Mice

PLXNC1-/- mice—originally provided by Amgen (Amgen, Thousand Oaks, California, USA)—were established on a C57BL/6 background and bred in our local animal facilities at the University of Tübingen, Germany. These PLXNC1-/- mice show a stable deletion of PLXNC1-expression in all body tissues and have been used researching the receptor by various researchers for over a decade [16, 18, 22]. C57BL/6 mice provided by Charles River (Charles River, Sulzfeld, Germany) were backcrossed with PLXNC1-/- mice to obtain a wild-type genotype as littermate controls.

In vivo mouse models

Animal experiments were performed in accordance with the German Animal Welfare Law and approved by the appropriate local authorities (i.e. Regierungspräsidium Tübingen). Polymicrobial sepsis was induced in respective mice by 50% ligation of the caecum and puncture using a 20 gauges needle. For sham surgery, the cecum was carefully removed and replaced without ligation or puncture.

The model of E. coli-induced sepsis was established by intraperitoneal injection of viable bacteria (5 × 107 CFUs/mouse; ATCC, #19138, Manassas, Virginia, USA).

In corresponding experiments, animals were euthanized 24 h after CLP. Peritoneal lavage fluid, blood, and organ tissue samples were retrieved for further analysis.

Histological assessment

Liver and colon tissue samples were retrieved from mice after indicated experiments. Samples were fixed with 4% formalin overnight after harvesting, and embedded in paraffin and stained with H&E. A Leica DM5000B microscope (Leica Microsystems, Wetzlar, Germany) was used to acquire images of tissue sections. Liver damage was assessed based on the Suzuki score [23], and intestinal damage was assessed based on Chiu’s score [24] by two independent, blinded examiners.

Cytokine measurement and LDH assay

Concentrations of cytokines (IL-6, TNF-α, IL-1ß, and keratinocyte-derived chemokine [KC]) in peritoneal lavage and cell culture supernatants were determined with ready-to-use murine sandwich ELISAs (R&D Systems, Minneapolis, Minnesota, USA). LDH activity was measured in cell culture supernatants using a CytoTox 96© Non-Radioactive Cytotoxicity Assay Kit according to the manufacturer's instructions (Promega, Madison, Wisconsin, USA). IL-18 concentration was measured using a murine IL-18 ELISA kit (MBL Life Science, #7625, Japan) according to the manufacturer’s instructions. ELISA plates were read in an Infinite® M200 Pro Plate Reader (Tecan, Männedorf, Switzerland).

Caspase-11 measurement

Caspase-11 concentration in peritoneal lavage fluid and cell culture supernatants was measured using a commercially available ELISA kit (Abbexa Ltd., abx255239, Cambridge, UK) in accordance with the manufacturer’s instructions.

Protein isolation and Western blot analysis

Peritoneal lavage fluid was harvested 24 h after CLP and centrifuged at 2000 rpm to precipitate the cellular fraction. The cellular fraction was then reconstituted in ice-cold acetone and methanol. In cell culture experiments, cells were lysed in RIPA buffer with protease inhibitor (1:100). Proteins were separated via SDS‒PAGE and transferred to a 0.2 µm PVDF membrane for analysis of caspase-11 (Novus Bio, #14D9, Littleton, Colorado, USA), caspase-1 (AdipoGen, #mAB Casper-1, Rockland, Maine, USA), ASC (LSBio, #LS-C413166, St. Louis, USA), GSDMD (Abcam, #ERP19828, Cambridge, UK), NLRP3 (LSBio, LS-C148764, St. Louis, Missouri, USA). Species-matched alkaline phosphatase-conjugated secondary antibodies were used. Protein detection was performed using a BCIP/NBT substrate or visualized by enhanced chemoluminescence (Amersham Pharmacia Biotech, Amersham, UK).

GAPDH expression was determined using an anti-GAPDH antibody (Sigma‒Aldrich, #G9545, St. Louis, Missouri, USA). Labeled protein bands were visualized by enhanced chemoluminescence.

Isolation of BMDMs

BMDMs were obtained from femurs and tibias of mice after euthanasia. Bones were washed out with PBS using a 27 gauges needle, and then cultured in a 10 cm Petri dish at 37 °C for a minimum of 6 days in RPMI 1640 medium (Sigma‒Aldrich, St. Louis, USA) supplemented with 10 ng/ml murine GM-CSF (R&D Systems, 415-ML, Minneapolis, Minnesota, USA). For stimulation in culture, cells were plated in a 48-well plate at a density of 5 × 105 cells/well.

Isolation and differentiation of human peripheral blood monocytes/macrophages

Human peripheral blood cells (PBMCs) were isolated from healthy donors at the Blood Bank of Eberhard Karls University of Tübingen by gradient centrifugation using Histopaque-1077 (MilliporeSigma, Burlington, Vermont, USA). In short, 20 ml of blood were mixed with PBS at a 1:1 ratio, and carefully layered onto 15 ml of Histopaque-1077 before being centrifuged (30 min, 1500 U/min, 20 °C). PBMCs—gathered in an opaque phase between erythrocytes at the bottom and the plasma phase above—were collected and cultured in RPMI 1640 medium supplemented with 10 ng/ml human recombinant GM-CSF (Miltenyi Biotec, #130–093-866, Bergisch Gladbach, Germany) at 37 °C in a 5% CO2 atmosphere for a minimum of 6 days for differentiation into monocytes/macrophages.

Stimulation of noncanonical inflammasome (caspase-11)

BMDMs from PLXNC1-/- and wild-type control mice were primed overnight with 50 ng/ml LPS from E. coli subtype O111:B4 (Sigma‒Aldrich, L4392-1MG, St. Louis, Missouri, USA) to induce upregulation of caspase-11, as well as of pro-IL-1β, and pro-IL-18 [5, 25]. Samples were then transfected with the same LPS for actual caspase-11 activation at a total concentration of 2 µg/ml LPS in the presence of Lipofectamine 2000© (3 µl/ml; Thermo Fisher, #11668019, Waltham, Massachusetts, USA) according to the manufacturer's instructions [25]. After 6 h, supernatants were collected for LDH activity and cytokine level measurements, and cells were lysed in RIPA buffer for protein extraction.

Digitonin assay

BMDMs from C57BL/6 wild-type mice were prepared and transfected with LPS in a 12-well plate as described above. After stimulation, cells were detached with Accutase© Cell Detachment Solution (InvivoGen, #00-4555-56, San Diego, California, USA) and pelleted by centrifugation. Resulting cell pellets were resuspended in 60 µl of digitonin buffer (Sigma‒Aldrich, #D141, St. Louis, Missouri, USA) before being centrifuged for separation of the cytosolic fraction and the membrane fraction. The supernatant constituting the cytosolic fraction was resuspended in 5 × Laemmli buffer; the pellet containing the membrane fraction was resuspended in PBS before adding 5 × Laemmli buffer. Proteins were then separated via SDS‒PAGE and transferred onto a 0.2 µm PVDF membrane. Plexin C1 expression was visualized using a polyclonal anti-PLXNC1 antibody (R&D, #AF5375, Minneapolis, Minnestoa, USA) and a BCIP/NBT substrate.

Transfection of SL4c-d ex vivo and in vivo

SL4c-d was custom-produced by ProImmune Ltd. (Oxford, UK) based on the following amino acid sequence: BSACRGDQGGESSLSVSKWNTF. For ex vivo experiments, 3 µg/ml SL4c-d was transfected into BMDMs from C57BL/6 wild-type mice using an Xfect™ Protein Transfection Kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. Two hours after SL4c-d transfection has been completed, media was changed, and cells were primed with LPS (50 ng/ml) overnight. To complete activation of the noncanonical inflammasome, cells have then been transfected with LPS as described above.

To achieve cellular internalization of SL4c-d in vivo, SL4c-d was coupled to the cell-penetrating peptide Arg9 (Genaxxon, P2286.9505, Ulm, Germany) at a 1:1 ratio. First, 5 µg of SL4c-d and 5 µg of Arg9 per animal were dissolved together in 500 µl of sterile PBS and incubated for 30 min; before i.v. injection, the total volume was adjusted to a maximum of 5 ml/kg using sodium chloride. Mice received one i.v. injection of SL4c-d/Arg9 or Arg9 into the tail vein, respectively, directly after CLP. For survival analysis, animals received one injection every 24 h [26, 27].

Protein kinase A activity assay

PKA activity was measured using a colorimetric activity assay kit (Thermo Fisher, EIAPKA, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.

AST assay

Murine serum AST levels were determined using an Aspartate Aminotransferase Colorimetric Assay (Cayman Chemical, #701640, Ann Arbor, Michigan, USA) according to the manufacturer’s instructions.

Next-generation sequencing

NGS of RNA was performed on murine BMDMs after LPS transfection as described above. RNA was isolated utilizing an RNeasy Micro Kit (QIAGEN, #74034, Hilden, Germany) according to the manufacturer’s protocol. Isolated RNA was processed by MFT Services and the Core Unit for Applied Genomics, Tübingen. For data analysis, raw gene expression data were filtered with the criterion of a minimum expression value of 1 counts per million (cpm) in at least three samples. The samples were analyzed with respect to their pairwise similarity. Spearman rank correlation and hierarchical clustering analyses were performed to determine relationships between values. Differential gene expression analysis was conducted based on a gene expression data set filtered with the abovementioned criterion. A statistical model incorporating pairwise relationships and group properties of samples was applied. Genes identified as up- or downregulated in the corresponding group according to the log fold change (logFC) value, p value, and false discovery rate (FDR) were imported into Ingenuity Pathway Analysis (IPA) software (QIAGEN, Hilden, Germany) to identify altered pathways and regulatory mechanisms. Log10 transcripts per kilobase million (TPM) values were used to produce detailed heatmaps including individual values for visualization.

Coimmunoprecipitation

Co-IP was performed as described in the instruction manual of the Thermo Scientific™ Pierce™ Coimmunoprecipitation Kit (cat. number 24149).

Human peripheral blood monocytes/macrophages were cultured in a 10 cm Petri dish. Cells were lysed in 500 µl of lysis/wash buffer, mixed well by pipetting on ice for 5 min and incubated on a rotator at 4 °C for 30 min. After centrifugation at 13000×g for 10 min, the supernatant was transferred to a new tube, and the protein concentration was measured by processing with a Pierce™ BCA Protein Assay Kit (Thermo Fisher, #23225, Waltham, Massachusetts, USA) and analyzed in an Infinite® M200 Pro Plate Reader (Tecan, Männedorf, Switzerland). Preclearing of lysates using control agarose resin was performed as described in the instruction manual. For antibody immobilization, 10 μg of affinity-purified antibody specific for either Plexin C1 (Santa Cruz Biotechnology #sc390216, Santa Cruz, California, USA) or ADCY4 (Invitrogen, PA5-101283, Waltham, Massachusetts, USA) was prepared for binding by adjusting the volume to 200 μl. As an IgG control, 10 µg of mouse IgG (Dako, X0943, Glostrup, Denmark) or 10 µg of rabbit IgG (Thermo Fisher Scientific, #31235, Waltham, Massachusetts, USA) was used. For co-IP analyses, 1 mg of protein was diluted in 200 µl of IP lysis/wash buffer, added to the appropriate resin and incubated with rocking overnight at 4 °C. Prey proteins were eluted in 60 μl of elution buffer. Samples were analyzed by Western blotting via BCIP/NBT method as described above.

Phospho explorer antibody array

Twenty-four hours after CLP, peritoneal lavage fluid was collected from PLXNC1-/- animals and their littermate controls. Proteins were isolated and pooled in accordance with the instructions for the Phospho Explorer Antibody Array (FullMoon BioSystems, PEX100, Sunnyvale, California, USA). For each antibody, the average signal intensity of two replicates was normalized to the median signal of all antibodies in the array. The presented fold change represents the ratio of the signal in PLXNC1-/- animals to the signal in their littermate controls. Data analysis was performed with the IPA software (QIAGEN). Pathways were verified and updated based on recent literature, the KEGG database (hsa04150, hsa04064, hsa04151) and the Reactome database (R-HSA-165159, R-HSA-5676590, R-HSA-198203). A schematic image picturing the respective pathway was constructed using biorender.com based on the data produced by the IPA software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism© 7.0 (GraphPad, San Diego, California, USA). Unpaired Student’s t test was applied for comparisons between two groups. The results are presented as the means ± SEMs. A p value < 0.05 was considered significant.

During survival experiments, animals were monitored for a maximum of 96 h; overall survival was examined by creation of a Kaplan-Meyer curve to estimate survival probability, and with a log-rank-test to analyze probable significance. As required by the German Animal Welfare Law and the respective German government bodies, a statistical analysis has been performed beforehand to determine the number of animals needed to proof or rule out our respective hypothesis.