In vivo augmentation af Gut-homing regulerende T-celle induktion
Summary
Her præsenterer vi en protokol for in vivo augmentation af Gut-homing regulatoriske T-celle induktion. I denne protokol er dendritiske celler konstrueret til lokalt at producere høje koncentrationer af det aktive vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og den aktive vitamin a (RETINSYRE eller RA) de Novo.
Abstract
Inflammatorisk tarmsygdom (IBD) er en inflammatorisk kronisk sygdom i mave-tarmkanalen (GUT). I USA er der ca. 1.400.000 IBD-patienter. Det er almindeligt anerkendt, at en dysreguleret immunrespons på tarm bakterier initierer sygdommen og forstyrrer slimhinde epitelial barrieren. Vi har for nylig vist, at Gut-homing regulerende T (Treg) celler er en lovende behandling for IBD. Derfor indeholder denne artikel en protokol for in vivo augmentation af tarm-homing Treg celle induktion. I denne protokol er dendritiske celler konstrueret til at producere lokalt høje koncentrationer af to molekyler de Novo, aktiv vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og aktiv vitamin a (retinsyre eller RA). Vi valgte 1,25 (OH)2d og RA baseret på tidligere resultater, der viser, at 1,25 (Oh)2d kan inducere ekspression af regulerende molekyler (f. eks. Forkhead box P3 og interleukin-10), og at RA kan stimulere ekspression af tarm-homing receptorer i T-celler. For at generere sådanne konstruerede dendritiske celler bruger vi en lentiviral vektor til at transduce dendritiske celler til at over udtrykke to gener. Et gen er cytochrom P450 familie 27 familien B medlem 1, der koder 25-hydroxyvitamin D 1α-hydroxylase, som fysiologisk katalyserer syntesen af 1,25 (Oh)2D. Det andet gen er aldehyd dehydrogenase 1 familiemedlem a2, der koder retinaldehyd dehydrogenase 2, som fysiologisk katalyserer syntesen af RA. Denne protokol kan anvendes til fremtidig undersøgelse af Gut-homing Treg celler in vivo.
Introduction
Inflammatorisk tarmsygdom (IBD) er en inflammatorisk kronisk sygdom i mave-tarmkanalen (GUT). I USA er der ca. 1.400.000 IBD-patienter. Det er almindeligt anerkendt, at en dysreguleret immunrespons på tarm bakterier initierer sygdommen og forstyrrer slimhinde epitelial barrieren1,2. Af denne grund, i øjeblikket tilgængelige U.S. Food and Drug Administration (FDA)-godkendte lægemidler hæmmer funktionerne af inflammatoriske mediatorer eller blokere homing af immunceller i tarmen. Men, de inflammatoriske mediatorer og immunceller, der er målrettet er også nødvendige for immunforsvaret. Som et resultat, de inflammatoriske mediator hæmmere kompromittere systemiske immunforsvar og immuncelle homing blokkere svække tarm immunforsvar, som begge kan føre til alvorlige konsekvenser3,4. Desuden kan immuncelle homing blokkere også blokere for homing af regulerende T (Treg) celler i tarmen og dermed kan forværre den allerede kompromitterede tarm immuntolerance i IBD patienter. Desuden kan blokering af Treg-celle homing i tarmen også føre til systemisk immunsuppression på grund af ophobning af Treg celler i blodet5. Endelig virker inhibitorer og blokkere forbigående og kræver dermed hyppige administrationer. Hyppig administration af disse hæmmere og blokkere kan yderligere forværre de uheldige bivirkninger.
For nylig foreslog vi en ny strategi, der potentielt kan afbøde eller endda eliminere bivirkningerne forbundet med nuværende lægemidler til IBD behandling6. Denne strategi forstærker induktion af tarm-homing Treg celler i perifert lymfevæv6. Rationalet bag denne strategi er, at Gut-homing Treg celler specifikt hjem til tarmen og dermed ikke vil kompromittere systemiske immunforsvar. Desuden, da treg celler kan potentielt danne hukommelse7,8, Gut-homing treg celler kan potentielt give en stabil kontrol af kronisk tarmbetændelse i IBD patienter og dermed, behandling bør ikke behøver at blive administreret så hyppigt. Da denne strategi desuden forstærker induktion af tarmationstreg-celler in vivo, er der ingen bekymring for in vivo-ustabilitet i et stærkt proinflammatorisk miljø, der er forbundet med adoptiv overførsel af in vitro-genererede treg-celler9,10. I denne henseende, in vitro genereret treg celler er en af de foreslåede strategier for behandling af autoimmune sygdomme11,12,13 og transplantation afvisning14,15. Endelig, i denne strategi, dendritiske celler (DCs) er konstrueret til at producere lokalt høje koncentrationer af to molekyler de Novo: aktiv vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og aktive vitamin a (retinsyre eller RA). Vi valgte 1,25 (Oh)2d og RA fordi 1,25 (Oh)2d kan inducere ekspression af regulerende molekyler (f. eks, forkhead box P3 [foxp3] og interleukin-10 [Il-10])16,17 og at RA kan stimulere ekspression af Gut-homing receptorer i T-celler18. Fordi både 1,25 (Oh)2D og RA også kan tolerize DCS28,29, vi grunden til, at de konstruerede DCS vil være stabilt vedligeholdt i en tolerogenic status in vivo og dermed omgå in vivo ustabilitet bekymringer, der er forbundet med in vitro genereret tolerogenic DCS (toldcs)19,20,21. I denne henseende er toldc’er også en af de foreslåede strategier for in vivo augmentation af treg celle funktioner19,20,21. For at støtte vores ræsonnement, har vi vist, at de konstruerede DCs, ved in vivo levering, kan forøge induktion af Gut-homing Treg celler i perifert lymfoid væv6.
En yderligere fordel ved vores foreslåede strategi er, at 1,25 (OH)2D har også andre funktioner, der potentielt kan gavne IBD patienter. Disse andre funktioner omfatter evnen af 1,25 (OH)2D at stimulere udskillelsen af antimikrobielle stoffer22 og undertrykker carcinogenese23. Infektioner og kræft er ofte forbundet med IBD24,25.
For at generere de DCs, der kan producere lokalt høje koncentrationer af både 1,25 (OH)2D og RA de Novo, bruger vi en lentiviral vektor til at ingeniør DCS til at over udtrykke to gener. Et gen er cytochrom P450 familie 27 familien B medlem 1 (CYP27B1), der koder 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), som fysiologisk katalyserer syntesen af 1,25 (Oh)2D. Det andet gen er aldehyd dehydrogenase 1 familiemedlem a2 (ALDH1a2), der koder retinaldehyd dehydrogenase 2 (RALDH2), som fysiologisk katalyserer syntesen af RA6.
Fordi in vivo augmentation af Gut-homing Treg celle induktion er potentielt vigtigt i behandlingen af IBD, i den følgende protokol vil vi detaljeret de procedurer for generering af de 1α-hydroxylase-RALDH2-overudtrykker DCs (DC-CYP-ALDH celler), der kan anvendes til den fremtidige undersøgelse af Gut-homing Treg celler in vivo.
Protocol
Representative Results
Discussion
I denne artikel beskriver vi brugen af DC-CYP-ALDH celler, for at forstærke induktion af Gut-homing Treg celler i perifert lymfevæv. Vores data har vist, at DC-CYP-ALDH-cellerne kan syntetisere lokalt høje koncentrationer af de Novo af både 1,25 (OH)2D og RA in vitro i nærværelse af tilsvarende substrater (dvs. 25 [Oh] d og retinol, hhv.). Da tilstrækkelige blodkoncentrationer på 25 (Oh) D og retinol let kan opnås gennem d-vitamin og et tillægsspørgsmål til henholdsvis patienter med mangler<sup cla…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Kontoret for den assisterende forsvarsminister for Sundhedsanliggender gennem peer reviewed medicinsk forskning program under Award No. W81XWH-15-1-0240 (XT). Meninger, fortolkninger, konklusioner, og anbefalinger er dem af forfatteren og er ikke nødvendigvis godkendt af Department of Defense. Dette arbejde blev også delvist understøttet af forsknings innovations stipendier fra Institut for medicin på Loma Linda University (681207-2967 [XT og GG], 681205-2967 [XT] og 325491 [DJB]).
Materials
| 10 mL syringes | ThermoFisher Scientific | Cat# 03-377-23 | |
| 100 mm x 20 mm culture dishes | Sigma-Aldrich | Cat# CLS430167 | |
| 12-well culture plates | ThermoFisher Scientific | Cat# 07-200-82 | |
| 150 mm x 25 mm culture dishes | Sigma-Aldrich | Cat# CLS430559 | |
| 25-hydroxycholecalciferol (25[OH]D) | Sigma-Aldrich | Cat# H4014 | |
| 293T cells | ATCC | CRL-3216 | |
| 2-mercaptoethanol | ThermoFisher Scientific | Cat#: 21985023 | |
| 6-well culture plates | ThermoFisher Scientific | Cat# 07-200-83 | |
| ALDEFLUOR kit | Stemcell Technologies | Cat# 01700 | |
| Anti-CYP27B1 | Abcam | Cat# ab95047 | |
| BD FACSAria II | BD Biosciences | N/A | |
| CaCl2 | Sigma-Aldrich | Cat# C1016 | |
| CM-10-D cell culture medium | DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| CM-10-R cell culture medium | RPMI 1640 medium (no glutamine) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| CM-4-D cell culture medium | DMEM medium containing 4% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| Corning bottle-top vacuum filters, 0.22 mM, 500 mL | Sigma-Aldrich | Cat# CLS430513 | |
| Corning bottle-top vacuum filters, 0.45 mM, 500 mL | Sigma-Aldrich | Cat# CLS430514 | |
| Dissecting scissor | ThermoFisher Scientific | Cat# 08-940 | |
| DMEM medium | ThermoFisher Scientific | Cat# 11960044 | |
| Fetal bovine serum | ThermoFisher Scientific | Cat# 16000044 | |
| Forceps | ThermoFisher Scientific | Cat# 22-327379 | |
| Gibco ACK lysing buffer | ThermoFisher Scientific | Cat# A1049201 | |
| Glycerol | Sigma-Aldrich | Cat# G5516 | |
| Goat anti-rabbit IgG | Abcam | Cat# ab205718 | |
| HEPES | Millipore | Cat# 391340 | |
| Lenti-CYP-ALDH | Custom-made | 1.6-kb mouse CYP27B1 and ALDH1a2 cDNAs were amplified by PCR using a plasmid containing the CYP27B1 cDNA and a plasmid containing the ALDH1a2 cDNA respectively (GeneCopoeia). The amplified CYP27B1 cDNA fragment with a 5' KOZAK ribosome entry sequence was cloned into the pRRL-SIN.cPPt.PGKGFP.WPRE lentiviral vector (Addgene). The resulting construct was designated as lenti-CYP-GFP. The amplified ALDH1a2 cDNA fragment was cloned into the lenti-CYP-GFP to replace the GFP and was designated as lenti-CYP-ALDH. This bicistronic plasmid expresses CYP27B1 controlled by SFFV promoter and ALDH1a2 controlled by PGK promoter. | |
| L-glutamine | ThermoFisher Scientific | Cat#25030081 | |
| Lipopolysaccharide | Sigma-Aldrich | Cat# L3755 | |
| Murine GM-CSF | Peprotech | Cat# 315-03 | |
| Murine IL-4 | Peprotech | Cat# 214-14 | |
| Na2HPO4 | Sigma-Aldrich | Cat# NIST2186II | |
| NaCl | Sigma-Aldrich | Cat# S9888 | |
| Needles | ThermoFisher Scientific | Cat# 14-841-02 | |
| Nonessential Amino Acids | ThermoFisher Scientific | Cat#: 11140076 | |
| pCMVR8.74 | Addgene | Plasmid# 22036 | |
| Penicillin/Streptomycin | ThermoFisher Scientific | Cat#15140148 | |
| Phoshate Balanced Solution (PBS) | ThermoFisher Scientific | Cat#: 20012027 | |
| PMD2G | Addgene | Plasmid# 12259 | |
| Polypropylene tube, 15 mL | ThermoFisher Scientific | Cat# AM12500 | |
| Polypropylene tube, 50 mL | ThermoFisher Scientific | Cat# AM12502 | |
| Protamine sulfate | Sigma-Aldrich | Cat# P3369 | |
| Rabbit polycloncal IgG isotype control | Abcam | Cat# ab171870 | |
| Radioimmunoassay for 1,25(OH)2D measurement | Heartland Assays | ||
| RPMI 1640 medium, no glutamine | ThermoFisher Scientific | Cat# 21870076 | |
| Sodium pyruvat | ThermoFisher Scientific | Cat#: 11360070 | |
| Sorvall Legend XTR Centrifuge | ThermoFisher Scientific | Cat# 75004521 | |
| Sterile Cell strainers, 40 mm | ThermoFisher Scientific | Cat# 07-201-430 | |
| Sterile storage bottles, 500 mL | ThermoFisher Scientific | Cat# CLS431432 |
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