The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.
Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.
The mammalian gastrointestinal tract harbors hundreds of species of bacteria that exist in a symbiotic relationship with the host1. The immune cells present in the local milieu enforce a peaceful coexistence with these microbes and establish a protective barrier against pathogen invasions. Thus, bi-directional interactions between the immune cells and the microbiota are critical to establish a commensal community that educates the host immune system and sets the threshold for immune reactivity to pathogens. Changes in the microbial composition, or dysbiosis, can disturb the immune homeostasis and perturb regulatory circuits that restrain intestinal inflammations leading to immune-mediated diseases such as Type 1 Diabetes and IBD2,3.
The period immediately after birth is a unique developmental window during which the intestinal microbial communities begin to establish at the same time the immune system matures4. The postnatal microbiota is not stable, with shifts in the community composition occurring naturally and frequently5. The immune cells that interact with the microbiota reside in two distinct anatomical locations in the intestine – the lamina propria and the intestinal epithelium6. Numerous types of immune cells are present in the intestine, including lymphocytes (such as T cells, B cells, and innate lymphoid cells) as well as myeloid cells (which include dendritic cells, monocytes, and macrophages). These cells, also known as hematopoietic cells, perform a multitude of functions that preserve the intestinal barrier and maintain homeostasis.
In addition to their regulatory functions at intestinal sites, immune cells of the mucosa may also carry microbial messages to the extra-intestinal sites to regulate systemic immunity7,8,9. This is an area of growing research interest and highlights the need for methods to identify immune cells that migrate out of intestinal tissues in order to probe their function. The protocol reported here utilizes a commercially available mouse model in which a photoconvertible fluorescent protein is exploited to label cells. PhAMexcised mice ubiquitously express a green fluorescent Dendra2 protein that is irreversibly switched to red fluorescence upon activation by ultraviolet (UV) light10. Using a fiber optic cannula to deliver 405 nm light into the colon of newborn mice, we demonstrate that photoconverted hematopoietic cells, which have originated in or transited through the colon can be found in the spleen.
All animal procedures were performed with the approval of and in compliance with the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital.
CAUTION: This protocol involves the use of a class 3b laser (LG3). LG3 laser safety goggles must always be used when operating this laser. Appropriate training and safety guidelines must be followed to avoid the risk of injury.
1. Design and Assembly of Laser
2. Photoconversion of Cells in the Colon
NOTE: Male and female mice were exposed to intracolonic 405 nm light 1 – 2 day(s) after their birth and were sacrificed prior to 1 week of age.
3. Isolation of Intestinal Lymphocytes
4. Isolation of Lymphocytes from the Spleen
5. Identification of Dendra-r+ Cells Using Flow Cytometry
A fiber-optic cable was used to deliver 405 nm light into the colons of 2-day old PhAMexcised mice. In previous experiments, a 30 s exposure was determined to give a maximal photoconversion of colon cells with minimal cytotoxicity (Figure 1A). Therefore, sequential 30 s exposures of different segments of the colon were carried out as described in the protocol. Following the laser exposure, the mice were immediately euthanized, and the photoconversion of the cells was determined. Single-cell preparations of the total colonic tissue were stained with fluorescent-conjugated anti-CD45 antibody and the flow cytometric analysis was performed to detect photoconversion. Unconverted Dendra2-green (Dendra-g) protein has excitation and emission maxima at 490 and 553 nm respectively and can be detected in the FITC channel, whereas photoconverted Dendra2-red (Dendra-r) protein has excitation and emission maxima at 507 and 573 nm respectively and may be detected in the PE channel of a flow cytometer. Once irreversibly switched to its red form, Dendra2 is highly photostable and useful for long-term tracking applications.
No Dendra-r+ cells were detected in unlasered mice (Figure 1B Left). However, 405 nm light exposure resulted in distinct Dendra-r+ cell populations in both the CD45neg and CD45+ cell compartments (Figure 1B Right). To determine if the colonic photoconversion protocol resulted in the background labeling of cells at extra-intestinal sites, we harvested spleens from newly photoconverted mice (T = 0) and assayed for Dendra-r+ cells by flow cytometry. No Dendra-r+ cells were detected in the CD45neg or CD45+ cell compartments (Figure 1C), suggesting that the intra-colonic laser beam did not penetrate enough to photolabel cells in the spleen. Thus, any Dendra-r+ cells detected in the spleen after allowing time for the migration (T = 3d, 5d) should have originated from the colon.
Figure 1: In vivo photoconversion of colonic tissue. Cells of the colon from 2-day old PhAMexcised pups were exposed to intra-colonic 405 nm light for 30 s as described. The photoconversion and labeling of the cells were determined immediately after the procedure (T = 0) by a flow cytometric analysis of single-cell preparations of the colonic tissue and spleen. (A) This representative flow cytometric dot plot shows the expression of CD45 (x-axis) and the staining of the live-dead dye (y-axis) in single-cell preparations from the colons of control and lasered mice. Panels B and C are representative flow cytometric dot plot overlays which show the expression of the Dendra2-green protein (Dendra2-g, horizontal-axis) and Dendra2-red protein (Dendra2-r, vertical-axis) in control-unexposed and exposed CD45+ (hematopoietic) and CD45neg (non-hematopoietic) cells in (B) the colon and (C) the spleen. Please click here to view a larger version of this figure.
Data from a typical photoconversion experiment to identify migratory cells in the spleen are shown in Figure 2. The photoconversion of the cells in the colons of 2-day old PhAMexcised mice was performed as in Figure 1. Mice were euthanized 3 and 5 days after the exposure and the presence of Dendra-r+ cells in the spleen was determined by flow cytometry. There were no Dendra-r+ cells in the CD45neg cell subset, consistent with the view that the CD45neg cells are primarily epithelial and other structural cells of the intestine. Some CD45+ hematopoietic cells expressed Dendra-r, suggesting that they had been photoconverted in the colon and migrated to the spleen.
Figure 2: CD45+ hematopoietic cells migrate from the colon to the spleen. Newborn (day 0 – 2) PhAMexcised mice were exposed to 405 nm light to photoconvert Dendra2 protein by intra-colonic exposure. The representative flow cytometry plots show the expression of the Dendra2-green protein (Dendra2-g, horizontal-axis) and Dendra2-red protein (Dendra2-r, vertical-axis) in CD45neg and CD45+ spleen cells of unexposed 5-days-old mice and exposed mice analyzed at 3 and 5 days (T = 3d, 5d) after the photoconversion. Please click here to view a larger version of this figure.
The identification and characterization of cells that interact with and are influenced by the microbiota in the colon are important and should facilitate understanding of how information from the mucosal microenvironment is relayed to the rest of the body. One method for studying gut-related cell migration requires the isolation of gut-associated cells followed by an adoptive transfer into recipient mice to determine their tissue-homing patterns and function12,13. This approach is limited, as only specific cell types that were transferred may be tracked. Further, artifacts resulting from the isolation of the cells from the tissues and the purification process may hinder the in vivo migratory process. More recently, an endoscopic photoconversion protocol to label intestinal cells using Kaede mice was reported14. This study found a broad trafficking of leukocytes to and from the intestine in adult mice.
Our report describes a method to label cells in the colon of newborn mice and track their migration to extra-intestinal sites such as the spleen. The PhAMexcised mouse ubiquitously expresses a photoconvertible green fluorescent Dendra2 protein that is irreversibly switched to red fluorescence upon its activation by UV light. The exposure of colons of newborn mice to 405 nm light labeled both CD45+ hematopoietic and CD45neg non-hematopoietic cells. CD45+Dendra2-red+ cells were detected in the spleen 3 to 5 days after the photoconversion, indicating the extra-intestinal migration of hematopoietic cells.
The rate-limiting step for this assay is the fraction of the colonic area that is exposed to 405 nm light. The fragility of the newborn colon and the presence of stool, albeit minimal at this early age, limit the length of the fiber optic cannula that can be inserted. In previous experiments, a longer cannula could not be successfully inserted more than 5 mm. Lubricants were avoided due to the risk of the variable light refraction of the laser beam. The outward movement of the cannula every 30 s helped maximize the colonic area exposed to the laser light. Importantly, a cannula with a large NA value (NA = 0.5) was used to generate a wide cone of light in the colon.
This protocol has been standardized for newborn (day 0 – 2) PhAMexcised pups. Given the changes in content and length of the colon with the age of the mice, the technique will have to be optimized accordingly when using older pups. Primarily, modifications to the total duration of the photoconversion and the length of the insertion of the fiber optic cannula will be necessary.
This approach to understanding the migratory behavior of cells in the colon is facilitated by flow cytometric analysis. Multi-parameter flow cytometry may be further used to identify and phenotype the migrant immune cell types within the CD45+ cell subset. Dendra2-r+ cells may also be sorted by FACS for downstream applications such as gene expression profiling or proteomics analyses. While directed to the understanding of hematopoietic cell subsets in the colon in this study, the method may be adapted to study cellular migration from additional tissue sites including the brain and the skin.
The authors have nothing to disclose.
Nitya Jain was supported by an NIH/NIAID Career Transition Award 1K22AI116661-01.
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 |
Altro | |||
Razor blades | VWR | 55411-050 | Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades |