Method details

Peritoneal cells, including macrophages, were harvested by peritoneal lavage with PBS, as previously described.⁵⁷ Peritoneal macrophages (2.0∗10⁵ cells) were stimulated with LPS (2 μg/mL) (Santa Cruz), S100A8 (0.3 μg/mL) (R&D), or S100A8/A9 (0.65 μg/mL) (R&D). Hepa1-6 murine hepatoma cells (5.0∗10⁴ cells) (RIKEN Bioresource Center, RCB1638) were stimulated with S100A8 (0.3 μg/mL) (R&D) or S100A8/A9 (0.65 μg/mL) (R&D).

Mice were injected intraperitoneally with PBS (100 μL), LPS (12.5 μg/g body weight (BW)) or S100A8 (0.1 μg/gBW). Peritoneal cells were harvested at 4 hours. Serum samples were collected after 24 hours. Peritoneal cells were harvested at 4 hours and survival rate was assessed at 48 or 72 hours after treatment were established. As practical models of sepsis, intraperitoneal injection of Escherichia coli (2.0∗10⁹ CFU per mouse, DH5α) and cecal ligation and puncture (CLP) were performed. Mice underwent CLP with a 21-g needle to induce polymicrobial sepsis; the procedure was performed as previously described.⁵⁸ Immediately and 24 hours after CLP, mice were fluid resuscitated with PBS (20 μL/gBW) or S100A8 (0.1 μg/gBW) injection. Core body temperature was monitored using a rectal thermometer (Natsume Seisakusho Co., KN-91-AD-1687-M).

Total RNA was isolated from each tissue using an RNase-free DNase and RNeasy Kit (Qiagen). cDNA was prepared using High-Capacity cDNA Reverse-Transcription Kits (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed by using Expression Assays (7900 Real-Time PCR System, Applied Biosystems) with THUNDERBIRD SYBR qPCR Master Mix (TOYOBO). Data were standardized according to the expression level of β-actin. The primer sequences are shown in Table S1.

The serum S100A8 levels in mice were determined using a Mouse S100A8 ELISA Kit (Abcam, catalog no. ab213886). To measure the serum cytokine levels in mice, we used a TNF alpha Mouse Uncoated ELISA Kit (Thermo Fisher Scientific, catalog no. 88-7324-22), an IL-1 beta Mouse Uncoated ELISA Kit (Thermo Fisher Scientific, catalog no. 88-7013-22), and an IL-6 Mouse Uncoated ELISA Kit (Thermo Fisher Scientific, catalog no. 88-7064-22). Blood glucose levels were evaluated with Glutest Neo Super (Sanwa Chemical Co.). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and triglyceride levels were assayed by enzymatic methods (Wako Pure Chemical Industries).

Peritoneal cells from myelomonocytic cell-specific S100A8 KO mice and control mice were stained with an Alexa Fluor 488-labeled anti-F4/80 (6F12) antibody (BD Biosciences) or a PE-labeled anti-Ly6G (RB6-8C5) antibody (BD Biosciences). Neutrophils and macrophages were identified as Ly6G-positive and F4/80-positive cells, respectively, by flow cytometry. Flow cytometry was performed using a FACSCanto II (BD Biosciences).