Figure 5: Infection-derived lipids stimulate the IMD pathway.
From: Infection-derived lipids elicit an immune deficiency circuit in arthropods
(a) Reference structures for the three lipids used in stimulation studies: (1) POPG, (2) PODAG and (3) MPPC. (b,c) Triplicate samples of 1 × 106 S2* cells were primed with 20-hydroxyecdysone (1 μM) and stimulated with 0.01–1 ng of indicated lipids, A. phagocytophilum (MOI 50) and positive controls for the Toll pathway (S. aureus) and the IMD pathway (E. coli peptidoglycan). Quantitative PCR (qPCR) quantifying diptericin and im1 transcripts are shown. (d) Triplicate samples of 1 × 106 S2* cells or (e) Five replicates of 1x105 ISE6 cells were incubated with 1 ng of indicated lipids before A. phagocytophilum infection at MOI 50. Bacterial load was quantified by qPCR and normalized to either rp49 (Drosophila) or β-actin (ISE6 tick cells). Data are represented as the mean±s.e.m. Analysis of variance-Dunnet. *P<0.05. NS, not significant. (−), non-stimulated. ISE6 (1 × 106) cells were stimulated with (f) diaminopimelic-type peptidoglycan (PGN) (10 μg/ml), A. phagocytophilum (MOI 50) and indicated lipids (1 ng) at indicated times or (g) the indicated ranges of A. phagocytophilum and lipids for 15 min. Lysates were probed against an I. scapularis Relish polyclonal antibody (Rel-N 41 kDa). β-Actin (45 kDa) was used as a loading control. (b–e) Data are representative of 3–5 biological replicates, as indicated, and two technical replicates. (f–g) Western blots (WBs) shown represent one of three biological replicates. See also Supplementary Figs 5, 11 and 12 and Supplementary Tables 2 and 3.
