Bacterial Vesicles Thaiv Viral Replication hauv Macrophages Ntawm TLR4-TRIF-axis
Sep 27, 2023
Abstract
Cov kab mob Gram-negative ib txwm tso tawm nano-qhov loj me ntawm cov txheej membrane vesicles (OMVs), uas yog qhov tseem ceeb ntawm kev sib txuas lus thiab kab mob. OMV uptake los ntawm tus tswv tsev cell qhib TLR signaling ntawm thauj PAMPs. Raws li ib qho tseem ceeb nyob rau hauv lub cev tiv thaiv kab mob, alveolar macrophages yog nyob rau ntawm cov huab cua-cov ntaub so ntswg interface uas lawv muaj xws li thawj kab ntawm kev tiv thaiv tawm tsam inhaled microorganisms thiab hais. Txog niaj hnub no, me ntsis paub txog kev sib cuam tshuam ntawm alveolar macrophages thiab OMVs los ntawm cov kab mob pathogenic. Lub cev tiv thaiv kab mob rau OMVs thiab cov txheej txheem hauv qab no tseem tsis tau paub. Ntawm no, peb tau tshawb xyuas cov lus teb ntawm thawj tib neeg macrophages rau cov kab mob vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) thiab pom qhov sib piv NF-κB ua kom thoob plaws txhua lub hlwv. Hauv qhov sib piv, peb piav qhia txog qhov sib txawv ntawm hom I IFN teeb liab nrog lub sijhawm ntev STAT1 phosphorylation thiab muaj zog Mx1 induction, thaiv tus kab mob khaub thuas A tus kab mob replication tsuas yog rau Klebsiella, E.coli, thiab Salmonella OMVs. OMV-induced antiviral cov teebmeem tsis tau tshaj tawm rau endotoxin-dawb Clear coli OMVs thiab Polymyxin-kho OMVs. LPS stimulation tsis tuaj yeem ua raws li cov xwm txheej tiv thaiv kab mob no, thaum TRIF knockout tshem tawm nws. Qhov tseem ceeb, supernatant los ntawm OMV-kho macrophages induced antiviral teb nyob rau hauv alveolar epithelial hlwb (AEC), qhia OMV-induced intercellular kev sib txuas lus. Thaum kawg, cov txiaj ntsig tau raug lees paub hauv tus qauv ex vivo kab mob nrog thawj tib neeg lub ntsws cov ntaub so ntswg. Hauv kev xaus, Klebsiella, E.coli, thiab Salmonella OMVs induce antiviral immunity in macrophages of TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs, and lung tissue. Cov kab mob gram-tsis zoo no ua rau muaj kev tiv thaiv kab mob hauv lub ntsws los ntawm OMVs, uas muaj peev xwm txiav txim siab thiab cuam tshuam loj heev rau cov kab mob thiab kis kab mob.
cistanche cog-nce kev tiv thaiv kab mob
Ntsiab lus
Extracellular vesicles, Outer membrane vesicles, Bacterial and viral co-infection, Pneumonia, Macrophage, Alveolar epithelial cell, Antiviral innate tiv thaiv
Taw qhia
Sab nrauv vesicles (OMVs) ib txwm tso tawm los ntawm cov kab mob gram-tsis zoo, uas ntsuas txog 300 nm inch. Cov kab mob Gram-zoo sib npaug tuaj yeem tso cov vesicles (MVs). Raws li qhov tshwm sim ntawm lawv cov biogenesis, cov lipid bilayer membrane cov qauv muaj, ntxiv rau cov ntsiab lus periplasmic, cov khoom tseem ceeb ntawm cov kab mob (sab nrauv) daim nyias nyias xws li lipids, proteins, thiab, nyob rau hauv cov kab mob gram-tsis zoo, lipopolysaccharides (LPS) [1]. Ntxiv rau lawv lub luag haujlwm hauv kev sib txuas lus ntawm cov kab mob, OMVs tau cuam tshuam nrog cov kab mob pathogenic thiab kev thauj mus los ntawm kev ua phem, xws li VacA los ntawm Helicobacter pylori, Shiga toxin los ntawm Shigella dysenteriae lossis ClyA los ntawm enterohemorrhagic Escherichia coli [2]. Cov OMVs no tau pom los tswj cov kab mob epithelial thiab lub cev tiv thaiv kab mob. Lub xub ntiag ntawm cov kab mob endotoxin ntawm OMV nto ua ke nrog lwm cov kab mob sib txuas nrog cov qauv molecular (PAMPs) ua rau lawv muaj zog stimulators rau lub cev tiv thaiv kab mob, raws li lawv tseem tuaj yeem lees paub los ntawm lawv cov receptors [3]. Kev koom tes ntawm Tolllike receptors (TLRs) thiab Nod-zoo li receptors nyob rau hauv lub cev tiv thaiv kab mob tuaj yeem ua rau kev tso tawm ntawm pro-inflammatory thiab immunoregulatory cytokines, nrog rau kev nrhiav neeg ntawm neutrophils los yog kev cuam tshuam ntawm nruj junctions hauv epithelial cell txheej [4]. Ib qho tseem ceeb hauv lub cev tiv thaiv kab mob hauv lub ntsws yog alveolar macrophages (AMs), uas nyob ntawm huab cua-cov ntaub so ntswg hauv lub alveoli. Los ntawm kev noj cov tshuaj inhaled, AMs sawv cev rau thawj kab ntawm kev tiv thaiv kab mob thiab cov kab mob los ntawm phagocytosis thiab degradation. Thaum cov kab mob ntsib, macrophages nthuav tawm cov tshuaj tiv thaiv kab mob rau lub cev tiv thaiv kab mob thiab tso tawm pro-inflammatory cytokines. Yog li, lawv tuaj yeem ua rau hom I thiab II alveolar epithelial cells (AECs) thiab lwm cov kab mob tiv thaiv kab mob (Cov ntaub ntawv ntxiv 1: Fig. S1A). AMs tuaj yeem qhib tau los ntawm ntau lub PAMPs los ua kom intracellular signaling thiab induction ntawm cov noob qhia txawv txawv ntawm ntau lub cev tiv thaiv kab mob [5, 6], ua rau MyD88 lossis TRIF signaling, uas ob qho tib si, dhau los, ua rau muaj cov noob sib txawv (Cov ntaub ntawv ntxiv 1 : Fig. S1B). Ntxiv mus, lawv ua tiav lwm yam dej num hauv pulmonary homeostasis thiab pathogenesis, ua rau lawv lub hauv paus teeb liab hub thiab orchestrator hauv lub ntsws tiv thaiv kab mob. Cov kab mob khaub thuas yog nyob hauv tsev neeg ntawm Orthomyxoviridae thiab ua rau muaj kab mob ua pa sab saud thiab sab qis, xws li mob me mus rau mob hnyav. Txawm hais tias feem ntau cov kab mob khaub thuas raws caij nyoog yog qhov txwv tus kheej, qee cov neeg mob muaj mob ntsws thiab mob ua pa nyuaj uas kwv yees ua rau muaj txog 650,000 txhua xyoo kev tuag thoob ntiaj teb [7]. Influenza A virus (IAV) feem ntau kis kab mob hauv cov hlab ntsha epithelial thiab rov ua dua nyob rau hauv. Tus kab mob kis tau ua rau tus qauv lees paub receptor (PRR) ua kom thiab teeb liab ntawm ntau qhov kev hloov pauv, xws li cov khoom siv hluav taws xob nuclear kappa B (NF-κB) thiab interferon regulatory factor (IRF)-3/7, uas induce hom I thiab III. Interferons (IFN) ntau lawm. Qhov no tseem ceeb IFN cov lus teb yog li ntawd induces cellular "antiviral state" nyob rau hauv ib tug auto- thiab paracrine yam [8]. Tsis tas li ntawd, ob peb lwm cytokines thiab chemokines tau zais thaum kis kab mob. Qhov kev tswj hwm nruj tswj hwm tus tswv tsev tiv thaiv cytokine network nrhiav cov neeg tiv thaiv kab mob hauv lub cev mus rau qhov chaw kis kab mob thiab orchestrates innate thiab adaptive tiv thaiv teb [9]. Txawm li cas los xij, IAV tseem tuaj yeem kis tau AMs, ua rau txo qis ntawm AMs thaum lub sijhawm kis mob hnyav, uas yuav tsum tau rov tsim kho dua los daws cov kab mob. Kev ua kom lub cev tiv thaiv kab mob hauv lub cev thiab kev tso tawm ntawm pro-inflammatory cytokines ua rau kev nrhiav neeg ntxiv hauv cov hlwb, tswj tus kab mob thiab pib kho cov ntaub so ntswg [10, 11]. Ntawm no, peb txheeb xyuas cov lus teb ntawm macrophages rau cov kab mob extracellular vesicles thiab lawv cov kev cuam tshuam rau cov kab mob IAV tom ntej.
cistanche ntxiv cov txiaj ntsig-yuav ua li cas ntxiv dag zog rau lub cev
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Khoom siv thiab cov txheej txheem
Tshuaj thiab tshuaj tiv thaiv
Ham's F12 nruab nrab tau txais los ntawm GE Healthcare Europe (Freiburg, Lub teb chaws Yelemees). RPMI-1640, DMEM, GlutaMAX, Penicillin/Streptomycin, thiab FCS tau yuav los ntawm Life Technologies (Darmstadt, Germany). PBS tau txais los ntawm Capricorn Scientific GmbH (Ebsdorfergrund, Lub teb chaws Yelemees). Opti-MEM tau txais los ntawm Thermo Fisher Scientific (Frankfurt, Lub teb chaws Yelemees). Phorbol 12-myristate 13-acetate (PMA) yog muab los ntawm Sigma-Aldrich Chemie (Munich, Lub teb chaws Yelemees). LPS (Salmonella Minnesota R595, qib TLR) tau txais los ntawm Enzo Life Sciences (Lausen, Switzerland). Pam3CSK4 tau yuav los ntawm Invivogen (San Diego, USA). JAK inhibitor Ruxolitinib tau txais los ntawm Biozol Diagnostics Vertrieb GmbH (Eching, Lub teb chaws Yelemees). Polymyxin B tau yuav los ntawm Merck Millipore (Billerica, USA). Antibodies tau txais los ntawm Abcam (Cambridge, UK): Mx1 (ab95926), influenza nucleoprotein (9G8); Cell Signaling (Cambridge, UK): phospho-IRF-3 (Ser396)(4D4G), phospho-TBK-1 (Ser172)(D52C2), TBK-1 (61223S), phospho- STAT1 (Tyr)(58D6), STAT1 (D1K9Y), nas los tiv thaiv luav (L27A9), IRAK-1 (4359S), phospho-p38 (Tr180/Tyr182)(9211S), p38 (9212S); Thermo Fisher Scientific: tshis los tiv thaiv nas (Alexa rau 488); ProteinTech: anti-IRF-3 (66670-1) los yog Santa Cruz Biotechnology (Heidelberg, Lub teb chaws Yelemees): -actin (C4), anti-nas (mIgGκBPHRP, sc-516102). Tag nrho lwm cov tshuaj siv tau yog qib tshuaj ntsuam xyuas thiab tau los ntawm cov khoom lag luam.
Kab lis kev cai thiab OMV/MV npaj
L. pneumophila strain Corby wildtype tau ua raws li tau piav qhia yav dhau los [12]. K. pneumoniae (#700721/ MGH78578), E.coli (#25922), thiab S. enterica serovar Typhimurium (#14028) tau txais los ntawm American Hom Culture Collection (Rockville, MD, USA). ClearColi ™ BL21 yog los ntawm BioCat GmbH (Heidelberg, Lub teb chaws Yelemees). S. pneumoniae D39 Δcps tau ua siab zoo los ntawm Sven Hammerschmidt. Cov kab mob tau loj hlob rau ntawm daim phiaj agar thaum hmo ntuj (MacConkey: Kp, Ec, Sal; LB: Clear coli; blood agar plates: Sp) thiab tom qab ntawd siv los ua kua dej tawm (LB: Kp, Ec, Sal, Clear coli; THY: Sp) . Cov kab mob tau loj hlob mus txog rau theem logarithmic lig ntawm 37 degree nyob rau hauv tas li co (MaxQ 6000, Thermo Fisher Scientific, Karlsruhe, Lub teb chaws Yelemees; tshwj tsis yog rau Sp). Cov kab mob kab mob ces spun down peb zaug (4,500 × g, 15 min, 4 degree; Multifuge X3R, Thermo Fisher Scientific). Cov kab mob uas tseem tshuav tau raug tshem tawm los ntawm kev ua kom tsis muaj menyuam los ntawm 0.22 µm pores. Lub supernatant tau concentrated nrog 100 kDa molecular hnyav txiav tawm lim (Merck KGaA, Darmstadt, Lub teb chaws Yelemees) thiab cov hlwv tau purified los ntawm ultracentrifugation los yog loj cais chromatography (SEC). Rau ultracentrifugation, lub supernatant yog ultracentrifuged ntawm 100,000 × g, 3 h, 4 degree. Tom qab ntxuav cov tau txais OMV / MV pellet nrog sterile PBS, ultracentrifugation tau rov qab thiab cov vesicle pellet tau yaj hauv sterile PBS. Cov ntsiab lus ntawm cov protein tau txiav txim siab los ntawm Pierce BCA cov khoom siv tshuaj ntsuam xyuas protein raws li cov chaw tsim khoom cov lus qhia (Thermo Fisher Scientific), thiab cov protein sib npaug tau siv rau kev sim stimulation. Rau SEC, lub supernatant yog concentrated rau 500 µL thiab loaded ntawm thawj / 70 nm Gen 2 kab (IZON Science LTD, Lyon, Fabkis), uas tau pre-ntxuav nrog 10 mL PBS raws li lub chaw tsim tshuaj paus raws tu qauv. Cov vesicles raug eluted siv sterile PBS thiab feem ntawm 500 µL tau sau. Vesicles tau eluted nyob rau hauv feem 7-12, uas tau txiav txim los ntawm nano-flow cytometry (nFCM; NanoFCM Co., Ltd, Nottingham, UK). Cov vesicle-uas muaj cov feem sib xyaw ua ke tau tsom mus rau 200-400 µL siv cov lim dej molecular hnyav (Merck KGaA). Lub vesicle concentration ntawm tag nrho cov kev npaj tau txiav txim los ntawm nFCM thiab sib npaug ntawm cov vesicles tau siv rau kev sim stimulation. OMV/MV kev npaj ntawm ob txoj kev ua kom huv yog kuaj xyuas cov kab mob sib kis los ntawm plating thiab khaws cia hauv aliquots ntawm -20 degree .
NFC
Rau nFCM tus Nano Analyzer, (NanoFCM Co., Ltd, Nottingham, UK) nruab nrog lub 488 nm laser tau calibrated nrog 200 nm polystyrene hlaws (NanoFCM Co.) nrog lub ntsiab lus txhais ntawm 2.08 × 10^8 particles / ml thiab kuj siv los ua ib qho kev siv rau particle concentration. Tsis tas li ntawd, monodisperse silica hlaws (NanoFCM Co.) ntawm plaub qhov sib txawv tau siv los ua tus qauv siv loj los ntsuas qhov loj ntawm cov vesicles. Freshly lim (0.1 µm) 1 × PBS tau soj ntsuam raws li lub teeb liab tom qab thiab rho tawm los ntawm lwm qhov kev ntsuas. Txhua qhov kev faib tawm histogram lossis dot zaj duab xis tau muab los ntawm cov ntaub ntawv khaws cia rau 1 feeb nrog tus qauv siab ntawm 1 kPa. OMV cov qauv tau diluted nrog lim (0.1 µm) 1 × PBS, ua rau cov khoom suav hauv qhov zoo tshaj plaws ntawm 2,500–12,000 cov xwm txheej. Particle concentration thiab loj faib tau suav nrog nFCM software (NF Profession V1.08).
Transmission electron microscopy (TEM)
Carbon-coated tooj liab daim phiaj (400 mesh) tau hydrophilized los ntawm glow discharging (PELCO easiGlow, Ted Pella, USA). Tsib µL ntawm cov qauv tau muab tso rau hauv daim phiaj hydrophilized, thiab stained nrog 2% (w / v) uranyl acetate tom qab ntxuav luv luv nrog ob chav dej distilled. Cov qauv raug txheeb xyuas nrog JEOL JEM-2100 kis tau tus mob electron microscope siv qhov nrawm nrawm ntawm 120 kV. Cov duab tau txais nrog lub koob yees duab F214 FastScan CCD (TVIPS, Gauting, Lub teb chaws Yelemees).
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Cell kab lis kev cai
Tib neeg cov kab ntawm tes (THP-1, A549, BEAS2B, Calu-3, HCC827) tau txais los ntawm American Type Culture Collection. Madin-Darby Canine Kidney II hlwb (MDCK II) tau yuav los ntawm ECACC-Sigma-Aldrich (Darmstadt, Lub Tebchaws Yelemees). THP-1 Dual™ (Dual) thiab THP-1 Dual™ TRIF-KO (TRIF−/−) hlwb tau txais los ntawm Invivogen Europe (Toulouse, Fabkis). Cells tau cog qoob loo hauv Ham's F12, DMEM lossis RPMI-1640 ntxiv nrog 1 mM sodium pyruvate, 2 mM glutamine nrog rau 10% heatinactivated FCS ntawm cell kab lis kev cai. THP-1 Dual™ hlwb tau cog rau hauv RPMI{17}} ntxiv nrog 1% Penicillin/Streptomycin, 2 mM glutamine, 10% heat-inactivated FCS, 100 µg/mL Normocin™ thiab 25 mM HEPES tsis. THP-1, Dual, thiab TRIF-/- cells tau sib txawv hauv cov hlwb zoo li macrophage los ntawm kev sib ntxiv ntawm 20 nM PMA rau 24 teev.
Cloning ntawm Mx1 rau SparQ vector
Cov txheej txheem coding ntawm Mx1 tau tsim los ntawm THP-1 cDNA los ntawm Phusion PCR siv Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, USA) raws li cov chaw tsim khoom cov lus qhia. Ib qho HAtag tau ntxiv los ntawm fusing HA coding sequence rau qhov rov qab primer (sense: 5′-atcggaTTCGAAATGGTTGTT TCCGAAGTGGAC-3}′, antisense: 5′-tccgatGCGGCC GCTTAAGCGTAATCTGGAACATCGTAGATGGAAGGAGTTAA{5}}GACGA Lub PCR fragment nrog rau SparQ vector (Addgene, Watertown, USA) tau zom nrog BstbI thiab NotI txwv enzymes (Thermo Fisher Scientific) thiab ligated nrog T4 DNA Ligase (New England Biolabs) rau hauv SparQ vector.
Kev hloov pauv ntawm HEK293T hlwb thiab cov khoom tsim Lentivirus
HEK293T hlwb tau hloov pauv nrog SparQ vector diluted hauv Opti-MEM uas muaj cov kab ke rau Mx1 thiab GFP ib ntus, cov kab mob ntim kab mob vector psPAX2, thiab lub hnab ntawv plasmid pVSV-G (Addgene) nrog Lipofectamine 2000 (Thermo Fisher Scientific) raws li qhov chaw tsim khoom raws tu qauv. Lentivirus tau tsim thiab muaj tus kab mob supernatant tau sau txhua hnub rau 72 teev. Lub supernatant tau lim siv lub lim 0.45 µm thiab THP{10}} hlwb raug hloov pauv (saib hauv qab). Ib qho khoob SparQ vector yam tsis muaj Mx1 ib ntus tau siv rau kev hloov pauv ntawm cov hlwb los tsim cov kab tswj hwm ntawm tes (VC=vector tswj).
Kev hloov pauv ntawm THP-1 cell
THP-1 cov hlwb tau hloov nrog cov kab mob lentivirus los ntawm cov lim dej supernatant ntawm HEK293T cell kab lis kev cai (saib saum toj). Polybene (4 µg / mL, Sigma-Aldrich) tau ntxiv los txhim kho qhov kev ua tau zoo. Cells raug incubated mus txog rau hnub. GFP-zoo hlwb raug txheeb xyuas los ntawm kev ntws cytometry.
Kev rho tawm thiab sib txawv ntawm BDMs
Tib neeg monocytes los ntawm cov neeg noj qab haus huv tau raug cais los ntawm kev sib nqus CD14 zoo xaiv los ntawm peripheral ntshav mononuclear hlwb thiab sib txawv rau hauv cov ntshav-derived macrophages (BDM) nyob rau hauv muaj 1% tib neeg ABserum raws li yav dhau los tau piav qhia [13].
OMV / MV stimulation ntawm macrophages
Tib neeg macrophages los yog txawv THP-1 hlwb tau incubated nrog purified OMVs/MVs (1 µg/mL txhua rau vesicles purified los ntawm ultracentrifugation thiab ib tug multiplicity ntawm vesicles (MOV) ntawm 1,000 rau SEC-purified vesicles) txog li 20 teev hauv kev tshaj tawm. Daim ntawv thov inhibitor ntxiv tau ua 1 h ua ntej kev kho vesicle. Te-stimulated macrophages yog siv rau cov protein lossis RNA cais lossis kev sim kab mob tom ntej. Cov tau txais cytokine-muaj supernatant tau siv rau ELISA lossis LDH tsom xam lossis lim dej kom tsis muaj menyuam thiab siv rau kev txhawb nqa ntawm epithelial cell.
Virus purification thiab virus titration
Kev ntxuav tus kab mob thiab kab mob titration tau ua nyob rau hauv MDCK II hlwb raws li tau piav ua ntej [14]. Tus kab mob titer tau txiav txim los ntawm plaque assay raws li tau piav qhia yav dhau los [15]. Luv luv, cov hlwb MDCK II tau kis tus kab mob sib kis los yog cov ntaub so ntswg supernatant (hauv MDCK II kab lis kev cai nruab nrab yam tsis muaj FCS). Tus kab mob no tau tso cai rau adsorb rau cov hlwb rau 1 h ntawm 37 degree. Kaum, lub inoculum tau hloov nrog cov xov xwm tshiab (ntxiv nrog 1 ug / mL TPCK kho trypsin (rau IAV) thiab 1% Avicel® PH-101 (Sigma-Aldrich)). MDCK II hlwb tau stained nrog crystal violet los yog cov kab mob uas muaj kab mob tau stained nrog thawj cov tshuaj tiv thaiv (1:2,{11}}) (nas anti-influenza A nucleoprotein, MCA400, Bio-Rad, Lub teb chaws Yelemees) thiab cov tshuaj tiv thaiv thib ob ( 1:4,{17}}) (Tshis Anti-Ntaus IgGHRP, 170–6516, Bio-Rad, Lub teb chaws Yelemees) rau cov tshuaj plaque. Cov plaques tau raug suav los ntawm tus kheej thiab qhia tias yog cov quav hniav sib sau ua ke (pfu) ib mL.
cistanche cov txiaj ntsig rau cov txiv neej-ua kom muaj zog tiv thaiv kab mob
Kev kis kab mob vais lav
Tom qab 20 h pre-stimulation nrog cov kab mob vesicles los yog cytokine-muaj supernatant, hlwb tau kis tus kab mob IAV los yog vesicular stomatitis virus (VSV). Macrophages tau kis tus kab mob H1N1 (A/WSN/33) lossis VSV, thaum lub hlwb epithelial tau kis H1N1 (A/Hamburg/2009/PDM) ob qho tib si hauv xov xwm tshiab yam tsis muaj FCS. Tom qab 1 teev ntawm inoculation, tus kab mob dawb raug tshem tawm thiab cov xov xwm tau hloov nrog TPCK-trypsin-muaj xov xwm rau IAV lossis cov xov xwm dawb paug rau VSV. Cells tau incubated mus txog rau 48 teev kom tso cai rau multicyclic replication.
Kev npaj ntawm tib neeg PCLS
Cov ntaub so ntswg ntawm lub ntsws tau txais los ntawm cov neeg mob uas tau txais kev kho mob lobe vim mob ntsws cancer ntawm Hannover Medical School (MHH, Hannover, Germany). Tsuas yog cov ntaub so ntswg los ntawm macroscopically thiab microscopically tsis muaj kab mob ntawm lub ntsws tau siv rau kev sim. Tib neeg precision-txiav lub ntsws (PCLS) tau npaj raws li tau piav ua ntej [16]. Ob daim ntaub so ntswg rau ib qhov dej tau cog rau hauv 24- lub phaj zoo nyob rau hauv submerged tej yam kev mob (DMEM / F12 ntxiv nrog 1% Penicillin / Streptomycin) ntawm 37 degree , 5% CO2 thaum hmo ntuj.
IAV infection of PCLS
PCLS tau txhawb nqa nrog 1 µg / mL ntawm OMVs purified los ntawm L. pneumophila los yog K. pneumoniae diluted hauv DMEM / F12 ntxiv nrog 1% Penicillin / Streptomycin ntawm 37 degree, 5% CO2 rau 20 h. Cov xov xwm raug muab tshem tawm thiab PCLS raug inoculated nrog 25, 000 pfu/well hauv 250 µL ntawm tus kab mob npaws A/California/04/2009(H1N1pdm). PCLS tau tsim nyob rau ntawm 35 degree thiab rocked txhua 15 feeb thaum lub sij hawm inoculation kom muaj homogenous kab mob. Tom qab ntawd, cov inoculum raug muab pov tseg thiab hloov los ntawm DMEM / F12 ntxiv nrog 1% Penicillin / Streptomycin. Tom qab incubation rau 48 teev, tus supernatant tau sau rau kev kuaj kab mob, LDH tso, thiab cytokine kom muaj nuj nqis. Cov qauv rau kev ntsuas cytokine tau ntxiv nrog 0.2% protease inhibitor cocktail (P1860, Sigma-Aldrich, Munich, Lub teb chaws Yelemees) thiab khaws cia ntawm 80 degree kom txog thaum kev soj ntsuam. PCLS tau khov thiab khaws cia ntawm 80 degree kom txog thaum RNA purification raws li tau piav qhia yav dhau los [17].
LDH
LDH tso tawm kev soj ntsuam tau ua raws li cov chaw tsim khoom cov lus qhia siv Pierce™ LDH Cytotoxicity Assay Kit tau los ntawm Roche (Mannheim, Lub teb chaws Yelemees) lossis Cytotoxicity Detection KitPLUS (LDH) (Roche, Merck). Qhov nqus tau ntsuas tau siv lub microplate nyeem ntawv infinite F200Pro (Tecan, Männedorf, Switzerland).
ELISA
Cytokines CXCL8/IL-8, CXCL10/IP-10, thiab IL-1 tau raug ntsuas los ntawm cell-free supernatant siv cov khoom muag ELISA (R&D Systems, Wiesbaden, Germany) raws li cov chaw tsim khoom. cov lus qhia.
QUANTI-blue™ kev xeem
Rau kev txiav txim siab tus neeg sau xov xwm kev ua haujlwm ntawm SEAP hauv xov tooj ntawm tes kab lis kev cai supernatant los ntawm THP-1 Dual™ thiab coj mus rau TRIF-/- cells, QUANTI-Blue™ kev soj ntsuam tau ua raws li tus neeg tsim khoom raws tu qauv. Luv luv, cell culture supernatant tau sau tom qab stimulation ntawm cov hlwb nrog OMVs. Ua ntej, QUANTI-Blue™ Solution thiab cell culture supernatant tau muab tso rau hauv cov rog hauv qab 96-zoo phaj. Tom qab incubation ntawm 37 degree rau 150 min, optical ceev ntawm 630 nm tau txiav txim nyob rau hauv ib tug microplate nyeem infinite F200Pro.
QUANTI-luc™ assay
Txhawm rau txiav txim siab tus neeg sau xov xwm kev ua haujlwm ntawm Lucia luciferase hauv cell kab lis kev cai supernatant los ntawm THP{{0}} Dual ™ thiab sib raug TRIF-/- hlwb, QUANTI-Luc™ kev soj ntsuam tau ua raws li cov chaw tsim khoom raws tu qauv. Luv luv, cell culture supernatant tau muab tso rau hauv cov rog dawb-hauv qab 96-zoo phaj (BRAND GmbH & Co. KG, Wertheim, Germany). QUANTI-Luc ™ tshuaj ntsuam xyuas tau ntxiv thiab kev ntsuas luminescence tau ua tiav tam sim nrog 0.1 s lub sijhawm nyeem ntawv hauv microplate nyeem ntawv tsis kawg F200Pro.
Western blot
Rau kev txiav txim siab ntawm cov protein qhia lossis phosphorylation, Western Blot tau ua raws li tau piav qhia yav dhau los [18].
Kev npaj RNA thiab PCR ntawm lub sijhawm
Rau kev txheeb xyuas cov noob caj noob ces, kev cais RNA tau ua los ntawm phenol-chloroform extraction thiab thim rov qab raws li tau piav qhia yav dhau los [12]. Tus nqi ntawm PCR lub sijhawm tiag tiag tau ua tiav hauv ViiA7 (Thermo Fisher Scientific) nrog Luna Universal qPCR Master Mix (New England BioLabs) thiab cov khub primer tshwj xeeb. Los ntawm kev siv 2−ΔΔCT txoj kev [19], x-fold induction tau suav thiab cov txiaj ntsig tau zoo rau cov kab ke tswj hwm. 18S: fwd: 5′-GACTCTTTCGAGGCCCTGTA-3′, rev: 5′-CACCAGACTTGCCCTCCAAT-3′ CXCL8: fwd: 5′-ACTGAGAGTGATTGATTGAG TGGAC-3′, rev: CTTTGCACCA {20}}′ IFI44: fwd: 5′-TATCCAGAGAAGCAGCAGCTAC-3′, rev: 5′-ATAGAAGAGGCTAGCCGCTTC-3′ IFIT1: fwd: 5′-ATGCAGGAAGGAACATGACAACC{{31:5}', ′-TCTGGACACTCCATTCTATAGCG-3′ IFNA1: fwd: 5′-ACAGGAAGGACCTTGATGATGATGTC-3′, rev: 5′-TCTGCTGGATCAGTCATGG-3′ IFNB: fwd: 5′-ACAAGTACTAC , rev: 5′-TGTCCTTGAAGGCAGTATTCAAG-3′ IL1B: fwd: 5′-AGCTCGCCAGGTGAAAATGATGG-3′, rev: 5′-CAGGTCCTGAGGAAGGAAGCACTTC-3′ IL12GATCAGCAGAC: fwd: 58}}′, rev: 5′-CACCATTTCTCCAGGGGCAT-3′ Mx1: fwd: 5′-GGGCTTTGGAATTCTTGTGGC-3′, rev: 5′-CCTTGGAATGGTGGTGGGAT{{68}:′ 5NP': fwd -GAAATTTCAAACAGCTGCACAAAG -3′, rev: 5′-AATATGAGTGCAGACCGTGC-3′ RPS18: fwd: 5'-GCGGCGGAAAAATAGCCTTTTG-3′, rev: 5′-GATACACGTTCCTT{8}}
Immunofluorescence
THP{{0}} cov hlwb tau sib txawv ntawm cov ntawv npog los ntawm qhov sib ntxiv ntawm 20 nM PMA thiab incubated nrog OMVs/MVs rau 20 h. Cells tom qab kis tau tus kab mob H1N1 (A/WSN/33; ntau tus kab mob (MOI) 0.1) rau 4 h. Tom qab 15 min fixation nrog 3% paraformaldehyde, swb tau ntxuav peb zaug nrog PBS thiab permeabilized nrog 0.2% triton X-100 hauv TBS (10 min, chav tsev kub). Tom qab thaiv (1% BSA hauv TBS / 0.2% triton X-100), cov hlwb raug tsim nrog -NP antibody (1: 250, hauv kev thaiv kev daws). Cov tshuaj tiv thaiv theem nrab (1: 1000) ua ke nrog DAPI (1: 2000) tau muab tso rau hauv qhov tsaus ntuj rau 1 teev. Mounted coverlips tau soj ntsuam ntawm lub fluorescence microscope (AxioVision, Zeiss, Jena, Lub teb chaws Yelemees).
Hais lus ncaj ncees
Kev sim nrog tib neeg lub ntsws cov ntaub so ntswg tau txais kev pom zoo los ntawm pawg neeg saib xyuas kev ncaj ncees ntawm Hannover Medical School (MHH, Hannover, Lub teb chaws Yelemees) thiab ua raws li Txoj Cai ntawm Kev Ncaj Ncees ntawm Lub Koom Haum Kho Mob Ntiaj Teb (tus naj npawb 2701-2015). Txhua tus neeg mob lossis lawv cov txheeb ze tau sau ntawv tso cai rau kev siv cov ntaub so ntswg rau kev tshawb fawb. Txhua tus neeg pub ntshav tau lees paub sau ntawv tso cai (Ethics pom zoo tus lej: 161/17).
Fig. 1 Tus cwj pwm ntawm cov kab mob vesicles thiab cov lus teb hauv tib neeg macrophages. Kev sib cais ntawm cov kab mob extracellular vesicles los ntawm cov proteins dawb los ntawm qhov loj cais chromatography los ntawm cov kab mob sib txawv (Legionella pneumophila (Lp), Klebsiella pneumoniae (Kp), Escherichia coli (Ec), Salmonella enterica serovar Typhimurium (Sal), thiab Streptococcus (Sal) ). Vesicle concentration nyob rau hauv txhua feem yog txiav txim los ntawm nano-flow cytometry (nFCM) thiab cov proteins raug xam los ntawm BCA. B Vesicle loj faib ntawm purified OMVs/MVs tau txiav txim los ntawm NFC. C TEM cov duab ntawm purified OMVs/MVs. Scale bar=50 nm. D-G BDMs tau txhawb nqa nrog OMVs / MVs (1 µg / mL txhua) los ntawm cov kab mob sib txawv los yog sab laug tsis kho kom tswj tau ntev txog 48 teev. D CXCL8 tso tawm tau txiav txim los ntawm ELISA thiab tau piav qhia hauv ng/mL. Kev nthuav qhia ntawm IL1B (E) thiab IL12B (F) tau txiav txim siab los ntawm qPCR, cov txiaj ntsig tau zoo li qub rau RPS18 thiab tau piav qhia txog cov hlwb uas tsis tau kho. G Tom qab 1 h incubation nrog cov kab mob vesicles, kev qhia thiab phosphorylation ntawm IRAK-1, p38, thiab TBK-1 tau txiav txim los ntawm Western Blot. Cov neeg sawv cev tau tshwm sim ntawm plaub qhov kev ywj pheej ntawm kev ywj pheej rov ua dua tau qhia. Bars sawv cev qhov tseem ceeb + SEM los ntawm peb (B) mus rau plaub (D-F) kev sim ywj pheej. Statistics: 2- txoj kev ANOVA (DF); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns=not significant; n=3–4
Kev txheeb cais
Cov ntaub ntawv tau pom tias yog qhov tseem ceeb + SEM rau tsawg kawg peb qhov kev sim tshuaj lom neeg ywj pheej. Prism 6.07 (GraphPad, La Jolla, USA) tau siv. Qhov kev xeem ANOVA ib-lossis ob-txoj kev tau ua rau cov qauv tsis sib xws. P-tus nqi Tsawg dua lossis sib npaug rau 0.05 tau suav tias yog qhov tseem ceeb. Yog tias tsis qhia lwm yam, cov kev ntsuam xyuas tau ua nrog rau kev tswj hwm (*).
Muaj cov ntaub ntawv thiab cov ntaub ntawv
Tag nrho cov ntaub ntawv tsim los yog tshuaj xyuas thaum lub sijhawm kawm no suav nrog hauv kab lus no thiab nws cov ntaub ntawv ntxiv.
Cov txiaj ntsig
Pro-inflammatory activation ntawm macrophages los ntawm OMVs / MVs
Txhawm rau kuaj lub cev tiv thaiv kab mob hauv lub cev ntawm tib neeg thawj cov ntshav-derived macrophages (BDM) rau OMVs / MVs los ntawm cov kab mob sib txawv, cov hlwv los ntawm Legionella pneumophila (Lp), Klebsiella pneumoniae (Kp), Escherichia coli (Ec), Salmonella enterica serovar Typhimurium (Sal ) thiab Streptococcus pneumoniae (Sp) raug cais tawm ntawm qhov loj cais chromatography (SEC). Cov feem cuam tshuam tau txheeb xyuas cov vesicles los ntawm nano-flow cytometry (nFCM) thiab rau qhov muaj cov proteins los ntawm BCA (Fig. 1A). Cov vesicles los ntawm feem 7-12 tau sib sau ua ke thiab siv rau kev sim. OMVs/MVs muaj qhov sib piv qhov loj me me (Fig. 1B), tsis txawv qhov nruab nrab qhov loj me (Cov ntaub ntawv ntxiv 1: Fig. S2A), tau concentrated sib npaug (Cov ntaub ntawv ntxiv 1: Fig. S2B), thiab tau pom los ntawm kev xa hluav taws xob. microscopy (TEM; Fig. 1C). BDMs tau txhawb nqa nrog OMVs / MVs txog li 48 h, sib npaug rau qhov sib npaug ntawm cov kab mob sib txawv ntawm cov kab mob sib txawv (Cov ntaub ntawv ntxiv 1: Fig. S2C). Incubation ntawm BDMs nrog vesicles tsis yog cytotoxic (Cov ntaub ntawv ntxiv 1: Fig. S2D). OMVs/MVs broad induced CXCL8 qhia thiab tso tawm los ntawm macrophages (Ntxiv cov ntaub ntawv 1: Fig. S3A thiab 1D), thaum IL1B thiab IL12B mRNA induction yog lub sij hawm thiab hom-dependent (Fig. 1E + F). Kev tso tawm ntawm IL-1 yog nyob ntawm hom kab mob uas cov kab mob vesicles raug cais tawm (Cov ntaub ntawv ntxiv 1: Fig. S3B). Txawm hais tias phosphorylation ntawm p38 tau sib piv hauv BDMs tom qab 1 teev ntawm vesicle incubation, tsuas yog OMVs los ntawm gram-negative bacteria-induced degradation of IRAK-1 thiab phosphorylation of TBK-1 tab sis tsis MVs los ntawm gram-positive (Fig . 1G thiab Cov ntaub ntawv ntxiv 1: S4A-D).
Fig. 2 Cov kab mob extracellular vesicles qhib hom I interferon teb hauv BDMs. BDMs tau txhawb nqa nrog OMVs / MVs (1 µg / mL txhua) los ntawm cov kab mob sib txawv los yog sab laug tsis kho raws li kev tswj hwm. Phosphorylation thiab kev qhia ntawm IRF-3 tau txiav txim siab tom qab 1 teev ntawm OMV/MV incubation los ntawm Western Blot. Cov neeg sawv cev tshwm sim ntawm peb qhov kev ywj pheej ntawm kev ywj pheej yog qhia. Kev nthuav qhia ntawm IFNA1 (B), IFNB (C), IFIT1 (E), thiab IFI44 (F) tau ntsuas los ntawm qPCR, cov txiaj ntsig tau zoo li qub rau RPS18 thiab tau piav qhia txog cov hlwb tsis kho. D Phosphorylation ntawm STAT1 tau txiav txim siab los ntawm Western Blot tom qab 2 teev ntawm OMV / MV stimulation. Cov neeg sawv cev tshwm sim ntawm plaub yam kev ywj pheej ntawm kev ywj pheej yog qhia. G Phosphorylation ntawm STAT1 thiab kev qhia ntawm Mx1 tau txiav txim siab los ntawm Western Blot tom qab 20 teev ntawm cov kab mob vesicle stimulation. Cov neeg sawv cev ntawm plaub qhov kev ywj pheej ntawm kev ywj pheej yog piav qhia. Bars sawv cev qhov tseem ceeb + SEM los ntawm plaub qhov kev sim ywj pheej. Statistics: 2- txoj kev ANOVA; *p<0.05, **p<0.01, ****p<0.0001; n=4
Dhau li ntawm kev ua kom NF-κB thiab AP-1 thiab lawv cov phiaj xwm nqes hav, PRR signaling tuaj yeem ua rau kev ua kom IRFs. Yog li, phosphorylation ntawm IRF -3 tau txiav txim siab. Kp/Ec/SalOMVs nce phosphorylation ntawm IRF-3 hauv BDMs (Fig. 2A), uas ua rau kev qhia ntawm IFN I (Fig. 2B+C), downstream phosphorylation ntawm STAT1 (Fig. 2D thiab Ntxiv cov ntaub ntawv 1: S4E) thiab induction ntawm interferon-stimulated noob (ISGs; IFIT1, IFI44 thiab Mx1; Fig. 2E-G). Raws li cov xwm txheej phosphorylation feem ntau yog luv luv thiab paub tias ua lub luag haujlwm tseem ceeb hauv kev hloov lub cev tiv thaiv kab mob hauv thiab ntawm, phosphorylation ntawm STAT1 kuj tau tshuaj xyuas 20 h tom qab qhov sib ntxiv ntawm Kp / Ec / SalOMVs (Fig. 2G thiab Ntxiv cov ntaub ntawv 1: S4F+G). Cov kev sim tau pom tias OMVs tsis yog tsuas yog ua kom muaj kev cuam tshuam rau cov teeb meem hauv macrophages tab sis kuj muaj cov kab mob tiv thaiv kab mob.
OMV pre-incubation alters IAV replication nyob rau hauv macrophages
Mx1 yog qhov paub zoo txog tshuaj tiv thaiv kab mob inhibiting IAV replication [20], uas tau qhia raws li Kp / Ec / SalOMV kev kho mob ntawm macrophages. Yog li peb xav tias OMV kev kho ua ntej hloov pauv tus kab mob sib kis. IAV kev sim kab mob tau teeb tsa hauv THP-1 hlwb, uas qhia cov qib zoo sib xws ntawm Mx1 thaum kho Kp/Ec/SalOMV ntawm mRNA thiab protein ntau (Cov ntaub ntawv ntxiv 1: Fig. S5A+B) thiab induce phosphorylation ntawm STAT1 , thaum tag nrho STAT1 protein tseem nyob ruaj khov (Cov ntaub ntawv ntxiv 1: Fig. S5C), nrog rau H1N1 strain (A/WSN/33) uas kis tau thiab rov ua dua hauv macrophages [21]. Kev txhawb nqa ua ntej ntawm THP-1 hlwb nrog LpOMV/SpMV nce IAV replication 24 h tom qab kis kab mob (pi) piv rau kev tswj tus kab mob (–-), thaum kho ua ntej nrog Mx1-inducing OMVs (Kp /Ec/Sal) blocked IAV replication (Fig. 3A). Thaum TLR2/1 agonist Pam3CSK4 ua ntej kev kho mob tuaj yeem ua raws li cov txiaj ntsig tau pom nrog LpOMV / SpMV, uas yog ob qho tib si paub txog teeb liab ntawm TLR2/1 [12, 22, 23], soluble LPS raws li TLR4 agonist tsis rov tsim dua Kp / Ec. /SalOMV effect (Cov ntaub ntawv ntxiv 1: Fig. S5D). Qhov sib txawv ntawm IAV load tom qab kev kho mob ua ntej kuj tseem tuaj yeem pom los ntawm immunofluorescence staining tiv thaiv tus kab mob nucleoprotein (NP) (Fig. 3B). Kp/Ec/SalOMV pre-treatment blocked IAV replication and the NP-positive area was significantly txo (Fig. 3C). Qhov no tsis tuaj yeem pom tom qab LpOMV / SpMV kho ua ntej. Txhawm rau tshawb xyuas yog tias Mx1 induction txaus rau qhov pom tau thaiv IAV replication, Mx1 tau ruaj khov overexpressed (pom niam txiv) hauv THP-1 macrophages los ua OMV pre-stimulation (Cov ntaub ntawv ntxiv 1: Fig. S5E). Tus kab mob IAV ntawm Mx1oex hlwb ua rau txo qis ntawm immunofluorescent NP-zoo cheeb tsam 4 h pi (Fig. 3D) thiab hauv qhov txo qis tus kab mob replication 6 h pi piv rau qhov khoob vector tswj (VC) (Daim duab 3E) txawm tias tsawg dua. piv nrog OMV prestimulation (Fig. 3A). Hauv kev xaus, Kp / Ec / SalOMV tau qhib cov macrophages nyob rau hauv ib qho classical pro-inflammatory yam thiab induced antiviral noob uas ua rau kom IFN- / receptor (IFNAR), downstream phosphorylation ntawm STAT1 thiab kev qhia ntawm Mx1, uas tuaj yeem cuam tshuam ncaj qha rau IAV replication ( Fig. 4A). Txhawm rau kom paub meej qhov tseem ceeb ntawm JAK / STAT teeb liab rau qhov pom kev cuam tshuam ntawm cov tshuaj tiv thaiv kab mob, JAK inhibitor (JAKi) tau siv ua ntej vesicle stimulation. JAK inhibition tsis hloov Kp/SalOMV-induced CXCL8 kev qhia (Fig. 4B), tab sis txo qis Mx1 ntawm mRNA (Fig. 4C) thiab qib protein ntau txawm tias muaj kab mob IAV (Fig. 4D thiab Ntxiv cov ntaub ntawv 1: S6A). Raws li, JAKi thim rov qab OMV induced viral replication blockade (Fig. 4E).
Fig. 3 Mx1-inducing OMVs block influenza A virus replication in THP-1 cells. Tus kab mob Influenza A replication nyob rau hauv sib txawv THP-1 hlwb. Cells tau raug kho ua ntej nrog OMVs/MVs (1 µg/mL txhua) lossis tsis kho rau kev tswj hwm (–). Tom qab 20 h ua ntej kev kho mob, cov hlwb tau kis tus kab mob A/WSN/33(H1N1) (MOI 0.001) rau 24 thiab 48 teev. IAV replication raug txiav txim los ntawm qPCR tiv thaiv IAV-NP normalized rau 18S. Qhov txiaj ntsig txhais tau tias ± SEM ntawm peb mus rau tsib qhov kev sim ywj pheej tau pom. B Differentiated THP{17}} cov hlwb tau kho ua ntej nrog OMVs/MVs (1 µg/mL txhua) lossis sab laug tsis kho rau kev tswj hwm (–). Tom qab 20 teev ua ntej incubation, hlwb tau kis tus kab mob A/WSN/33(H1N1) (MOI 0.1) rau 4 teev. Tom qab kho, cov hlwb tau stained nrog ib qho -influenza NP antibody (daj) thiab DAPI (xiav). Cov neeg sawv cev tshwm sim los ntawm plaub qhov kev sim tshuaj lom neeg ywj pheej tau pom. C Quantification of NP positive (NP+) area from (B). Bars sawv cev qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. D THP-1 cells stably overexpressing Mx1 (Mx1oex) thiab khoob vector tswj (VC) hlwb tau kis tus kab mob npaws A/WSN/33(H1N1) (MOI 0.1) rau 4 h. Tom qab kho thiab immunofluorescence staining nrog -influenza NP, NP + cheeb tsam tau txheeb xyuas thiab tau piav qhia txog VC hlwb. Bars qhia tau hais tias qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. E Mx1oex thiab VC hlwb tau kis A/WSN/33(H1N1) (MOI 0.001) rau 6 h. Viral replication tau txiav txim siab los ntawm kev soj ntsuam cov quav hniav thiab cov txiaj ntsig tau piav qhia raws li cov kab mob sib sau ua ke (pfu) ib mL. Bars sawv cev qhov tseem ceeb + SEM ntawm plaub qhov kev sim ywj pheej. Statistics: 2- txoj kev ANOVA (A), 1- txoj kev ANOVA (C), unpaired t-test (D + E); *p<0.05, **p<0.01, ****p<0.0001; n=3–5
Antiviral teb rau OMVs yog TLR4-TRIF-dependent
Txhawm rau txheeb xyuas qhov cuam tshuam nrog PRR, kev lag luam tiv thaiv kab mob agonists tau siv ib leeg lossis ua ke nrog LPS los ua OMVs. Txawm li cas los xij, tsis muaj qhov tsom iav Mx1 induction tau pom nrog KpOMVs (Cov ntaub ntawv ntxiv 1: Fig. S7), qhia txog qhov sib txawv thiab ua kom ntev ntev ntawm macrophages los ntawm OMVs vim lawv cov ligand muaj pes tsawg leeg lossis kev nthuav qhia spatial rau PRRs. Te koom nrog kev tiv thaiv kab mob receptors tau soj ntsuam ntxiv los ntawm THP-1 cov xov tooj ntawm tes rau NF-κB- thiab IRF-signalling (Fig. 5A). Raws li kev kho KpOMV, THP-1 cov hlwb tau pom muaj kev nthuav qhia ntau ntxiv ntawm Mx1 protein (Fig. 5B thiab Ntxiv cov ntaub ntawv 1: S6B), uas tau ploj hauv TRIF tsis txaus (TRIF−/−) THP-1 hlwb ( Daim duab 5B thiab cov ntaub ntawv ntxiv 1: S6B). Ib yam li ntawd tau pom zoo rau IRF reporter (Fig. 5C), thaum NF-κB reporter activation thiab CXCL8 induction tau siab nyob rau hauv ob qho tib si THP-1 thiab TRIF-/- hlwb raws li stimulation (Fig. 5D + E). STAT-dependent IFIT1, IFI44, thiab Mx1 gene qhia tau tshwm sim los ntawm KpOMV stimulation hauv THP-1 cov xov tooj ntawm tes (Fig. 5E), thaum nws tau txo qis hauv qhov sib npaug ntawm TRIF-/- hlwb (Fig. 5E). Kev kis kab mob IAV ntxiv tom qab kev txhawb nqa ua ntej ua rau txo qis tus kab mob rov ua dua, qhov kev tshem tawm TRIF cawm IAV replication (Fig. 5F). Txhawm rau tshawb xyuas seb KpOMVs elicit cov lus teb dav dav dav dav hauv macrophages, cov hlwb tau kis tus kab mob vesicular stomatitis (VSV), uas rov ua dua hauv cov hlwb tswj, tab sis raug thaiv hauv KpOMV cov hlwb ua ntej kho thiab tuaj yeem cawm tau los ntawm TRIF deletion thiab (Fig. 5G). Txij li thaum kev lees paub ntawm LPS ntawm TLR4 yog qhov tseem ceeb rau endocytosis thiab TRIF-ua kom ua haujlwm, LPS ntawm OMV nto tau nruab nrab los ntawm lipopeptide tshuaj tua kab mob Polymyxin B (PB), ua rau 50% txo Mx1 induction (Fig. 6A + B thiab cov ntaub ntawv ntxiv 1. : S6C), tab sis tsis muaj qhov sib txawv tseem ceeb hauv NF-κB-dependent CXCL8 transcription (Fig. 6C). Viral replication tau cawm mus rau qib basal raws li KpOMV + PB pre-stimulation (Fig. 6D). Raws li inhibition ntawm LPS kev lees paub los ntawm PB tsis txaus los thaiv tag nrho Mx1, OMVs los ntawm endotoxin-dawb Clear coli (Cc) raug cais tawm thiab siv rau kev txhawb nqa ua ntej. Cc qhia txog qhov hloov pauv lipid A thiab tsis tuaj yeem ua rau lub tshuab ua haujlwm TLR4 / MD2 complex [24]. CcCOMVs tsis tau ntxias Mx1 (Fig. 6A+B thiab Ntxiv cov ntaub ntawv 1: S6C) thiab tsis txo IAV replication hauv macrophages (Fig. 6D). Nws yog qhov paub zoo tias kev cais tawm ntawm cov hlwv ntawm cov hlab ntsha ntawm qhov sib txawv ntawm qhov sib txawv ultracentrifugation ua rau muaj kev sib cais ntawm cov kab mob (xws li cov proteins lossis cov kab mob ntawm tes) nrog rau cov hlwv. Raws li MISEV2018 cov lus qhia [25], cov kev sim tseem ceeb tau rov ua dua nrog OMVs cais los ntawm kev sib xyaw ntawm ultrafiltration (UF) thiab qhov loj me tsis suav nrog chromatography (SEC). Kev txhawb nqa ntawm THP-1 cov neeg sau xov xwm nrog UF-SEC purified KpOMVs ua rau induction ntawm Mx1 (Cov ntaub ntawv ntxiv 1: Fig. S8A) thiab IRF reporter (Fig. S8B), thaum nws poob hauv TRIF−/− macrophages ( Cov ntaub ntawv ntxiv 1: Fig. S8A+B). IAV replication raug thaiv nyob rau hauv KpOMV pretreated hlwb thiab cawm thaum TRIF deletion (Ntxiv cov ntaub ntawv 1: Fig S8C). NF-κB kev tshaj tawm xov xwm tau raug ntxias los ntawm KpOMVs tsis hais txog TRIF xwm txheej ntawm cov hlwb (Cov ntaub ntawv ntxiv 1: Fig. S8D).
Daim duab 4 Inhibition ntawm JAK-signalling cawm tus kab mob khaub thuas A tus kab mob replication hauv macrophages. Ib qho OMV qhib TRIF-IRF-signalling nyob rau hauv macrophages, uas nyob rau hauv lem induces IFN- qhia thiab tso tawm nrog tom qab IFNAR-signalling. IFNAR teeb liab ntawm JAK / STAT thiab ua rau muaj kev qhia ntawm ISGs, ib qho yog Mx1. Qhov no ua rau qhov thaiv ntawm IAV replication. B–E THP-1 cov hlwb tau ua ntej incubated rau 1 h nrog 10 µM JAK inhibitor (JAKi) ua ntej ntxiv OMVs (1 µg/mL; Kp/Sal) rau 2{{22 }} h. B–C CXCL8 (B) thiab Mx1 (C) qhia tau txiav txim siab los ntawm qPCR thiab cov txiaj ntsig tau zoo li qub rau RPS18 thiab qhia txog tus txheeb ze rau kev tswj tsis tau kho. Bars qhia tau hais tias qhov tseem ceeb ntawm peb mus rau plaub qhov kev sim ywj pheej + SEM. D+E OMV cov hlwb ua ntej kho tau ntxiv nrog IAV (MOI 0.001). D Western blot qhia Mx1 protein qhia ntawm 0-3 h tom qab kis kab mob (pi). E Viral replication tau txiav txim siab los ntawm cov tshuaj plaque 24 teev tom qab kis kab mob. Bars qhia tau hais tias qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. Statistics: 1-way ANOVA (B+C+E); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; *compared to DMSO control, # as depicted in the graph; ns=not significant; n=4
Fig. 5 OMVs induce antiviral signaling nyob rau hauv macrophages ntawm TRIF. A THP-1 Dual reporter cells induce the expression of secreted embryonic alkaline phosphatase (SEAP) after activation of the NF-κB pathway and the expression and secretion of Lucia luciferase when activation of IRF pathway. Tsis tas li ntawd, TRIF (grey bars) tau diferentiated thiab tom qab ntawd txhawb nrog −/- hlwb muaj qhov ruaj khov knockout ntawm lub adapter molecule TRIF. (BF) THP-1 xov tooj ntawm tes (=Dual; dub bars) thiab TRIF−/− cells KpOMV (1 µg/mL) rau 20 h lossis sab laug tsis kho rau kev tswj. Tom qab lub sijhawm qhia, supernatant, RNA thiab / lossis cov protein tau sau. B Tus Neeg Sawv Cev Western Blot duab ntawm Mx1 protein qhia. C + D Lucia reporter act (C) thiab SEAP reporter act (D) tau txiav txim siab hauv cell culture supernatant. Tib lub supernatant tau siv los txiav txim qhov kev ua ntawm ob tus neeg sau xov xwm. E txheeb ze mRNA kev qhia rau STAT- thiab NF-κB-nyob ntawm lub hom phiaj cov noob (los ntawm sab saum toj mus rau hauv qab: IFIT1, IFI44, Mx1, CXCL8) tau txiav txim siab los ntawm qPCR thiab cov txiaj ntsig tau normalized rau RPS18 thiab qhia txog tus txheeb ze rau kev tswj tsis tau Dual. Fold hloov tau log2 hloov. Cells tau kis tus kab mob A/WSN/33(H1N1) (MOI 0.1) rau 24 h (F) los yog VSV (MOI 0.1) rau 12 h (G). Viral replication tau txiav txim siab los ntawm kev soj ntsuam plaque. Bars qhia qhov tseem ceeb ntawm plaub (C–F) rau fve (G) kev sim ywj pheej + SEM. Statistics: 2-way ANOVA (C–G); *p<0.05, **p<0.01, ****p<0.0001; * compared to unstimulated Dual control, # as depicted in the graph; ns=not signifcant; n=4–5
Daim duab 6 OMV-induced Mx1 qhia tau ploj tom qab LPS inhibition. THP-1 hlwb raug incubated rau 20 h nrog OMVs (1 µg/mL; Kp los yog Clear coli (Cc)) ib leeg los yog ua ke nrog 20 µg/mL Polymyxin B (PB) los yog sab laug tsis kho. rau kev tswj. Ib qho Mx1 protein qhia tau txiav txim los ntawm Western Blot. Cov neeg sawv cev ntawm peb qhov kev sim tshuaj lom neeg ywj pheej tau pom. Mx1 B thiab CXCL8 C qhia tau txiav txim los ntawm qPCR. Bars qhia tau hais tias qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. D THP-1 hlwb raug incubated rau 20 h nrog OMVs (1 µg/mL; Kp los yog Cc) ib leeg los yog ua ke nrog 20 µg/mL PB los yog sab laug tsis kho rau kev tswj thiab ces kis tau tus kab mob A/WSN/33. (H1N1) (MOI 0.001). Tus kab mob replication tau txiav txim siab los ntawm cov tshuaj plaque 24 teev tom qab kis kab mob. Bars qhia tau hais tias qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. Statistics: 1- txoj kev ANOVA B–D; *p<0.05, **p<0.01, ****p<0.0001; *compared to control, #compared to KpOMV; ns=not significant; n=3–4
IAV replication-inhibiting effect ntawm OMVs yog hloov mus rau AECs
Txij li thaum macrophages tsis yog hom cell tseem ceeb rau IAV replication nyob rau hauv lub ntsws thiab raws li IFN kuv kuj ua paracrine, kev hloov pauv ntawm IAV replication nyhuv rau AECs tau sim, vim cov hlwb no, tsis zoo li macrophages, tsis ncaj qha teb rau OMVs/MVs ( Cov ntaub ntawv ntxiv 1: Fig. S9). Supernatant (SN) los ntawm KpOMV-kho macrophages induced Mx1 hauv A549 hlwb, nyob rau hauv sib piv rau LpOMV-SN (Fig. 7A), thaum CXCL8 yog induced tom qab ob leeg (Fig. 7B). KpOMVSN txo tus kab mob replication (Fig. 7C), uas yog nyob rau hauv txoj kab nrog macrophage thwmsim thiab qauv ntawm Mx1 induction. Txhawm rau kom paub meej qhov kev cia siab ntawm qhov pom ntawm IAVlimiting nyhuv ntawm JAK / STAT-signalling, KpOMV-SN tau ua ke nrog JAKi, thaiv Mx1 induction raws li KpOMV-SN-kev kho mob (Daim duab 7A) thiab kho IAV replication (Fig. 7C). Txhawm rau ua raws li txoj cai tswj hwm txoj kev tiv thaiv kab mob hauv lub ntsws, ex vivo IAV kab mob ntawm tib neeg precision-cut lung slices (PCLS) tau teeb tsa. Lp/KpOMVs tsis yog cytotoxic rau PCLS (Fig. 8A) thiab kev sib xyaw ua ke ntawm OMV pre-stimulation thiab IAV kab mob ua rau Mx1 induction (Fig. 8B) thiab CXCL10 tso tawm (Fig. 8C). KpOMVs tab sis tsis LpOMVs txo qhov IAV replication hauv PCLS (Fig. 8D). Te pom cov teebmeem raug tshem tawm los ntawm kev kis kab mob nrog UV inactivated IAV (Fig. 8A–C). Peb qhov kev tshawb pom ua rau tus qauv ntawm lub ntsws o nyob rau hauv AMs teb rau OMVs los ntawm gram-tsis zoo cov kab mob nrog induction ntawm IFN I, inducing antiviral teb nyob rau hauv ib tug autocrine yam hauv AMs thiab nyob rau hauv ib tug paracrine yam nyob rau hauv AECs nyob rau hauv vitro thiab nyob rau hauv ib tug ex vivo PCLS qauv. (Fig. 8E).
Fig. 7 Influenza A virus replication-inhibiting effect of OMVs is transferable to AECs. THP-1 hlwb tau incubated nrog LpOMV los yog KpOMV rau 20 h los yog sab laug tsis kho rau kev tswj. Lub supernatant (SN) tau lim dej tsis huv thiab siv rau kev txhawb nqa ua ntej ntawm A549 hlwb rau 20 h ib leeg lossis ua ke nrog 10 µM JAKi. A+B Mx1 (A) thiab CXCL8 B qhia tau txiav txim los ntawm qPCR thaum lub sijhawm kis tus kabmob (0 h pi). Bars qhia tau hais tias qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM normalized rau cov hlwb tsis kho. C Pre-kho A549 hlwb ntxiv tau tus kab mob npaws A/Hamburg/5/2009(H1N1pdm) (MOI 0.01) rau 24 teev. Viral replication tau txiav txim siab los ntawm kev soj ntsuam plaque. Bars yog qhov tseem ceeb ntawm plaub qhov kev sim ywj pheej + SEM. Statistics: 1- txoj kev ANOVA; **p<0.01, ****p<0.0001; * compared to control-SN; # compared to KpOMV-SN; n=4
Kev sib tham
Peb pom tias vesicles los ntawm cov kab mob sib txawv ua rau muaj qhov sib txawv ntawm cov cim hauv macrophages uas tuaj yeem txwv IAV rov ua dua hauv kev kis kab mob tom ntej. Raws li cov ntaub ntawv, cais OMVs los ntawm Lp, Kp, Ec, thiab Sal, thiab MVs los ntawm Sp tau muab piv rau qhov loj [26–28]. Macrophages incubated nrog cov kab mob vesicles tsis qhia cytotoxicity [29, 30], tab sis induced ib tug pro-inflammatory teb. Tag nrho cov vesicles ua rau p38 phosphorylation thiab tso tawm ntawm CXCL8 thiab IL-1 los ntawm thawj tib neeg macrophages, thaum muaj qhov sib txawv ntawm qhov induction ntawm IFN kuv thiab lawv cov downstream signaling. Peb thiab lwm tus twb tau pom tias LpOMVs teeb liab ntawm TLR2 [12, 22] thiab tseem SpMVs tau ua rau muaj qhov txawv txav hauv macrophages [31]. LpOMVs/SpMVs activated pro-inflammatory NF-κB hom noob, raws li tau piav qhia rau cov kab mob ntawm keeb kwm [12, 22, 31]. Txawm li cas los xij, kev lees paub ntawm Kp/Ec/SalOMVs provoked, ntxiv rau NF-κB lub hom phiaj noob, qhov induction ntawm IRF{18}} phosphorylation nrog downstream IFNB qhia thiab ntev phosphorylation ntawm STAT1 ua ke nrog induction ntawm ISGs, sib cav rau active IFNAR-JAK/STAT signaling. OMVs nqa LPS ntawm lawv qhov chaw, tab sis qhov muaj peev xwm ua rau OMVs tsis tuaj yeem ua raws li LPS ntshiab. Peb thiab lwm tus tau pom tias macrophages muaj kev nkag siab ntau dua rau OMVs piv rau tib tus nqi ntawm LPS ntshiab [32, 33]. OMVs muaj cov lej ntawm kev tiv thaiv kab mob agonists uas xav tau rau kev txhawb nqa lub cev tiv thaiv kab mob [32]. Kev lag luam TLR thiab RIG-I agonists ib leeg lossis ua ke nrog LPS tsis tuaj yeem ua raws li cov txiaj ntsig. Nws tau tshwm sim ua ntej tias LPS-loaded liposomes ua rau lub sijhawm ua haujlwm ntev dua ntawm TRIF-IRF-3 hauv macrophages piv rau LPS dawb [34]. Los ntawm kev siv TRIF-/- macrophages, peb tau pom qhov kev vam khom ntawm ISG induction ntawm no adapter molecule. Ntxiv mus, endotoxin-dawb CcCOMVs lossis OMVs ua ke nrog LPSmasking PB tau txaus los tshem tawm cov lus teb ntawm macrophages. Qhov no qhia txog kev lees paub ntawm OMVs ntawm TLR4 nrog cov endocytosis tom qab thiab TRIF-signalling. Raws li, Kp / Ec / SalOMVs tuaj yeem ua rau muaj kev cuam tshuam rau kev ua haujlwm ntawm macrophages ntawm TLR4 ua rau MyD88 signaling thiab NF-κB nyob ntawm cov noob qhia nrog rau TRIF signaling nrog IFN kuv teb. Qhov no yog nyob rau hauv txoj kab nrog cov ntaub ntawv uas qhia tias TLR4 thiab TRIF ua ke yuav tsum tau rau lub immunogenicity ntawm Neisseria meningitidis OMVs nyob rau hauv nas [35]. Raws li hom I IFNs yog tus tswv tswj hwm ntawm cov lus teb tiv thaiv kab mob, peb xav tias kev lees paub ntawm Kp / Ec / SalOMVs cuam tshuam rau IAV replication. Peb pom tau hais tias macrophages zoo thaiv IAV thiab VSV replication tom qab TRIF-activate OMV pre-treatments, thaum TRIF-/- cawm tus kab mob replication. Txhawm rau txheeb xyuas qhov cuam tshuam ntawm IFNAR signaling, JAK1/2 inhibition ua ke nrog OMVs, ua tiav STAT1- nyob ntawm Mx1 induction thiab cawm tus kab mob rov ua dua hauv macrophages. Kev sib xyaw ntawm KpOMVs nrog PB lossis daim ntawv thov ntawm endotoxin-dawb CcCOMVs tsis txo IAV replication hauv macrophages vim lawv tsis muaj peev xwm inducing antiviral noob. Raws li extracellular vesicle npaj tau los ntawm ultracentrifugation muaj cov protein dawb thiab cov kab mob ntawm tes txuas nrog rau cov hlab ntsha, OMV kev npaj tau ntxiv los ntawm kev sib xyaw ntawm ultrafiltration thiab loj cais chromatography [25].
Suav tshuaj ntsuab cistanche cog-Antitumor
Cov kev npaj cov vesicle ntshiab tau sib npaug tuaj yeem ua rau TRIF-IRF signaling nrog downstream Mx1 induction thiab thaiv cov kab mob sib txuas sib cav rau cov nyhuv ncaj qha vesicle. Txawm hais tias ATMs yog thawj kab ntawm kev tiv thaiv hauv lub ntsws, feem ntau ntawm IAV kab mob thiab rov ua dua hauv tib neeg lub ntsws tshwm sim hauv AECs [36]. Yog li, AECs tau txhawb nqa nrog OMVs, tab sis lawv tsis teb rau qhov kev txhawb nqa zoo li macrophages. Tsuas yog bronchial epithelial cell kab BEAS2B teb nrog CXCL8 qhia, tab sis tsis muaj Mx1 induction. Txij li thaum peb tuaj yeem pom cov kab mob tshuaj tiv thaiv kab mob nyob ntawm TLR4 thiab TRIF hauv macrophages, peb xav tias plaub cov kab mob epithelial cell tsis qhia TLR4 mus rau qhov zoo sib xws li macrophages thiab tias lawv ua tsis tau qhov endocytose OMVs thaum ua kom TLR4. Qhov no yog nyob rau hauv txoj kab nrog cov ntaub ntawv qhia tias cov tib neeg AECs cais tsis teb rau LPS raws li khub AMs tau ua [37]. Raws li peb txuas cov tshuaj tiv thaiv kab mob raws li OMV pre-incubation rau IFN kuv, peb siv lub supernatant ntawm OMV-stimulated macrophages rau pre-stimulate AECs. Qhov nruab nrab nruab nrab ntawm KpOMV-stimulated macrophages, tsis zoo li cov hlwv ncaj qha stimulation, induced Mx1 qhia nyob rau hauv cov hlwb thiab txo IAV replication nyob rau hauv cov kev sim kab mob tom ntej. Supernatant los ntawm LpOMV-kho macrophages induced CXCL8 nyob rau hauv epithelial hlwb tab sis ua rau tsis muaj Mx1 induction thiab kev hloov hauv viral replication. Txhawm rau muab cov txiaj ntsig tau pom nrog cov xov xwm txias rau IFN I, tus supernatant tau ua ke nrog JAK1 / 2 inhibitor, thaiv Mx1 induction, thiab tsis muaj kev cuam tshuam rau cov kab mob sib kis. Txij li kev sib cuam tshuam ntawm ntau hom ntawm tes hauv tib neeg lub ntsws yog qhov nyuaj thiab tsis tuaj yeem ua tiav los ntawm cov xov xwm txias, peb tau txuas ntxiv mus rau qhov chaw siv tau ntawm tib neeg lub ntsws ntsws, uas tau siv dav rau kev tshawb fawb ntawm tus tswv tsev-cov kab mob sib cuam tshuam nrog rau kab mob khaub thuas thiab kab mob kab mob [38–41]. Kev sib piv cov qauv siv hauv vitro, ex vivo tib neeg lub ntsws cov ntaub so ntswg tswj hwm peb-seem qauv ntawm lub ntsws thiab tso cai rau kev sib koom ua ke ntawm lub cev ntawm cov neeg nyob hauv lub ntsws, tab sis nws tsis muaj peev xwm ntawm kev nkag mus ntawm cov kab mob tiv thaiv kab mob ntxiv. Kev kis kab mob sib law liag ntawm KpOMV-stimulated PCLS nrog IAV txo qis cov kab mob sib kis piv rau kev tswj tsis tau kho ua ntej. Raws li tag nrho cov AECs kuaj hauv vitro tsis teb rau cov kab mob ncaj qha vesicle stimulation, peb xav tias nyob rau hauv tus qauv ex vivo no, AMs yog hom xovtooj uas tseem ceeb tshaj plaws. Txij li thaum tus kab mob khaub thuas cais tawm tau raug xaiv rau cov kab mob tom ntej uas tsuas yog kis tau thiab rov ua dua hauv AECs, nws tuaj yeem xav tias qhov no yog cov tshuaj tiv thaiv kab mob paracrine los ntawm AMs thiab kis mus rau AECs. Raws li cov ntaub ntawv tau txais ntawm no, peb npaj cov qauv hauv qab no (Daim duab 8E): OMVs los ntawm cov kab mob gram-tsis zoo tuaj yeem ua rau tib neeg cov macrophages hauv cov txheej txheem pro-inflammatory thiab anti-virally prime lawv ntawm TLR4 thiab TRIF tom qab ua tiav endocytosis ntawm lub cov hlab ntsha. IFN Kuv tso tawm tuaj yeem ua rau macrophages thiab / lossis AECs tshuaj tiv thaiv kab mob ntawm IFNAR thiab JAK / STAT signaling. Kev kis kab mob tom qab ntawm cov hlwb no ua rau Mx1- txo qis hauv cov kab mob kis. Txij li thaum peb tsis muaj kev nkag mus rau thawj tib neeg AMs, cov kev sim tau ua siv tib neeg BDMs ua qauv. Xav txog qhov sib txawv ntawm cov hauv paus chiv keeb thiab kev loj hlob ntawm cov macrophages, nws yog qhov pom tau tias AMs yuav tau pom qhov tsis muaj zog tiv thaiv kab mob hauv qhov sib piv rau BDMs. Cov kab mob vesicles twb tau siv rau hauv cov tswv yim txhaj tshuaj tiv thaiv kab mob ntsws sib txawv (saib hauv [42]). Yog li ntawd, OMVs tsis yog tsuas yog sawv cev rau cov cuab yeej rau cov tswv yim txhaj tshuaj tiv thaiv kab mob tiv thaiv lawv cov kab mob hauv zos tab sis kuj tuaj yeem siv hauv zos los tiv thaiv kab mob los ntawm kev ua kom cov neeg nyob hauv lub cev tiv thaiv kab mob. Cov kev tshawb fawb yav tom ntej hauv vivo yuav tsum tau kuaj seb cov kev tshawb pom hauv vitro puas siv tau rau lwm cov kab mob kis. Tsis tas li ntawd, nws yuav tsum tau muab sau tseg tias tej zaum yuav tshwm sim endotoxic cov lus teb yuav tsum tau saib xyuas kom tsis txhob exaggerated tiv thaiv kab mob nyob rau hauv vivo. Qhov induction ntawm hom I IFN yuav tsum tau tswj kom nruj, raws li nws tau pom nyob rau hauv cov neeg noj qab haus huv uas nqus tau pa ntawm TLR7 agonist tau pib zoo tom qab thawj koob, tab sis ua rau muaj kev nce ntxiv TNF- thiab IFN I teb thiab cov tsos mob zoo li mob khaub thuas. , tom qab koob thib ob [43]. Txhawm rau tiv thaiv cov teebmeem tsis zoo, kev hloov pauv caj ces ntawm OMVs yuav xav tau, ua rau muaj kev tiv thaiv kab mob sib npaug thiab ua tiav kev siv tau zoo. Ua ke, peb nthuav tawm cov qauv ntawm yuav ua li cas OMVs tuaj yeem ua rau muaj kev cuam tshuam cov kab mob hauv tib neeg macrophages thiab yuav siv li cas los tiv thaiv IAV replication.
Fig. 8 KpOMV txo tus mob khaub thuas A tus kab mob replication nyob rau hauv tib neeg precision-txiav lub ntsws slices. Tib neeg PCLS raug tsim los nrog 1 µg/mL OMVs (Lp/Kp) rau 20 teev thiab tom qab ntawd kis tus kab mob khaub thuas A/California/04/2009(H1N1pdm) rau 48 teev. UV inactivation ntawm tus kab mob ua haujlwm los tswj. Cytotoxicity tau txiav txim los ntawm qhov ntau ntawm LDH tso tawm los ntawm PCLS thiab tau piav qhia hauv % piv rau tag nrho cov lysis. B Mx1 kev qhia tau ntsuas los ntawm qPCR thiab nthuav tawm txheeb ze rau RPS18 thiab tsis kho PCLS. C CXCL10 tso tawm tau txiav txim los ntawm ELISA thiab tau piav qhia hauv ng / mL. D Viral replication tau txiav txim siab los ntawm kev soj ntsuam cov quav hniav thiab tau piav qhia hauv pfu/mL. Bars qhia tau hais tias qhov tseem ceeb ntawm peb mus rau tsib qhov kev hloov pauv lom neeg + SEM. E Cov qauv tsim rau induction ntawm kev tiv thaiv kab mob ntawm OMVs hauv lub ntsws. Statistics: 1- txoj kev ANOVA (AC), Friedman-test (D); *p<0.05, ***p<0.001, ****p<0.0001; * compared to IAV infected, but not pre-treated PCLS, # as depicted in the graph; ns=not significant; n=5
Cov ntaub ntawv
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