Animal experiments were performed with adult 6- to 16-week-old mice with equal numbers of males and females. All mice were provided sterile pellet food and water ad libitum. C57BL/6N mice were purchased from Orient Bio (South Korea), and Gpr81−/− mice were generated as described previously23. CD45.1+ (B6. SJL-Ptprca Pepcb/BoyJ), LepR-cre (B6.129(Cg)- LepRtm2(cre)Rck/J), and tdTomato-loxP (B6. Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) mice were purchased from Jackson Laboratory and backcrossed in house. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Asan Biomedical Research Center (Approval No: PN 2018-12-288) and conducted in compliance with regulatory guidelines. Mice were maintained under specific pathogen-free conditions at the Asan Biomedical Research Center (Seoul, South Korea), and GF mice were maintained in the animal facility at POSTECH (Pohang, South Korea). All animal experiments were performed under anesthesia with a mixture of ketamine (100 mg/kg BW) and xylazine (20 mg/kg BW).
Purification of BM cells
BM plugs were flushed gently from the marrow cavity of two femurs and two tibias using a syringe fitted with a 23-gauge needle containing cold phosphate-buffered saline (PBS) to isolate BM mononuclear cells. Flushed cells were then gently drawn up into and expelled from a 26-gauge needle to dissociate clumps. Next, cells were filtered through a 40-µm strainer (BD Falcon), and recovered total BM cells were counted. Intact BM plugs from two femurs and two tibias were enzymatically digested in prewarmed digestion solution (DNase I [100 µg/mL, Roche], LiberaseDL [250 µg/mL, Roche] in HBSS plus Ca2+and Mg2+) and incubated at 37 °C for 30 min with gentle shaking (~120 r.p.m.) to isolate MSCs. After weak vortexing for 20 s, cells were allowed to settle for ~3 min, after which the supernatant was transferred to another tube on ice. The settled BM plugs were enzymatically digested repeatedly two times, as previously described. The collected cells were then counted with a 100-µm strainer (BD Falcon).
Total BM cells were collected from two femurs and two tibias and flushed gently to analyze the hematopoietic cells. Dissociated BM cells were then washed with 1% fetal bovine serum (FBS) in PBS. Cells were stained with any of the following antibodies for 20 min on ice: anti-CD16/32 (Invitrogen), lineage antibody cocktail-v450 (BD), anti-Sca-1 (Invitrogen, D7), anti-CD45 (BD, 30-F11), anti-CD45.1 (BD, A20 or Invitrogen, A20), anti-CD45.2 (Invitrogen, 104), anti-CD48 (Invitrogen, HM48-1), anti-CD150 (Invitrogen, 9D1 or BioLegend, TC15-12F12.2), anti-c-Kit (BioLegend, ACK2 or Invitrogen, ACK2), anti-CD34 (Invitrogen, RAM34), anti-CD41 (BioLegend, MWReg30), and anti-CD105 (BioLegend, MJ7/18). Goat anti-LepR-biotin polyclonal antibody (R&D Systems), Live/Dead dye (Invitrogen), anti-CD45 (BD, 30-F11), anti-Ter119 (Invitrogen, Ter119), anti-CD31 (BioLegend, MEC 13.3), and streptavidin (BD) were used to analyze the MSCs. Cells were then fixed and permeabilized using Cytofix/Cytoperm (BD) for 20 min during intracellular staining. Next, the cells were washed with Perm/Wash buffer (BD), incubated with rabbit anti-SCF polyclonal antibody (LSbio), and stained with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen). Flow cytometry data were collected using LSR II (BD Biosciences), and the files were analyzed using FlowJo software (Tree Star).
Reconstitution of BM cells
Recipient (CD45.2+) mice were treated with 10 Gy of total-body irradiation (cesium source irradiator; Precision X-Ray, North Branford, CT). The next day, recipient Gpr81+/− or Gpr81−/− mice received BM cells (1 × 107 cells) from donor WT mice (CD45.1+). Recipient mice were then analyzed 12 weeks after irradiation.
Administration of probiotics, L. plantarum, and lactate
Age- and sex-matched mice were orally administered probiotic VSL#3 (2 × 109 CFU, Sigma-Tau Pharmaceuticals) in 100 μL of PBS and drinking water containing dl-lactate (10 mM, Sigma-Aldrich) daily for 1 week. L. plantarum strains were grown at 37 °C in MRS broth (BD Difco), as previously described33. Both L. plantarum strains were kindly provided by Dr. Siqing Liu, National Center for Agricultural Utilization Research, US Department of Agriculture. GF mice were orally administered 2 × 109 CFU of WT L. plantarum or L. plantarum ΔldhD-ΔldhL strains.
Irradiation-induced injury model
Mice were irradiated with 6 Gy of total-body irradiation (cesium source irradiator; Precision X-Ray, North Branford, CT) and analyzed at 1–2 weeks postirradiation. During the busulfan-induced injury model, mice were injected intraperitoneally with busulfan (25 mg/kg, Sigma-Aldrich) and analyzed after 1 week.
Dissected bones were fixed in 4% paraformaldehyde for 1 day and then decalcified for 1 week in 0.5 M EDTA refreshed daily. The fixed tissues were dehydrated through a chain of graded ethanol baths and then embedded in paraffin. The paraffin-embedded blocks were cut into 5-μm-thick sections and stained with hematoxylin and eosin (H&E).
Bone sections were permeabilized in PBS containing 0.5% Triton X-100 at RT for 20 min and blocked with 5% BSA in PBS at RT for 1 h. The sections were then stained with primary antibodies against DAPI (Invitrogen), anti-VE-cadherin (R&D, 162709), anti-CD31 (BD, MEC 13.3), anti-SDF-1α (R&D, 79018), anti-endomucin (Abcam, V.7C7.1), rabbit anti-SCF polyclonal (Abcam), and goat anti-LepR-biotin polyclonal (R&D Systems) overnight at 4 °C. Sections were then washed with PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG (Invitrogen), Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Invitrogen), DyLight 488-conjugated streptavidin (BioLegend), or DyLight 647-conjugated streptavidin (BioLegend) at RT for 1 h. Fluorescence images of all samples were captured on an LSM 710 confocal microscope (Carl Zeiss). To measure the number of SCF+ cells, we counted LepR+SCF+ cells attached to endothelial cells.
Enzymatically digested BM cells were seeded in eight-well Lab-Tek II Chamber slides (5 × 104 cells/well, Sigma-Aldrich). Cells were cultured in α-MEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) for 3 days and then treated with sodium l-lactate (5 mM, Sigma-Aldrich), 3,5-DHBA (0.2 mM, Sigma-Aldrich) or 3-OBA (3 mM, Sigma-Aldrich). Plated MSCs were fixed with Cytofix/Cytoperm (BD) for 20 min at 4 °C and subsequently washed with Perm/Wash buffer (BD) during immunocytochemistry. After blocking with 5% BSA in PBS for 1 h, MSCs were stained with DAPI, goat anti-LepR-biotin polyclonal (R&D Systems), anti-endomucin (Abcam, V.7C7.1), anti-SDF-1α (R&D, 79018), and rabbit anti-SCF polyclonal (Abcam) antibodies overnight at 4 °C. After one wash with Perm/Wash buffer, cells were stained with DyLightTM 488-conjugated streptavidin (BioLegend) or Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Invitrogen) as secondary antibodies. All samples were viewed under a confocal laser scanning microscope (Carl Zeiss).
Colony-forming unit (CFU) assay
BM cells were suspended in MethoCult media (StemCell) as per the manufacturer’s instructions. Cells were then dispensed into 24- or 48-well culture plates (Thermo Scientific) using a syringe with a 16-gauge blunt needle (StemCell) and incubated at 37 °C in a 5% CO2 incubator. CFU-E and CFU-GM were counted after 2 and 12 days of culture, respectively. During the CFU-F analysis, BM MSCs were seeded in a six-well culture plate (Thermo Scientific) (1 × 106 cells/mL) and cultured with α-MEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) and 10 µM ROCK inhibitors (Sigma). Cells were then washed with PBS to prevent the spread of hematopoietic cells and cultured with fresh medium. Colonies were counted after 7 days of culture by staining with 1% crystal violet (Sigma).
Complete blood count analysis
Blood cells were analyzed using the ADVIA 2120i Hematology System (Siemens). Peripheral blood obtained from retro-orbital plexus bleeding was placed immediately into a microtainer blood collection tube (BD) with K2 EDTA. Samples were then gently mixed and stored on ice until analysis.
RNA was extracted using the RNeasy mini kit (Qiagen) from the total BM cells isolated from the femur and tibia. Thereafter, RNA was converted to cDNA using Superscript II reverse transcriptase and oligo (dT) primers (Thermo Fisher). The cDNA was then used as the template for real-time qPCR conducted using SYBR green chemistry (Affymetrix) on an ABI 7500 real-time qPCR system (Applied Biosystems). The primer sequences used for real-time qPCR were as follows: Scf, 5′-GCCAGAAACTAGATCCTTTA-3′ and 5′-CATAAATGGTTTTGTGA CACTGACTCTG-3′; SDF-1α, 5′-GAGCCAACGTCAAGCATCTG-3′ and 5′-CGGGTC AATGCACACTTGT-3′; and β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′.
Whole BM cells were homogenized with Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, 0.05% sodium azide, 1% Tween-80, and protease inhibitor cocktail, Roche) and centrifuged at 11,000 × g for 10 min. The supernatant was collected, and the SCF concentration was measured with an SCF ELISA Kit (Abcam) as per the manufacturer’s instructions. Absorbance was measured at 450 nm using a spectrophotometer microplate reader (Bio-Rad).
Lactate concentration analysis
Serum was obtained from retro-orbital plexus bleeding. The lactate concentration in serum was measured using a lactate assay kit (Biovision) as per the manufacturer’s instructions. Absorbance was measured at 570 nm using a spectrophotometric microplate reader (Bio-Rad).
Statistical analyses were performed with Prism software (GraphPad), and two-tailed t-tests were performed for pairwise and two independent group comparisons. Data are presented as the mean ± SEM, and a P value of <0.05 was considered statistically significant. The exact value of n, representing the number of mice in the experiments depicted, is indicated in the figure legends. Any additional technical replicates are described within the “Materials and methods” and “Results” sections.