Immunomodulatory effects of ITA-LNP in vitro and in vivo. (A) Immunoblot probing for IL-1β in BMDMs treated with 100 ng/mL LPS in the presence of various doses of ITA-LNPs. (B) Schematic of the experiments in C57BL/6 mice subjected to LPS-induced endotoxemia. (C) Kaplan-Meier survival analysis after LPS-induced (15 mg/kg) endotoxemia in C57BL/6 mice pretreated with Ctrl/ITA-LNPs or anti–IL-1β mAb. (D) Rectal temperature measured in the mice from panel C before and 6 hours after LPS injections. (E-F) The levels of IL-1β and IL-6 in the serum from mice of a separate cohort injected as in panel B but with a sublethal dose of 8 mg/kg LPS; n = 10 to 15 mice per group. (G) NETs in primary human neutrophils incubated for 5 hours in vitro with 50 μM LNPs as indicated and in the presence of 100 nM PMA followed by immunofluorescence analysis; n = 6 for Ctrl-LNP and ITA-LNP without PMA; n = 8 for Ctrl-LNP with PMA; n = 9 for ITA-LNP with PMA; and n = 4 for PBS groups with or without PMA. (H) Cytokine analysis in supernatants from experiments in panel G. Statistical analysis is through pairwise t test with Holm post hoc test. N.d., not detected; PBS, phosphate-buffered saline; ROI, region of interest; T, temperature.
Figure 2.

Immunomodulatory effects of ITA-LNP in vitro and in vivo. (A) Immunoblot probing for IL-1β in BMDMs treated with 100 ng/mL LPS in the presence of various doses of ITA-LNPs. (B) Schematic of the experiments in C57BL/6 mice subjected to LPS-induced endotoxemia. (C) Kaplan-Meier survival analysis after LPS-induced (15 mg/kg) endotoxemia in C57BL/6 mice pretreated with Ctrl/ITA-LNPs or anti–IL-1β mAb. (D) Rectal temperature measured in the mice from panel C before and 6 hours after LPS injections. (E-F) The levels of IL-1β and IL-6 in the serum from mice of a separate cohort injected as in panel B but with a sublethal dose of 8 mg/kg LPS; n = 10 to 15 mice per group. (G) NETs in primary human neutrophils incubated for 5 hours in vitro with 50 μM LNPs as indicated and in the presence of 100 nM PMA followed by immunofluorescence analysis; n = 6 for Ctrl-LNP and ITA-LNP without PMA; n = 8 for Ctrl-LNP with PMA; n = 9 for ITA-LNP with PMA; and n = 4 for PBS groups with or without PMA. (H) Cytokine analysis in supernatants from experiments in panel G. Statistical analysis is through pairwise t test with Holm post hoc test. N.d., not detected; PBS, phosphate-buffered saline; ROI, region of interest; T, temperature.

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