Figure 5. L. pneumophila infection regulates IL-17A and IL-17F production by neutrophils.

A. Flow cytometric analysis was performed on cells obtained from whole lung digests at 24, 48, or 72 h postinfection with L. pneumophila (Lp) (10⁷ CFU/mouse) as described in Materials and Methods. Total numbers of IL-17A and IL-17F positive cells from each lung following saline challenge or infection. For experiments A-B, a total of 5-8 mice/group were used. *, p<0.05; **, p<0.01; ***, p<0.001 (compared with 24 h L. pneumophila infected mice). B. CD4, γδ, NK1.1, and CD8 cells were purified from the spleen whereas neutrophils were isolated from digested lungs of L. pneumophila-infected mice at 24 h using magnetic selection (positive selection for γδ cells; negative selection for CD4, CD8, NK1.1 cells and neutrophils). Production of IL-17A and IL-17F was determined by ELISA of the culture supernatants following stimulation of 1 × 10⁴ cells with PMA and ionomycin for 5 h. For experiment B, a total of 6-8 mice were used to isolate cells. C-F. Neutrophils express IL-17A and RORγt following L. pneumophila infection. Neutrophils were purified from the lungs (C-D) or BALF (E-F) and stained with antibodies against IL-17A and RORγt. Shown is a representative image of 6 slides with similar results and 500 cells were counted in lung sections or BALF cytospins and plotted. *, p<0.05; **, p<0.01; ***, p<0.001 (compared with samples with saline challenge).