Chemicals and animals

LPS from Escherichia coli (055:B5) was obtained from Sigma (St. Louis, MO). Diff-Quik was from Sysmex (Kobe, Japan). An antibody against actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against histone H3 (citrulline R2 + R8 + R17) and neutrophil elastase were purchased from Abcam (Cambridge, UK). L-carnosine, luminal-based chemiluminescent probe (L-012), Evans Blue Dye, zymosan, isoflurane and formalin neutral buffer solution were from WAKO Pure Chemicals (Tokyo, Japan). 4,6-diamidino-2-phenylindole (DAPI) was purchased from Dojindo (Kumamoto, Japan). RNeasy® kit was obtained from Qiagen (Hilden, Germany), PrimeScript® 1st strand cDNA Synthesis Kit was from Takara Bio (Ohtsu, Japan), and SsoFast™ EvaGreen Supermix was from Bio-Rad (Hercules, CA). Mounting medium for immunohistochemical analysis (VECTASHIELD™) was purchased from Vector Laboratories (Burlingame, CA). Mayer’s haematoxylin, 1% eosin alcohol solution and mounting medium for histological examination (malinol) were from MUTO Pure Chemicals (Tokyo, Japan). Novo-Heparin (5000 units) for injection was from Mochida Pharmaceutical (Tokyo, Japan). A549 cells or RAW264 cells were purchased from the American Type Culture Collection (Manassas, VA) or RIKEN BioResource Center (Tsukuba, Japan). ICR mice (6–7 weeks old, male) were purchased from Charles River (Yokohama, Japan). The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and were approved by the Animal Care Committee of Musashino University.

Treatment of mice with LPS and carnosine

Mice anesthetized with isoflurane were given a single intratracheal injection of LPS (1 mg/kg) in 0.9% NaCl (2 ml/kg), zymosan (1 mg/kg) in 0.9% NaCl (2 ml/kg) or 0.2 M hydrochloric acid (2 ml/kg) using a P200 micropipette via the mouth. During administration, the nostrils of the mice were blocked with a finger, so that the solutions were inhaled from the mouth into the respiratory tract as the mice breathed.

Mice were orally administered carnosine (10, 50, 100 mg/kg) in 0.9% NaCl by syringe using a sonde needle. The first administration of carnosine was given immediately before LPS administration (except for Supplementary Fig. S3). In control experiments, we examined the effect of administering carnosine alone, and found that it did not affect the lung histology, the protein concentrations or number of leukocytes in BALF, the plasma levels of malondialdehyde (an indicator of ROS), or the number of neutrophils in blood (Supplementary Fig. S5). The levels of malondialdehyde in the plasma were determined using TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI). Measurement of neutrophil number was performed by the LSI Medience Corporation Central Laboratory (Tokyo, Japan), using a Sysmex XT-2000iV™ automated haematology analyser (Sysmex, Kobe, Japan).

Evaluation of lung permeability

To quantitatively examine lung permeability, Evans blue dye (30 mg/kg) was intravenously administered 2 h before the mice were sacrificed. Tissue samples were cut into pieces and incubated with formamide solution at 60 °C for 24 h. Samples were centrifuged to obtain supernatants, the absorbances of which were measured at 620 nm to determine the amount of Evans blue dye present.

Preparation of BALF

BALF was collected by cannulating the trachea and lavaging the lung twice with 1 ml of sterile 0.9% NaCl containing 50 units/ml heparin. Approximately 1.8 ml of BALF was routinely recovered from each mouse and the total cell number was counted using a hemocytometer. After centrifugation with a Cytospin®4 (Thermo Fisher Scientific, Waltham, MA), cells were stained with Diff-Quik reagents and the ratio of neutrophils to total cell number was determined. The amount of protein and double-stranded DNA (dsDNA) present in the BALF was evaluated by the Bradford method and by using a Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific).

Measurement of ROS by in vivo imaging analysis

In vivo imaging of ROS in mice was performed as described previously49,57, with some modifications. We used an imaging system (Lumazone in vivo imaging system; Shoshin Em, Okazaki, Japan), which contains a chamber equipped with an electron-multiplying CCD camera. Mice were intravenously administered with the luminescent probe, L-012, in saline (75 mg/kg). At 5 min after the L-012 injection, mice were euthanized and the lungs were rapidly dissected and imaged (5 min exposure). All data were analyzed using SlideBook 6 software (Intelligent Imaging Innovations, Inc., Denver, CO).

Real-time reverse transcription (RT) polymerase chain reaction (PCR) analysis

Total RNA was extracted from lung tissue using an RNeasy kit according to the manufacturer’s protocol. Samples were reverse-transcribed using the PrimeScript® kit described above. The synthesized cDNA was used in real-time PCR experiments with SsoFast EvaGreen Supermix and analyzed with a Bio-Rad (Hercules, CA) CFX96™ real-time system and CFX Manager™ software. Specificity was confirmed by electrophoretic analysis of reaction products and by the inclusion of template- or reverse transcriptase-free controls. To normalize the amount of total RNA present in each reaction, hypoxanthine phosphoribosyltransferase 1 (HPRT1) cDNA was used as an internal standard. Primers were designed using Primer3 or Primer-BLAST websites. Primers sequences will be provided upon request.

Immunoblotting analysis and measurement of MPO activity

BALF or homogenized lung samples were prepared. BALF samples (2 μL) were then applied to NuPAGE® Novex 4–12% Bis-Tris Gel (Thermo Fisher Scientific) and subjected to electrophoresis. After western blotting, proteins were detected with their respective antibodies and chemiluminescent staining using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific). Band intensities were quantitated by using ImageJ software (version 1.39 u). The MPO activity of homogenized lung samples was measured using an MPO assay kit (BioVision, Milpitas, CA) according to the manufacturer’s protocol.

Histological and immunohistochemical analyses and TUNEL assay

Tissue samples were fixed in 10% neutral buffered formalin for 24 h, and then embedded in paraffin before being cut into 4 μm thick sections. Sections were stained first with Mayer’s hematoxylin and then with 1% alcoholic eosin (H&E staining). Slides were mounted with malinol and visualized with a microscope and digital camera (Olympus DP71; Tokyo, Japan).

For immunohistochemical analysis, sections were incubated with Tris–EDTA buffer (pH 9.0) or proteinase K (20 μg/ml) for antigen retrieval and then incubated with DAKO® peroxidase blocking reagent for removal of endogenous peroxidase activity. Sections were blocked with 3% goat serum or 5% goat serum for 10 min, incubated overnight with rabbit anti-histone H3 antibody (1:100 dilution) or for 2 h with rabit anti-neutrophil elastase antibody (1:100 dilution) in DAKO® Antibody Diluent, and then incubated with a DAKO® EnVision peroxidase-labelled polymer–goat anti-rabbit immunoglobulin conjugate for 1 h. Then, 3,3′-diaminobenzidine was applied to the sections for colour development. The sections were finally counterstained with Mayer’s haematoxylin. Slides were mounted with malinol and visualized with a microscope and digital camera (Olympus DP71; Tokyo, Japan).

For the TUNEL assay, sections were incubated first with proteinase K (20 μg/ml) for 15 min at 37 °C, then with terminal deoxynucleotide transferase and biotin-14-ATP for 1 h at 37 °C, and finally with an Alexa Fluor 488–streptavidin conjugate and DAPI (5 μg/ml) for 2 h. Slides were mounted with Vectashield and inspected with the aid of a microscope and digital camera (Olympus DP71).

Cell culture

A549 cells (a human type II pulmonary epithelial cell line) and RAW264 cells (a mouse macrophage-like cell line) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. All cells were cultured in a humidified atmosphere of 95% air with 5% CO 2 at 37 °C. Viable cell number was quantified using a WST-based cell counting kit (Dojindo, Kumamoto, Japan) or CellTiter-Glo® 2.0 (Promega Corporation, Madison, WI). The levels of ROS in vitro were quantified using dihydroethidium (DHE), an indicator of superoxide (Thermo Fisher Scientific).

Statistical analysis

All values are expressed as the mean ± S.E.M. Two-way ANOVA followed by Dunnett’s test or the Student’s t-test for unpaired results was used to evaluate differences between three or more groups or between two groups, respectively. Kaplan–Meier plots were used to describe survival data and log-rank tests was performed to assess statistical differences. SPSS24 software was used for all statistical analyses. Differences were considered to be significant for values of P < 0.05.