Inhibition of platelet activation suppresses reactive enteric glia and mitigates intestinal barrier dysfunction during sepsis

Animals

All animal experiments in this study were approved by the Ethics Committee for Animal Experimentation of the Xi’an Jiaotong University (Xi’an, China, 2018-107). Male C57BL/6 mice (6–8 weeks old) were obtained from Experimental Animal Center of Xi’an Jiaotong University (Xi’an, China) and raised in standard cages under controlled conditions with a 12-h light/dark cycle at 21 ± 2 °C temperature and 60–70%.

Sepsis model and drug administration

Cecal ligation and puncture (CLP) was performed to establish the sepsis model with some modifications as described previously (Rittirsch et al. 2009). Briefly, we anesthetized the mice with isoflurane inhalation and injected lidocaine subcutaneously local to incision as an analgesic before surgery. The cecum was exteriorized through a 1–2 cm longitudinal midline abdominal incision. Then 75% of the cecum was ligated and punctured twice with a 21-G needle to externalize feces. All mice were subcutaneously injected with 1 mL 0.9% normal saline for fluid resuscitation and administrated a single dose of antibiotics (ceftriaxone at 30 mg/kg and clindamycin at 25 mg/kg) immediately after CLP. Subsequently, mice were temporarily placed on a heating pad to maintain the body temperature until fully recovered from anesthesia. The sham-operated mice underwent the same procedure without ligation and puncture. In the additional groups of mice, cilostazol (10 mg/kg) was diluted from 0.5% carboxymethyl cellulose sodium salt (CMC) and administrated orally 2 h prior to and at 12 h after CLP to inhibit platelet activation (Chang 2015), compound 6,877,002 (10 μmol/kg) was injected intraperitoneally 2 h prior to and at 12 h after CLP to block CD40L–CD40 signaling pathway (Zarzycka et al. 2015) and GSNO was administrated intraperitoneally at 10 mg/kg/day for 7 days before CLP to verify its function on intestinal hyperpermeability (Savidge et al. 2007). 24 h after CLP, mice were sacrificed, and blood or tissue samples were collected for further experiments.

Intestinal barrier permeability test

The intestinal barrier permeability was evaluated by fluorescein isothiocyanate (FITC)-dextran test 24 h after CLP. Briefly, overnight fasted mice were orally gavaged with 25 mg/mL of 4 kDa FITC-Dextran (0.5 mg/g body weight; Sigma-Aldrich). After 1.5 h, plasma was collected, and plasma fluorescence was measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

Water content

Intestinal tissues were excised and weighed immediately (wet weight) and dried at 60 °C for 48 h before weighing (dry weight). The water content represented the degree of intestinal edema and was calculated as follows: water content (%) = (wet weight − dry weight)/wet weight × 100%.

Bacterial content

The mesenteric lymph nodes, lung and liver tissues were homogenized, and the supernatant was obtained by centrifugation. After serial dilutions, 500 μL of each dilution was evenly spread onto blood agar plates, incubated at 37 °C for 24 h, and the colony forming units (CFUs) were counted.

Histological damage score analysis

The histological damage score was estimated after formalin fixation, paraffin embedding and hematoxylin/eosin staining of sections. The images were observed by a light microscope, and a representative field was chosen for assessment. The intestinal damage score was graded as follows: 0, normal mucosal villi; (1) minor subepithelial space and capillary congestion; (2) extensive subepithelial space with little epithelial layer lifting from the lamina propria; (3) massive epithelial layer lifting from the lamina propria; (4) villi detachment and hemorrhage (Bi et al. 2020). The histological damage scores were recorded and analyzed by two investigators blinded to the experimental treatments. Grading of intestinal injury was measured as an average score. Statistical analysis of histological data was performed by an investigator blinded to the experimental treatments.

Echocardiography

Echocardiography was performed 24 h after the CLP using Vevo 3100 with a 400 MHz probe (FUJIFILM SonoSite, lnc. JAPAN). The mice (prior removal of hair from the precardiac region) were anesthetized with isoflurane inhalation and the limbs were fixed. The long axis section of the left ventricle was evaluated, and the left ventricle movement was detected with M-mode echocardiography. The following four parameters were measured: left ventricular end diastolic diameter (LVEDD), left ventricular end systolic dimension (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). All data were processed under the same parameters and analyzed by investigator blinded to the experimental treatments. The value of each measurement index was the average value of three consecutive cardiac cycles.

Complete blood counts

Whole blood samples were collected from the mice via eyeball enucleation in K2EDTA tubes for complete blood counts analysis. Complete blood counts, including platelets, white blood cells (WBC), neutrophil (NEUT), monocyte (MONO) and lymphocyte (LYMPH), were measured using an auto hematology analyzer (Mindray, BC-6800Plus, China).

Flow cytometry

To analyze platelet surface CD40L, blood was collected in sodium citrate and stained with 1 μL of FITC anti-mouse CD41 Antibody (BioLegend) and 2.5 μL of PE anti-mouse CD154 (CD40L) Antibody (BioLegend) at room temperature in the dark, according to the manufacturer’s instructions. Then, the blood samples were incubated with a red blood cell lysis solution (Solarbio, China) and analyzed by flow cytometry (BD Biosciences, U.S.A.). Data analysis was performed using FlowJo (Ashland, OR). Scatter and staining with the FITC-anti-CD41 and PE-anti-CD40L antibodies were used to gate platelet populations. Cells were first gated by regions within a side scatter area (SSC-A) versus forward scatter area (FSC-A) plot, then by gating those populations in a SSC-A versus FITC-A plot. Activated platelets were defined as FITC-anti-CD41-A positive and PE-anti-CD40L-A positive (Additional file 1: Fig. S1).

Soluble CD40L determination

Soluble CD40L (sCD40L) levels in serum was measured using ELISA kits (Cloud-Clone Corp, Wuhan, China) according to the manufacturer’s recommendations.

Immunofluorescence staining

Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.

Western blot analysis

Protein was extracted from intestinal tissues and cells using RIPA buffer containing protease inhibitor cocktail and measured using a BCA protein assay kit. An equivalent of protein samples was separated by 12% SDS-PAGE and transferred to PVDF membranes for western blot analysis. After blocking with 5% skimmed milk for 1 h, the membranes were probed overnight at 4 °C with the following different primary antibodies: rabbit anti-GFAP (1:5000, GeneTex, Irvine, CA, USA), rabbit anti-CD40 (1:1000, Abcam, Cambridge, UK), rabbit anti-ZO-1 (1:1000, Affinity, USA), rabbit anti-occludin (1:5000, ABclonal, Wuhan, China), mouse anti-TRAF1 (1:100, Santa Cruz, CA), mouse anti-TRAF2 (1:100, Santa Cruz, CA), mouse anti-TRAF3 (1:100, Santa Cruz, CA), mouse anti-TRAF4 (1:100, Santa Cruz, CA), mouse anti-TRAF5 (1:100, Santa Cruz, CA), mouse anti-TRAF6 (1:100, Santa Cruz, CA), and rabbit anti-GAPDH (1:5000, ABclonal). Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The immunoreactive bands were detected using a chemiluminescence imaging system ChemiScope 6000 (Clinx, Shanghai, China), and the intensity was analyzed with ImageJ.

Primary EGCs and astrocytes culture

As mentioned previously, primary EGCs were prepared from the intestinal tract of embryonic days 17–19 fetal mice (Li et al. 2021) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Ltd., NY, USA) containing 10% fetal bovine serum (FBS), 1% glutamine (Sigma), and 1% penicillin and streptomycin solution (Pen Strep) (Gibco) at a density of 4 × 104 cells/cm2. After 1 week, EGCs were isolated using 1 U/mL dispase (Roche, Basel, Switzerland) to eliminate the fibroblasts or epithelial cells and identified by GFAP and Sox 10 immunoreactivity.

As described previously, primary astrocytes were prepared from postnatal days 1–3 mice brains (Zhu et al. 2020) and cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS), 1% glutamine (Sigma), and 1% penicillin and streptomycin solution (Pen Strep) (Gibco) at a density of 4 × 104 cells/cm2. After 1 week, the cells were shaken (200 rpm, 37 °C) overnight to eliminate nonspecific glia, such as microglia, and the astrocytes were identified by GFAP and Iba-1 immunoreactivity.

Stimulation and siRNA transfection

To induce the cell sepsis model, EGCs were stimulated by single lipopolysaccharide (LPS) (10 μg/mL, E. coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA), CD40L (1 μg/mL, PeproTech, Rocky Hill, NJ, USA) or LPS + CD40L. After 24 h, the cell culture supernatants were collected as EGCs-conditioned medium for cytokine measurement and Caco-2 cell treatment.

For the siRNA transfection, CD40-siRNA, TRAF6-siRNA, negative control siRNA (NC-siRNA), and siRNA-Mate transfection reagent were purchased from GenePharma Co. Ltd, (Shanghai, China). EGCs were transfected with CD40-siRNA, TRAF6-siRNA or NC-siRNA using siRNA-Mate according to the manufacturer’s protocols for 72 h before LPS and CD40L stimulation.

Caco-2 culture and in vitro permeability assay

Caco-2 cells (CL-0050, Procell Life Science & Technology Co., Ltd., Wuhan, China) were cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS), 1% glutamine (Sigma), and 1% penicillin and streptomycin solution (Pen Strep) (Gibco). For in vitro permeability assay, Caco-2 cells were seeded at a density of 4 × 106 cells/cm2 in 6.5-mm transwell dishes with 0.4 μm pore polycarbonate membrane inserts (3413; Corning). After 1 week of culture, transepithelial electrical resistance (TEER) was measured using an Electrical Resistance System (ERS) (Millipore) to confirm cell polarization and establish barrier characteristics (> 450 Ω/cm2). Then, Caco-2 cells were stimulated with EGCs-conditioned medium and GSNO (50 µM, Sigma) for 24 h before Western blot and immunofluorescence staining. Transepithelial electrical resistance was measured as Ω cm2 every hour after stimulation by subtracting the background resistance and multiplying with the monolayer surface area to calculate the relative value.

Cytokine and GSNO measurements

TNF-α, IL-1β and GSNO were measured using ELISA kits as recommended by the manufacturer. Briefly, the supernatants were harvested from intestinal tissue homogenate, serum and EGCs-conditioned medium, and the impurity was removed by centrifugation at 12,000g for 10 min. TNF-α (EMC102a.96) and IL-1β (EMC001b.96) ELISA kits were obtained from NeoBioscience (Shenzhen, China) to measure TNF-α and IL-1β production in vivo and in vitro. GSNO (ml08365974) ELISA kit was obtained from Mlbio (Shanghai, China) to measure GSNO production in vitro.

Bioinformatics analysis

Bioinformatics analysis was performed using public databases. These data were downloaded from the GSE156905 dataset of the Gene Expression Omnibus (GEO) database. The single-cell sequencing data (GSE156905) were sourced from distal colon of adult mouse.

The “Read10×” function in the package was used to analyze single-cell sequencing data. Seurat objects were created using the “CreateSeuratObject” function. The “FilterCells” function was used to filter the data and remove the effects of dead cells and cell adhesion. The specific parameters used in the subsequent analysis are as follows: NFEATURE RNA > 200, nFEATURE RNA < 2500 and PERCENT.mt < 5, leaving 1384 cells and 17,047 genes. Data were normalized using the “NormalizeData” function with the following parameters: normalize. Method = “LogNormalize” and scale.factor = 1000. The “Find variable genes” function was used to calculate highly variable genes and set to default values. The “ScaleData” function was further used to normalize the data and remove the source of variation. The Uniform Manifold Approximation and Projection (UMAP), was obtained via analysis using the “BiocManager” packets in the R language.

Screening and annotation of cell marker genes in different subpopulations. The screening and annotations of different cell subpopulations were compared among all cell subpopulations and obtained genes per subgroup. The identification of each cell was based on the differential expression of characteristic genes among the various cell clusters. GFAP has long been a specific marker of astrocyte (Yang and Wang 2015), which shares many characteristics with EGCs (Gulbransen and Sharkey 2012; Jessen and Mirsky 1980; Yang and Wang 2015). Given that accumulating evidences depict that GFAP can be used for identifying EGCs (Rao et al. 2017), we applied it as a marker for EGCs. Subsequently, the cell tag gene was analyzed in the R language using the “CellMarker” website (http://biocc.hrbmu.edu.cn/CellMarker/), which determines the cell type corresponding to each subpopulation.

KEGG enrichment analysis

All microarray data were downloaded from the GEO database (http://www.ncbi.nih.gov/geo). The raw data were downloaded as MINiML files. The differentially expressed mRNA was studied using the limma package in the R software. The adjusted P-value was analyzed to correct the false positive results in GEO datasets. “Adjusted P < 0.05 and Log (Fold Change) > 1 or Log (Fold Change) < − 1” were defined as the threshold for the differential expression of mRNAs. To further confirm the underlying function of potential targets, the data were analyzed by functional enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis is a practical resource for studying gene functions and associated high-level genome functional information. To better understand the carcinogenesis of mRNA, ClusterProfiler package (version: 3.18.0) in R was employed to enrich the KEGG pathway.

Statistical analysis

Statistical analysis was performed using a GraphPad Prism software (version 7.00, U.S.A.). The survival rates were analyzed using the log-rank (Mantel–Cox) test and reported as percentages (%). The other data were reported as mean ± standard error of the mean (SEM). Gaussian distribution was evaluated by Shapiro–Wilk normality test. The statistical significance of the differences between groups, except for the survival rate, was evaluated by Student’s t-test or one-way analysis of variance (ANOVA) (Dunnet correction for multiple comparisons). Nonparametric Kruskal–Wallis tests were performed if data did not belong to the Gaussian distribution. Differences were considered significant at a P value less than 0.05 (*), less than 0.01 (**), less than 0.001 (***), or less than 0.0001 (****).

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