Tib neeg Amniotic Mesenchymal Stem Cells Alleviate AGVHD Tom Qab Allo-HSCT Los Ntawm Kev Tswj Kev Sib Raug Zoo ntawm Gut Microbiota Thiab Kev Tiv Thaiv Kab Mob

Abstract

Mob graft-tiv thaiv tus tswv tsev kab mob (aGVHD) tom qab allogeneic hematopoietic qia cell transplantation ua rau ib qho kev nyuaj siab tshaj plaws. Lub plab microbiota dysbiosis tuaj yeem txuas ntxiv nrog aGVHD thiab mesenchymal qia hlwb (MSCs) muaj peev xwm kho tau zoo rau aGVHD. Txawm li cas los xij, seb hAMSCs puas cuam tshuam rau lub plab microbiota thaum lub sij hawm aGVHD mitigation tseem tsis paub. Raws li, peb tau nrhiav los txhais cov teebmeem thiab cov txheej txheem hauv qab ntawm tib neeg amniotic membrane-derived MSCs (hAMSCs) tswj lub plab microbiota thiab plab hnyuv tiv thaiv hauv aGVHD. Los ntawm kev tsim humanized aGVHD nas qauv thiab kev kho mob hAMSCs, peb pom tias hAMSCs tau kho cov tsos mob ntawm aGVHD, thim rov qab lub cev tsis sib haum xeeb ntawm T cell subsets thiab cytokines, thiab rov ua kom lub plab zom mov. Tsis tas li ntawd, qhov sib txawv thiab muaj pes tsawg leeg ntawm plab microbiota tau txhim kho thaum kho nrog hAMSCs. Spearman qhov kev txheeb xyuas kev sib raug zoo tau pom tias muaj kev sib raug zoo ntawm lub plab microbiota thiab cov nqaij sib txuas nruj nreem, lub cev tiv thaiv kab mob thiab cytokines. Peb cov kev tshawb fawb tau pom tias hAMSCs txo qis aGVHD los ntawm kev txhawb lub plab microbiota normalization thiab tswj kev cuam tshuam ntawm plab microbiota thiab plab hnyuv teeb meem, tiv thaiv kab mob.

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Ntsiab lus

Mob graft-versus-host disease · Amniotic mesenchymal stem cells · Gut microbiota · Intestinal barrier · Intestinal immunity

Taw qhia

Lub hom phiaj tseem ceeb ntawm allogeneic hematopoietic qia cell transplantation (allo-HSCT) yog lub zog tiv thaiv qog cov lus teb vim yog graft-vsus-leukemia (GVL) cov nyhuv uas yog tshwm sim los ntawm kev lees paub ntawm peptides tshwm sim los ntawm qhov sib txawv ntawm genomic polymorphisms ntawm tus neeg mob thiab pub. Txawm li cas los xij, tus kab mob kis mob hnyav-tiv thaiv tus tswv tsev (aGVHD) yog nyob ib puag ncig kev txhim kho ntawm cov neeg tau txais cov ntaub so ntswg puas tsuaj vim qhov kev tawm tsam los ntawm allo-reactive pub dawb T hlwb thiab sawv cev rau qhov teeb meem loj loj hauv alloHSCT [1, 2]. Cov kab mob plab microbial tau raug tshaj tawm los ua rau cov lus teb inflammatory thiab sib cuam tshuam nrog cov kab mob sib kis [3, 4]. Ntau cov kev tshawb fawb tau qhia tias muaj kev cuam tshuam hauv plab microbiota yog qhov tseem ceeb ua rau aGVHD tom qab allo-HSCT, thiab dhau los ua lub hom phiaj tshiab rau kev kho mob [5, 6]. Tshwj xeeb, plab microbiota dysbiosis hauv aGVHD feem ntau yog tshwm sim los ntawm kev poob ntawm cov kab mob hauv plab hnyuv thiab qhov kev loj hlob ntawm cov kab mob kis tau zoo [6]. Hauv plab hnyuv GVHD, qhov cuam tshuam mucosal barrier pib ua kom cov tswv tsev antigen-tam sim no hlwb thiab pub T hlwb ua rau T-cell sib txawv raws li hom kab mob -1 thiab hom -17 paradigms ntawm tus nqi ntawm tolerogenic regulatory T- cell qauv [7]. Feem ntau ntawm lub hauv paus ntawm 16S ribosomal RNA (16S rRNA) sequencing qhia tias ntau hom kab mob plab microbiota tau txuam nrog GVHD, thiab piv txwv li, butyrate-ua clostridia yog txuam nrog kev saib xyuas ntawm epithelial barrier muaj nuj nqi thiab attenuation ntawm mob GVHD [8] ]. Mesenchymal qia hlwb (MSCs) tau txais kev saib xyuas ntau nyob rau hauv kaum xyoo dhau los vim lawv tus kheej rov ua dua tshiab thiab muaj peev xwm sib txawv ntawm ntau hom, nrog rau cov khoom siv tshuaj tiv thaiv kab mob [9]. MSCs tau xav tias yuav ua rau cov ntaub so ntswg puas los ntawm kev sib npaug ntawm cov kab mob thiab cov kab mob rov ua dua tshiab nyob rau hauv lub xub ntiag ntawm qhov mob hnyav thiab tau siv dav los kho cov kab mob tiv thaiv kab mob, uas cov ntawv thov kev kho mob tau zoo tshaj plaws yog koom nrog hauv kev kho GVHD [10]. Tib neeg amniotic mesenchymal qia hlwb (hAMSCs) yog cov kab mob perinatal uas muaj embryonic qia cell zoo li kev sib txawv muaj peev xwm thiab cov neeg laus qia cell zoo li immunomodulatory zog. Sib koom phenotypes zoo ib yam li MSCs ib txwm, hAMSCs tau dhau los ua qhov muaj txiaj ntsig ntawm cov qia hlwb vim qhov yooj yim, tsis muaj kev cuam tshuam, thiab kev nyab xeeb ntawm cov ntaub so ntswg tau, muaj ntau cov cell yield, thiab tsis tshua muaj kev ncaj ncees thiab kev ncaj ncees tsis sib haum xeeb piv nrog cov qia hlwb los ntawm lwm qhov chaw. Tib neeg AMSCs muaj qhov zoo ntawm kev tiv thaiv kab mob qis thiab tsis muaj qog nqaij hlav, ua rau lawv qhov chaw zoo tshaj plaws rau kev kho ntawm tes [11, 12]. Kev sim hauv vitro tau lees paub tias hAMSCs tau nthuav tawm lub peev xwm loj dua thiab muaj peev xwm loj hlob mus ntev dua li cov pob txha pob txha-derived MSCs [13, 14]. Ntau cov kev tshawb fawb tau pom tias hAMSCs tuav lub peev xwm los kho ntau yam kab mob, nrog rau cov kab mob plab hnyuv, osteoarthritis, thiab kab mob autoimmune [11]. Peb txoj kev tshawb fawb tsis ntev los no tau pom tias hAMSCs ameliorated aGVHD los ntawm kev tswj qhov sib npaug ntawm T effector thiab Treg hlwb [15]. Txawm li cas los xij, cov nyhuv thiab cov txheej txheem ntawm hAMSCs ntawm lub plab microbial teeb meem hauv GVHD tsis paub. Ntawm no, peb tsom mus tshawb xyuas qhov muaj feem cuam tshuam ntawm hAMSCs ntawm aGVHD nrog rau lub plab microecosystem. Nws yuav yog thawj txoj kev tshawb fawb los nthuav qhia lub plab microbiota thiab nws txoj kev sib raug zoo nrog cov hnyuv tiv thaiv kab mob tom qab hAMSCs kev kho mob ntawm aGVHD.

Cov ntaub ntawv thiab cov txheej txheem

Kev cais tawm, kab lis kev cai, thiab kev txheeb xyuas ntawm hAMSCs

Cov hAMSCs tau muab rho tawm raws li tau piav qhia dhau los [15]. Lub hlwb thib peb (P3) tau sau los ntawm kev txheeb xyuas phenotype los ntawm staining nrog cov tshuaj tiv thaiv kab mob CD34 (581, BD Pharmingen, USA), CD45 (HI30, BD Pharmingen, USA), HLA-DR (G46-6, BD Pharmingen . BD FACS Canto II). Cov hlwb raug kuaj xyuas lawv cov kev sib txawv ntawm cov kab sib txawv uas siv qhov nruab nrab induction (BGscience, Tuam Tshoj), thiab stained nrog roj Red-O thiab Alizarin Liab rau adipogenesis thiab osteogenic sib txawv, raws li. hAMSCs hauv nqe 3 txog 6 tau siv rau kev sim.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Human PBMCs Collection

Cov qauv ntshav plasma tau txais los ntawm cov neeg ua haujlwm pabcuam noj qab haus huv nrog sau ntawv tso cai. Tib neeg peripheral ntshav mononuclear hlwb (PBMCs) raug cais tawm ntawm cov ntshav peripheral los ntawm Ficoll-Hypaque (Tianjin Haoyang, Tuam Tshoj) ceev centrifugation, ntxuav nrog PBS, tshem tawm hauv cov ntshav liab lysis tsis (Solarbio, Tuam Tshoj) ntawm 4 degree rau 15 min. Ntxuav dua thiab muab tshem tawm hauv PBS rau kev txhaj tshuaj rau hauv NPG nas.

Cov nas

8-10 lub lis piam ntawm hnub nyoog thiab 25-30 g txiv neej thiab poj niam NPG nas tau yuav los ntawm Beijing Vitalstar Biotechnology Co., Ltd. thiab rov kho dua nyob rau hauv SPF cov xwm txheej ntawm 12 h lub teeb-tooj ​​tsaus ntuj nrog kub thiab av noo. Tag nrho cov txheej txheem tsiaj tau pom zoo los ntawm Southern Medical University Institutional Animal Care and Use Committee (No. L2019132) raws li National Health and Medical Research Council of China Guidelines on Animal Experimentation.

Mob GVHD Tsiaj Qauv thiab Kev Kho Mob

Tus qauv aGVHD nas tau tsim raws li tau piav qhia yav dhau los [15]. Rau kev kho mob, 5 × 105 hAMSCs rau ib tus nas (ntawm no hAMSCs pawg) thiab PBS (ntawm no aGVHD pab pawg) tau txhaj ntawm tus Tsov tus tw leeg rau hauv txhua tus nas nyob rau peb hnub tom qab hloov pauv. Hauv txhua tus qauv nas, nas raug kuaj txhua ob hnub rau kev mob hnyav thiab qhov hnyav hloov. Txhua tus nas tau qhab nia rau cov yam ntxwv xws li kev poob phaus, hunched posture, ruffled fur, daim tawv nqaij mob, txo kev txav mus los, thiab raws plab thiab tom qab ntawd muab qhab nia raws li aGVHD cov qhab nia kho mob tau yoog los ntawm ib qho uas tau piav qhia los ntawm Cooke [16]. Qee qhov xwm txheej, cov nas hauv pawg aGVHD tau raug kev txom nyem los ntawm aGVHD hnyav, thiab cov nas nyob hauv pawg hAMSCs nyob rau hnub thib peb tom qab kev kho mob tau txi rau cov ntshav thiab lub hom phiaj ntawm lub cev nrog rau lub siab, lub ntsws, lub ntsws, thiab cov hnyuv. Cov quav ntawm cov nas no tau sau ua ntej kev txi.

GFP-labeled hAMSCs thiab hauv vivo Tracing

GFP-labeled hAMSCs tau tsim thiab txheeb xyuas raws li tau piav qhia yav dhau los [15]. Hauv luv luv, peb tau hloov pauv cov fluorescence protein (GFP) noob rau hauv hAMSCs nrog lentivirus ua vector. 5 × 105 GFP-labeled hAMSCs raug tshem tawm hauv 500 μL PBS tau txhaj rau hauv aGVHD nas ntawm tus Tsov tus tw leeg. Tom qab 24 teev thiab 72 teev, cov nas tau euthanized los sau cov hnyuv thiab tom qab ntawd tsim cov seem khov. DAPI counterstaining tau siv los paub qhov txawv ntawm cov neeg tau txais cov hlwb.

Kev ntsuam xyuas Histopathological

Lub hom phiaj ntawm aGVHD tau sau thaum lub sijhawm necropsy thiab tom qab ntawd ua tiav rau paraffin-embedded blocks los tsim 5-μm-thick seem rau hematoxylin thiab eosin staining (H&E). Cov qhab nia histological tau soj ntsuam raws li cov ntaub so ntswg puas tsuaj thiab lymphocyte infiltration [17]. Rau immunohistochemistry, ntu tau deparafnized, rehydrated, thiab kho nrog 3% H2O2 hauv methanol rau 20 min kom inactivate endogenous peroxidase kev ua. Tom qab ntawd cov ntu tau raug rau cov tshuaj tiv thaiv antigen thiab kho nrog serum albumin rau 1 teev. Tom qab ntawd, cov seem tau tsim nrog luav los tiv thaiv tib neeg CD45 cov tshuaj tiv thaiv kab mob (EP322Y, Abcam, England) lossis ZO-1 cov tshuaj tiv thaiv ((EPR19945-296, Abcam, England) rau 1 h ntawm 37 degree . Cov tshuaj tiv thaiv tau kuaj pom nrog HRP-conjugated Secondary antibody (Genentech, USA) thiab pom nrog DAB.

cistanche tsob nroj-nce kev tiv thaiv kab mob

16S rRNA Sequencing ntawm Fecal Microbiota

Fecal genomic DNA tau muab rho tawm los ntawm 0.1 g khov fecal kuaj siv EZNA® quav DNA Kit (Omega BioTek, Norcross, GA, US) raws li cov chaw tsim khoom raws tu qauv. Siv genomic DNA ua tus qauv, tshwj xeeb primers (338F: 5'-ACTCCTACGGGAGGCAGCA-3', thiab 806R: 5'-GGACTACHVGGGTWTCTAAT-3') nrog barcodes thiab PremixTaq (TaKaRa, Tuam Tshoj) tau siv los ua kom nrov lub V3-V4 hypervariable cheeb tsam ntawm cov kab mob 16S rRNA noob. Tom qab amplification thiab purification, tag nrho cov genome ntawm cov qauv yog sequenced ntawm lub Illumina Hiseq los yog Miseq high-throughput sequencing platform los ntawm Magigene Technology Co., Ltd. (Guangzhou, Suav teb) kom tau txais cov ntaub ntawv raw hauv FASTQ hom. Kev soj ntsuam cov ntaub ntawv ib ntus tau ua tiav siv Magichand platform.

Flow Cytometric Analysis

Lub aGVHD lub hom phiaj lub cev thiab cov ntshav peripheral ntawm cov nas tau txais thiab npaj rau hauv kev ncua ib leeg. Cells tau stained nrog monoclonal antibodies tiv thaiv CD3 (UCHT1, Biolegend, USA), CD4 (RPA-T4, Invitrogen, USA), CD8 (RPA-T8, Biolegend, USA), CD25 (BC96, Biolegend, USA) los yog isotype-matched tswj IgG (eBioscience, USA) rau 30 min ntawm chav tsev kub hauv qhov tsaus ntuj. Tom qab ntawd cov hlwb tau ua tiav nrog Lysing Buffer (BD Pharm Lyse™) ntawm 4 degree hauv qhov tsaus ntuj rau 15 feeb. Intracellular Foxp3 (236A / E7, Invitrogen, USA) staining tau ua raws li cov chaw tsim khoom pom zoo (Fixation / Permeabilization Solution Kit; eBioscience). Tom qab ntawd polychromatic flow cytometric tsom xam tau ua ntawm Flow Cytometer.

Cytokines Quantitation

Cov theem ntawm cytokines hauv cov ntshav thiab cov ntaub so ntswg supernatant ntawm nas tau txiav txim siab siv Cytometric Bead Array (CBA) Human Th1/Th2/Th17 cytokine kit (BD Pharmingen, USA) raws li cov chaw tsim khoom cov lus qhia. Luv luv, 50 μL ntawm txhua tus qauv supernatant tau tsim nrog 50 μL ntawm cov tib neeg sib xyaw Th1 / Th2 / Th17 cytokine ntes hlaws thiab 50 μL ntawm tib neeg Th1 / Th2 / Th17 PE nrhiav pom reagent ntawm chav tsev kub hauv qhov tsaus ntuj, tom qab ntawd tshem tawm. rau kev ntsuas cytometry 3 h tom qab.

Quantitative Real-time PCR

Tag Nrho RNA tau muab rho tawm los ntawm nas cov ntaub so ntswg tom qab kev sib cais siv Trizol Reagent (TaKaRa, Tuam Tshoj). Tag Nrho RNA tau siv los ua tus qauv los thim rov qab cDNA. -actin tau siv los ua kev siv sab hauv. Qhov kev qhia mRNA tau ntsuas los ntawm qPCR los ntawm kev siv cov primers hauv qab no: ZO-1-F, 5'-ACCACCAACCCGAGAAGAC-3', thiab ZO- 1-R, 5'-CAGGAGTATGGACGCACA-3 '; Occludin-F, 5'-TTGAAAGTCCACCTCCTTTACAGA-3', and OccludinR, 5'-CCGGATAAAAAGAGATAGTACGCTGG-3'; -actin-F, 5'-GGCTGTGTATTCCCCTCCATCG-3', thiab -actin-R, 5'-CCAGTTGGTAACAATGCCATGT-3'. Txoj kev 2-ct tau siv los ntsuas RNA qhia theem.

Enzyme-Linked Immunosorbent Assay (ELISA)

Cov theem ntawm D-LA thiab DAO hauv plasmas tau ntsuas siv cov khoom siv ELISA (Jingmei, Tuam Tshoj) raws li cov chaw tsim khoom cov lus qhia. Luv luv, cov plasma supernatants sau tau muab tso rau hauv 96-cov tshuaj tiv thaiv tshwj xeeb-coated daim hlau, raws li. Kev nqus tau raug kaw ntawm 450 nm siv tus nyeem ntawv microplate (Multiskan MK3, Thermo Fisher Scientific).

Kev txheeb cais

Cov ntaub ntawv tau nthuav tawm raws li txhais tau tias ± SD. Cov qauv ywj pheej t-test tau ua thiab pom pom siv GraphPad Prism8.0. 16S rRNA sequencing cov ntaub ntawv txheeb xyuas tau ua los ntawm Magichand platform (http://cloud.magigenecom/). Kev txheeb xyuas kev sib raug zoo ntawm plab microbiota thiab kev tiv thaiv kab mob tau ua los ntawm R pob (v3.6.3) los tso saib qhov sib txheeb matrix. P tus nqi<0.05 were considered statistically significant. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. not signifcant.

Cov txiaj ntsig

Kev txheeb xyuas ntawm HAMSCs

Cov hAMSCs nthuav tawm polygon lossis fibroblast-zoo li morphology hauv kab lis kev cai (Daim duab 1A) thiab tau zoo heev rau kev nthuav tawm ntawm MSC cov cim tshwj xeeb CD90, CD105, thiab CD73, tab sis tsis zoo rau CD34, CD45, HLA-DR, thiab CD11b (Daim duab . 1C). Raws li kev sib txawv ntawm ntau haiv neeg, peb cov txiaj ntsig tau pom tias hAMSCs tau pom adipocytic thiab osteoblastic sib txawv (Fig. 1B).

hAMSCs Qhov Tseem Ceeb Tshaj Plaws aGVHD Cov tsos mob hauv nas

Peb thawj zaug soj ntsuam cov txiaj ntsig kho mob ntawm hAMSCs ntawm aGVHD. Piv nrog rau aGVHD pab pawg, hAMSCs tau txais txiaj ntsig zoo heev aGVHD, raws li pom los ntawm kev poob qis (Daim duab 2B) thiab txo qis aGVHD cov qhab nia kho mob (Daim duab 2C). Tsis tas li ntawd, pawg hAMSCs muaj qhov muaj sia nyob ntau dua thaum piv rau aGVHD nas (Fig. 2D). Kev soj ntsuam ntawm pathological histology tau pom tias aGVHD ua rau cov ntaub so ntswg puas thiab leukocyte infiltration. hAMSCs, raws li tau pom los ntawm H&E, txo qis cov ntaub so ntswg cuam tshuam thiab infiltration ntawm exogenous T hlwb hauv aGVHD lub hom phiaj hauv nruab nrog cev (Fig. 2E). Raws li qhov tshwm sim, cov txiaj ntsig ntawm IHC kuj tau lees paub tias hAMSCs txhim kho cov kab mob pathological tsis zoo (Daim duab 2F). Cov txiaj ntsig no tau qhia tias hAMSCs muaj peev xwm los kho cov tsos mob aGVHD hauv aGVHD nas.

hAMSCs cuam tshuam tus tswv tsev tiv thaiv hauv aGVHD nas

Txhawm rau nkag siab txog cov txheej txheem immunomodulatory hauv qab uas hAMSCs txo aGVHD, peb ntsuas qhov ua kom thiab nthuav dav ntawm T hlwb hauv cov ntshav peripheral thiab lub hom phiaj hauv nruab nrog cev. Flow cytometry assay tau qhia tias hAMSCs tau zoo heev inhibited tus naj npawb ntawm CD3+CD4+T thiab CD3+CD8+T hlwb hauv cov ntshav thiab lub hom phiaj ntawm lub cev (Fig. 3A, B). Ntxiv mus, qhov kev faib ua feem ntawm CD4+CD25+Foxp3+Tregs hauv cov ntshav thiab lub hom phiaj lub cev ntawm cov nas hauv pawg hAMSCs tau ntau dua li cov hauv pawg aGVHD (Fig. 3C) . Peb tom ntej no tau tshawb xyuas qhov cuam tshuam ntawm hAMSCs ntawm cytokine profile (Fig. 4). hAMSCs kev tswj hwm ua rau muaj kev txo qis hauv IL-17A, IFN- , TNF, IL-6, thiab IL{13}} qib hauv cov hom phiaj hauv nruab nrog cev thiab ntshav piv rau cov pab pawg aGVHD, tshwj tsis yog rau qib TNF hauv tus po. Nyob rau lub sijhawm no, ntxiv rau IL-10 qib hauv lub siab, hAMSCs kho cov nas pom tau nce qib ntawm IL-10 thiab IL{16}} hauv lub hom phiaj lub cev thiab cov ntshav. Ua ke, hAMSCs tau ua lub luag haujlwm tiv thaiv hauv aGVHD los ntawm kev thim rov qab lub cev tsis muaj zog ntawm T cell subsets thiab cytokine cua daj cua dub.

Cov txiaj ntsig ntawm cistanche tubulosa- ua kom muaj zog tiv thaiv kab mob

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hAMSCs Ameliorated Intestinal Barrier Damage nyob rau hauv aGVHD nas

Txhawm rau tshawb xyuas seb hAMSCs puas tuaj yeem kho cov kab mob plab hnyuv hauv aGVHD, peb xub pom seb hAMSCs puas tuaj yeem txav mus rau hauv cov hnyuv. Raws li pom hauv daim duab 5A, GFP-labeled hAMSCs tau kuaj pom hauv cov hnyuv tom qab txhaj tshuaj rau hAMSCs thiab tuaj yeem nkag mus thiab nkag mus rau hauv cov hnyuv. Tom qab ntawd peb tsim ib qho kev ntsuam xyuas histopathological ntawm cov hnyuv me. Nyob rau hauv aGVHD pawg, cov hnyuv tau eroded thiab necrotic, lub plab hnyuv villi tau pom tseeb puas, lub villi tau tawg thiab loj leukocytes infiltrated lub plab hnyuv mucosa, submucosa, thiab lamina propria. Lub plab hnyuv villi tau raug teeb tsa tsis tu ncua, thiab tsis muaj cov hnyuv villus tawg lossis detachment ntawm villous epithelium tom qab hAMSCs kho (Fig. 5B). Tib neeg lymphocyte infiltration kuj tau soj ntsuam los ntawm immunohistochemistry hauv cov hnyuv hauv aGVHD pawg, qhov kev kho mob hAMSC tuaj yeem txo lawv cov infiltration (Fig. 5C). Txhawm rau ntsuas qhov kev tiv thaiv ntawm hAMSCs ntawm txoj hnyuv mucosal kev ncaj ncees, peb tau ua immunohistochemistry txhawm rau txheeb xyuas cov nyhuv ntawm ZO-1. Hauv pab pawg kho mob hAMSCs, qhov qhia txog qib ntawm cov protein sib txuas nruj ZO-1 tau nce ntau dua piv nrog rau pawg aGVHD (Fig. 5D). Peb kuj tau tshuaj xyuas mRNA qhia theem ntawm plab hnyuv nruj hlws ris proteins (TJs) thiab plasma qib ntawm D-lactic acid (D-LA) thiab diamine oxidase (DAO) los txiav txim siab txoj hnyuv ua haujlwm thiab plab hnyuv permeability. Thaum noj cov tshuaj intravenously, hMASCs tuaj yeem nce qib mRNA qhia ntau ntawm ZO-1 thiab Occludin, cov protein tseem ceeb ntawm TJs (Fig. 5E, F), thaum txo qis ntawm D-LA thiab DAO (Fig. 5G, H). Li no, thaum aGVHD tshwm sim, cov plab hnyuv teeb meem raug puas tsuaj nrog cov hnyuv nruj hlws ris cov proteins txo qis thiab cov hnyuv permeability-nce. Lub caij no, hAMCSs tuaj yeem thim rov qab qhov cuam tshuam ntawm txoj hnyuv.

Fig. 1 Phenotypic thiab functional characterization of hAMSCs. (A) Cov neeg sawv cev micrographs ntawm hAMSCs los ntawm nqe lus 0 mus rau 3 (tsis muaj xim, 100 ×). (B) Multilineage diferentiation peev xwm ntawm hAM SCs. Adipogenic diferentiation ntawm hAMSCs (sab laug) tau stained nrog Roj Liab-O, thiab osteoblastic diferentiation ntawm hAMSCs (txoj cai) tau stained nrog Alizarin Liab (staining, 100 ×). (C) Flow cytometric tsom xam qhia hAMSCs zoo rau CD90, CD105 thiab CD73 thaum tsis zoo rau CD34, CD45, CD11b thiab HLA-DR

Fig. 2 hAMSCs alleviated aGVHD hauv NPG nas. (A) Daim duab schematic qhia txog lub sijhawm tswj hwm ntawm hAMSCs hauv aGVHD nas qauv. (B) Kev hloov ntawm lub cev hnyav hauv ntau pawg (n=10). (C) aGVHD cov qhab nia kho mob (raws li kev poob phaus, hunched posture, rufed fur, tawv nqaij, txo kev txav mus los thiab raws plab) hauv cov pab pawg sib txawv (n=10). (D) Ciaj sia nyob ntawm ntau pawg (n=10). (E) Cov duab sawv cev ntawm H&E staining (400 ×) thiab histology qhab nia (raws li cov ntaub so ntswg destruc tion thiab lymphocyte infltration) (n=4). (F) Cov neeg sawv cev ntawm micro scopic duab ntawm immunohistochemistry (400 ×) thiab qhov feem ntawm CD45 zoo cheeb tsam (n=4). Cov nqi tau nthuav tawm raws li qhov txhais tau tias ± SD

Fig. 3 hAMSCs inhibited pub T cell expansion thaum txhim kho Tregs tiam lossis nthuav dav hauv vivo. (A) Flow cytometry analysis of CD3+CD4+T cells(n=6), (B) Flow cytometry analysis of CD3+CD8+ T cells(n=6), (C) Flow cytometry analysis of CD4+CD25+Foxp3+Tregs (n=6). Cov nqi tau nthuav tawm raws li qhov txhais tau tias ± SD

Fig. 4 hAMSCs downregulated pro-inflammatory cytokines IL-17A, IFN-, TNF, IL-6, IL-2, thiab upregulated anti-inflammatory cytokines IL-10 thiab IL -4 hauv vivo (n=5). Cov nqi tau nthuav tawm raws li qhov txhais tau tias ± SD

Gut Microbiota Alternations Tom qab hAMSCs Kev Kho Mob hauv GVHD nas

16S rRNA sequencing tau ua nyob rau hauv cov kab mob fecal DNA cais los ntawm ntau pawg nas. Cov -diversity indexes, suav nrog OUT, Chao1, thiab Shannon indexes, tshwm sim zoo ib yam, thiab hAMSCs kho nas harbored microbiota nrog ntau dua -diversity txheeb ze ntawm pawg aGVHD (Fig. 6A). Tus Thawj Saib Xyuas Kev Ua Haujlwm (PCoA) raws li Un weighted-unifrac nrug, Manhattan nrug, thiab Bray-Curtis metric nrug, yielded dispersed cov ntsiab lus ntawm ob pawg, txawm tias qhov sib txawv tsis tseem ceeb (Fig. 6B). Tom qab ntawd, peb tau soj ntsuam cov toj roob hauv pes ntawm lub plab microbiota txhawm rau tshawb xyuas qhov muaj peev xwm sib txawv ntawm ob pawg. Tag nrho ntawm 773 OTUs raug xam, thiab cov pab pawg hAMSCs muaj OTUs siab dua piv nrog cov pab pawg aGVHD (Fig. 6C). Hais txog cov kab mob muaj pes tsawg leeg nyob rau theem phylum, Firmicutes thiab Bacteroidetes yog ob qho tseem ceeb tshaj plaws phylum (Fig. 6D). Peb kuj tau teeb tsa ib daim ntawv qhia kev sib piv rau kev tsom xam ntawm plab microbiota ntawm ob pawg (Fig. 6E). Cov genus ntawm Odoribacter thiab Ruminococcus _1 tau nthuav tawm qhov kev nplua nuj nyob hauv cov pab pawg hAMSCs, uas yog cov microbes tseem ceeb los tswj txoj hnyuv homeostasis (Fig. 6F, G). Txhawm rau kom paub meej tias cov kab mob twg tau hloov pauv los ntawm kev kho mob hAMSCs thiab ua rau muaj kev cuam tshuam rau tus kab mob kev loj hlob tawm tsam aGVHD, peb tau ua qhov kev sib piv hauv chav kawm siab uas siv cov kev ntsuam xyuas kab sib cais (LDA) ntawm qhov loj me (LEfSe) uas tau kuaj pom qhov sib txawv ntawm qhov tseem ceeb ntawm cov kab mob hauv zej zog ntawm ob pawg. Raws li qhia hauv (Fig. 6H, I), Streptococaceae (tsev neeg thiab cov genus Streptococcus), Paludibacteraceae, F0058, thiab Delftia yog hom kab mob tseem ceeb uas ua rau lub plab microbiota dysbiosis hauv pawg aGVHD. Txawm li cas los xij, cov kab mob muaj txiaj ntsig Lachnospiraceae, Roseburia, Ruminococcaceae, Ruminiclostridium, Oscillibacter, thiab Clostridia (chav kawm thiab txiav txim Clostridiales) tau nthuav tawm cov txheeb ze ntxiv hauv pawg hAMSCs, uas yuav cuam tshuam nrog hAMSCs-mediated alleviation of aGVHD. Ua ke, qhov sib txawv thiab muaj pes tsawg leeg ntawm plab microbiota tau txhim kho tom qab hAMSCs kev tswj hwm thiab tau txav mus rau hauv ib qho kev sib tw tag nrho uas muaj txiaj ntsig zoo rau lub cev.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

hAMSCs tswj cov kev sib cuam tshuam ntawm Gut Microbiota thiab plab hnyuv tiv thaiv kab mob

Txhawm rau nkag siab txog kev sib raug zoo ntawm plab microbiota thiab plab hnyuv mucosal barrier, kev sib raug zoo ntawm TJs thiab plab microbiota ntawm qib genus tau txheeb xyuas los ntawm Spearman qhov kev txheeb xyuas kev sib raug zoo. Raws li pom hauv daim duab 7, muaj kev sib raug zoo ntawm ZO-1 thiab cov kab mob uas muaj txiaj ntsig, Ruminococcaceae_UCG.014, Muribaculum, Ruminococcus_1, thiab Ruminiclostridium_9. Cov txheeb ze ntawm cov kab mob muaj txiaj ntsig, Roseburia, Odoribacteria, Ruminococcus_1, thiab Ruminococcaceae_UCG.014 tau pom muaj kev sib raug zoo nrog Occludin. Peb tom ntej no tau ua qhov kev txheeb xyuas kev sib raug zoo ntawm plab microbiota thiab plab hnyuv tiv thaiv kab mob los ntawm Spearman txoj kev sib raug zoo (Fig. 7). Peb tau pom tias qhov nce ntawm Roseburia, Muribaculum, thiab Ruminococcus _1, tau cuam tshuam tsis zoo nrog rau feem pua ​​​​ntawm CD3 +CD4+T hlwb. Tsis tas li ntawd, qhov nce ntawm cov kab mob muaj txiaj ntsig, Ruminococcus_1 thiab Ruminiclostridium _9, tau muaj kev cuam tshuam zoo nrog feem pua ​​​​ntawm Tregs qhov tsis zoo nrog IL-17. Qhov ntau ntawm Lactobacillus thiab Candidatus{16}}Arthromitus tau cuam tshuam tsis zoo nrog IFN- . Muaj qhov cuam tshuam tsis zoo ntawm kev nplua nuj ntawm Muribaculum thiab IL-2. Qhov nce hauv Candidatus{19}}Saccharimonas abundance thiab txo qis hauv Escherichia. Shigella abundance tau zoo correlated nrog IL-10 qib (Fig. 7). Cov txiaj ntsig ntawm kev txheeb xyuas kev sib raug zoo tau qhia tias hAMSCs txhim kho qhov chaw mob ntawm tus tswv tsev thiab tswj cov hnyuv homeostasis uas yuav cuam tshuam nrog lub plab microbiota hloov kho, thiab yog li tiv thaiv aGVHD.

Fig. 5 hAMSCs ameliorated plab hnyuv barrier dysfunction nyob rau hauv aGVHD nas. (A) GFP-labeled hAMSCs nkag mus rau hauv cov hnyuv. (B) Cov duab sawv cev ntawm H&E staining ntawm txoj hnyuv (400 ×) thiab cov qhab nia histology (raws li cov ntaub so ntswg puas tsuaj thiab lymphocyte infiltration) (n=4). (C) Cov neeg sawv cev ntawm cov duab microscopic ntawm immunohistochemistry (400 ×) ntawm cov hnyuv thiab qhov feem ntawm CD45 thaj chaw zoo (n=4). (D) Cov neeg sawv cev ntawm cov duab microscopic ntawm immunohistochemistry (400 ×) ntawm cov hnyuv thiab qhov feem ntawm ZO-1 thaj chaw zoo (n=4). (E) mRNA qhia theem ntawm ZO-1 thiab Occludin (n{11}}). (F) Plasma theem ntawm D-LA thiab DAO (n{13}}).Cov nqi tau nthuav tawm raws li qhov txhais tau tias ± SD

Kev sib tham

Kev tsom xam microbial Advanced tau muab kev nkag siab tshiab rau hauv kev sib cuam tshuam ntawm tus tswv tsev thiab lub plab microbiota [18]. Lub plab microbiota tuaj yeem hloov pauv tom qab allo-HSCT thiab muaj feem cuam tshuam nrog aGVHD, qhia tias lub plab microbiota yuav yog lub hom phiaj tshiab rau kev kho aGVHD [5]. Ntau cov kev tshawb fawb suav nrog peb txoj kev tshawb fawb tsis ntev los no tau tshawb nrhiav thiab lees paub qhov kev kho mob ntawm MSCs ntawm aGVHD [15, 19, 20]. Txawm li cas los xij, qhov cuam tshuam ntawm plab microbiota nyob rau lub sijhawm no tseem tsis meej. Hauv txoj kev tshawb no, peb tau tshawb xyuas qhov cuam tshuam ntawm hAMSCs ntawm aGVHD thiab cov txheej txheem muaj peev xwm nyob rau hauv vivo. Raws li cov kev tshawb fawb yav dhau los [15, 21–23], peb cov txiaj ntsig tau qhia tias hAMSCs muaj kev tiv thaiv zoo ntawm aGVHD hauv cov nas, raws li pom los ntawm cov tsos mob aGVHD txo qis thiab muaj sia nyob ntev. Peb kuj tau pom tias hAMSCs tau nthuav tawm cov tshuaj tiv thaiv kab mob los ntawm kev txo tus neeg pub dawb T cell expansion thaum txhim kho Tregs tiam hauv aGVHD. Lub caij no, hAMSCs tuaj yeem txo qis cov tshuaj tiv thaiv kab mob cytokines thiab txhim kho cov tshuaj tiv thaiv cytokines hauv vivo, txhawm rau tiv thaiv cov lus teb inflammatory. Cov kab mob plab hnyuv, cov qauv ntom ntom tsim los ntawm cov monolayer ntawm plab hnyuv epithelial hlwb, tswj nws txoj kev ncaj ncees thiab tiv thaiv kev hloov pauv ntawm luminal microbiota hauv lub xeev noj qab haus huv [24]. Thaum lub sij hawm allo-HSCT, cov khoom ua kom txias ua rau lub plab zom mov tsis zoo, uas yog thawj kauj ruam hauv kev txhim kho aGVHD, vim nws tso cai rau kev hloov pauv ntawm cov kab mob hla cov kab mob thiab ua rau cuam tshuam cov kab mob hauv plab hnyuv homeostasis [24, 25]. Cov ntaub ntawv pov thawj pom tau tias MSCs muaj peev xwm kho cov ntaub so ntswg, uas tau ua pov thawj los kho cov kab mob plab hnyuv siab rau hauv cov nas colitis [26]. Tight junction proteins, suav nrog ZO-1 thiab Occludin, muaj lub luag haujlwm tseem ceeb hauv plab hnyuv muaj nuj nqi thiab kev hloov pauv ntawm plab hnyuv microbiota hla lub plab hnyuv tsis zoo provokes o [27]. D-LA yog muab los ntawm cov kab mob hauv plab, thiab DAO feem ntau yog tsom rau hauv plab hnyuv mucosa. Yog li, qhov siab ntawm cov ntshav plasma D-LA thiab DAO tuaj yeem cuam tshuam qee qhov kev hloov pauv hauv plab hnyuv permeability thiab plab hnyuv ua haujlwm [28]. Tam sim no, peb pom tias theem kev qhia ntawm TJs tau nce siab heev tom qab hAMSCs tswj hwm, thaum qib plasma ntawm D-LA thiab DAO txo qis, qhia tias hAMSCs tuaj yeem ua kom lub plab zom mov tsis ua haujlwm thiab rov qab plab hnyuv permeability. Lub plab yog ib qho ntawm cov kabmob uas cuam tshuam loj tshaj plaws los ntawm aGVHD thiab kev tshawb fawb tau qhia tias cov kab mob, tshwj xeeb tshaj yog lub plab microbiota plays lub luag haujlwm tseem ceeb hauv aGVHD pathogenesis thiab kev loj hlob [5, 6]. Raws li tau tshaj tawm hauv cov kev tshawb fawb yav dhau los, kev poob ntawm cov kab mob sib txawv ntawm allo HSCT cov neeg mob tau cuam tshuam nrog kev tuag ntau ntxiv los ntawm aGVHD [29, 30]. Fecal domination los ntawm opportunistic pathogens Enterococcus thiab Proteobacteria kuj tau txuas rau GVHD nce ntxiv [6, 31]. Tau ntau xyoo dhau los, kev saib xyuas tau nce ntxiv rau kev sib raug zoo ntawm lub plab microbiota thiab MSCs kev kho mob hauv cov kab mob o. MSCs tau raug pov thawj rau ameliorate plab microbiota dysbiosis ntawm rheumatoid mob caj dab, sepsis, thiab inflammatory plob tsis so tswj kab mob [32–34]. Txhawm rau tshawb xyuas ntxiv txog cov txheej txheem uas hAMSCs ameliorate aGVHD, peb tshawb xyuas qhov cuam tshuam ntawm hAMSCs ntawm lub plab microbiota. Hauv txoj kev tshawb fawb tam sim no, hAMSCs kev tswj hwm tau pom muaj txiaj ntsig zoo rau - ntau haiv neeg ntawm cov zej zog microbial piv nrog tsis muaj kev hloov pauv, qhia tau hais tias kev nplua nuj ntawm plab microbiota tuaj yeem hloov pauv tom qab hAMSCs kho. Txawm li cas los xij, tsis muaj qhov sib txawv tseem ceeb hauv kev ntsuas kev sib txawv, tej zaum vim yog cov qauv me me ntawm ob pawg lossis kev kho tsis txaus ntawm hAMSCs. Kev tsom xam siv qhov sib piv ntawm cov phiaj xwm hauv genus qhia tau hais tias hAMSCs kev tswj hwm tau nce ntau ntawm cov kab mob muaj txiaj ntsig, Odoribacteria thiab Ruminococcus _1 hauv plab hnyuv, uas yog cov saw hlau luv fatty acids (SCFAs) cov neeg tsim khoom nrog cov khoom tiv thaiv kab mob [35 , 36] ib. Txhawm rau txheeb xyuas cov kab mob tseem ceeb hauv qab nruab nrab los ntawm kev hloov pauv hAMSCs, LEfSe kev tshuaj xyuas tau ua los ntawm ob pawg. Piv rau aGVHD pab pawg, hAMSCs kev kho mob tau nce ntau ntawm qee cov kab mob uas muaj txiaj ntsig zoo cuam tshuam rau cov tshuaj tiv thaiv kab mob, xws li Lachnospiraceae, Roseburia, Ruminococcaceae, Ruminiclostridium, Oscillibacter, Clostridia, uas tau piav qhia tag nrho kom muaj txiaj ntsig zoo rau cov plab hnyuv microbiotaSC. 37–42] ib. Yog li ntawd, peb cov kev soj ntsuam tau qhia tias tom qab hAMSC kev tswj hwm, lub plab microbiota dysbiosis tau ameliorated, uas yog tshwm sim los ntawm kev txhim kho ntau haiv neeg thiab cov txheeb ze ntau ntxiv ntawm cov kab mob muaj txiaj ntsig.

Daim duab 6 Gut microbiota alternations in aGVHD nas. (A) kev sib piv ntawm -diversity. (B) kev sib piv-diversity. (C) Venn daim duab ntawm OTUs. (D) txheeb ze cov kab mob abundance ntawm phylum theem. (E) Heatmap ntawm hom abundance ntawm genus theem. (F) Kev txheeb ze ntawm Odoribacter ntawm qib genus. (G) Cov txheeb ze ntau ntawm Ruminococcus_1 ntawm qib genus. (H) Cladogram raws li kev txheeb xyuas LEfSe. (I) LDA cov qhab nia suav los ntawm cov yam ntxwv sib txawv ntawm cov pab pawg aGVHD thiab hAMSCs. (AI) n=3 nas rau ib pab. Cov nqi tau nthuav tawm raws li qhov txhais tau tias ± SD

aGVHD yog cov txheej txheem inflammatory nyuaj uas tau pib los ntawm kev puas tsuaj ntawm txoj hnyuv hauv plab ua rau muaj kev ncaj ncees ua raws li kev hloov ntawm txoj hnyuv microbiota thiab lawv cov khoom. Kev lees paub ntawm kev puas tsuaj cuam tshuam nrog cov qauv molecular thiab cov kab mob sib txuas nrog cov qauv molecular los ntawm cov tshuaj tiv thaiv kab mob uas nthuav tawm cov hlwb induces pro-inflammatory teb nrog rau kev ua kom T hlwb thiab cytokine cua daj cua dub ua rau mob plab hnyuv puas tsuaj thiab txhawb txoj kev loj hlob ntawm aGVHD [24, 43]. Ntawm qhov tod tes, lub plab microbiota thiab microbiota-derived metabolites xws li SCFAs ua lub luag haujlwm tseem ceeb hauv kev tswj cov plab hnyuv siab thiab plab hnyuv homeostasis nrog rau kev txhim kho lub cev tiv thaiv kab mob, thiab ntsuas tus tswv tsev tiv thaiv nrog microbial Cheebtsam thiab metabolites [24, 4] . Kev sib txuas lus ntawm tus tswv tsev thiab lub plab microbiota ntawm lub plab hnyuv kab mob tswj cov mucosal thiab lub cev tiv thaiv kab mob thiab, hauv cov xeev pathological, tuaj yeem ua rau GVHD [6, 45, 46]. Nws tau pom tias lub plab microbiota muaj feem cuam tshuam nrog kev nthuav qhia ntawm cov nqaij sib txuas ceev ceev [47]. Txoj kev tshawb fawb tam sim no tau pom tias mRNA qhia ntawm TJs ZO-1 thiab Occludin yog qhov cuam tshuam tsis zoo rau cov txheeb ze ntawm cov kab mob uas muaj txiaj ntsig zoo thaum muaj kev sib raug zoo rau cov kab mob uas muaj txiaj ntsig, qhia tias lub plab microbiota tseem ceeb los tswj kev ncaj ncees ntawm cov kab mob. plab hnyuv barrier. Tsis tas li ntawd, lub plab microbiota kuj tseem ceeb heev hauv kev tswj lub plab hnyuv tiv thaiv kab mob [47]. Peb pom tias qee cov kab mob muaj txiaj ntsig tau cuam tshuam tsis zoo nrog feem pua ​​​​ntawm CD3+CD4+T hlwb, tab sis muaj kev cuam tshuam zoo nrog feem pua ​​​​ntawm Tregs. Ntawm no, peb tau nthuav tawm tias cov kab mob muaj txiaj ntsig, feem ntau cov neeg tsim khoom SCFA, tau cuam tshuam tsis zoo rau cov kab mob cytokines tab sis muaj kev cuam tshuam zoo nrog cov tshuaj tiv thaiv kab mob cytokines, thaum cov kab mob ua rau muaj teeb meem sib cuam tshuam. Hauv cov ntsiab lus, qhov sib txawv tseem ceeb hauv plab microbiota ntawm cov kev kho mob pom zoo ib daim duab tag nrho ntawm kev sib cuam tshuam ntawm plab microbiota thiab txoj hnyuv, kev tiv thaiv kab mob. Tseem muaj ntau qhov kev txwv tsim nyog los tham hauv peb qhov kev sim. Ua ntej, txawm hais tias peb txoj kev tshawb fawb tau tshawb xyuas qhov cuam tshuam ntawm hAMSCs ntawm kev tiv thaiv kab mob, kab mob plab hnyuv, thiab plab microbiota hauv aGVHD, lub hauv paus txheej txheem tseem yuav tsum tau txheeb xyuas hauv kev tshawb fawb ntxiv. Thib ob, plab microbiota depletion thiab fecal microbiota hloov pauv yog xav tau los daws qhov kev sib raug zoo ntawm cov plab hnyuv microbiota, plab hnyuv teeb meem, thiab kev tiv thaiv. Thib peb, kev tshawb fawb ntxiv los tshawb xyuas qhov cuam tshuam ntawm hAMSCs ntawm microbiota-derived SCFAs thiab lwm yam metabolites yog lav. Hauv kev xaus, peb cov kev tshawb fawb pom tias hAMSCs txo qis aGVHD los ntawm kev txhawb nqa lub plab microbiota normalization thiab tswj kev cuam tshuam ntawm plab microbiota thiab plab hnyuv teeb meem, tiv thaiv kab mob. Peb txoj kev tshawb fawb yog thawj qhov systematically los txheeb xyuas cov teebmeem thiab cov txheej txheem hauv qab ntawm hAMSCs tswj lub plab microbiota thiab plab hnyuv tiv thaiv kab mob hauv aGVHD.

Daim duab 7 Kev txheeb xyuas kev sib txheeb ntawm plab microbiota thiab kev tiv thaiv kab mob hauv plab. Spearman qhov kev txheeb xyuas kev sib raug zoo tau ua los ntawm cov txheeb ze ntau ntawm 15 qhov sib txawv ntawm lub plab microbiota (ntawm qib genus) thiab mRNA qhia theem ntawm TJs, feem pua ​​​​ntawm cov kab mob hauv lub cev nrog rau cov concentration ntawm cytokines hauv cov hnyuv ntawm ob pawg. Spearman r qhov tseem ceeb ntawm -0.5 (xiav) txog 0.5 (liab)

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