Figure 3.

Analysis of Th17 response in Ehrlichia-infected C57BL/6 mice. Wild type C57BL/6 mice were intraperitoneally infected either with E. muris (EM) or Ixodes ovatus Ehrlichia (IOE) species. Serum and splenocytes were isolated at the 5^th or 7^th day post infection (p.i.) and analyzed by ELISA or flow cytometry techniques. (A) Quantification of pro-inflammatory cytokines related to Th17 differentiation (GM-CSF, IL-1 β and IL-6) in serum as measured by ELISA assay. Asterisks represent relevant differences between samples and uninfected controls. (B) Flow cytometry analysis of ex-vivo experimental sets, in which splenocytes isolated from infected or uninfected mice were stimulated with ehrlichial antigens. Cells from IOE-infected animals were treated with IOE sonicate (IOE - IOE ag) and cells from EM-infected animals were stimulated with EM sonicate (EM - EM ag). Cells from naive controls were either incubated with IOE (naive - IOE ag) or EM (naive - EM ag) antigens. After gating on splenocytes, NK1.1 × CD3 flow cytometry plots were built and three regions of analysis were considered for the assessment of intracellular IL-17 expression (evidenced in SSC × IL-17 zebra plots): NK1.1⁺CD3⁻ (conventional NK cells), NK1.1⁺CD3⁺ (NKT cells); and CD3⁺NK1,1⁻ (considered as T cells). Values represent percentage that correspond to the population in the assigned regions. Quantitative analysis of IL-17⁺ cells in the considered cell subpopulations as percentages (C) and absolute numbers (D). (E) Serum levels of IL-17 measured by ELISA at the 7^th day p.i. All quantitative analyses are expressed as mean and standard deviation. Asterisks represent relevant statistical difference between groups. Data is representative of at least three independent experiments.