Modulation Ntawm Aryl Hydrocarbon Receptor Signaling Pathway Impacts On Junín Virus Replication Part 1

Abstract:

Junín tus kab mob (JUNV), ib tug tswv cuab ntawm tsev neeg Arenaviridae, yog tus etiological tus neeg sawv cev ntawm Argentine hemorrhagic fever, ib tug kab mob endemic nyob rau hauv cheeb tsam nyob deb nroog ntawm Argentina tsis muaj cov tshuaj kho mob tshwj xeeb. Aryl hydrocarbon receptor (AHR) yog qhia nyob rau hauv ob peb cov ntaub so ntswg mammalian thiab tau qhia tias yog ib tug sensor ntawm ligands los ntawm txawv qhov chaw thiab ib tug modulator ntawm lub cell tiv thaiv teb.

Tus kab mob Junin yog ib yam kab mob tsawg heev uas cuam tshuam rau tib neeg lub cev tiv thaiv kab mob thiab ua rau lub cev tiv thaiv kab mob hnyav. Txawm li cas los xij, kev tiv thaiv tsis yog tib lub tswv yim uas tsis tuaj yeem tiv thaiv tus kab mob Junin.

Ua ntej, kev so kom txaus thiab pw tsaug zog yog qhov tseem ceeb los ua kom koj lub cev tsis muaj zog. Thaum pw tsaug zog, lub cev tso tawm cov tshuaj hu ua cytokines uas pab lub cev tiv thaiv kab mob ua haujlwm zoo dua rau cov kab mob thiab lwm yam kev hem thawj.

Qhov thib ob, kev noj zaub mov zoo kuj yog tus yuam sij rau kev txhim kho kev tiv thaiv. Noj cov zaub mov uas muaj cov vitamins C thiab D, xws li citrus txiv hmab txiv ntoo thiab nceb, tuaj yeem pab txhawb koj lub cev tiv thaiv kab mob.

Tsis tas li ntawd, kev tawm dag zog me ntsis kuj tuaj yeem txhim kho kev tiv thaiv. Kev tawm dag zog kom zoo tuaj yeem ua rau muaj cov qe ntshav dawb hauv lub cev, yog li txhim kho lub peev xwm ntawm lub cev.

Thaum kawg, lub hlwb zoo kuj tseem ceeb heev rau kev noj qab haus huv ntawm lub cev tiv thaiv kab mob. Kev ntxhov siab thiab kev ntxhov siab tuaj yeem cuam tshuam rau lub cev tiv thaiv kab mob, yog li nws tseem ceeb heev kom nyob twj ywm thiab zoo.

Hauv luv luv, txawm hais tias tus kab mob Junin muaj kev cuam tshuam rau lub cev tiv thaiv kab mob, los ntawm kev txhim kho peb txoj kev ua neej thiab tswj tus cwj pwm zoo, peb tuaj yeem txhim kho peb txoj kev tiv thaiv kab mob thiab tiv thaiv thiab teb cov kab mob tawm tsam. Los ntawm qhov kev xav no, peb yuav tsum txhim kho peb txoj kev tiv thaiv. Cistanche tuaj yeem txhim kho kev tiv thaiv zoo, vim tias cov hmoov tshauv nqaij muaj ntau yam khoom siv roj ntsha, xws li polysaccharides, ob lub nceb, Huang Li, thiab lwm yam. Cov khoom no tuaj yeem txhawb kev tiv thaiv kab mob ntau yam ntawm cov hlwb hauv lub cev, ua kom lawv lub cev tiv thaiv kab mob.

Nyem cistanche deserticola ntxiv

Qhov zoo siab, cov kev tshawb fawb tsis ntev los no tau qhia tias kev ua kom muaj zog lossis kev nyuaj siab ntawm AHR txoj kev taw qhia tuaj yeem ua lub luag haujlwm hauv qhov tshwm sim ntawm ntau haiv neeg kis kab mob. Hauv tsab ntawv tshaj tawm tam sim no, cov txiaj ntsig ntawm kev kho tshuaj kho mob ntawm AHR ntawm JUNV hauv vitro kis tau raug tshuaj xyuas. Qhov kev tshuaj ntsuam microarray thawj zaug tau pom tias txoj hauv kev AHR tau nthuav tawm ntau dhau hauv cov kab mob siab JUNV.

Concomitantly, tus kab mob ntawm Vero thiab Huh -7 hlwb nrog JUNV hom IV4454 thiab Candid #1 tau cuam tshuam loj heev nyob rau hauv ib koob tshuaj raws li kev kho mob nrog CH223191, ib tug tshwj xeeb AHR antagonist, raws li tau pom los ntawm kev kuaj kab mob, tiag- Lub sij hawm RT-PCR thiab immunofluorescence nrhiav pom cov kab mob kis kab mob. Tsis tas li ntawd, lub luag haujlwm tiv thaiv kab mob ntawm AHR hauv kev kis tus kabmob JUNV zoo li tsis muaj kev ywj pheej ntawm IFN-I txoj hauv kev. Peb qhov kev tshawb pom txhawb nqa qhov kev pom zoo ntawm kev kho tshuaj kho mob ntawm AHR raws li lub hom phiaj muaj peev xwm rau kev tswj hwm ntawm AHF.

Ntsiab lus:

Aryl hydrocarbon receptor; Tus kab mob Junín; arenavirus; Argentine hemorrhagic kub taub hau; lub hom phiaj kho mob.

1. Taw qhia

Tam sim no, muaj ntau cov pov thawj txhawb nqa tias aryl hydrocarbon receptor (AHR) yog ib qho tseem ceeb modulator ntawm tus tswv lub cev tiv thaiv kab mob thiab cov yam ntxwv no pab txhawb rau qhov sib txawv ntawm kev kho mob tshwm sim ntawm tus kab mob ntawm tus kheej thiab cov pej xeem theem [1,2]. AHR yog ib qho cytoplasmic receptor ubiquitously qhia nyob rau hauv cov ntaub so ntswg mammalian, ua hauj lwm raws li ib tug sensor ntawm ntau yam endogenous thiab exogenous ligands nrog txawv keeb kwm xws li cov pa phem, noj, microbiome, thiab host metabolism [3-5].

Qhov tseeb, nws tau pom zoo tias AHR muaj kev cuam tshuam loj rau kev sib cuam tshuam ntawm ib puag ncig yam xwm txheej thiab kis kab mob [1,2]. Thaum AHR lees paub lub ligand molecule, nws tau hloov mus rau lub nucleus qhov twg nws heterodimerizes nrog aryl hydrocarbon receptor nuclear translocator thiab tswj cov kev qhia ntawm xenobiotic metabolism genes (CYP1A1, CYP1A2, CYP1B1).

AHR tseem tuaj yeem cuam tshuam nrog lwm yam kev hloov pauv, xws li NF-kB, los hloov kho ntau txoj hauv kev qhia, suav nrog cov koom nrog hauv kev tiv thaiv kab mob [6–8]. AHR tuaj yeem hloov kho qhov kev qhia ntawm cov tshuaj tiv thaiv kab mob thiab hloov txoj kev sib txawv ntawm cov kab mob dendritic thiab T-cells [9-11].

Cov kev tshawb fawb tsis ntev los no tau ua los ntawm peb pab pawg tshawb fawb tau pom tias kev ua kom lub AHR teeb liab txoj hauv kev ua lub luag haujlwm tiv thaiv kab mob thaum lub sij hawm Zika (ZIKV), dengue (DENV), [12] thiab mob hnyav ua pa mob coronavirus 2 (SARS-CoV{{ 4}}) kab mob [13]. Nco ntsoov, nws tau lees paub tias AHR inhibition ua rau txo qis tus kab mob rov ua dua tshiab hauv cov qauv kab mob no, qhia tias AHR sawv cev rau kev cog lus tshuaj tiv thaiv kab mob.

Junín virus (JUNV) yog ib lub hnab ntim RNA genome virus los ntawm tsev neeg Arenaviridae [14]. Txhua ntu RNA encodes rau ob lub proteins nyob rau hauv cov lus qhia. Ntawm ib sab, ntu loj (L RNA) encodes rau RNA-dependent RNA polymerase (L) thiab matrix protein (Z).

Ntawm qhov tod tes, qhov me me (S RNA) encodes rau nucleoprotein (NP) thiab rau glycoprotein precursor (GPC), uas yog cleaved los ntawm proteases los tsim peb subunits (GP1, GP2, thiab lub teeb liab peptide SSP) uas yog. tuav ua ke los tsim glycoprotein complex [14]

JUNV yog tus etiological tus neeg sawv cev ntawm Argentine hemorrhagic fever (AHF) [15], ib qho kab mob endemic cuam tshuam rau cov neeg ua haujlwm nyob deb nroog hauv thaj av Pampa ntawm Argentina. Nco ntsoov, tus yam ntxwv ntawm AHF yog qhov dav ntawm cov tsos mob tshwm sim los ntawm cov neeg mob. Asymptomatic lossis mob me, nrog cov tsos mob zoo li mob khaub thuas, tshwm sim hauv 70-80 feem pua ​​​​ntawm cov kab mob. Txawm li cas los xij, cov mob hnyav cuam tshuam nrog hemorrhagic thiab neurological teeb meem nrog kev tuag ntawm 20 mus rau 30 feem pua ​​​​ntawm cov neeg mob hauv tsev kho mob [15].

Qhov tseem ceeb, ib qho tshuaj tiv thaiv kab mob hu ua Candid #1 tau ua pov thawj tias muaj txiaj ntsig zoo hauv kev tiv thaiv tus mob hnyav ntawm AHF [15,16]. Txawm li cas los xij, tsuas yog kev kho mob uas muaj tawm tsam tus kab mob no yog kev tswj hwm lub cev tsis muaj zog plasma los ntawm cov neeg mob convalescent; ib qho kev kho mob uas tau pom tias txo cov neeg mob tuag tsuas yog siv li ntawm 8 hnub tom qab pib cov tsos mob thawj zaug [17].

Txawm hais tias nyob rau xyoo tas los no ntau daim ntawv pov thawj tau sau los ntawm cov txiaj ntsig ntawm AHR thaum kis kab mob, tseem tsis muaj pov thawj pom tseeb ntawm nws lub luag haujlwm thaum kis tus kabmob arenavirus [2].

Raws li cov pej xeem nyob deb nroog raug cuam tshuam los ntawm AHR ligands, xws li cov pa phem los ntawm kev lag luam ua liaj ua teb xws li agrochemicals, tshuaj tua kab, thiab fossil fuel combustion by-products [18], peb tau soj ntsuam hauv txoj haujlwm no qhov cuam tshuam ntawm in vitro AHR pharmacological modulation ntawm lub cellular teb rau JUNV kab mob thiab muaj peev xwm siv AHR raws li ib tug neeg sib tw lub hom phiaj rau kev kho mob nyob rau hauv AHF kab mob.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kab Kab

Cov kab hauv qab no tau siv: Huh-7 (JCRB Cell Bank #0403) thiab HepG-2 ATCC HB8065, ob tug tib neeg hepatocyte-derived carcinoma cell kab; Vero ATCC CCL-81, liab-derived raum epithelial cell kab. Txhua kab ntawm tes tau cog qoob loo hauv Eagle's Yam Ntxim Saib Ntxim Ua Qhov Nruab Nrab (MEM) (Gibco, ThermoFisher Scientific, Waltham, MA, USA) ntxiv nrog 5 feem pua ​​​​ntawm cov me nyuam mos liab ntshav (NBCS) (Gibco, ThermoFisher Scientific, Waltham, thiab MA) ntxiv, USA 50 µg/mL gentamycin (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Gibco, ThermoFisher Scientific, Waltham, MA, USA), thiab buffered nrog HCl/NaHCO3. Cov kab ntawm tes tau raug coj los ua thiab khaws cia ntawm 37 ◦C nrog 5 feem pua ​​​​CO2 cua.

2.2. Kab mob Viral

Cov kab mob hauv qab no tau siv: cov kab mob IV4454 tau txais los ntawm cov neeg mob me me ntawm AHF [19] thiab cov tshuaj tiv thaiv-attenuated Candid # 1 hom [20]. Vero hlwb tau siv ob qho tib si rau cov kab mob hauv Tshuag thiab rau tus kab mob titration los ntawm cov txheej txheem plaque assay.

Txhua qhov kev ua haujlwm nrog cov kab mob kis tau ua tiav hauv Biosafety Level 2 cov chaw thiab tau pom zoo los ntawm Lub Chaw Haujlwm Saib Xyuas Kev Noj Qab Haus Huv thiab Kev Nyab Xeeb ntawm Lub Tsev Kawm Ntawv ntawm Kev Tshawb Fawb, University of Buenos Aires. Txhua tus neeg ua haujlwm koom nrog cov txheej txheem kis kab mob tau txhaj tshuaj nrog Candid-1 muab los ntawm Instituto Nacional de Enfermedades Virales Humanas Dr. JI Maiztegui (Pergamino, Buenos Aires, Argentina).

2.3. Microarray Analysis

Tag nrho ntawm 5 × 105 lub hlwb HepG2 tau muab noob rau hauv 25 cm2 kab lis kev cai flasks thiab kis JUNV IV4454 ntawm ntau tus kab mob (MOI) ntawm 1 lossis mock-infected. RNA tau muab rho tawm thiab huv si siv RNAeasy (Qiagen, Hilden, Lub teb chaws Yelemees) ntawm 24 thiab 48 h tom qab kis kab mob (pi). RNA concentrations tau ntsuas siv Nanodrop. Gene qhia profiles ntawm HepG2 hlwb tau tus cwj pwm siv microarray tsom xam (Affymetrix Human U133 Plus 2.0 GeneChips) txhawm rau txheeb xyuas cov tswv tsev cov noob transcriptionally tswj los ntawm kev kis kab mob.

Tag nrho tus naj npawb ntawm cov noob caj noob ces tau tswj hwm los ntawm kev kis kab mob tau suav rau txhua lub sijhawm los ntawm kev txheeb xyuas cov noob uas pom, yam tsawg kawg, ib qho 1.6-fold hloov hauv kev qhia thiab 95 feem pua ​​​​qhov tshwm sim ntawm kev qhia txawv (p {{ 3}.05).

Lub WebGestalt software (http://www.webgestalt.org, nkag mus rau 3 Lub Xya Hli 2020), uas ua haujlwm rau Wikipathways database tau siv rau Gene Ontology thiab txoj hauv kev tshaj tawm kev txheeb xyuas. Cov kev sim rov ua dua tau ua nyob rau hnub sib txawv (n=3 kev sim ywj pheej ib yam).

2.4. Me Me Molecule Compounds

Synthetic AHR-specific competitive antagonist CH223191, 1-Methyl-N-[2-methyl4-[2-(2-methyl phenyl)diazenyl]phenyl]{ {9}}H-pyrazole-5-carboxamide (Tocris Bioscience, Bristol, United Kingdom), tau solubilized nyob rau hauv dimethyl sulfoxide (DMSO) thiab siv nyob rau hauv ib tug concentration ntawm 1.25, 2.5, 5, 10, 20, 40, thiab 80 µM.

Lub tryptophan metabolite L-kynurenine, (2S)-2-Amino-4-(2-aminophenyl)-4-oxobutanoic acid (Sigma-Aldrich, St. Louis, MO, USA) yog Solubilized nyob rau hauv DMSO thiab siv nyob rau hauv ib tug concentration ntawm 2.5, 5, 10, 20, 40, 80, 160, thiab 320 µM.

Tag nrho cov kev kho mob tau ua nyob rau hauv tsis muaj lub teeb, muab hais tias cov tebchaw muaj photosensitivity.

2.5. Cell Viability Assay

Rau kev txiav txim siab ntawm cell viability Huh-7 thiab Vero cell monolayers tau loj hlob hauv 96 qhov chaw zoo rau 24 teev hauv MEM 5 feem pua ​​NBCS. Cell monolayers ntawm 90 feem pua ​​​​ntawm qhov sib npaug tau kho nrog qhov sib txawv ntawm CH223191 (1.25 µM txog 80 µM) thiab kynurenine (5 µM txog 320 µM) rau 72 teev. Mock tau kho nrog tib qhov concentration ntawm DMSO raws li cov koob tshuaj ntau tshaj plaws ntawm ligand kev kho mob thiab raug suav tias yog 100 feem pua ​​​​ntawm cov cell viability.

Tag nrho cov kev kho mob tau ua nyob rau hauv quadruplicate. Cell viability raug soj ntsuam los ntawm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. . Louis, MO, USA) assay. Absorbance tau ntsuas ntawm 570 nm hauv FLUOstar OPTIMA microplate nyeem ntawv. Cell viability feem pua ​​yog txiav txim los ntawm relativizing kho absorbance tau siv Mock absorbance qhov tseem ceeb.

Tsis tas li ntawd, kev loj hlob muaj peev xwm thiab morphology ntawm cov hlwb kho tau raug kawm nyob rau hauv lub teeb tsom iav. Cov duab tau thaij thaum 72 teev nrog Nikon Eclipse TS100 microscope; Loj: 100 ×; NPE: 0.25.

2.6. Kev kho mob thiab kis kab mob

Vero thiab Huh{{0}} cell monolayers raug pretreated nrog CH223191 thiab kynurenine diluted hauv MEM 1.5 feem pua ​​NBCS rau 1 h lossis 24 h, feem, ntawm 37 ◦C. CH223191 tau siv nyob rau hauv ntau qhov sib txawv ntawm 1.25 thiab 20 µM, thaum kynurenine tau siv ntawm qhov ntau ntawm 5 thiab 80 µM. Tom qab ntawd, tus kab mob kis tau ua nrog IV4454 lossis Candid # 1 strain ntawm MOI ntawm 0.5. Tom qab 1 teev, cov inocula raug tshem tawm thiab cov hlwb raug nthuav tawm rau ob qho tib si rau 48 teev ntawm cov lus hais. Thaum 48 h pi supernatants tau sau rau cov tshuaj plaque thiab cov cell monolayers tau ua tiav rau qhov tsis ncaj ncees lawm immunofluorescence lossis RT-qPCR.

2.7. Indirect Immunofluorescence

Huh-7 monolayers kho nrog CH223191 lossis kynurenine thiab kis JUNV raws li tau hais los saum toj no tau kho ntawm 48 h pi nrog methanol rau 10 min ntawm −20 ◦C, ntxuav peb zaug nrog PBS, thiab khaws cia hauv PBS tshiab ntawm 4 ◦C mus txog thaum ua.

Cov tshuaj tiv thaiv hauv qab no tau siv: thawj nas monoclonal anti-NP antibodies (1:200) (NA05-AG12) [21] thiab theem nrab Alexa Fluor 488 conjugated anti-nas tshis antibodies (1:200) (A{{ 10}}) (ThermoFisher Scientific, Waltham, MA, USA). Cell nuclei raug stained nrog 40,6-diamidino-2- phenylindole (DAPI) (1:1000) (Sigma-Aldrich, St. Louis, MO, USA). Microscopy thiab yees duab cov ntaub ntawv tau txais siv Olympus IX71 fluorescence microscope nrog QImaging Exi-Aqua lub koob yees duab txuas (pixel loj: 6.45 µm; thaj chaw photodetector: 1392 × 1040 pixels2).

Kev txheeb xyuas cov ntaub ntawv fluorescence tau ua los ntawm Fiji software (version 1.53v) (National Institutes of Health, Bethesda, MD, USA). Kev ceev kom muaj nuj nqis ntawm DAPI-stained nucleus thiab NP-zoo hlwb tau ua manually los ntawm lub cell counter plugin suav nrog hauv Fiji software (version 1.53v). Cov hlwb raug suav hais tias yog qhov zoo yog tias NP tus kab mob antigen staining tuaj yeem kuaj pom. Qhov feem pua ​​​​ntawm NP-zoo hlwb tau qhia raws li qhov piv ntawm NP-zoo hlwb thiab tag nrho cov hlwb hauv txhua daim teb thiab tom qab ntawd qhov nruab nrab feem pua ​​raug txiav txim siab thiab nthuav tawm.

Qhov kev siv fluorescence ntawm NP-zoo hlwb tau los ntawm kev ntsuas qhov nruab nrab grey tus nqi ntawm ib tus neeg lub hlwb thiab rho tawm cov fluorescence tom qab. Viral foci tau delimited los ntawm kev xav txog cov pab pawg ntawm NP-zoo hlwb ncig los ntawm NP-tsis zoo hlwb. Viral foci tau suav rau ntawm 3 cov duab sib txawv los ntawm 3 qhov sib txawv xaiv qhov chaw thiab qhov nruab nrab foci loj yog txiav txim los ntawm cov xov tooj ntawm tes uas tsim lawv.

2.8. Viral Nucleic Acid Detection

Nucleic acid extraction tau ua los ntawm cov kab mob cell monolayers los ntawm kev siv Highway ADN/ARN PuriPrep-VIRUS cov khoom K1501 (Inbio Highway, Buenos Aires, Argentina), raws li cov chaw tsim khoom cov lus qhia. JUNV RNA viral load thiab cellular transcripts kom muaj nuj nqis tau ua los ntawm RT-qPCR. Kev hloov pauv rov qab tau ua los ntawm kev siv random primers thiab Moloney murine leukemia tus kab mob thim rov qab (M-MLV RT).

Rau kev nthuav dav fragment, cov primers hauv qab no tau siv: JUNV-np (Forward: 5 0 -GGC ATC CTT CAG AAC ATC-30, Rov qab: 50 -CGC ACA GTG GAT CCT AGGC{{4} } ), ahr (Forward: 50 -ATC CTT CCA AGC GGC ATA GAG ACC-30; Reverse: 50 -CAA AGA AGC TCT TGG CTC TCA GG-30 ), cyp1a1 ( Forward: 50 -TTCCGACACTCTTCCTTCGT-30; Rov qab: 50 -ATGGTTAGCCCATAGATGGG-30 ), -actin (Forward: 50 -TTA GTT GTT GCG TTA CAC CAC TTC TTG{17 }}; Reverse: 50 -TCA CCT TCA CCG TTC CAG TTT-30 ). PCR cov ntsiab lus ntawm lub voj voog yog raws li hauv qab no: thawj zaug denaturalization 2 min ntawm 95 ◦C; 45 amplification cycles muaj 30 sec ntawm 95 ◦C, 1 min ntawm 58 ◦C, thiab 1 min ntawm 72 ◦C; lub voj voog kawg ntawm 5 min ntawm 72 ◦C; Thaum kawg, melting curves tau ua. Kev kuaj pom tau ua tiav siv cov cuab yeej MyiQ2 (Bio-Rad Laboratories, Hercules, CA, USA). Cov txiaj ntsig tau tshaj tawm raws li 2−∆∆Ct (Fold change) thiab normalized siv -actin raws li cov noob tu tsev.

2.9. Plaque Assay

Viral yields tau txiav txim los ntawm cov txheej txheem plaque assay. Cell supernatants tau sau thiab serial dilutions tau npaj los kis Vero cell monolayers rau 1 h ntawm 37 ◦C. Tom qab tus kab mob adsorption, cov inocula raug tshem tawm, MEM 5 feem pua ​​​​NBCS ntxiv nrog 0.7 feem pua ​​​​methylcellulose tau ntxiv thiab cov hlwb raug incubated rau 1 lub lis piam ntawm 37 ◦C hauv 5 feem pua ​​CO2 incubator. Tom qab ntawd, cell monolayers tau kho nrog 10 feem pua ​​formaldehyde, ntxuav, thiab stained nrog crystal violet. Viral titer raug xam tom qab plaque forming unit (PFU) kom muaj nuj nqis.

2.10. Kev txheeb cais

Microarray txheeb cais tau ua tiav siv WebGestalt software (http://www. webgestalt.org/, nkag rau 3 Lub Xya Hli 2020). Cov ntaub ntawv tau piav raws li txhais tau tias ± SD Ib-Txoj Kev ANOVA nrog Dunnett's post hoc test los ntawm GraphPad Prism 8 (version 8.0.1) rau Windows 11 (GraphPad Software, San Diego, CA, USA) tau ua los ntsuas qhov tseem ceeb ntawm cov ntaub ntawv. p Tsawg dua lossis sib npaug li 0.05 tau suav tias yog qhov tseem ceeb.

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