This study was performed in strict accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology in Japan, 2006. All experimental procedures involving animals followed the Regulations for Animal Experiments and Related Activities at Tohoku University, Sendai, Japan and were approved by the Institutional Animal Care and Use Committee at Tohoku University. All methods were performed in accordance with the relevant guidelines and regulations, and all efforts were made to minimize the suffering of the animals. The study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org/).
CARD9 gene–disrupted knockout (KO) mice were generated and established as described previously46, and backcrossed to C57BL/6 J mice for more than eight generations. MyD88KO mice, established as described previously47, and backcrossed to C57BL/6 J mice, were purchased from OrientalBioService, Inc. (Kyoto, Japan). Wild-type (WT) C57BL/6 J mice, purchased from CLEA Japan (Tokyo, Japan), were used as a control. Male mice at 7 to 10 weeks of age were used in all experiments shown in Figs. 1, 2, 3 and 4. For the results shown in Supplementary Fig. 3, female mice were used in half the experiments, because there were not sufficient numbers of MyD88KO male mice for all analyses. Food and water were available ad libitum. All mice were kept under specific pathogen-free conditions in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine (Sendai, Japan). All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of Tohoku University. All experiments were performed under anesthesia, and all efforts were made to minimize the suffering of the animals.
Wound creation and tissue collection
Mice were anesthetized by intraperitoneal injection of 40 mg/kg sodium pentobarbital (Somnopentyl, Kyoritsu Seiyaku Corporation, Tokyo, Japan), and sustained by inhalation anesthesia of isoflurane (Isoflurane, Mairan Pharma, Osaka, Japan). The dorsal hair was shaved to fully expose the skin, which was then rinsed with 70% ethanol. Four full-thickness wounds extending to the panniculus carnosus were created on each mouse using a 6-mm-diameter biopsy punch (Biopsy Punch, Kai Industries Co., Ltd., Gifu, Japan) under sterile conditions and covered with a polyurethane film (Tegaderm Transparent Dressing, 3 M Health Care, St. Paul, MN, USA) and an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) for an occlusive dressing. At various time points, mice were sacrificed by overdose of isoflurane, and the wound tissue was collected by excising a 1-cm-square section of skin using scissors and a surgical knife. The tissue was then processed for histopathological analysis and measurement of cytokine concentrations.
Preparation of heat-killed Lactobacillus plantarum KB131
L. plantarum KB131 (International Patent Organism Depositary, Japan, NITE BP-03375) was stored at Bio-Lab Co., Ltd. L. plantarum KB131 was grown aerobically overnight at 37 °C in MRS broth (Difco, Detroit, MI, USA) and washed with distilled water, followed by centrifugation at 10,000×g for 3 min. The bacterial suspension in distilled water was heated at 105 °C for 30 min using an autoclave (HV-25IILB; Hirayama Manufacturing Corp., Saitama, Japan). Based on the recent reclassification of LAB, L. plantarum belongs to the Lactiplantibacillus genus48. We confirmed using species-specific primers that KB131 belonged to L. paraplantarum49. Wounds were created in accordance with the method described above. Immediately after wounding, a 5 μL suspension of heat-killed L. plantarum KB131 (125 μg/μg) or distilled water as a vehicle control was applied to the base of the wounds in mice.
Measurement of the wound area
Morphometric analysis was performed on digital images using a digital camera (G800, Ricoh, Tokyo, Japan). After the wounds were created, photographs were taken of each wound before dressing. At various time points, the polyurethane films were gently removed from the sacrificed mice, and the wounds were photographed. The wound area was quantified by tracing its margin and calculating the pixel area using AxioVision imaging software, Release 4.6 (Carl Zeiss Micro Imaging Japan, Tokyo, Japan). Wound healing was evaluated as the percent wound closure, which was calculated using the following formula: % wound closure = (1 − wound area at the indicated time point/wound area on day 0) × 100.
Histopathology and immunohistochemistry
The wounded tissues were fixed with 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin after caudocranial dissection, as previously described20,21,50. Sections were harvested from the central portion of the wound and stained with hematoxylin–eosin (HE) according to the standard method. The extent of re-epithelialization of each wound was measured in these HE-stained sections by measuring the distance from the normal wound margin to the edge of the epithelium. The re-epithelialization index was determined based on the percentage of new epithelium present in the total wound. Granulation area was determined on HE-stained sections. For immunohistochemistry, the sections were stained with anti-CD31 antibody (1:600 dilution; R&D Systems, Minneapolis, MN, USA), anti-α-smooth muscle actin (α-SMA) antibody (1:300 dilution; Dako, Santa Clara, CA, USA), or anti-F4/80 antibody (1:100 dilution; BioLegend, San Diego, CA, USA) after endogenous peroxidase and nonspecific binding were blocked. They were then incubated with peroxidase-conjugated secondary antibodies (Histofine® Simple Stain MAX-PO, Nichirei Bioscience, Tokyo, Japan). Control sections were treated with non-immune IgG in place of any of the first antibodies. The vascular density in granulation tissue and the number of macrophages in each of five random fields (each 0.03 mm2) were determined by counting the number of CD31-positive vessels and the number of F4/80-positive cells, respectively. The amount of myofibroblasts was determined by calculating the area of α-SMA-positive cells.
Measurement of cytokine, chemokine, and growth factor concentrations
The wounded tissues were homogenized with saline solution, and the concentration of cytokines and chemokines in the supernatants was measured using appropriate enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, San Diego, CA, USA, for TNF-α, IL-4, IL-5, IL-6, IL-10, IFN-γ, IL-12p40; R&D Systems, Minneapolis, MN, USA, for CXCL1, CXCL2, CCL2, CCL3, CCL4, CCL5, bFGF, TGF-β1, VEGF, and EGF). The results were expressed as the values per wound.
Preparation of leukocytes in the wound tissue
Mice were sacrificed at days 1, 3, or 5 after wound creation. The wound tissues were excised and teased apart using stainless-steel mesh in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 mM HEPES, 10% fetal calf serum (FCS) (BioWest, Nuaillé, France), 0.2 mg/mL Liberase TL, 2.5 mg/mL collagenase, 0.1 mg/mL DNase, and 2.0 mg/mL Dispase (Sigma-Aldrich). They were incubated for 2 h at 37 °C with vigorous shaking. After incubation, the tissue fragments and most dead cells were removed by passing the cells through a 70-μm cell strainer (BD Falcon, Bedford, MA, USA). After centrifugation, the cell pellet was resuspended in 4 ml of 40% Percoll (Pharmacia, Uppsala, Sweden) and layered onto 4 ml of 80% Percoll. After centrifugation at 1800 rpm for 20 min at 20 °C, the cells at the interface were collected, washed three times with staining buffer, and counted using a hemocytometer.
Analysis of leukocyte fraction using flow cytometric analysis
The cells obtained from the wounded tissues were stained with 7-aminoactinomycin D (7-AAD) (BioLegend), Pacific Blue-anti-CD45 monoclonal antibody (mAb) (clone 30-F11, BioLegend), APC-anti-CD11b mAb (clone M1/70, BioLegend), APC/Cy7-anti-Ly6G mAb (clone 1A8, BioLegend), PE-anti-F4/80 mAb (clone BM8, BioLegend), FITC-anti-CD3ε mAb (clone 145-2C11, BioLegend), FITC-anti-NK1.1 mAb (clone PK136, BioLegend), FITC-anti-T-cell receptor γδ (TCRγδ) mAb (clone GL3, BioLegend), and FITC-anti-CD45R/B220 mAb (clone RA3-6B2, BioLegend). Isotype-matched irrelevant IgG was used for control staining. To investigate macrophage subtypes, the cells were further stained with biotin-conjugated anti-mouse CD119 (IFN-γ R α chain) mAb (clone 2E2, BioLegend), followed by staining with APC/Cy7 Streptavidin (BioLegend). In addition, CD206 was stained according to the manufacturer’s methods. Briefly, leukocytes from wounded tissues were incubated in the presence of Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ), washed twice in BD perm/wash solution, and stained with APC-anti-CD206 antibody (clone C068C2; BioLegend) or control rat IgG. Neutrophils and macrophages were identified as CD45+CD11b+Ly6G+ cells and CD45+CD11b+F4/80+ cells, respectively. Lymphocytes were identified as CD45+ cells expressing CD3ε, NK1.1, TCRγδ, or B220. M1-type and M2-type macrophages were identified as CD45+F4/80+IFNGR1+ CD206– cells and CD45+F4/80+ IFNGR1–CD206+ cells, respectively. The stained cells were analyzed using a BD FACS Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). The number of neutrophils, macrophages, and lymphocytes was estimated by multiplying the total leukocyte number by the proportion of each fraction.
Data are expressed as the mean ± standard deviation (SD). Data analysis was performed using Welch’s t-test to compare two experimental groups, and a one-way analysis of variance (ANOVA) with post hoc Dunnett’s or Tukey–Kramer’s honestly significant difference (HSD) test was used for more than three experimental groups. A p-value less than 0.05 was considered to indicate statistical significance.