Murine sepsis model of CLP

All procedures were performed in accordance with strict institutional guidelines for animal care and use. Protocols were conducted in accordance with the National Institutes of Health guidelines. Protocol approvals were obtained from the Institutional Animal Care and Use Committee of Rhode Island Hospital (RIH) (No. 5025–17) and Washington University in St. Louis (WUSTL) (Nos. 20180135 and 19086600). Murine models performed at RIH and WUSTL were nearly identical. Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Mice (8–10 weeks old) were housed in a room at an ambient temperature of 22°C and a 12:12-h light:dark cycle. They were allowed to acclimatize in the animal facility for 1 week before their use in experiments. A number of pioneering studies have shown the impact of sex as a variable in experimental mice being subjected to septic challenge, with protective effects of the pro-estrus state (and other members of the estrogen family) seen in mature adult female mice when compared with males (36, 37). Thus to have uniform and predictable responses the use of male mice is preferred. Polymicrobial sepsis was induced in the CLP mouse model as previously described (38, 39). Briefly, mice were anesthetized with isoflurane, and a ventral midline incision was made below the diaphragm to expose the cecum. The cecum was ligated with 5–0 silk, punctured twice with a 21-gauge needle (RIH) or 27-gauge needle (WUSTL), and gently compressed to extrude a small amount of fecal content through the punctured holes. The cecum was returned to the abdomen and the incision was closed in layers with 6–0 Ethilon suture (Ethicon Inc, Somerville, NJ). Buprenorphine 0.05 mg/kg (Hospira Inc, Lake Forest, Ill) was used for analgesia at the end of the procedure. No antibiotics were used for this study. Then, all animals were resuscitated with 0.6 mL lactated Ringer’s-sodium chloride solution. At RIH, mice were treated at 3 and 9 h post-CLP with 200 μg LD01 injected intraperitoneally (IP) or 200 μg α-PD-1 Ab (RMP1–14 mAb; Bio X Cell, Lebanon, NH) was injected IP once at 3 h post-CLP. For survival studies, mice received two daily injections of LD01 on days 0, 1, and 2 following CLP or once daily injection of α-PD-1 Ab on days 0, 1, 2, 3, and 4 following CLP, and were monitored for 7 days. At WUSTL, LD01 and control peptide were administered at a dose of 200 μg/injection/mouse IP twice daily on days 0, 1, and 2 following CLP, with the first dose administered 3 h post-surgery. For survival studies, mice were monitored for 7 days post-CLP.