Figure 1. Chitin regulation of macrophage IL-17.

3% thioglycollate-elicited peritoneal macrophages (A-C, E) and bone-marrow derived macrophages (D) from wild-type (A-D) or Rag2/γC null mice (E) were incubated with chitin (40-70μm; 100μg/ml unless otherwise indicated) or vehicle controls for 8h unless otherwise noted. The effects of these treatments on the levels of supernatant IL-17 protein (A, B, Chitin: solid squares, controls: solid circles) were assessed by ELISA. In panel C macrophage intracellular IL-17 protein was assessed using FACS analysis. In this evaluation, the upper panel compares the expression of the intracellular IL-17 in cells incubated in medium alone (dark) or medium plus chitin (line) (mean fluorescent intensity; MFI). The staining with the isotype control is shown in light gray. The lower panel illustrates the MFI values (mean ± SEM) quantitating the intracellular IL-17 protein expression of cells incubated in medium alone (hatched bars) and medium plus chitin (solid bars). In panels D and E, cell debris was excluded by light-scatter cell gating (D, E; first column) and this gate was used for all other analysis. Macrophages were identified by surface marker staining using F4/80 and CD11b co-staining which revealed a macrophage purity greater than 90% for all tested conditions (D, E; second column). To specifically assess intracellular IL-17A, macrophages were fixed and permeabilized, Fc blocking and isotype controls were used to exclude unspecific staining (D, E; third column). In control (D, E; fourth column) and chitin (D, E; fifth column)-treated macrophages intracellular IL-17A staining is depicted with F4/80 co-staining. * p < 0.05, ** p < 0.01, *** p < 0.001 for chitin-treated versus control-treated cells.