacological feem ntau yog nyob ntawm ntau yam tsis muaj. Hauv cov qog nqaij hlav qog noj ntshav qis ntawm rapamycin piv rau cov koob tshuaj siab sib txawv inhibits mTORC1 thiab mTORC2 uas ua rau ib nrab lossis ua tiav inhibition ntawm cell-cycle mus [14]. Rau tag nrho peb cov ex-vivo BMDCs cov kev tshawb fawb hauv cov ntawv sau tam sim no peb tau xaiv cov koob tshuaj qis ntawm 10 ng / mL ntawm rapamycin raws li kev tshawb fawb yav dhau los nrog tolerogenic BMDCs [15,16]. Lub sij hawm muaj pes tsawg koob tshuaj (peb lossis ib qho) yog raws li peb cov ntawv tshaj tawm yav dhau los nrog kev nthuav tawm nas BMDCs thiab ntxiv nrog GMCSF [17,18]. Peb cov ntaub ntawv tam sim no qhia tau hais tias ob qho tib si ib leeg thiab ntau yam kev kho mob ntawm DCs nrog rapamycin induces tsis muaj zog immunogenic cov lus teb (qis cytokines thiab co-stimulatory molecules) tom qab LPS stimulation. Kev hloov ntawm ib leeg lossis ntau yam rapamycin kho DCs sib npaug tiv thaivlub raumlos ntawm IRI hauv syngeneic (C57BL / 6 BMDC → C57BL / 6 nas) lossis allogeneic (BALB / c BMDC → C57BL / 6 nas) qauv ntawm IRI. Txawm li cas los xij, ntau qhov kev kho mob ex-vivo nrog rapamycin (3 kev kho mob tshaj 7 hnub), ntxiv rau txo cov tshuaj tiv thaiv kab mob, kuj nce mitochondria tus lej thiab ua haujlwm (Seahorse Analyzer) piv rau DCs kho tsheb. Cov kev soj ntsuam sim no qhia DCs nrog cov lej mitochondria siab dua muaj peev xwm los ua kev tswj hwm, thiab kev hloov pauv ntawm cov DCs tuaj yeem tiv thaiv.lub raumlos ntawm kev raug mob ischemic. Raws li peb txoj kev tshawb fawb tsis ntev los no siv FTY720 kho DCs pom tau hais tias [18], tshuaj kho mob lossis mitochondrial hloov pauv uas ua rau cov lej mitochondria ntau dua hauv cov kab mob tiv thaiv kab mob (DCs lossis macrophages) kuj ua rau cov hlwb no muaj cov tshuaj tiv thaiv kab mob phenotype, ib qho cuam tshuam los ntawm kev saib xyuas ntawm mitochondria. oxidative phosphorylation (OXPHOS) thiab nrog txo kev hloov mus rau glycolysis rau lub zog tom qab stimulation. Rau cov teebmeem no, peb exogenously nce mitochondria tus lej hauv DCs los kuaj yog tias tus lej mitochondria siab dua tuaj yeem ua rau muaj kev tswj hwm phenotype los ntawm kev siv mitochondrial transplants. Hloov ntawm cov Mito-DCs tiv thaivlub raumlos ntawm IRI; Txawm li cas los xij, tsis zoo li Rapa-DCs cov Mito-DCs no tsis muaj qhov txo cov tshuaj tiv thaiv kab mob (MHCII, co-stimulatory molecules thiab cytokines) tom qab LPS stimulation, tejzaum nws qhia tau tias nce mitochondria tus lej tuaj yeem cuam tshuam cov kev hloov pauv uas tshwm sim los ntawm exogenous LPS stimulation. Nyob rau hauv txoj kev tshawb no thiab raws li yav tas los qhia [6,7,18], cov kab ke txhaj tshuaj DCs (tsheb-, rapamycin- lossis mitochondria-kho) ntawm koob tshuaj ntawm ib nrab lab feem ntau tsuas pom nyob rau hauv tus po thiab me me kom tsis muaj teeb meem pom. hauvlub raum. Hauv spleen zoo li FTY720-DCs, rapamycin thiab mitochondria nplua nuj DCs tuaj yeem ua rau muaj kev tiv thaiv kab mob uas ua rau muaj kev tiv thaiv los ntawmraumIRI. Cov fifindings seminal no muab cov pov thawj tshiab uas qhia tias nce mitochondrial tooj (exogenously los ntawm mitochondrial transplants los yog los ntawm pharmacological kev cuam tshuam (FTY720 los yog rapamycin)) nyob rau hauv lub cev tiv thaiv kab mob yuav yog ib tug tshiab kev kho mob hloov mus rau thaum kawg tswj lawv immunologic teb rau kev tiv thaiv thiab / los yog thaum ntxov kev kho mob.mob raum.
Cov ntaub ntawv thiab cov txheej txheem
Cov nasTag nrho cov txheej txheem tsiaj thiab tuav tau ua raws li National Institutes of Health Guide for the Care and Use of Laboratory Tsiaj, thiab Bajwa UTHSC raws tu qauv tooj, 18-080, hnub pom zoo 09/28/2018 tau pom zoo los ntawm University of Tennessee Health Science Center (UTHSC) Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Pawg. Laboratory txoj cai saib xyuas tsiaj ua raws li Public Health Service Policy on Humane Care and Use of Laboratory Animals. Tag nrho cov txiaj ntsig tau tshaj tawm raws li cov lus qhia ARRIVE [19]. Rau tag nrho cov kev kawm hloov pauv C57BL/6J thiab BALB/c nas tau yuav los ntawm Jackson Laboratory (Bar Harbor, ME, USA). Cov nas tau khaws cia hauv cov vaj tsev nyob hauv tus qauv vivarium nrog lub sijhawm 12- teev lub teeb / tsaus nti ntawm kev noj zaub mov thiab dej muaj pub dawb.
Lub raum Ischemia-Reperfusion Injury Tag nrho cov txheej txheem phais kom ntxaws tau ua raws li tau tshaj tawm yav dhau los [18]. C57BL/6 txiv neej nas (8-12 lub lis piam) raug rau 26-min ob sab ischemia tom qab 20- 24 teev reperfusion. Luv luv, txhua tus nas nas tau muab tshuaj loog nrog kev txhaj tshuaj intraperitoneal (ip) ntawm ketamine thiab xylazine sib xyaw, nrog rau kev txhaj tshuaj subcutaneous ntawm buprenorphine thiab muab tso rau ntawm lub ncoo sov kom tswj lub cev kub ntawm 34.5-36 ◦C. Cov nas tom qab tau randomized mus rau qhov kev txaj muag lossis IRI kev ua haujlwm. Ob sab flflank incision tau ua. Lub cev kub tau kuaj xyuas thiab khaws cia thoob plaws lub sijhawm ischemic. Shamoperated nas tau ua tib yam txheej txheem tshwj tsis yog rau lub nkoj clamping thiab kaw cov qhov txhab phais. Nas uas muaj ib tugraumnrog tsis muaj reperfusion 24 teev tom qab ischemia raug tshem tawm los ntawm txhua qhov kev soj ntsuam.
Kev soj ntsuam ntawm lub raum ua haujlwm thiab HistologyCov ntshav tau sau los ntawm tshuaj loog los ntawm retro-orbital sinus, thiab plasma creatinine (mg / dL) tau txiav txim siab los ntawm kev siv txoj kev enzymatic nrog kev hloov me me los ntawm cov chaw tsim khoom raws tu qauv (Diazyme Laboratories, Poway, CA, USA) thiab cov ntshav tso zis nitrogen (BUN. ) thiab kev ntsuas creatinine tau ua los ntawm QuantiChrom Urea Assay Kit (DIUR-100, BioAssay Systems, Hayward, CA, USA) raws li tau piav qhia yav dhau los [18,20]. Rau histology,lub raumtau kho thaum hmo ntuj hauv 4 feem pua PLP lossis 10 feem pua formalin thiab embedded hauv paraffin.Lub raumtau npaj rau H&E staining raws li tau piav qhia yav dhau los [18] thiab cov duab tau thaij nrog qhov ci ntsa iab / qhov sib txawv hloov kho nrog EVOS microscope (Thermo Fisher Scientifific, Waltham, MA, USA). Rau kev txheeb xyuas cov qhab nia ntawm tubular raug mob, ntu tau ntsuas los ntawm suav cov feem pua ntawm cov tubules uas pom cov cell necrosis, poob ntawm txhuam ciam teb, cam khwb cia tsim, thiab tubule dilation raws li hauv qab no: 0=ib txwm; 1=Tsawg dua lossis sib npaug li 10 feem pua ; 2=10 mus rau 25 feem pua ; 3=26 rau 50 feem pua ; 4=51 mus rau 75 feem pua ; 5=Ntau dua lossis sib npaug li 75 feem pua . Tsib mus rau 10 fifields los ntawm txhua lub medulla sab nrauv tau raug soj ntsuam thiab tau qhab nia hauv qhov muag tsis pom. Qhov kev hloov pauv ntawm histological tau qhia tias mob hnyav tubular necrosis (ATN), tau qhab nia raws li tau piav qhia yav dhau los [7,21]. Apoptotic hlwb raug kuaj pom los ntawm TUNEL kev soj ntsuam (Hauv Situ Cell Detection Kit, TMR Liab; Roche, Basel, Switzerland) raws li cov chaw tsim khoom cov lus qhia.
Bone Marrow (BM)-Derived-Dendritic Cell (DC) Kab lis kev cai thiab hloov pauv hloov8–1{12}} lub lis piam qub C57BL/6J los yog BALB/c WT txiv neej nas tau siv los tsim DCs los ntawm tag nrho BM precursors [22]. GMCSF-nplua nuj supernatant tau muab los ntawm J558L hlwb stably hloov nrog nas GMCSF. Lub xovtooj ntawm tes yog ib qho khoom plig pub dawb los ntawm Dr. Ira Mellman (Department of Biology, Yale University, New Haven, CT, USA). Luv luv, BM tshiab tau raug cais tawm nrog 6 ng/mL recombinant nas GMCSF (tag nrho ntawm 3 kev kho mob) rau 8 hnub hauv RPMI 1640 (Invitrogen, Carlsbad, CA, USA). Qhov koob tshuaj zoo tshaj plaws ntawm 10 ng / mL rapamycin tau txiav txim siab tom qab kuaj ntau qhov koob tshuaj (0.1-10 ng / mL). BMDC tau kho nrog 10 ng/mL rapamycin (Sigma, St. Louis, MO, USA) rau tag nrho peb txoj kev kho mob lossis 1-kev kho thaum hmo ntuj). BMDCs tau kho nrog TLR4 agonist lipopolysaccharide (LPS; Escherichia coli serotype 0111:B4: 100 ng/mL; Sigma-Aldrich); los yog lub tsheb (1 × PBS) rau 24 teev hauv kab lis kev cai nruab nrab rau kev tshawb fawb syngeneic (C57BL / 6J BMDCs → C57BL / 6J nas) thiab sab laug tsis kho rau kev tshawb fawb allogenic (BALB / c BMDCs → C57BL / 6J nas). Cells raug ntxuav, thiab 0.5 × 106 hlwb rau ib tus nas tau txhaj tshuaj (iv) txhaj rau nas naive 1 hnub ua ntej ob tograumIRI. BMDCs tau sau npe nrog MitoTracker CMXRos Liab lossis MitoTracker Deep Red (100 nM, 30 min @ 37 ◦C, Invitrogen) lossis Mitosox (5 µM; 10 min @ 37 ◦C; Invitrogen).
Quantitative Real-Time PCRCov txheeb ze mitochondrial DNA (mtDNA) qhia qib tau ntsuas raws li tau piav qhia yav dhau los [23]. Luv luv, tag nrho cov genomic DNA raug cais tawm thiab sib npaug (5 ng) tau siv rau RTPCR siv ND1 li surrogate primers rau mtDNA thiab HK2 primers rau nuclear DNA (nDNA) rau nas thiab ND6 rau tib neeg mtDNA raws li yav dhau los tau piav qhia [23,24]. Tag nrho RNA raug cais tawm thiab thim rov qab rau cDNA, thiab RT-PCR tau ua raws li tau piav qhia yav dhau los [7,25,26].
Mitochondria Isolation thiab QuantifificationMitochondria raug cais tawm ntawm HEK293 hlwb raws li tau piav qhia yav dhau los [18]. Luv luv, 60 cm daim hlau ntawm HEK293 hlwb tau sau los ntawm kev ntxiv 5 mL ntawm homogenization tsis (300 mmol / L sucrose, 10 mmol / L HEPES-KOH, 1 mmol / L EGTA-KOH, pH 7.4) nrog 1 mg Subtilisin A protease los ntawm Bacillus licheniformis (Sigma-Aldrich). Cov txheej txheem ntxaws ntxaws rau kev sib cais ntawm mitochondria tuaj yeem pom hauv peb cov ntawv sau yav dhau los [18].
2 Seahorse Flux BioanalyzerXya-hnub qub BMDCs raug xa mus rau Seahorse 24-cov ntaub so ntswg kab lis kev cai daim hlau, thiab cov pa oxygen noj npaum li cas (OCR) tau ntsuas, thiab cov kev ntsuas tau raug xam raws li tau piav qhia yav dhau los [25] nrog kev hloov kho hauv qab no. Tom qab ntsuas basal ua pa tus nqi, oligomycin (Sigma; 2 µM), FCCP (Sigma; 1.5 µM; carbonyl cyanide 4-} (triflfluoromethoxy)-phenylhydrazone (FCCP)), thiab electron thauj saw ( complex I thiab III) inhibitors, rotenone (Sigma; 0.5 µM), thiab antimycin A (Sigma; 0.5 µM)) tau txhaj tshuaj ua ntu zus thaum lub sijhawm kuaj. Basal mitochondrial respiration, ATP-txuas ua pa, pro ton leak (tsis-ATP txuas oxygen noj), kev ua pa siab tshaj plaws, tsis yog mitochondrial ua pa, khaws cia muaj peev xwm ua pa, tswj kev ua pa, thiab kev sib txuas ua haujlwm tau txiav txim siab hauv tag nrho cov hlwb raws li Hom thiab al. . [27]; N=4–5 qhov dej tau siv rau txhua pab pawg sim thiab cov kev sim tau rov ua dua tsawg kawg 3 zaug.
Flow Cytometric Analysis, Western Blot, thiab ELISAFlow cytometry cov ntaub ntawv tau txais tau ua tiav ntawm FACS Calibur (Becton Dickinson, San Jose, CA, USA) nrog Cytek {{0}}} xim flflow cytometry upgrade (Cytek Development, Inc., Fremont, CA, USA). Cov ntaub ntawv tau txheeb xyuas los ntawm FlowJo software 9.0 (Tree Star, Ashland, OR, USA) raws li tau piav qhia yav dhau los [6,7,28]. Rau ELISA media tau sau los ntawm BMDCs 24 teev tom qab kho nrog 100 ng / mL LPS. TNF , IL6, lossis IL10 qib tau ntsuas los ntawm kev siv cov khoom siv nas ELISA (Invitrogen) ua raws li tus neeg tsim khoom raws tu qauv thiab raws li tau piav qhia yav dhau los [18]. Veh- lossis Mito-DC kho nrog thiab tsis muaj LPS rau 6 lossis 24 teev tau siv los cais tag nrho cov protein uas siv RIPA lysis buffer supplemented nrog protease thiab phosphatase inhibitor cocktail (Thermo Fisher Scientifific, Vernon Hills, IL, USA). Rau GAPDH lysate supernatants tau boiled (10 min ntawm 100 ◦C) thiab lysates tau tso rau hauv chav sov li 10 min rau nas OXPHOS cocktail (Abcam, Cambridge, MA, USA) [18]. Ib txoj hauv kev ntxaws ntxiv tau piav qhia yav dhau los [18].
Cov ntaub ntawv thiab Kev Tshawb Fawb Txog Kev Tshawb FawbGraphPad Prism 8 (GraphPad Inc., San Diego, CA, USA) tau siv los txheeb xyuas thiab nthuav tawm cov ntaub ntawv. Cov ntaub ntawv raug txheeb xyuas, tom qab hloov pauv yog xav tau los tsim kom muaj kev faib tawm ib txwm, los ntawm 2-tailed t test lossis 1- txoj kev ANOVA nrog kev soj ntsuam tom qab hoc raws li qhov tsim nyog. Ob-tailed unpaired t test tau siv los tshuaj xyuas ob pawg. p Tsawg dua lossis sib npaug rau 0.05 tau siv los qhia qhov tseem ceeb.
Cov txiaj ntsig
Kev kho ntau yam nrog Rapamycin (Rapa-M) Induces Ntau dua Mitochondria Numbers thiab Tsawg Immunogenic DCsC57BL/6 WT DCs raug cais tawm thiab nthuav tawm rau 8 hnub nyob rau hauv lub xub ntiag ntawm GMCSF thiab lub tsheb (1X PBS) los yog rapamycin (10 ng / mL), tag nrho ntawm peb txoj kev kho mob. DCs tau kho nrog 100 ng / mL LPS thaum hmo ntuj. LPS kho DCs tau siv rau RNASeq lossis sau nrog ntau yam mitochondrial dyes. Cov ntaub ntawv RNASeq tau lees paub cov ntaub ntawv dhau los uas kev kho mob rapamycin induces immunogenic DCs tsawg nrog qis pro-inflammatory cytokines uas tswj innate thiab adaptive immune teb nrog IL10 raws li txoj kev hloov loj (Cov Khoom Siv Ntxiv S1A, B thiab Table S1). Piv nrog rau kev kho tsheb, kev kho mob rapamycin tau nce mitochondrial cov ntsiab lus hauv BMDCs (Daim duab 1A) raws li kuaj nrog MitoTracker CMXRos Liab (50 nM) nrog thiab tsis muaj LPS. Ib yam li ntawd, muaj qhov tseem ceeb dua daim ntawv lo nrog MitoTracker Deep Red (100 nM) thiab tseem ceeb txo qis nrog MitoSox (5 µM) thiab tom qab hmo ntuj LPS stimulation hauv Rapa-M-DC piv rau Veh-DC (Daim duab 1B, C). Semi-quantification ntawm histograms MFI rau tag nrho cov mitochondrial dyes (Daim duab 1D). Tsis tas li ntawd, Veh- thiab Rapa-M-DCs tau staining siv JC-1 los xyuas seb kev kho Rapamycin hloov lub peev xwm ntawm mitochondria. Kev kho ntau yam nrog rapamycin tsis hloov lub hauv paus mitochondria membrane muaj peev xwm piv rau lub tsheb kho BMDCs raws li qhia los ntawm immunoflfluorescence (Cov Khoom Siv Ntxiv S2A) thiab flflow cytometry (Cov Khoom Siv Ntxiv S2B, C).
(Veh) thiab sau npe nrog (A) MitoTracker CMXRos (50 nM) lossis (B) MitoSox (5 µM) lossis (C) MitoTracker Deep Red (100 nM) . (D) Semi-quantifified tsom xam ntawm MFI ntawm mitochondria dyes sau npe flflow cytometry cov ntaub ntawv. (E) DCs tau cog rau hauv Seahorse XF-24e analyzer, txhawb nqa nrog thiab tsis muaj LPS rau 24 teev, thiab cov pa oxygen noj (OCR) tau txiav txim siab thaum cov kev kho mob ua ntu zus nrog oligomycin (1), FCCP (2) thiab Antimycin A / rotenone (3). (F) Quantification ntawm basal OCR thiab ATP ntau lawm. (G) mRNA qib ntawm Pgc1a thiab Tfam hauv Veh-DC thiab Rapa-M-DC kho nrog 100 ng / mL LPS lossis sab laug tsis muaj zog rau 24 teev. (H) Flow cytometry tsom xam ntawm MHC II, co-stimulatory molecules (CD40/80/86) thiab PDL1 qhia tau txiav txim siab nrog thiab tsis muaj LPS stimulation. (I) ELISA ntawm IL10, IL6, thiab TNF los ntawm Veh-DC thiab Rapa-M-DC kho nrog 100 ng / mL LPS rau 24 teev. (J–L) mRNA theem ntawm Il10, Tnfa, thiab Il6 hauv Veh-DC thiab Rapa-M-DC kho nrog 100 ng / mL LPS lossis sab laug tsis muaj zog rau 24 teev. * p Tsawg dua lossis sib npaug rau 0.05, ** p Tsawg dua lossis sib npaug rau 0.01, thiab *** p Tsawg dua lossis sib npaug rau 0.001, ib-txoj kev ANOVA ua raws li Tukey's post-test. Cov ntaub ntawv sawv cev txhais tau tias ± SEM ntawm triplicates.
Hloov kho mitochondrial muaj nuj nqi, bioenergetic tsom xam tau soj ntsuam siv Seahorse Bioanalyzer. Raws li yav dhau los tau tshaj tawm [18], LPS blunted oxygen noj piv rau Veh tswj hauv Veh-DCs raws li xav tau (Daim duab 1E, xiav rau kab dub). Qhov zoo siab, rapa-M DCs nrog lossis tsis muaj LPS muaj ntau dua basal OCR (Daim duab 1E, ntsuab rau xiav kab ntawm lub sijhawm xoom). Rapa-M-DC tau pom qhov ua tsis tiav los ua kom muaj peev xwm ua pa siab tshaj plaws tom qab FCCP txhaj tshuaj hauv cov hlwb tsis muaj zog uas tau tuav nrog LPS stimulation, qhia tias rapamycin ablates lub peev xwm ua pa, raws li peb tau tshaj tawm ua haujlwm nrog FTY-DC [18]. LPS kho DCs tau txo ATP ntau lawm raws li ntsuas los ntawm OCR (xiav rau dub) piv rau Veh-DCs. Rapa-M-DC pom tau tias muaj txiaj ntsig ntau dua ATP production piv rau Veh-DC hauv ob qho tib si tsis muaj zog thiab LPS-stimulated DCs (Daim duab 1F). Rapa-M-DCs tso tawm kom siab mRNA qib rau peroxisome proliferator-activated re ceptor gamma co-activator 1-alpha (Pgc1a) piv rau Veh-DCs (Daim duab 1G) thiab cov lus qhia noob rau Pgc1a thiab mitochondrial transcription factor A (Tfam ) tau khaws cia hauv Rapa-M-DCs tom qab LPS stimulation. Cov ntaub ntawv no qhia tias kev nthuav tawm ntawm BMDCs nyob rau hauv lub xub ntiag ntawm rapamycin nce mitochondrial cov ntsiab lus, basal OCR, thiab ATP ntau lawm.
Txhawm rau kuaj yog tias rapamycin ntxiv rau kev nce mitochondria tus lej thiab kev ua haujlwm kuj ua rau muaj kev hloov pauv hauv DC co-stimulatory molecules tom qab LPS tau sim. Qhov zoo siab, tom qab kev kho LPS, Rapa-M-DC tau txo qis qis ntawm cov lus qhia ntawm co-stimulatory antigen nthuav qhia molecules (CD80, CD86 thiab CD40), MHCII, thiab PDL1. piv rau Veh-DC (Daim duab 1H, xiav vs. liab). Qhov zoo siab, Rapa-M-DCs muaj cov lus qhia zoo sib xws rau PDL1 piv rau Veh-DCs tab sis muaj qhov nce ntxiv hauv PDL1 / CD86 piv tom qab LPS stimulation piv rau LPS kho Veh-DCs qhov twg PDL1 / CD{{ 18}} txo qis (Veh/Veh 0.59 ± 0.03 vs. Veh/LPS 0.43 ± 0.{{33 }}02, p < 0.001="" thiab="" rapa/veh="" 0.56="" ±="" 0.001="" vs="" rapa/lps="" 0.73="" ±="" 0.02,="" p="">< 0.05).="" tsis="" muaj="" kev="" hloov="" pauv="" tseem="" ceeb="" hauv="" cd11c="" qhia="" tau="" pom="" ntawm="" veh-="" thiab="" rapa-m-dcs,="" txawm="" hais="" tias="" rapa-m-dc="" muaj="" qis="" sab="" tawg="" teeb="" liab="" qhia="" qhov="" me="" me="" ntawm="" cov="" hlwb="" tom="" qab="" lps="" stimulation="" (cov="" ntaub="" ntawv="" tsis="" qhia).="" rapa="" m-dcs="" muaj="" cov="" txheeb="" ze="" mtdna="" ndna="" ntau="" dua="" piv="" rau="" veh-dcs="" (105.7="" ±="" 8.7="" vs="" 196.8.9="" ±="" 49.3),="" qhia="" txog="" kev="" kho="" rapamycin="" nce="" dc="" mitochondria="" tus="" lej.="" rapamycin="" txo="" qis="" cov="" noob="" qhia="" ntawm="" lps-induced="" il6,="" il10,="" thiab="" tnfa="" thiab="" protein="" ntau="" ntawm="" tnf="" thiab="" il6="" tab="" sis="" khaws="" cia="" siab="" dua="" il10="" qib="" piv="" rau="" veh-dc="" kho="" nrog="" lps="" (daim="" duab="" 1i-l).="" lwm="" cov="" noob="" uas="" paub="" tias="" tau="" tswj="" hwm="" los="" ntawm="" rapamycin="" (autophagy="" lossis="" o)="" yog="" teev="" nyob="" rau="" hauv="" cov="" khoom="" siv="" ntxiv="" s2.="" cov="" noob="" tom="" qab="" lps="" qhia="" nyob="" rau="" hauv="" ntsuab="" tau="" down-regulated="" thiab="" nyob="" rau="" hauv="" liab="" tau="" up-regulated="" piv="" rau="" cov="" kev="" kho="">
Hloov ntawm Rapa-M-DC Tiv Thaiv raum los ntawm Ischemic InjuryTxhua DCs tau qhib nrog 100 ng / mL LPS ua ntej hloov pauv hauv txhua qhov kev tshawb fawb syngeneic (C57BL / 6 BMDC → C57BL / 6 nas). Ib nrab lab DCs tau txhaj 1 hnub ua ntej 26-min ob sabraumIRI. Schematic ntawm kev kawm tsim (Daim duab 2A). Raws li kev tswj hwm, nas tau txhaj nrog 1 × PBS raws li tsis muaj cell (NC) tswj. Piv rau NC thiab Veh-DC kho cov nas, Rapa-M-DC kho cov nas muaj txiaj ntsig zoo tiv thaiv cov nas.lub raumlos ntawm kev raug mob, plasma creatinine (Daim duab 2B); Cov kev hloov zoo sib xws hauv BUN tau pom hauv cov nas no [Sham 41.30 ± 3.4 vs NC 159.75 ± 3.2 vs Veh-DC 151.82 ± 1.5 vs Rapa-M-DC 114.68 ± 16.3; p <0.05 nc="" or="" veh-dc="" vs="" rapa-m-dc].="" kev="" hloov="" pauv="" ntawm="" morphological="" (daim="" duab="" 2c,="" d)="" kev="" tshawb="" fawb="" txog="" kev="" ua="" haujlwm.="" cov="" nas="" kho="" nrog="" rapa-m-dcs="" muaj="" qis="">raummRNA theem ntawm Kim1 [Veh DC 0.016 ± 0.009 vs Rapa-M-DC {{2{{23} }}}. ± 0.15 vs Rapa-M-DC 0.0005 ± 0.0002] piv rau Veh-DC kho nas. Hauv cov kev tshawb fawb no, thiab raws li tau pom yav dhau los [6,7,17,18], Veh-DC tau kho cov nas ntawm ib koob ntawm ib nrab lab muaj kev hloov pauv hauvlub raum ua haujlwmthiabraumcytokine profile. Hauv kev tshawb fawb syngeneic hloov pauv, nas kho nrog Rapa-M-DCs tau txo qis apoptosis (terminal deoxynucleotidyl transferase-mediated digoxigenin deoxyuridine nick-end labeling [TUNEL]) piv rau cov nas kho nrog Veh-DC (Veh-DC 3.8 ± 1.4 vs Rapa- M-DC 1.1 ± 0.6; p < 0.05);="" cov="" duab="" sawv="" cev="" los="" ntawm="" tunel="" tsom="" xam="" tau="" pom="" hauv="" cov="" khoom="" siv="" ntxiv="" s3.="" interestingly,="" tsis="" muaj="" kev="" hloov="" pauv="" tseem="" ceeb="" ntawm="" tus="" naj="" npawb="" ntawm="" infiltrating="" neutrophils="" hauv="" rapa-m-dc-kho="" nas="" piv="" rau="" veh-dc="" (cov="" ntaub="" ntawv="" tsis="" qhia),="" qhia="" tias="" rapa-m-dc-kho="" nas="" yuav="" tsis="" muaj="" kev="" hloov="" pauv="" hauv="" chemokines,="" txawm="" hais="" tias="" kev="" ua="" haujlwm="" ntawm="" cov="" infifiltrating="" innate="" lub="" cev="" tiv="" thaiv="" kab="" mob="" tuaj="" yeem="" hloov="">
Kev Kho Mob Ib Leeg Nrog Rapamycin (Rapa-S) Induces Less Immunogenic DCs yam tsis hloov Mitochondrial DynamicsPeb tom ntej no sim yog tias ib qho kev kho mob nrog rapamycin txaus los ua kom muaj kev tswj hwm phenotype yog li ua kom muaj peev xwm ua kom muaj kev tswj hwm DCs phenotype ntau qhov chaw kho mob. WT DCs raug cais tawm thiab nthuav tawm rau 8 hnub nyob rau hauv lub xub ntiag ntawm GMCSF, tag nrho ntawm peb txoj kev kho mob. Xya-hnub qub DCs tau kho nrog rapamycin (10 ng/mL) ua ntej kev kho mob nrog 100 ng/mL LPS rau kev tsim tawm hmo ntuj thiab sau hnub tom qab nrog MitoTracker CMXRos Liab (50 nM). Piv rau kev kho tsheb, kev kho mob Rapa-S tsis hloov cov ntsiab lus mitochondrial hauv BMDCs (Daim duab 3A) nrog thiab tsis muaj LPS. Ib yam li ntawd, tsis muaj kev hloov pauv hauv kev sau npe nrog MitoTracker Deep Red (100} nM) thiab MitoSox (5 µM) thiab tom qab hmo ntuj LPS stimulation hauv Rapa-S-DC piv rau Veh-DC (Daim duab 3B, C). Semi-quantification ntawm histograms MFI rau tag nrho cov mitochondrial dyes (Daim duab 3D). Ib yam li ntawd, rau ntau qhov kev ntsuam xyuas kev kho mob, Veh- thiab Rapa-S-DCs tau staining siv ua tiav siv JC{{20}} los xyuas seb ib qho kev kho mob rapamycin hloov pauv mitochondria daim nyias nyias. Kev kho mob ib leeg nrog rapamycin tsis hloov lub hauv paus mitochondria membrane muaj peev xwm piv rau lub tsheb kho BMDCs raws li qhia los ntawm immunoflfluorescence (Cov Khoom Siv Ntxiv S2A) thiab flflow cytometry (Cov Khoom Siv Ntxiv S2B, C). LPS txo qis oxygen noj piv rau Veh tswj hauv Veh-DCs raws li xav tau (Daim duab 3E, xiav rau kab dub). Rapa-S-DCs tsis muaj kev hloov pauv hauv basal OCR (Daim duab 3E, ntsuab rau xiav kab ntawm lub sijhawm xoom). Thaum ATP ntau lawm, LPS txo ATP ntau lawm raws li ntsuas los ntawm OCR (xiav rau dub) hauv Veh-DCs thiab Rapa-S-DC (Daim duab 3F). Txawm li cas los xij, Rapa-S-DCs tau nthuav tawm cov qib siab mRNA rau Pgc1a piv rau Veh tswj, thiab cov qib no tau khaws cia hauv Rapa-S-DCs tom qab LPS (Daim duab 3G), me me rau tsis muaj kev hloov pauv hauv cov ntawv teev lus ntawm Tfam. Tom ntej no, peb sim yog tias ib qho kev kho mob nrog rapamycin ua rau muaj kev hloov pauv hauv DC co-stimulatory molecules tom qab LPS. Interestingly, tom qab LPS kho ment, Rapa-S-DC tau txo qis qhov qhia tawm theem ntawm co-stimulatory antigen nthuav qhia molecules (CD80, CD86, thiab CD40), MHCII, thiab PDL1 piv rau Veh-DC (Daim duab 3H). Rapa-S-DCs muaj cov lus qhia zoo sib xws rau PDL1 piv rau Veh-DCs thiab muaj qhov cuam tshuam zoo sib xws hauv PDL1 / CD86 piv tom qab LPS stimulation piv rau Veh-DCs (Veh/Veh 0). 59 ± 0.03 vs Veh / LPS 0.43 ± 0.002, p < 0.001="" thiab="" rapa-s="" veh="" 0.69="" ±="" 0.02="" vs="" rapa-s="" lps="" 0.56="" ±="" 0.008,="" p="">< 0.05).="" kev="" kho="" ib="" leeg="" nrog="" rapamycin="" txo="" qis="" cov="" noob="" qhia="" ntawm="" lps-induced="" il6,="" il10,="" thiab="" tnfa="" nrog="" rau="" cov="" protein="" ntau="" ntawm="" il10="" nrog="" me="" me="" lossis="" tsis="" muaj="" kev="" hloov="" pauv="" hauv="" tnf="" thiab="" tom="" qab="" kho="" nrog="" lps="" (daim="" duab="" 3i-l).="" lwm="" cov="" noob="" caj="" noob="" ces="" raug="" tswj="" hwm="" los="" ntawm="" rapamycin="" (autophagy="" [beclin,="" atg7,="" atg9,="" lc3b="" thiab="" lamp2]="" los="" yog="" mob="" [il1b,="" il12p40,="" tlr4,="" nos2,="" hif1a="" thiab="" ho1])="" tsuas="" yog="" tswj="" ib="" feem="" piv="" rau="" lub="" tsheb="" dcs="" tom="" qab="" lps.="" khoom="" table="">
Hloov pauv ntawm Rapa-S-DC tiv thaiv raum los ntawm Ischemic InjuryTxhua DCs tau qhib nrog 100 ng / mL LPS lossis kho nrog rapamycin (10 ng / mL) ua ntej hloov pauv hauv txhua qhov kev tshawb fawb syngeneic (C57BL / 6 BMDC → C57BL / 6 nas). Ib nrab lab tus tsov ntxhuav DCs tau txhaj 1 hnub ua ntej ob sabraumIRI. Schematic ntawm kev kawm tsim (Daim duab 4A). Raws li kev tswj hwm, nas tau txhaj nrog 1 × PBS raws li tsis muaj cell (NC) tswj. Piv rau Veh-DC kho cov nas, Rapa-S-DC kho cov nas tseem ceeb tiv thaivlub raumlos ntawm kev raug mob plasma creatinine (Daim duab 4B); Cov kev hloov pauv zoo sib xws hauv BUN tau pom hauv cov nas no [Veh-DC 151.82 ± 1.5 vs Rapa-S-DC 139.53 ± 3.8]. Kev hloov pauv ntawm morphological (Daim duab 4C, D) kev tshawb fawb txog kev ua haujlwm. Cov nas kho nrog Rapa-S-DCs muaj qis duaraummRNA theem ntawm Kim1 [Veh-DC 0. -S-DC 0.004 ± 0.002], Ngal [Veh DC 2.14 ± 1.2 vs Rapa-S-DC 0.14 ± 0.008] and Tnfa [Veh-DC 0.196 ± 0.15 vs Rapa-S-DC 0.0017 ± 0.001] piv rau Veh-DC kho nas.
Allogeneic BMDC Hloov ntawm Rapa-M-DC lossis Rapa-S-DC Sib npaug tiv thaiv raum los ntawm Ischemic InjuryIb nrab lab DCs tau txhaj tshuaj 1 hnub ua ntej ob tograumIRI rau allogeneic (BALB / C BMDC → C57BL / 6 nas) hloov cov kev tshawb fawb. Schematic ntawm kev kawm tsim (Daim duab 5A). Raws li kev tswj hwm, nas tau txhaj nrog 1x PBS raws li tsis muaj xov tooj ntawm tes (NC) tswj. Muab piv rau Veh-DC cov nas kho, Rapa-M-DC, lossis Rapa-S-DC cov nas kho, tiv thaiv cov nas ntau heev.lub raumlos ntawm kev raug mob (Daim duab 5B). Kev hloov pauv ntawm morphological (Daim duab 5C, D) kev tshawb fawb txog kev ua haujlwm. Cov nas uas kho nrog rapamycin DCs muaj qis duaraummRNA theem ntawm Kim1 [Veh-DC 0. 005 ± 0.0{{30}}2 vs Rapa-S-DC 0. }05 ± 0.0002], Ngal [Veh-DC 0.57 ± 0.3 vs RapaM-DC 0.19 ± 0.11 vs Rapa- S-DC 0.05 ± 0.01] thiab Tnfa [Veh-DC 0.037 ± 0.02 vs Rapa-M-DC 0.007 ± 0.003 vs Rapa-S-DC 0.008 ± 0.005] piv rau Veh-DC kho nas. Hauv kev tshawb fawb allogeneic, cov nas kho nrog Rapa-M-DC lossis Rapa-S-DC tau txo qis apoptosis TUNEL piv rau cov nas kho nrog Veh-DC (Veh-DC 16.7 ± 8.2 vs Rapa-M-DC 7.2 ± 2.5 vs Rapa- S-DC 9.1 ± 0.1).
Exogenous Mitochondria Loaded DCs (Mito-DC) nrog siab dua Mitochondrial Dynamics thiab Ntau Immunogenic Phenotype tom qab LPS StimulationWT DCs raug cais tawm thiab nthuav tawm rau 8 hnub nyob rau hauv lub xub ntiag ntawm GMCSF, tag nrho ntawm 3 kev kho mob. Xya-hnub qub DCs raug kho thaum hmo ntuj nrog tib neeg exogenous mito chondria (10 µg/mL). Piv nrog rau kev kho tsheb, mitochondria kho DC Mito-DCs muaj ntau dua mitochondria. Mitochondria raug cais tawm ntawm HEK293 tib neeg kab ntawm tes. BMDCs tau txheeb xyuas qhov muaj tib neeg mitochondria hauv nas WT DCs siv tib neeg mtDNA primers rau NADH dehydrogenase (ND). Cov neeg txheeb ze [mtDNA] cov ntsiab lus hauv Veh-DC yog {{10}}.26 ± 0.10 piv rau 16.8 ± 0.59; p < 0.001,="" nce="" ntawm="" 63-fold="" hauv="" genomic="" dna="" cais="" los="" ntawm="" bmdcs.="" ib="" qho="" kev="" txwv="" ntawm="" qhov="" kev="" ntsuam="" xyuas="" tam="" sim="" no="" yog="" tias="" nws="" ntsuas="" tib="" neeg="" lossis="" moue="" mtdna="" txheeb="" ze="" rau="" nuclear="" dna="" thiab="" tus="" nqi="" me="" me="" ntawm="" tib="" neeg="" mtdna="" kuaj="" pom="" hauv="" veh-dc="" tuaj="" yeem="" yog="" vim="" me="" me="" sib="" tshooj="" ntawm="" nd="" hauv="" pcr="" kev="" soj="" ntsuam="" lossis="" muaj="" peev="" xwm="" txij="" li="" peb="" muaj.="" qhia="" txog="" kev="" txheeb="" ze="" rau="" nas="" nuclear="" dna.="" txhawm="" rau="" txiav="" txim="" siab="" yog="" tias="" cov="" ntsiab="" lus="" mitochondrial="" siab="" dua="" kuj="" hloov="" pauv="" mitochondrial="" muaj="" nuj="" nqi,="" bioenergetic="" tsom="" xam="" tau="" ua="" tiav="" siv="" seahorse="" bioanalyzer.="" lps="" blunted="" oxygen="" noj="" piv="" rau="" veh="" tswj="" hauv="" veh-dcs="" raws="" li="" xav="" tau="" (daim="" duab="" 6a,="" xiav="" rau="" kab="" dub).="" mito-dcs="" muaj="" ntau="" dua="" basal="" ocr="" (daim="" duab="" 6a,="" ntsuab="" rau="" xiav="" kab="" ntawm="" lub="" sijhawm="" xoom).="" thaum="" kho="" nrog="" uncoupler="" fccp,="" mito-dc="" tau="" pom="" tias="" tsis="" ua="" kom="" muaj="" peev="" xwm="" ua="" pa="" siab="" tshaj="" plaws="" hauv="" cov="" hlwb="" uas="" tsis="" muaj="" zog="" uas="" tau="" khaws="" cia="" nrog="" lps="" stimulation,="" qhia="" tias="" kev="" kho="" mob="" nrog="" mitochondria="" ablates="" qhov="" muaj="" peev="" xwm="" ua="" pa="" (tej="" zaum="" vim="" tias="" twb="" nyob="" rau="" qhov="" siab="" tshaj="" ocr="" hauv="" lub="" xeev="" basal).="" thaum="" atp="" ntau="" lawm,="" lps="" txo="" atp="" ntau="" lawm="" raws="" li="" ntsuas="" los="" ntawm="" ocr="" (xiav="" rau="" dub)="" hauv="" veh-dcs.="" mito-dc="" pom="" tau="" tias="" muaj="" txiaj="" ntsig="" ntau="" dua="" basal="" ocr="" thiab="" atp="" ntau="" lawm="" piv="" rau="" veh-dc="" hauv="" lps-stimulated="" dcs="" (daim="" duab="" 6b).="" mito-dcs="" tso="" tawm="" kom="" siab="" mrna="" qib="" rau="" pgc1a="" thiab="" nthuav="" dav="" tfam="" qis="" dua="" piv="" rau="" veh="" tswj="" (daim="" duab="" 6c).="" qhov="" sib="" txawv="" ntawm="" pgc1a="" thiab="" tfam="" nyob="" rau="" hauv="" cov="" txheej="" txheem="" ntawm="" kev="" sim="" tom="" qab="" lps="" stimulations="" piv="" rau="" cov="" ntaub="" ntawv="" nyob="" rau="" hauv="" daim="" duab="" 1="" thiab="" 3="" yog="" tej="" zaum="" vim="" yog="" siv="" balb/c="" bmdcs.="" piv="" rau="" c57bl="" 6="" bmdcs,="" dcs="" los="" ntawm="" balb="" c="" muaj="" kev="" hloov="" pauv="" tseem="" ceeb="" hauv="" mitochondria="" thiab="" cytokine="" ntau="" lawm="" tom="" qab="" stimulation="" [29,30].="" qhov="" ua="" tau="" vim="" yog="" cov="" kev="" hloov="" pauv="" hauv="" dcs,="" keeb="" kwm="" peb="" tau="" siv="" lub="" sijhawm="" ischemic="" ntau="" dua="" hauv="" balb="" c="" nas="" piv="" rau="" c57bl="" 6="" kom="" muaj="" qhov="" zoo="" sib="">raumraug [6,18]. Cov ntaub ntawv no qhia tias kev hloov pauv ntawm exogenous noj qab haus huv mitochondria ua rau muaj zog mitochondrial cov ntsiab lus, basal OCR, thiab ATP ntau lawm. Qhov no qhia txog lub peev xwm rau kev tiv thaiv kab mob phenotype hauv DCs tom qab kev kho mob nrog exogenous mito chondria, raws li qhia hauv peb txoj kev tshawb fawb dhau los [18]. Tom ntej no, peb sim yog tias DCs kho nrog exogenous mitochondria uas ua rau muaj cov lej mitochondrial ntau ntxiv thiab ua haujlwm kuj ua rau muaj kev hloov pauv hauv DC co-stimulatory molecules tom qab LPS. Zoo kawg nkaus, nrog thiab tsis muaj LPS stimulation, Mito-DC muaj qhov qhia tau ntau dua ntawm cov co-stimulatory antigen nthuav qhia molecules (CD80, CD86 thiab CD40), MHCII, thiab PDL1 piv rau Veh-DC (Daim duab 6D). Tsis tas li ntawd, Mito-DCs tau qhia qib siab dua rau PDL1 piv rau Veh-DCs; Txawm li cas los xij, lawv kuj muaj qhov nce hauv PDL1/CD{17}} piv tom qab LPS stimulation piv rau LPS kho Veh-DCs (cov ntaub ntawv tsis qhia). Mito-DC tau muaj qhov cuam tshuam loj heev ntawm cov noob qhia ntawm LPS-induced Tnfa, tab sis ntau dua cov noob qhia ntawm Il6 thiab Il10 piv rau Veh-DC thiab qis dua cov protein ntau ntawm TNF thiab IL10 tab sis khaws cia siab dua IL6 qib piv rau Veh-DC kho nrog LPS (Daim duab 6E. -H). Lwm cov noob uas paub tias tau tswj hwm los ntawm mitochondria (autophagy [Beclin, Atg7, Atg9, Lc3b thiab Lamp2] los yog mob [Il1b, Il12p40, Tlr4, Nos2, Hif1a thiab Ho1]) tau raug tswj ib nrab tom qab LPS stimulation khoom (Tshuaj ntxiv). Veh-DC thiab Mito-DCs tau kho nrog LPS rau 6 thiab 24 teev. Cov txheeb ze hloov pauv hauv mitochondrial complex tau ntsuas. Mito-DC kho nrog thiab tsis muaj LPS muaj qhov tseem ceeb ntawm cov protein ntau ntawm cov mitochondrial complexes IV, II, thiab kuv thiab qis dua theem ntawm Complexes V thiab III piv rau Veh-DCs (Daim duab 6I). Semi-quantitative tsom xam ntawm cov txheeb ze protein qhia ntawm mitochondrial com plexes rau GAPDH (Daim duab 6J). Tag nrho ntev blots yog muab los ua cov duab ntxiv (Cov Khoom Siv Ntxiv S4-S7).
Daim duab 6. Mitochondria loaded DCs (Mito-DC) muaj ntau dua mitochondrial muaj nuj nqi thiab immunogenicity tom qab LPS stimulation piv rau Veh-DCs. Xya-hnub qub Veh-DC thiab tau tsim nrog 10 µg/mL HEK293 mitochondria ib hnub ua ntej kho nrog 100 ng/mL LPS ntxiv 24 teev lossis sab laug tsis muaj zog ( Veh). (A) 24 teev tom qab sib ntxiv ntawm mitochondria, DCs tau cog rau hauv Seahorse XF-24e analyzer, txhawb nrog thiab tsis muaj LPS rau 24 teev, thiab cov pa oxygen noj npaum li cas (OCR) tau txiav txim siab thaum cov kev kho mob ua ntu zus nrog oligomycin (1 ), FCCP (2) thiab antimycin A/rotenone (3). (B) Quantifification ntawm basal OCR thiab ATP ntau lawm. (C) mRNA theem ntawm Pgc1a thiab Tfam hauv Veh-DC thiab Mito-DC kho nrog 100 ng / mL LPS lossis sab laug tsis muaj zog rau 24 teev. (D) Flow cytometry tsom xam ntawm MHCII, co-stimulatory molecules (CD40/80/86) thiab PDL1 qhia tau txiav txim siab nrog thiab tsis muaj LPS stimulation. (E) ELISA ntawm IL10, IL6, thiab TNF los ntawm Veh-DC thiab Mito-DC kho nrog 100 ng / mL LPS rau 24 teev. (F–H) mRNA theem ntawm Tnfa, Il6, thiab Il10 hauv Veh-DC thiab Mito-DC kho nrog 100 ng / mL LPS lossis sab laug tsis muaj zog rau 24 teev. Cov ntaub ntawv sawv cev txhais tau tias ± SEM, * p Tsawg dua lossis sib npaug rau 0.05, ** p Tsawg dua lossis sib npaug rau 0.01, thiab *** p Tsawg dua lossis sib npaug rau 0.001, ib-txoj kev ANOVA ua raws li Tukey's post-test. Cov ntaub ntawv sawv cev txhais tau tias ± SEM ntawm triplicates.
Allogeneic BMDC Hloov ntawm Mito-DC Tiv ThaivLub raumlos ntawm Ischemic Injury Ib nrab ntawm ib lab DCs tau txhaj 1 hnub ua ntej ob sabraumIRI rau Mito-DC allo geneic (BALB / C BMDC → C57BL / 6 nas) hloov cov kev tshawb fawb. Schematic ntawm kev kawm tsim (Daim duab 7A). Raws li kev tswj hwm, nas tau txhaj nrog 1x PBS raws li tsis muaj xov tooj ntawm tes (NC) tswj. Piv nrog rau Veh-DC kho cov nas, Mito-DC kho cov nas tseem ceeb tiv thaiv cov nas.lub raumlos ntawm kev raug mob (Daim duab 7B). Kev hloov pauv ntawm morphological (Daim duab 7C, D) kev tshawb fawb txog kev ua haujlwm. Cov nas kho nrog Mito-DCs muaj qis duaraummRNA theem ntawm Kim1 [Veh-DC 0.04 ± 0.02 vs Mito-DC 0.{14}}2 ± 0.01], Ngal [Veh-DC 0.57 ± 0.3 vs Mito-DC 0.39 ± {{29} }.20] thiab Tnfa [Veh-DC 0.037 ± 0.02 vs Mito-DC 0.0028 ± 0.0013] piv rau Veh-DC kho nas. Hauv kev tshawb fawb allogeneic, cov nas kho nrog Mito-DC tau txo cov apoptosis TUNEL piv rau cov nas kho nrog Veh-DC (Veh-DC 16.7 ± 8.2 vs Mito-DC 3.0 ± 1.2).
Kev Sib Tham Hauv txoj kev tshawb fawb tam sim no peb pom muaj kev tiv thaiv los ntawmraumIRI raug ntxias los ntawm kev hloov pauv ntawm rapamycin-kho DCs (txawm ib zaug lossis ntau zaus) hloov kho DC kev ua haujlwm uas yog nyob ntawm cov txheeb ze hloov pauv hauv mitochondria dynamics. Ob leeg ib leeg thiab ntau yam kev kho mob nrog rapamycin induces txo immunogenic (qis cytokines thiab co-stimulatory molecules) DCs. Hloov ntawm Rapa-M-DCs lossis Rapa-S-DCs tiv thaivlub raumlos ntawm ischemic reperfusion raug mob, qhia qhov txo qis immunogenic xeev ntawm rapamycin hloov DCs induces kev tiv thaiv. Tsis tas li ntawd, kev hloov pauv ntawm Veh-DC uas muaj kev nce ntxiv hauv mitochondria tus lej kuj tseem tiv thaiv.lub raumlos ntawm IRI (Daim duab 8A–D)). Los ntawm cov kev tshawb fawb no peb tuaj yeem txiav txim siab tias kev txhaj tshuaj tsawg dua immunogenic DCs yog lub hauv paus rau kev txhawb nqa kev tiv thaiv los ntawm IRI cov txheej txheem uas tuaj yeem ua tiav los ntawm kev kho tshuaj (rapamycin) lossis hloov pauv ntawm kev noj qab haus huv exogenous mitochondria. Raws li yav dhau los tau tshaj tawm [31] thiab tau lees paub hauv peb cov kev tshawb fawb RNAseq, tag nrho, tsis muaj qhov tseeb tolerogenic kos npe cuam tshuam nrog rapamycin-modulated DCs. Txawm hais tias rapamycin DCs qhia qee qhov kev hloov pauv tseem ceeb hauv cov noob tom qab LPS stimulation piv rau lub tsheb DCs, cov no tau txwv rau RapaM-DCs uas muaj qis cytokine-mediated signaling thiab cytokine ntau lawm (TNF-signaling ntawm NF-kB thiab IFN ntau lawm) txhua yam tseem ceeb hauv ob qho tib si. innate thiab adaptive immune teb. Raws li yav dhau los tau tshaj tawm thiab tau lees paub hauv peb txoj kev tshawb fawb, IL-10 kev taw qhia tau pom tias yog txoj hauv kev tseem ceeb uas txawv hauv rapamycin-kho DCs nrog FDR ntawm 5.87 × 10 4 (Cov Khoom Siv Ntxiv S1), lub cim cytokine ntawm tolerogenic kos npe hauv DCs.
Keeb kwm, tolerogenic DCs (Tol-DC) raug txheeb xyuas raws li kev loj hlob tsis zoo lossis ib nrab tsis paub qab hau nrog kev qhia qis ntawm co-stimulatory (CD80, CD86, CD40) nrog nce IL10 ntau lawm nrog qis IL12 thiab IFN secretion. , lub peev xwm qis dua rau prime T hlwb thiab muaj peev xwm induce Treg tom qab proinflammatory stimulation [32,33]. Kev tswj hwm lossis tolerogenic DCs tuaj yeem sib txawv hauv vitro los ntawm kev siv cov tshuaj tiv thaiv kab mob lossis cov tshuaj sib xyaw xws li FTY720 [18] lossis rapamycin (txoj kev tshawb fawb tam sim no), lossis tshuaj tiv thaiv kab mob cytokines xws li IL10 [34] lossis los ntawm kev hloov kho caj ces.
Lub luag haujlwm ntawm Dendritic Cells nyob rau hauv mob raum mob (AKI)Txawm hais tias cov tshuaj kho mob tseem ceeb tau ua rau kev tiv thaiv lossis kev kho mob ntau yam kev raug mob tsis muaj rau AKI, yog li nws tseem yog lub nra hnyav rau kev noj qab haus huv [35]. Tsis tas li ntawd, txhawm rau tiv thaiv lossis txo cov kev tiv thaiv kev tiv thaiv kab mob sib kis los ntawm kev tsis lees paub lossis kab mob autoimmune siv cov tshuaj tiv thaiv kab mob tshwj xeeb yog cuam tshuam nrog cov kev mob tshwm sim tsis zoo. Yog li, kev siv lub cev tiv thaiv kab mob xws li DCs uas tuaj yeem ua lub hom phiaj hauv lub cev thiab hloov lub cev tiv thaiv kab mob yog qhov zoo. Tsis tas li ntawd, DCs yog cov hlwb tseem ceeb hauv kev tiv thaiv kab mob lossis kev ua siab ntev, thiab lub tswv yim ntawm kev siv ex-vivo kev tswj hwm lossis kev zam rau DCs hauv kev kho mob ntawm tes rau kev mob qog noj ntshav, autoimmune disorders thiab hloov pauv tau raug tshawb xyuas [36]. Pharmacological (FTY720 [18]) los yog cov tswv yim lom neeg (kev hloov kho caj ces) ua rau muaj kev tswj hwm lossis tolerogenic DCs (Tol-DC) [37], uas yog cov maturation resistant los yog muaj kev loj hlob qis los yog hloov pauv uas thaum kawg qhia cov qib qis ntawm MHC I. los yog MHC II thiab co-stimulatory molecules xws li CD40, CD80, thiab CD86. Siv CD11c-DTR transgenic nas peb tau luam tawm yav dhau los cov kev tshawb fawb uas pom tau tias depletion ntawm DCs nrog diphtheria toxin tseem ceeb tiv thaiv nas.lub raumlos ntawm ischemia reperfusion raug mob (IRI) [7,38], thiab qhov hloov pauv ntawm koob tshuaj nce ntxiv hauv DC cov lej ua rau hnyav dua.mob raum[7], qhia tias DCs tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev tswj hwm AKI. Nws yog ib qho tsim nyog hais tias hauv AKI, tus qauv ntawm AKI uas tau siv (kev siv tshuaj khomob siv cisplatin lossis ischemia) dictates lub luag haujlwm DCs ua si los hloov cov tshuaj tiv thaiv kab mob. Hauv cisplatin induced AKI, DC ablation los ntawm kev siv cov nas transgenic los yog ntawm liposomal chlodronate txhaj ua rau muaj kev raug mob ntau dua li IL-10 tsim los ntawm DCs hauv cov qauv no yog qhov kawg tsim nyog rau dampen nephrotoxic raug mob [39,40].
Rapamycin: Inflammation, Immune Cells, Ischemia Reperfusion Injury (IRI)Cov kev tshawb fawb luam tawm yav dhau los tau qhia txog kev tiv thaiv lub luag haujlwm ntawm rapamycin hauv AKI qauv siv IRI cov kev tshawb fawb uas koom nrog tswj NKT cell infifiltration thiab ua haujlwm li no ameliorating.mob raum[41], hauv txoj kev tshawb no nas tau kho qhov ncauj peb zaug nrog rapamycin 24 h, 1 h ua ntej ischemia thiab 12 h tom qab ischemia. Kev noj cov nas nyob hauv qhov ncauj ib hnub ua ntejraumIRI thiab txhua hnub tom qab (txog li 7 hnub) ua rau ntau duamob raumvim inhibition ntawmlub raumtubular cell proliferation ntawm 1 thiab 3 hnub tom qab ischemia [42]. Qhov no tau tshwm sim raws li nyob rau hauv tus qauv nas ntawm unilateral ureteral obstruction (UUO), qhov twg rapamycin kho amelioratedraumfifibrosis los ntawm ncaj qha inhibiting mTOR signaling hauv myofifibroblasts thiab interstitial macrophages [43]. Cov kev tshawb fawb no qhia tias rapamycin tuaj yeem muaj qhov cuam tshuam tsis zoo hauv ntau yam mob hnyav thiab mob ntevraumqauv. Txawm li cas los xij, ntau yam kev tshawb fawb tau qhia txog lub luag haujlwm tsis zoo rau rapamycin hauv cov qauv tsiaj ntawm IRI. Kev txhaj tshuaj ntawm qhov ncauj nrog rapamycin (4 mg / hnub) rau 7 hnub hauv Yorkshire npua ua rau muaj kev cuam tshuam ntawm endothelial-dependent vasorelaxation thiab nce myocardial necrosis hauv tus qauv ntawm cov hlab ntsha occlusion [44]. Txawm li cas los xij, hauv cov nas, kev kho mob hnyav nrog rapamycin (ib teev ua ntej ischemia, txhaj tshuaj intraperitoneal) ua rau muaj kev tiv thaiv cardio los ntawm kev tswj hwm JAK2-STAT3 teeb liab los tiv thaiv myocardial infarction [45]. Nyob ntawm txoj kev (gavage lossis subcutaneous lossis intraperitoneal), koob tshuaj (0.1–6 mg/kg/hnub), lub sijhawm (thaum ntxov lossis ncua sijhawm) [46], thiab hom qauv siv (lub siab [47] , cev [48] orraum[41,49]), rapamycin tau pom tias muaj kev tiv thaiv [47] lossis tsis tiv thaiv [50].
Cell Therapy thiab RapamycinKev nthuav tawm ntawm nas BMDCs lossis tib neeg CD14 ntxiv rau cov monocytes uas peb xav tias mob ntev ua rau muaj lwm txoj kev tiv thaiv lossis kev tswj hwm phenotype hauv DCs uas tswj hwm ob qho tib si hauv lub cev thiab hloov lub cev tiv thaiv kab mob. Hauv cov kab mob ua rau tuag taus-tiv thaiv tus tswv tsev, DCs tsim nrog rapamycin tau txo qis MHC II thiab co-stimulatory molecules tab sis muaj cov pej xeem ntau dua ntawm IL12 ua tom qab LPS stimulation. Cov IL12 no tsim Rapa-M-DC txhim kho alloreactive T cell apoptosis, ib qho kev koom tes nrog IFN [51]. Ib yam li ntawd, CD14 ntxiv rau monocytes propagated nyob rau hauv lub xub ntiag ntawm rapamycin kuj ua tau siab dua IL12p70 nrog rau IL27 qib uas tswj NK cell muaj nuj nqi, yog li hloov allogeneic T cell teb los ntawm IFN [52]. Hauvraum,Kev hloov pauv ntawm rapamycin-kho Tregs tau txo qis T cell, myeloid cell, thiab myofifibroblast cov lus teb, ua rau muaj kev txhim kho mob thiab mob ntev.mob raumpiv rau kev kho cov nas ncaj qha nrog Rapamycin [53]. Ib yam li ntawd, kev hloov pauv hloov ntawm rapamycin-kho myeloid-derived suppressor cells (MDSCs) rau hauv cov nas tau txhim kho.lub raum ua haujlwmnrog txo qis histological puas tsuaj, thiab tiv thaiv kab mob hauv lub cev [54]. Cov kev tshawb fawb no muab pov thawj tias kev kho mob ntawm tes uas cuam tshuam nrog kev kho cov hlwb nrog rapamycin ua ntej txhaj tshuaj tiv thaiv kev mob tshwm sim uas yuav cuam tshuam nrog kev kho mob rapamycin. Tsis tas li ntawd, peb tau sim siv cov metformin lossis cov tshuaj sib xyaw xws li 5-aminoimidazole-4-carboxamide-1- -4- ribofuranoside (AICAR) los txhawb kev tswj hwm phenotype hauv nas DCs los ntsuas lawv lub luag haujlwm hauv kev hloov pauv.raumIRI. Zoo li lub tswv yim noj tshuaj siv nrog rapamycin (Rapa-S-DC), peb tau kho BMDCs nrog cov tshuaj metformin tsawg (1 mM) lossis AICAR (1 mM) hnub 7 nrog rau LPS. Peb tau txhaj tshuaj 0.5 x 106 ntawm metformin-DCs lossis AICAR-DCs ib hnub ua ntejraumIRI. Cov nas uas tau kho nrog metformin-DCs lossis AICAR-DCs tau tiv thaiv qhov tseem ceeb piv rau NC lossis Veh-DCs kho cov nas (tsis tau tshaj tawm kev soj ntsuam, Bajwa lab). Yog li, ntau yam tshuaj lossis cov tshuaj sib xyaw ua ke uas ua rau muaj kev hloov pauv hauv metabolic lossis qhib txoj hauv kev AMPK yuav tsim nyog kuaj los ntawm ib leeg lossis ua ke. Nws yog ib qho tsim nyog sau cia tias kev siv ntau cov tshuaj sib xyaw ua ke lossis cov tshuaj sib xyaw ua ke (metformin lossis AICAR) uas tuaj yeem ua kom muaj txiaj ntsig ntxiv rau kev tiv thaiv cov teebmeem ntawm rapamycin tseem tsis tau tshawb nrhiav thaum ntsuas lawv siv hauv BMDCs. Hauv cov qog nqaij hlav cancer, kev sib txuas ntawm AICAR nrog rapamycin txhim kho qhov ua tau zoo ntawm rapamycin kom ntxiv dag zog rau mTORC2 kom induce apoptosis [55]. Hauv T hlwb, kev sib xyaw ua ke ntawm metformin thiab rapamycin kuj tau raug kuaj raws li lub peev xwm metabolic checkpoint regulator los cuam tshuam T cell function [56]. Yog li, cov kev kho mob sib xyaw ua ke no yuav tsum tau sim yav tom ntej nrog tolerogenic lossis kev tswj hwm BMDCs uas muaj peev xwm ncaj qha lossis tsis ncaj ntawm cov chaw kuaj mob metabolic.
Rapamycin: Lub luag haujlwm ntawm Mitochondria hauv Dendritic Cells thiab MacrophagesDC thiab macrophage ua haujlwm yog tswj hwm los ntawm mitochondrial metabolism. Hom 1 macrophages [57,58] thiab immunogenic DCs (TLR-mediated activation) muaj kev hloov pauv hauv metabolic uas mitochondrial oxidative phosphorylation yog inhibited los ntawm endogenously synthesized nitric oxide (NO) thiab cog lus rau siab aerobic glycolytic tus nqi [59]. Kev ua kom DCs lossis macrophages los ntawm ob peb TLR agonists (LPS lossis CpG) ua rau muaj kev nce ntxiv hauv glycolysis, ua raws li kev txo qis hauv OXPHOS thiab mitochondrial membrane peev xwm [59–61]. Qee lub luag haujlwm rau mitochondria tau pom nrog DCs kho hauv vitro nrog vitamin D; Cov DCs no tau nce OXPHOS, mitochondrial mass, thiab mROS ntau lawm [62], peb txoj kev tshawb fawb tsis ntev los no siv FTY720 [18] pom cov kev hloov pauv zoo sib xws hauv DC (siab dua OXPHOS thiab tsawg dua immunogenicity), kev kho mob hnyav nrog rapamycin tau pom tias txuas ntxiv lub cellular lifespan. DCs uas yog nyob ntawm kev khaws cia ntawm mitochondrial muaj nuj nqi [10] los ntawm inhibition ntawm NO. Raws li kev ua kom lub neej ntev ntawm DCs, tib pab pawg kuj tau tshaj tawm tias qhov mob hnyav lossis luv luv rau rapamycin hauv DCs kuj ua rau lawv muaj zog activators ntawm antigen tshwj xeeb CD8 ntxiv rau T hlwb, yog li txhim kho kev tswj ntawm B16 melanoma raws li kev txhaj tshuaj hauv nas [63]. Kev kho mob hnyav (90 min) ntawm tib neeg monocyte muab los ntawm DCs nrog rapamycin suppresses LPS induced immunostimulatory molecules thiab allo-stimulatory peev xwm uas koom nrog qhov tsis muaj peev xwm kho DCS los txhawb NF-kB signaling [64]. Hauv macrophages, rapamycin inhibits kev tsim tawm ntawm IL1 thiab IL -18Tom qab LPS stimulation, ib txheej txheem uas tseem yuav txo tau mitochondrial ROS (mtROS) los ntawm kev ncaj qha inhibiting NLRP3 inflammasome-p38 MAPK-NF-kB txoj kev [65]. rapamycin conditioned DC (Rapa-DC) los ntawm cov nas thiab tib neeg coj txawv txawv, piv rau nas Rapa-DC, tib neeg Rapa-DC tau ua pov thawj tias nws yog ib feem resistant rau maturation tom qab proin-fllammatory cytokines thiab tso saib heterogeneity hauv regulating effector T-cell expansion thiab ua haujlwm [66]. Ntau ntawm cov tshuaj tiv thaiv kab mob ntawm rapamycin-conditioned monocyte-derived DCs thiab lawv lub luag haujlwm hauv kev hloov pauv tau raug tshuaj xyuas zoo los ntawm Macedo li al. [67].
Peb qhov kev tshawb pom tam sim no qhia tau hais tias ib leeg lossis ntau yam rapamycin kho DCs muaj qhov txo qis ntawm immunogenic thiab immunostimulatory phenotype tom qab LPS stimulation. Kev hloov ntawm DCs no tiv thaivlub raumlos ntawm IRI. Rapamycin hloov pauv mitochondria dynamics; Tsuas yog ntau yam kev kho mob ua rau muaj tus lej mitochondria ntau dua. Cov ntaub ntawv no qhia tias DC mitochondrial dynamics (siab dua mitochondria cov ntsiab lus), nrog rau tsawg immunogenic phenotype induced los ntawm rapamycin, koom tes tiv thaiv.lub raumlos ntawm yog chemic raug mob. Txawm hais tias nthuav ntau dua, peb cov ntaub ntawv nrog exogenous mitochondria kho DCs qhia tias ntau dua mitochondria tus lej tuaj yeem kov yeej ntau lub xeev immunogenic ntawm DCs tom qab LPS stimulation los tiv thaiv.lub raumlos ntawm IRI. Nws yuav nthuav kom paub meej yog tias cov lej mitochondria siab dua hauv DCs hloov T cell teb, DCs muaj peev xwm hloov lub cev tiv thaiv kab mob raws li peb txoj kev tshawb fawb yav dhau los tau qhia. Peb cov ntaub ntawv ua ntej qhia tau hais tias, nyob rau hauv allogenic T cell proliferation kev tshawb fawb, DCs nrog ntau dua mitochondria tooj ua suppress CD4 T cell proliferation nyob rau hauv mixed lymphocyte tshua (MLR, cov ntaub ntawv tsis qhia). Tam sim no tsis paub meej yog tias DCs nrog cov lej mitochondrial siab dua tuaj yeem ua rau Treg cov lus teb uas tuaj yeem pab txhawb kev tiv thaiv.lub raumlos ntawm IRI. Cov no yuav yog lub hom phiaj ntawm peb cov kev tshawb fawb tom ntej.
Cov lus xaus Hauv cov ntsiab lus, peb tau pom tias BMDCs tuaj yeem tswj hwmmob raumlos ntawm ib tug mech anism uas koom nrog mitochondria biogenesis los ntawm ntau yam kev kho mob nrog rapamycin los yog exogenously nce mitochondria tooj los ntawm kev siv exogenous mitochondria. Peb xaus lus tias kev tswj hwm DCs (DCs nrog cov lej mitochondria siab dua) yuav muaj txiaj ntsig zooraumIRI zoo li hauv lwm lub xeev o xws li kev hloov pauv thiab cov kab mob autoimmune los tiv thaiv lossis kho kev raug mob.
