Determination of blood CIRP levels in human neonatal samples
After obtaining Institutional Review Board approval (Northwell Health IRB # 19-0511), serum samples were obtained from patients in a neonatal intensive care unit at a tertiary care children’s hospital. Serum samples were collected initially for patient care and the remaining volume in a sample after use for diagnostic testing was used for this study. The medical record was reviewed for demographics and clinical and diagnostic information. Serum was stored at -80°C until analysis. eCIRP levels in the serum were measured using a human CIRP ELISA kit (American Research Products, Waltham, MA) according to manufacturer’s instructions.
House-bred male and female C57BL/6 mice were kept in a 12 h light/dark, temperature-controlled room and fed standard Purina rodent diet. Females were closely observed to ensure an accurate date of birth for all litters. Five to seven-day old neonatal mice were used for all in vivo experiments. Sex determination via external characteristics is difficult to reliably determine at this age and weight (three to four grams). As such, pups were not identified as either male or female and both genders were therefore used in all experiments. Pups remained with their mothers throughout all experimental timeframes and could breastfeed ad libitum. All experimental procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Care and Use Committee of the Feinstein Institutes for Medical Research.
Murine model of neonatal sepsis
Neonatal sepsis was induced with cecal slurry (CS) as originally developed by Wynn et al and modified as previously described by us.[34, 35] Sepsis was induced in neonatal mice via by administration of 0.525 mg/g body weight (BW) intraperitoneal CS. Pups were removed and returned to their cage with their mothers as a group. Sham mice received an intraperitoneal injection of an equivalent volume of 5% dextrose. At 16 hours after CS injection, pups were anesthetized using 2.5% isoflurane anesthesia, underwent echocardiogram as described below, and were subsequently euthanized by cardiac puncture. Blood, heart, and lungs were collected. Heart and lungs were immediately flash-frozen in liquid nitrogen. Blood was centrifuged at 1,000 rotations per minute (rpm) for 10 minutes and serum was collected. Serum, heart, and lungs were then stored at -80°C until analysis. For the survival study, pups received a diluted dose of CS (0.175 mg/g BW) and were monitored for seven days for survival.
In vivo administration of rmCIRP and M3
Recombinant murine CIRP (rmCIRP) was produced as previously described. rmCIRP at a dose of 10 mg/kg BW or an equivalent volume of normal saline was administered intraperitoneally. M3 (10 mg/kg BW) or vehicle (normal saline) was given i.p. at the time of cecal slurry injection. For the survival study, an additional group of mice received M3 2 h after CS injection.
Isolation of primary murine neonatal cardiomyocytes
Neonatal cardiomyocytes were isolated from 0 to 2-day old neonatal C57BL/6 mice using the Pierce Primary Cardiomyocyte Isolation kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Cells were cultured in DMEM from the kit supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37°C.
Measurements of reactive oxygen species, mitochondrial depolarization, and mitochondrial calcium levels
Cardiomyocyte and mitochondrial reactive oxygen species (ROS), mitochondrial depolarization, and mitochondrial calcium levels were assayed as described previously by Joseph et al.[36, 37] Cardiomyocytes were plated in 96-well plates. Plated cells were pre-treated with 10 µg/mL M3 peptide or equivalent volume additional media for 20 minutes. Cells were then stimulated with either PBS as a control or rmCIRP at various doses for 4 h to assess ROS and mitochondrial depolarization or 1.5 h to determine calcium levels. To measure total ROS, cardiomyocytes were loaded with 25 μM 2′,7′-dichlorofluorescin diacetate (DCF) (Sigma-Aldrich, St Louis, MO) for 30 minutes in the dark. Excess DCF was removed by washing and DCF fluorescence was recorded at excitation/emission wavelengths of 490/530 nm. To measure mitochondrial ROS, MitoSOX Red, a mitochondrial superoxide indicator, (5 uM, ThermoFisher Scientific) was added to cardiomyocytes and incubated in the dark for 30 minutes. Excess MitoSOX Red was removed, and fluorescence was recorded at excitation/emission wavelengths of 525/620 nm. Tetramethylrhodamine methyl (TMRM) ester (1 nM, ThermoFisher Scientific) was used to assess changes in mitochondrial membrane potential. Cardiomyocytes were stained with TMRM ester for 30 minutes and florescence was recorded at excitation/emission 540 nm/590nm. Due to its internal positive charge, TMRM preferentially accumulates inside mitochondria. As the mitochondria depolarizes, less TMRM is trapped. Therefore, the TMRM signal is proportional to the inner membrane potential of the mitochondria. Finally, changes in mitochondrial calcium were determined using a 30 minute incubation with 10 μM Rhod-2-AM (ThermoFisher Scientific) followed by a 1 hour washout- and cytosolic quenching with 0.5 mM manganese (Trevigen, Gaithersburg, MD). Fluorescence was measured at 552 nm (excitation)/581 nm (emission).
Enzyme-linked immunosorbent assay (ELISA)
Cardiomyocyte supernatant was analyzed by ELISA kits for interleukin (IL)-6 and tumor necrosis factor-α (TNF-a) (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. Cardiac and lung tissue was crushed in liquid nitrogen, and equal weights of powdered tissues (~50 mg) were dissolved in 500 µl of lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 2 mM each of EDTA and EGTA), and subjected to sonication on ice. Protein concentration was determined by the BioRad protein assay reagent (Hercules, CA). Equal amounts of proteins (250-500 µg) were loaded into respective ELISA wells for the assessment of IL-6 and IL-1β (Invitrogen, Carlsbad, CA). Serum was analyzed using a Bio-Plex Pro Mouse Cytokine Th17 Panel A -6-Plex kit (BioRad).
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)
To examine sepsis-associated lung and cardiac inflammation, the lung and heart mRNA expression of IL-6 and IL-1 β were measured. TREM-1 expression in neonatal murine cardiomyocytes was assessed after 24 hours of rmCIRP stimulation. Total RNA was extracted from cells and tissue using Trizol reagent (Invitrogen). cDNA was synthesized using MLV reverse transcriptase (Applied Biosystems, Foster City, CA). PCR reactions were carried out in 20 μl of a final volume of 0.08 μM of each forward and reverse primer, cDNA, water, and SYBR Green PCR master mix (Applied Biosystems). Amplification was performed in a Step One Plus real-time PCR machine (Applied Biosystems). Mouse β-actin or GAPDH mRNA was used as an internal control for amplification for lung and cardiac tissue, respectively, and relative gene expression levels were calculated using the DDCT method. Relative expression of mRNA was expressed as fold change in comparison with sham tissues or PBS treated cells.
Two hours after injection of rmCIRP or 16 hours after sepsis induction with CS, cardiac function in neonatal mice was assessed by transthoracic echocardiography. Echocardiography was conducted using a 40 MHz center frequency transducer coupled to a Vevo®3100 Imaging System (Fujifilm VisualSonics, Toronto, ON, Canada). Sedation was induced with 2.5% isoflurane and maintained with 0.5-1% isoflurane for the duration of the echocardiogram. Mice were maintained on a heated table during this time. Parasternal long axis views were taken in B and M modes. VevoLab (Fujifilm VisualSonics) software was used to determine cardiac parameters. VevoStrain (Fujifilm VisualSonics) software was used to measure myocardial strain and strain rate using speckle-tracking echocardiography. 
Data represented in the figures are expressed as mean ± SE. All data has been tested for normality using the Kolmogorov-Smirnov Test of Normality. Normally distributed data was analyzed using the two-tailed Student’s t test for two-group comparisons and One-way ANOVA for comparison among multiple groups with the significance between individual groups determined using the Tukey method. Nonparametric data was analyzed using one-way comparison among multiple groups with the Kruskal-Wallis test with a Dunn’s multiple comparison test. Significance was considered for p ≤ 0.05 between study groups. Data analyses were carried out using GraphPad Prism graphing and statistical software (GraphPad Software, San Diego, CA).