In Vivo Photolabeling af celler i tyktarmen til at vurdere vandrende potentiale af hæmatopoietisk celler i Neonatal mus
Summary
Protokollen beskrevet her udnytter en photolabeling tilgang i nyfødte mus til specifikt at identificere immunceller, der udvandrer fra tyktarmen til ekstra intestinal websteder. Denne strategi vil være nyttigt at studere vært-microbiome interaktioner tidligt i livet.
Abstract
Enterisk bakterielle samfund er etableret tidligt i livet og påvirke immun celle udvikling og funktion. Den neonatale mikrobiota er modtagelige for mange ydre påvirkninger herunder antibiotika brug og kost, som påvirker modtagelighed for autoimmune og inflammatoriske sygdomme. Lidelser såsom inflammatoriske tarm sygdom (IBD) er karakteriseret ved en massiv tilstrømning af immunceller tarmene. Dog kan immunceller betinget af mikrobiota desuden emigrere ud af tarmene til at påvirke immunrespons på ekstra intestinal websteder. Der er således behov for at identificere og karakterisere celler, der kan bære mikrobielle beskeder fra tarmene til distale websteder. Her, beskriver vi en metode til at mærke cellerne i tyktarmen af nyfødte mus i vivo der giver deres identifikation på ekstra intestinal websteder efter overflytningen.
Introduction
Pattedyr mavetarmkanalen havne hundredvis af arter af bakterier, der findes i et symbiotisk forhold med vært1. Immunceller til stede i det lokale miljø håndhæve en fredelig sameksistens med disse mikrober og oprette en beskyttende barriere mod patogenet invasioner. Bi-directional interaktion mellem immunceller og mikrobiota er derfor kritisk at etablere et commensal samfund, der uddanner vært immunsystemet og angiver tærsklen for immun reaktivitet til patogener. Ændringer i den mikrobielle sammensætning eller dysbiosis, kan forstyrre den immun homøostase og forurolige regulerende kredsløb, der hæmmer intestinal betændelser fører til immun-medierede sygdomme som Type 1 Diabetes og IBD2,3 .
Perioden umiddelbart efter fødsel er en unik udviklingsmæssige vindue hvor de intestinale mikrobielle samfund begynder at etablere samtidig immunforsvaret modnes4. Den postnatale mikrobiota er ikke stabil, med skiftehold i Fællesskabet sammensætning forekommer naturligt og ofte5. De immunceller, der interagerer med mikrobiota bosat i to forskellige anatomiske steder i tarmen – lamina propria og den intestinale epitel6. Mange typer af immunceller er til stede i tarmen, herunder lymfocytter (som T-celler, B-celler og medfødte lymfoide celler) samt myeloide celler (som omfatter dendritiske celler, monocytter og makrofager). Disse celler, også kendt som hæmatopoietisk celler, udføre et væld af funktioner, der bevarer den intestinale barriere og opretholde homeostase.
Ud over deres lovgivningsmæssige funktioner på intestinal websteder, kan immunceller af slimhinden endvidere foretage mikrobielle meddelelser til de ekstra intestinal steder at regulere systemisk immunitet7,8,9. Det er et område af voksende forskningsinteresse og fremhæver behovet for metoder til at identificere immunceller, der vandrer ud af tarmens væv for at sonde deres funktion. Protokollen rapporteret her udnytter et kommercielt tilgængelige musemodel, hvor en photoconvertible fluorescerende proteiner er udnyttet til etiketten celler. PhAMskåret ud mus udtrykke allestedsnærværende en grøn fluorescerende Dendra2 protein, der er uigenkaldeligt skiftede til rød fluorescens ved aktivering af ultraviolet (UV) lys10. Ved hjælp af en fiber optic kanyle til at levere 405 nm lys ind i tyktarmen af nyfødte mus, viser vi, at photoconverted hæmatopoietisk celler, som har oprindelse i eller passeres gennem tyktarmen kan findes i milten.
Protocol
Representative Results
Discussion
Identifikation og karakterisering af celler, der interagerer med og er påvirket af mikrobiota i tyktarmen er vigtige og bør lette forståelsen af hvordan oplysninger fra den slimhinde mikromiljø videresendes til resten af kroppen. En metode til at studere gut-relateret celle migration kræver isolering af gut-associerede celler efterfulgt af en adoptiv overførsel til recipient mus til at bestemme deres væv-homing mønstre og fungere12,13. Denne tilgang er be…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Flemming Ørholt Jain blev støttet af en NIH/NIAID karriere overgang pris 1K22AI116661-01.
Materials
| Laser | |||
| Light Emitting Diode (LED) | THORLABS | M405FP1 | CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1 |
| LED driver | THORLABS | LEDD1B | Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616 |
| Optogenetics patch cable | THORLABS | M87L01 | 1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&pn=M87L01#11454 |
| Fiber optic cannula | Doric lenses | MFC_480/500-0.5_5mm_ZF1.25_C45 | 5 mm long cannula with an outer diameter of 500 µm and an inner diameter of 480 µm. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036 |
| Power supply | THORLABS | KPS101 | Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101 |
| LG3 laser safety goggles | THORLABS | LG3 | Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&pn=LG3#2523 |
| Red light | Electron Microscopy Sciences | 74327-10 | 15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences |
| Intestinal cell isolation | |||
| Isoflurane | Patterson Veterinary | 07-893-1389 | CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood. https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3 |
| 1X HBSS | Gibco | 14025076 | Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&searchTerm=14025076&searchType=RAPID&matchedCatNo=14025076 |
| Calf Serum | Hyclone AZM | 197696 | |
| EDTA | Invitrogen | 15575020 | 0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020 |
| DTT | Sigma | 10197777001 | CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en®ion=US |
| HEPES | Gibco | 15630080 | 1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080 |
| Petri dish | Corning | 353004 | https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&searchTerm=08772F&searchType=RAPID&matchedCatNo=08772F |
| 70 micron cell strainer | Falcon | 352350 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712 |
| Micro magnetic stir bar | Fisherbrand | 1451364 | Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364 |
| Magnetic stir plate | Corning Laboratory Stirrers | 440826 | https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&persist=true&ip=no&gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE |
| Collagenase | Roche | 5401020001 | https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en®ion=US&gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE |
| DNase I | Sigma | 10104159001 | https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en®ion=US |
| 1X PBS | Gibco | 20012-027 | https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027 |
| Pipet aid | Thermo Scientific | 14387165 | https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165 |
| 10 mL serological pipet | Falcon | 357530 | https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659 |
| 25 mL serological pipet | Falcon | 357515 | https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659 |
| 15 mL conical centrifuge tube | Thermo Scientific | 339651 | https://www.thermofisher.com/order/catalog/product/339650 |
| 50 mL conical centrifuge tube | Thermo Scientific | 339653 | https://www.thermofisher.com/order/catalog/product/339650 |
| Single cell suspension | |||
| Eppendorf tubes | Seal-Rite | 1615-5500 | Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx |
| Tissue homogenizer | Kimble | K7495400000 | Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000 |
| Homogenizer tips | Kimble | 7495210590 | Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590 |
| ACK lysing buffer | Gibco | A10492-01 | https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01 |
| 40 micron cell strainer | Falcon | 08-771-1 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711 |
| Antibodies | |||
| BV786 anti-mouse CD45 | BD | 564225 | Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225 |
| Live/Dead | Invitrogen | L34962 | https://www.thermofisher.com/order/catalog/product/L34962 |
| Other | |||
| Razor blades | VWR | 55411-050 | Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades |
References
- Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 489, 231-241 (2012).
- Paun, A., Yau, C., Danska, J. S. The Influence of the Microbiome on Type 1 Diabetes. Journal of Immunology. 198, 590-595 (2017).
- Mathis, D., Benoist, C. Microbiota and autoimmune disease: the hosted self. Cell Host Microbe. 10, 297-301 (2011).
- Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny. Immunity. 46, 350-363 (2017).
- Jain, N., Walker, W. A. Diet and host-microbial crosstalk in postnatal intestinal immune homeostasis. Nature Reviews in Gastroenterology and Hepatology. 12, 14-25 (2015).
- Mowat, A. M., Agace, W. W. Regional specialization within the intestinal immune system. Nature Reviews in Immunology. 14, 667-685 (2014).
- Macpherson, A. J., Uhr, T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science. 303, 1662-1665 (2004).
- Mowat, A. M. Anatomical basis of tolerance and immunity to intestinal antigens. Nature Reviews in Immunology. 3, 331-341 (2003).
- Diehl, G. E., et al. Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells. Nature. 494 (3), 116-120 (2013).
- Pham, A. H., McCaffery, J. M., Chan, D. C. Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics. Genesis. 50, 833-843 (2012).
- Conway, K. L., et al. ATG5 regulates plasma cell differentiation. Autophagy. 9, 528-537 (2013).
- Buzoni-Gatel, D., Lepage, A. C., Dimier-Poisson, I. H., Bout, D. T., Kasper, L. H. Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii. Journal of Immunology. 158, 5883-5889 (1997).
- Guo, X., Muite, K., Wroblewska, J., Fu, Y. X. Purification and Adoptive Transfer of Group 3 Gut Innate Lymphoid Cells. Methods in Molecular Biology. 1422, 189-196 (2016).
- Morton, A. M., et al. Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut. Proceedings of the National Academy of Sciences of the United States of America. 111, 6696-6701 (2014).
