Human subjects
Altogether, data of 27 cases suffering from P.A.pneumonia in the ICU was collected from The First Affiliated Hospital of Chongqing Medical University. Pneumonia caused by P. aeruginosa was diagnosed from X-ray films by the presence of continuous infiltration, accompanied by at least one of the symptoms of hypothermia (< 35.0°C) or fever (≥ 38.0°C), pleuritic chest pain, cough, altered breathing sounds upon auscultation, and dyspnea [18]. Definite causative pathogens were confirmed for isolates of P. aeruginosa obtained either from sputum culture, or good-quality sputum containing < 10 epithelial cells and > 25 polymorphonuclear cells in each power field (100× magnification), and the dominant growth in sputum culture containing ≥ 106 colony-forming units (CFUs)/mL [19]. We obtained patient data, such as the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, levels of procalcitonin (PCT), and oxygenation index (PaO2/FiO2). This study excluded cases undergoing organ transplantations, or those with cancer, infected with human immunodeficiency virus (HIV), or taking immunosuppressants over the past eight weeks. Table 1 shows the patient data. Additionally, we also collected control samples from normal subjects (n = 17) without any underlying disease from the Medical Examination Center of The First Affiliated Hospital of Chongqing Medical University and another 22 patients admitted to the ICU with no P.A. pneumonia. The Clinical Research Ethics Committee of the Chongqing Medical University approved our study protocols. Each participant provided informed consent for participation in line with the Declaration of Helsinki.
Human serum cytokine measurements
Blood samples were collected through venipuncture from patients infected by P. aeruginosa, other ICU cases, or healthy people. The blood samples were centrifuged for 10 min at 1000 g and 4°C immediately after collection. Serum samples were then divided evenly into the Eppendorf (EP) tubes and preserved at -80 °C for subsequent experiments. The ELISA test kit (R&D Systems, California, USA, # DY9110–05) was used for measuring IL-38 levels according to a previous method [17]. Serum levels of IL-6, IL-8, IL-10, IL-17, IL-1β, and TNF-α were detected at the Clinical Testing Center of The First Affiliated Hospital of Chongqing Medical University.
Animals
We obtained 8–12-week-old male C57BL/6 mice weighing 20–24 g from the Laboratory Animal Center of the Chongqing Medical University (Chongqing, China) under the license number SYXK (Chongqing, China) 2018–0003. All animals were housed under specific pathogen-free (SPF) conditions (relative humidity (RH) between 50%-60%, temperature at 24°C, and light/dark cycle of 12 h/12 h). The mice had free access to food and water and were healthy (without infection) during the experiment. We randomly divided 20 mice into four groups (healthy control group, P.A.pneumonia model group, P.A.pneumonia + rIL-38 group, and P.A.pneumonia + anti-IL-38 group), each group with five mice.
Each animal was treated according to the guidelines for the care and use of laboratory animals in China. Our experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chongqing Medical University.
Pseudomonas aeruginosa-induced pneumonia mouse model
To perform experiments in vivo, 5 mg/kg xylazine and 100 mg/kg ketamine were used to anesthetize the animals, followed by the intranasal infection of P. aeruginosa (5 × 107 contained in 50 µL PBS) to simulate the natural pattern of human pneumonia infection. To analyze the animal survival, we monitored the survival rate of mice for 10 days post-infection. Each experiment was performed following the guidelines of the Institutional Animal Care and Use Committee of the Chongqing Medical University.
In vivo administration of recombinant murine IL-38
Recombinant murine IL-38 protein (1 µg, Adipogen, #AG-40B-0101) [15] was administered by intratracheal injection (i.t.) 2 h after bacterial infection. The mouse IgG was similarly injected as a control vehicle.
IL-38 suppression in vivo
For blocking IL-38 in vivo, intranasal injection of 50 µg rat anti-mouse IL-38 antibody (R&D Systems, California, USA, #MAB7774) [15] dissolved in 100 µL PBS was performed 2 h post-infection. The controls were injected with a mouse IgG antibody.
Bronchoalveolar Lavage Fluid (BALF) and Lung Tissue Collection
After 24 or 72 h of P. aeruginosa i.t., mice were sacrificed after being anesthetized and the BALF and lung tissues were harvested immediately [20]. Then, the chest was tapped, the right bronchus was bundled, and the left lung was rinsed. The right lung was resected, then the upper right lobe was harvested for counting bacterial quantity, and the remaining tissue samples were immediately stored at -70 °C until further analysis.
Determination of Lung Bacterial Burden
The upper right lung lobe was collected in an aseptic environment and homogenized using sterile saline (1 ml) by a tissue homogenizer in a vented hood. The lung homogenate was obtained by gradient dilutions, where 10 mL samples were added on the dry tryptic soy-base blood agar plates in solutions of different dilutions and incubated at 37°C overnight. Then, we calculated the number of CFUs as the total CFUs/lung.
Measurement of inflammatory mediators
IL-38 concentration was measured by a murine IL-38 ELISA kit (R&D Systems, California, USA, #DY2427–05) or the human IL-38 quantification ELISA kit (R&D Systems, California, USA, #DY9110–05), as appropriate. We also measured inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-17A, and CXCL-1, in the BALF or lung homogenate, using the Mice Cytokine Magnetic Bead Panel Kit (eBioscience, USA) according to specific instructions.
Histology
After inflation by 0.5% agarose, 10% formalin was used to fix lung tissues for 24 h, followed by formalin fixation, paraffin embedding, and slicing into 6-µm sections for immunohistochemical (IHC) staining. A pathologist assessed the pathology score of the lung surface according to previous descriptions. Typically, the pathologist was blinded to the information regarding edema, bronchitis, intra-alveolar inflammation, interstitial inflammation, endothelialitis, pleuritis, and the proportion of the lung surface showing syncretic inflammatory infiltrate.
TUNEL assay
The TUNEL assay was performed to measure cell apoptosis by using the In Situ Cell Apoptosis Detection Kit I, POD (Roche, Switzerland) according to the manufacturer’s protocol. Briefly, 4-µm sections were deparaffinized with xylene and rehydrated using gradient ethanol. The activity of endogenous peroxidase was blocked using 3% hydrogen peroxide (H2O2) for 10 min, and the sections were digested with proteinase K solution (10–20 µg/mL) for 15 min at 37°C. The sections were later rinsed with PBS and reacted with terminal deoxynucleotidyl transferase (TdT; dilution at 1:20) in a reaction buffer (digoxigenin-labeled nucleotides) at 37°C for 2 h. The sections were rinsed in a stop/wash buffer thrice, with 2 min of rinsing each time. Then, each section was incubated with the anti-digoxin antibody diluted at 1:100 for 30 min at 37°C, and later with ABC at 37°C for another 30 min. The 3,3’-Diaminobenzidine chromogen was used to incubate sections for approximately 20 min, and the sections were counterstained with hematoxylin to measure apoptosis. Subsequently, we randomized five fields of view (FOVs) in each section (400× magnification). Finally, we measured the proportion of TUNEL-positive cells in each of the five FOVs at 400× magnification.
Western blot (WB) analysis
Using the protein extraction kit (Beyotime, China), total protein was extracted from lung tissues or T lymphocytes. The BCA protein detection kit (Pierce, USA) was used to measure the protein content. After separating the proteins using 10% SDS-PAGE, they were transferred onto the nitrocellulose membranes. Next, 5% (w/v) skimmed milk in Tris-buffered saline containing 0.05% Tween-20 was used to block the membranes, followed by overnight membrane incubation using the β-actin antibody (#3700) or anti-TBK1/NAK (E9H5S) antibody (both obtained from the Cell Signaling Technology, USA, #51872) at 4°C. An imaging system (BIO-RAD) was used for band densitometry.
Flow cytometry
To separate the cells from the supernatant, we centrifuged the BALF at 400 g for 10 min and washed the separated cells with PBS before converting them into pellets, followed by flow cytometric analysis. Monoclonal antibodies Foxp3 and CD4 were used, which were stained using the anti-Foxp3-APC, anti-CD4-FITC, and Fixation/Permeabilization kits (eBioscience, USA) following specific protocols. Cells (105) were harvested by the FACScan flow cytometer (Becton Dickinson, USA) and analyzed by the Flow Jo software 7.6.
Cell purification and culture
Peripheral blood mononuclear cells (PBMCs) were isolated from the mouse spleen by the Lymphocyte Separation Medium (GE Healthcare, USA). Then, naïve CD4+ T cells were isolated from the collected PBMCs by magnetic-activated cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) with the use of the Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies, Canada) according to specific protocols. The purity of naïve CD4+ T cells (> 90%) was determined by flow cytometry. Cells were cultivated using the RPMI 1640 growth medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) at 5% CO2 and 37°C.
Treg cell subset generation
Treg cell subsets were produced by culturing Treg cells in L-glutamine (2 mM), β-mercaptoethanol (50 mM), anti-CD3 (5 µg/mL), anti-CD28 (2 µg/mL), IL-2 (50 U/mL), and TGF-β (2.5 ng/mL) for three days. To determine the role of IL-38 in induction, recombinant murine IL-38 protein was added to the culture medium. The levels of surface markers and intracellular staining were determined by flow cytometry.
Statistical analysis
Data were statistically analyzed in SPSS19.0 (IBM, Armonk, New York, USA) and are shown as mean ±SD or median (with interquartile ranges). Mann-Whitney U tests were performed to assess the differences between two specific groups, and one-way ANOVA was performed for comparing multiple groups. Survival curves were assessed by the log-rank (Mantel-Cox) test. Differences between and among groups were considered to be statistically significant at P < 0.05.