Animals

In line with the 3 R-principles, we performed a post-hoc analysis of jejunal tissue samples obtained from a previously published study [20]. All procedures were conducted in accordance with the current Animal Welfare Experimental Animal Ordinance with the approval of the Tübingen Regional Council (TVA Reg. No. 1345). Blunt AT was applied to male C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France). Eight- to 12-week-old mice weighing 20–30 g were selected either as experimental or sham animals (n = 5). Animals had free access to water, food, and nesting material, and were kept under a 12/12 h light-dark cycle.

Anesthesia and blunt AT

Opioid analgesia was provided with subcutaneous buprenorphine (Temgesic®, Boehringer, Mannheim, Germany) at a dose of 0.05 mg/kg body weight, administered 30 min prior to trauma induction or sham treatment. Throughout the ensuing observation period, supplementary doses were administered at 8, 16, and 20 h post-intervention. Anesthesia was induced with 4 vol% sevoflurane (Abbott, Wiesbaden, Germany) in 96 vol% oxygen and maintained via a nasal mask. The experimental apparatus for blunt trauma induction consisted of a two-part cylinder divided by a thin polyester membrane (DuPont, Wilmington, USA). A distance of 2.6 cm was set between the cylinder and the mice abdomen. The upper chamber, connected to a compressed air cylinder, was pressurized until the membrane ruptured at 0.83 ± 0.27 bar, releasing a targeted blast of compressed air that caused abdominal compression without external skin injuries. To prevent post-traumatic hypothermia, animals were placed in a heated cage during recovery from anesthesia. In addition to the experimental animals, sham animals underwent an identical experimental procedure except for the application of the AT. Accordingly, sham mice received buprenorphine injections and brief sevoflurane anesthesia, and tissues were collected 24 h afterwards.

Harvest and tissue preparation

Organ harvesting took place 24 h post-trauma or sham procedure after induction of deep sevoflurane anesthesia and exsanguination by cardiac puncture. The abdomen was opened for organ retrieval and after ligation, the jejunum was separated from the other intestinal sections. An approximately 3 cm long segment of the jejunum was fixed in 3.7% formaldehyde solution for 24 h, then rinsed, dehydrated through a graded alcohol series, and embedded in paraffin. Organ sections of 4 µm thickness were cut using a microtome for histological analysis. To preserve additional intestinal tissue, the lumen of the jejunum was rinsed thoroughly with cold PBS and stored at −80 °C. Bronchoalveolar lavage fluid (BALF) was collected by flushing the left lung with 0.5 mL sterile phosphatebuffered saline (PBS) and centrifugation for 10 min at 400 g and 4 °C, and then stored at −80 °C for further analysis. EDTA blood samples were centrifuged for 5 min at 800 g and 4 °C and 2 min at 13,000 g and 4 °C. Plasma was collected and stored at −80 °C.

Hematoxylin and eosin (H&E) and periodic acid-schiff (PAS)-Alcian blue staining

Jejunal sections were deparaffinized in xylene and rehydrated through a graded ethanol series. For H&E staining, sections were placed in hematoxylin solution modified according to Gill III° (Sigma Life Sciences, St. Louis, USA) for 6 min, rinsed in water and 0.5% hydrochloric acid, and stained with 1% eosin solution (Morphisto, Frankfurt am Main, Germany) for 30 s. For PAS-Alcian blue staining, tissue sections were stained with Alcian blue solution (Sigma Life Sciences, St. Louis, USA) for 5 min, followed by periodic acid solution for 10 min, Schiff’s reagent for 15 min and hematoxylin solution modified according to Gill III° for 20 s. Rinsing was performed between all step. Finally, H&E- and PAS-Alcian blue-stained sections were dehydrated and mounted using Neo-Mount™ mounting medium (Sigma Life Sciences, St. Louis, USA).

Immunohistochemical staining

For immunohistochemical staining, jejunal sections were deparaffinized and rehydrated, followed by antigen retrieval by boiling in either citrate buffer (pH 6.0; for MPO staining) or EDTA buffer (pH 9.0; for CDH1 and CD3 staining) for 7 min. After cooling, sections were rinsed in distilled water and Tris-buffered Saline (TBS), and subsequently permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, USA) for 10 min. To minimize autofluorescence, tissue sections were treated with Sudan Black B (Sigma-Aldrich, St. Louis, USA) for 20 min, followed by rinsing in TBS and TBST (TBS with 1% Tween-20). Non-specific binding sites were blocked using antigen block solution (see Suppl. Table 1). Primary antibodies were applied overnight at 4 °C at the following dilutions: 1:200 for MPO (Human/Mouse Myeloperoxidase/MPO Antibody, R&D Systems, Minneapolis, USA), 1:100 for CDH1 (E-cadherin [24E10] Rabbit IgG monoclonal antibody, Cell Signaling Technology, Boston, USA), and 1:250 for CD3 (Anti-CD3 antibody [CD3-12] Rat monoclonal antibody, Abcam, Cambridge, UK). After washing, sections were incubated with the corresponding secondary antibody for 60 min. For MPO, Donkey anti-Goat IgG (H+L), Alexa Fluor™ 568 (1:200); for CDH1, Alexa Fluor™ 488-conjugated Goat anti-Rabbit IgG (1:300); and for CD3, Goat anti-Rat IgG conjugated to Alexa Fluor™ 568 (1:300) were used (all antibodies from Thermo Scientific, Rockford, USA). The tissue sections were covered with Prolong Gold Antifade with DAPI (Life Technologies Corporation, Eugene, USA) and dried overnight at 4 °C. Imaging was performed using a Zeiss Axio Imager M1 light microscope with AxioVision SE64 Rel. 4.9 software (Zeiss, Oberkochen, Germany).

Histological analysis

QuPath-0.4.4 software [22] was used for quantification of mucus-covered areas and goblet cells. Images of PAS-Alcian blue-stained jejunal tissue sections were taken at 200x magnification. For subsequent autonomous area recognition by the software, six randomly selected images were used as training data by manually labeling mucus-covered areas. For each animal, ten villi were selected and included in the analysis.

Quantitative polymerase chain reaction (qPCR)

Jejunal tissue was gently pulverized using an RNase-decontaminated (RNaseZAP™, Sigma-Aldrich, St. Louis, USA) mortar and pestle on liquid nitrogen to prevent autolysis and kept on ice until RNA extraction. Total RNA was isolated using the RNeasy Plus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was determined using the Qubit™ RNA High Sensitivity Assay Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol.

Complementary DNA (cDNA) was synthesized from 500 ng of RNA per sample using the AffinityScript qPCR cDNA Synthesis Kit (Agilent, Santa Clara, USA) in accordance with the manufacturer’s instructions. The cDNA was diluted with RNase-free water and stored at −20 °C. To determine alterations in gene expression between sham and experimental group, qPCR analyses were performed in duplicate. The procedure was conducted according to the manufacturer’s instructions using the Brilliant III SYBR-Green qPCR Master Mix Kit (Agilent, Santa Clara, USA) and AriaMx Real-time PCR-System (Agilent, Santa Clara, USA). For evaluation of the qPCR results, the AriaMx HRM qPCR software (Agilent, Santa Clara, USA) was used. Primers used for gene expression analysis are listed in Suppl. Table 1. Relative fold-change expression levels were calculated using the 2-ΔΔCT method, with Actb serving as the reference gene.

Tissue homogenization and total protein concentration

Frozen jejunal tissue samples were lysed in 200 µl RIPA buffer using innuSPEED Lysis Tubes P (Innuscreen GmbH, Berlin, Germany) and homogenized with the SpeedMill Plus tissue homogenizer (Analytik Jena, Jena, Germany). Homogenates were shock-frozen, before they were thawed and centrifuged at 16,000 g for 15 min at 4 °C. The resulting supernatant was kept on ice for the subsequent steps. Total protein concentration was determined using the PIERCE BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

To measure the concentrations of C5a in plasma and BALF, a sandwich ELISA was performed using the C5a ELISA Kit Mouse Duo Set (R&D Systems, Minneapolis, USA). For the BALF samples, the BCA Protein Assay Kit (#23225; Thermo Fisher Scientific, Inc.) was used to determine the total amount of protein. The experiments were performed according to the manufacturer’s instructions.

Proteome profiler

Cytokine expression on a protein level was determined semi-quantitatively using the Proteome Profiler Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, USA). Membranes were imaged using the ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Hercules, USA), and analyzed with ImageLab software (Bio-Rad Laboratories, Hercules, USA). Pathway analysis of differentially regulated cytokines was performed using ShinyGO v0.82 (https://bioinformatics.sdstate.edu/go/).

Western blotting

Tissue homogenates prepared in RIPA buffer were diluted 1:2 with 2x Laemmli Sample Buffer, before incubation for 5 min at 95 °C and electrophoresis using Mini-PROTEAN® TGX™ Stain-Free Gels (4–20%) (Bio-Rad Laboratories, Hercules, USA). Proteins were transferred to Hybond P-PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, USA). To block non-specific antibody binding sites, membranes were incubated in 5% milk for 1.5 h, followed by overnight incubation at 4 °C with a 1:1500 dilution of CAMP polyclonal antibody (Proteintech, Manchester, UK) in 5% milk or a 1:500 dilution of Cleaved Caspase-3 (Asp175) Antibody (Cell Signaling Technology, Boston, USA) in 5% milk, respectively. Membranes were washed three times with TBST, incubated with StrepTactin-HRP-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, USA) for 60 min and washed again in TBST and TBS. Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, USA) was applied, and membranes were imaged using the ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Hercules, USA). ReBlot Plus Strong Antibody Stripping Solution (Merck KGaA, Darmstadt, Germany) was applied for 15 min, membranes were blocked with 5% BSA for 30 min and incubated overnight at 4 °C with β-Actin antibody (Cell Signaling Technology, Boston, USA) in a 1:1500 dilution with 5% BSA. Following rinsing with TBST, membranes were incubated with StrepTactin-HRP-conjugated secondary antibody for 60 min, rinsed again and Clarity Western ECL Substrate was applied for subsequent imaging.

Statistical analysis

Data were analyzed using GraphPad Prism version 10.0.0 for Windows (GraphPad Software, Boston, USA). Results are presented as individual values and mean ± standard error of the mean (SEM). Due to low animal numbers, a normal distribution was not assumed. Group differences were assessed using the Mann-Whitney U test for non-parametric comparison between two groups. Two-sided p-values < 0.05 were considered statistically significant.