a, RT-qPCR analysis of CXCL10, IL1B, CXCL8, CCL2, TNF, and ALPK1 expression in PMA-differentiated THP-1 cells treated with ADP-Hep, UDP-Hep, or UDSP-Hep for 12 h. Levels of mRNA were normalized to that of GAPDH (mean ± s.d., n = 3). b, Heatmap of cytokine concentrations in the sera of WT or Alpk1−/− C57BL/6 mice injected (i.v.) with PBS or an indicated dose of ADP-Hep or UDSP-Hep (n = 7 for untreated WT group, 5 for 5-µg ADP-Hep- and 500-µg UDSP-Hep-treated WT group, and 6 for other groups). c, e, WT or Alpk1−/− C57BL/6 mice or indicated strains of mice were immunized with OVA (100 μg per mouse) alone or in combination with aluminium (Alum, 2 mg per mouse) or an indicated dose of ADP-Hep or UDSP-Hep (c) or ADP-Hep (500 µg per mouse), or UDSP-Hep (20 µg per mouse) (e). Anti-OVA IgG production was measured by ELISA on day 21 after immunization, and the absorbance values are shown as mean ± s.e.m. (c, n = 6 for WT and 7 for Alpk1−/−; e, n = 6). Two-way ANOVA. d, Heatmap of cytokine concentrations in the sera of C57BL/6, 129, or BALB/c mice injected (i.v.) with PBS, ADP-Hep or UDSP-Hep at the indicated doses. Each lane indicates the average cytokine expression (n = 5–6 mice per group). f, RT-qPCR analyses of Cxcl10, Tnf, and Ifi205 expression in C57BL/6, 129, or BALB/c strain-derived BMDMs treated with ADP-Hep, UDP-Hep, or UDSP-Hep for 6 h. Levels of mRNA were normalized to that of Gapdh (mean ± s.d., n = 3). g, ALPK1−/− HEK293 T cells expressing eGFP-TIFA and Flag-ALPK1 derived from the C57BL/6 or 129 mice or humans were treated with ADP-Hep, UDP-Hep, or UDSP-Hep. TIFA phosphorylation was assessed by anti-pT9-TIFA immunoblotting. h, Serum concentrations of UDSP-Hep over time after a single s.c. or i.v. injection (2.5 mg kg−1) into C57BL/6 mice. Blood was collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, and 7 h after the UDSP-Hep injection; its concentrations were quantified by LC-MS/MS (mean ± s.e.m., n = 3). i, Local biodistribution after peri-tumoural injection of UDSP-Hep. The concentrations of UDSP-Hep in tumour tissues and tdLNs 15 min after the injection (2.5 mg kg−1) are shown (mean ± s.e.m., n = 4). Data are representative of two (h, i) or three (a–g) independent experiments.
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