GoKawiilMice
All animal experiment were performed in accordance with protocol approved by the Institutional Animal Care and Use Committee and Institutional Biosafety Committee at Princeton University. C57BL/6 (stock no. 000664), Lgr5eGFP−IRES-creERT2 (stock no. 008875), BALB/cJ (stock no. 000651) and ΔdblGATA (stock no. 033551) mice were purchased from The Jackson Laboratory. Germ-free C57BL/6 mice were purchased from Taconic Biosciences. For timed pregnancy, females were intraperitoneally injected with 2 IU PMSG followed by 2 IU hCG 46–48 h later and then co-housed with male breeder mice. This hormone-induced timed pregnancy model was used for all experiments unless otherwise specified. For all experiments, C57BL/6 male breeder mice were used unless otherwise specified. Tissue collection and analyses were performed at embryonic day 12.5 for pregnant mice or 12 days postpartum for lactating mice unless otherwise specified. For long-term experiments, pups were weaned at 3 weeks and female mice were maintained for 12 weeks post-lactation. Age-matched mice that were administrated with hormones and co-housed with male mice but did not get pregnant were used in each experiment as nulliparous controls. Animals were randomly assigned to experimental groups whenever possible, and sample processing and outcome assessment were performed in a blinded manner where feasible.
Bacteria and infections
Wild-type Y. pseudotuberculosis IP32777 was grown overnight in 2× YT medium (Sigma-Aldrich) at 25 °C with shaking at 200 rpm. A nalidixic-acid-resistant strain of S. Typhimurium, IR715, was cultured in LB at 37 °C with shaking at 200 rpm. L. monocytogenes strain 10403s was cultured overnight in brain heat infusion broth (Teknova) at 37 °C with shaking at 200 rpm. Overnight cultures were centrifuged at 4,000g, resuspended in PBS and adjusted to a density (absorbance at 600 nm) of 1.0 in PBS. Mice fasted overnight (12–16 h) in new cages were orally administered 200 μl of the indicated pathogen suspension. To determine bacterial burden in the maternal spleen and faecal samples, spleens were homogenized through 70 μm filters with PBS, whereas faecal samples were collected in pre-weighed tubes and homogenized in PBS. For Y. pseudotuberculosis, homogenates were serially diluted and plated onto Yersinia-selective agar with Yersinia-selective supplement (Millipore), and colonies were counted after incubation at 25 °C for 48 h. For S. Typhimurium, homogenates were serially diluted and plated onto LB agar with 50 mg ml–1 nalidixic acid and incubated overnight at 37 °C. For L. monocytogenes, homogenates were serially diluted and plated onto brain heart infusion agar (Sigma Aldrich) with 100 mg ml–1 streptomycin and incubated overnight at 37 °C. All lactating dams were infected between day 5 and 8 postpartum period.
Tissue processing
Mice were euthanized with CO 2 . Tissues (small intestine, bone marrow, lung, skin and spleen) were collected into complete medium (RPMI 1640 supplemented with 20 mM HEPES, 2 mM l-glutamine, 1 mM sodium pyruvate, 1X nonessential amino acids, 55 μM β-mercaptoethanol, 100 U ml−1 penicillin and 100 μg ml–1 streptomycin) with 3% FBS on ice until further processing. For compartmentalization analysis, we defined the first 10 cm adjacent to the stomach as proximal, the last 10 cm as distal and the intervening segment as middle. For cell isolation from the small intestinal lamina propria, Peyer’s patches were removed and tissues were opened longitudinally, washed with cold PBS twice, cut into 1 cm segments and placed immediately into 3% complete medium containing 5 mM EDTA and 0.154 mg ml−1 (1 mM) dithiothreitol. Tissues were then incubated for 20 min at 37 °C with continuous stirring at 400 rpm, vigorously shaken in RPMI containing 20 mM HEPES, 2 mM EDTA, 100 U ml–1 penicillin and 100 μg ml–1 streptomycin for 1 min 3 times to remove any remaining mucus and further digested with 10 ml of 0% medium containing 0.5 mg ml–1 DNase I (Sigma-Aldrich) and 0.1 mg ml–1 Liberase TL (Roche) for 25 min at 37 °C with constant stirring at 400 rpm. Lungs were diced and incubated in 2 ml of prewarmed medium containing 0.5 mg ml−1 DNase I and 0.1 mg ml–1 collagenase IV for 35 min at 37 °C. To isolate cells from the skin, ear pinnae were excised and separated into ventral and dorsal sheets, digested by placing the dermal side down in medium containing 0.5 mg ml–1 DNase I with 0.25 mg ml−1 Liberase TL and incubated for 90 min at 37 °C. Digested intestines, lung and skin were passed through 70 μm cell strainers and centrifuged at 1,500 rpm for 5 min. Leukocytes were enriched by resuspending the samples in 4 ml of 37.5% Percoll and centrifuged at 1,800 rpm for 5 min. For blood analysis, 200 μl blood was added with 1 ml of ACK lysing buffer and incubate on ice for 10 min with occasional shaking. The reaction was then quenched with 5 ml of 3% medium followed by spinning at 1,800 rpm for 5 min. The pellet was then resuspended in 3% medium. Cells were washed with HBSS once before downstream staining and analyses.
Flow cytometry analysis
Cells isolated from tissues were plated into 96-well plates with counting beads. To measure cytokine production potential, cells were re-stimulated in complete IMDM supplemented with 10% FBS, 50 ng ml–1 PMA, 5 μg ml–1 ionomycin and 3 μg ml–1 BFA for 2.5 h at 37 °C. Cells were washed twice with cold HBSS before staining. Surface staining was performed on ice for 20 min, followed by washing with HBSS once and 10 min of Live/Dead staining. Cells were then washed twice with HBSS and fixed for 1 h at room temperature using either eBioscience intracellular fixation buffer (00-5123-43, Invitrogen) if intracellular staining was needed or BD Cytofix Fixation buffer (554722, BD Biosciences) for surface staining only. For intracellular staining, fixed cells were washed twice with the corresponding permeabilization buffer and intracellular staining was performed overnight at 4 °C. Before flow analysis, cells were washed twice and resuspended in 200 μl cold PBS. Flow cytometry analysis was performed on a FACSymphony A3.
The following antibodies were used for flow cytometry: anti-CD90-2 (FITC; 1:200; 53-2.1), anti-CD90-2 (BV786; 1:400;3 0-H12), anti-MHCII (BV650; 1:500; M5/114.15.2), anti-CD64 (BV605; 1:100; X54-5/7.1), anti-CD103 (BV510; 1:100; 2E7), anti-Ly6C (APCCy7; 1:200; HK1.4), anti-F4/80 (AF700; 1:200; BM8), anti-PD-L1 (PE; 1:100; 10 F.9G2), anti-TCRγ/δ (BV650; 1:200; GL3), anti-IFNγ (BV605; 1:200; XMG1.2), anti-CD4 (BV510; 1:200; RM4-5), anti-IL17A (BV421; 1:200; TC11-18H10.1), anti-T-bet (PECy7; 1:200; 4B10), anti-CD48 (BV786; 1:200; HM48-1), anti-CD45R/B220 (BV421; 1:400; RA3-6B2), anti-CD135 (APC; 1:200; A2F10), anti-CD150 (PECF594; 1:200; TC15-12F12.2), anti-FcεRIα (AF700; 1:200; MAR-1), anti-CCR3 (PECy7; 1:200; J073E5), anti-cKit (PECy7; 1:200; 2B8) and anti-CD200R3 (PE; 1:100; Ba13) were from BioLegend; anti-CD11c (BV786; 1:200; N418), anti-B220 (BUV805/PerCPe710; 1:200; RA3-6B2), anti-CD11b (BUV737; 1:100; M1/70), anti-CD45 (BUV395/APCCy7; 1:200/1,000; 30-F11), anti-FOXP3 (FITC; 1:400; FJK-16s), anti-TCRb (BUV737; 1:100; H57-597), anti-CD44 (AF700; 1:100; IM7), anti-GATA3 (APC; 1:200; TWAJ), anti-IL-5 (PE; 1:200; TRFK5), anti-IL-13 (PE; 1:200; eBio13A), anti-cKit (PerCPe710; 1:200; 2B8), anti-CD34 (AF488; 1:100; RAM34), anti-SCA1 (BV605; 1:400; D7), anti-CD16/CD32 (AF700; 1:200; 93) and anti-CD127 (PECy7; 1:200; A7R34) were from eBioscience; and anti-Ly6G (BV650/APC; 1:200; 1A8), anti-Siglec-F (PECF594/BV510; 1:200; E50-2440), anti-CD8b (BUV395; 1:400; H35-17.2), anti-RORγt (PECF594; 1:400; Q31-378), anti-CD125 (PE; 1:100; T21) and anti-CD90-2 (BUV805; 1:200; 30-H12) were from BD Bioscience.
Imaging of intestinal tissues
... continue reading