Donor APC secretion of IL-23 in the colon is dependent upon an intact LPS/TLR4 signaling pathway. (A) Lethally irradiated Balb/c mice were transplanted with B6 BM (10 × 10⁶) alone (n = 11, ▫), IL-23^−/− BM alone (10 × 10⁶; n = 10, ), B6 BM plus spleen cells (0.4-0.6 × 10⁶; n = 18, ■), or BM and spleen cells from IL-23^−/− animals adjusted to yield the same dose of mature T cells (n = 18, □). Mice were bled 7 days after transplantation, and serum was assayed for endotoxin. Data are presented as the mean plus or minus SEM and are cumulative results from 3 experiments. (B) Lethally irradiated Balb/c mice were transplanted with B6 BM plus spleen cells (n = 18, ■) or IL-23^−/− BM and spleen cells (n = 18, □). Serum was obtained from mice 21 to 30 days after BMT and assayed for endotoxin. Data are cumulative results from 3 experiments and are presented as the mean plus or minus SEM. (C) Lethally irradiated Balb/c mice were transplanted with B6 BM plus spleen cells (■) or IL-23^−/− BM and spleen cells adjusted to yield the same T-cell dose (□). Groups of mice (n = 7-9/time point) were killed at the indicated times (days 4, 7, 14, 21, and 28), and colon tissue from each animal was cultured overnight in media. Supernatant was then analyzed for the amount of endotoxin. Data are derived from 2 experiments and are presented as the mean amount of endotoxin divided by the weight of cultured colon tissue (± SEM). (D) Lethally irradiated Balb/c mice were transplanted with B6 BM (10 × 10⁶) and spleen cells (n = 9-14, ■) or IL-23^−/− BM (10 × 10⁶) and spleen cells adjusted to yield the same dose of T cells (n = 9-14, □). Mice were killed 28 days posttransplantation. RNA was extracted from spleen, colon, and liver tissues obtained from these animals as well as from normal nontransplanted Balb/c mice (n = 4-6, ▫). Gene expression of TLR4 was analyzed as described in “Real-time quantitative polymerase chain reaction.” Data are normalized for 18S ribosomal RNA and presented as fold increase over 18S RNA plus or minus SEM. Data are cumulative results from 3 independent experiments for mice transplanted with B6 or IL-23^−/− marrow grafts. (E-G) Lethally irradiated Balb/c mice were transplanted with B10 BM (10 × 10⁶) and spleen cells adjusted to yield 0.4-0.5 × 10⁶ T cells (n = 9, ■) or TLR4^−/− BM (10 × 10⁶) and an equivalent number of splenic T cells (n = 10, □). Mice were killed 32 days after transplantation and examined for pathologic damage in the colon (E). Colon explant tissues from these same mice were cultured overnight in medium and assayed for LPS (F) and IL-23p19 (G) levels. Data are cumulative results from 2 experiments. *P ≤ .05; **P < .01.