<i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> bone marrow-derived macrophages are defective in both MyD88 and TRIF-dependent cytokine production.

Abstract

<p>(A) Bone marrow-derived macrophages (BMDMs) from B6, <i>Ripk3</i>^<i>-/-</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> were infected with <i>Yersinia</i> or <i>Salmonella</i> for 24hrs, and IL-6 and IL-12p40 cytokine levels present in supernatants were quantified by ELISA. (B) IL-6, IL-12p40 and TNF production was measured by ELISA from the indicated BMDMs treated with LPS (100 ng/mL) for 6hrs. (C) IL-1β expression was measured by flow cytometry from the indicated BMDMs treated with LPS (100 ng/mL) for 5hrs. (D) BMDMs were treated with LPS (100 ng/mL) and <i>Ifnb</i> mRNA was assayed by RT-qPCR at the indicated time points. (E) Resident peritoneal macrophages from indicated mouse genotypes were isolated and stimulated <i>ex vivo</i> with LPS (10 ng/mL) for 4 hrs. TNF production by large peritoneal macrophages (LPMs) was measured by flow cytometry. (F) IL-6, IL-12p40 and TNF production was measured by ELISA from BMDMs treated with Poly(I:C) (50 μg/mL) for 24 hrs, CpG (1 μg/mL) or Pam3CSK4 (1 μg/mL) for 6hrs.(G) BMDMs were treated with LPS (100 ng/mL) and <i>Il1b</i>, <i>Il12b</i> and <i>Il6</i> mRNA was assayed by RT-qPCR at the indicated time points. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by Student's unpaired two-tailed <i>t</i>-test. Representative of minimum of three independently performed experiments with triplicate samples for each condition. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s002" target="_blank">S2 Fig</a>.</p