Tregs, including those expanded by IL-33, restrain the development of proinflammatory attributes by granulocytes and macrophages. Foxp3-DTR B6 mice were administered 15 ng/g DT on day −11 through day −1 (every other day) concurrently with IL-33 (day −10 through day −1). On day 0, spleens were harvested and splenocytes stained for multicolor flow cytometric analysis. (A) Assessed total splenocyte numbers for indicated groups (n = 3-4 mice per group): untreated (Untr.), DT only (DT), IL-33 only (IL-33), concurrent IL-33 and DT (DT/IL-33). (B) Representative contour plots and graphical analysis assessing Treg depletion during IL-33 treatment. Plots show Foxp3 vs CD25 expression on CD4⁺-gated cells. Graph presents the average frequency of CD25⁺Foxp3⁺ cells. (C) Representative flow plots and graphical analysis of CD11b⁺ cells in response to Treg depletion during IL-33 administration. (D) Representative flow plots and graphs showing F4/80 vs Gr-1 on CD11b⁺ cells. All graphs depict averages and SD from 3 to 4 mice per group and are representative of 2 independent experiments. Indicated significant differences were calculated using an unpaired Student t test (*P < .05, **P < .01, ***P < .01). (E) CD3⁻CD11b⁺F4/80⁺Gr-1^lo and CD3⁻CD11b⁺ F4/80⁻ Gr-1^hi cells were flow sorted from the spleens of day 0 IL-33– or IL-33/DT-treated B6 Foxp3-DTR mice and assessed in an ex vivo suppression assay. Data represent the average and standard error of the mean (SEM) from 3 mice per group. Significant differences were calculated using an unpaired Student t test (*P < .05, **P < .01, ***P < .001, ****P < .0001). (F) Differential gene expression was also assessed by microarray between CD3⁻CD11b⁺F4/80⁺Gr-1^lo cell populations from day 0 IL-33– or IL-33/DT-treated B6 Foxp3-DTR mice (n = 3 mouse per group). Partek calculated fold change values and associated P and q values were assessed using Ingenuity Pathway Analysis (IPA) and identified IFNγ as an active upstream regulator in CD3⁻CD11b⁺F4/80⁺Gr-1^lo cells in the absence of Treg (z score, 2.137; P, 8.56E-06). The schematic is a graphical representation of the IFNγ signaling pathway in CD3⁻CD11b⁺F4/80⁺Gr-1^lo cells from IL-33–/DT- vs IL-33 only-treated mice. The level of upregulation is indicated by intensity of red color at that node. Gray nodes are part of network, but were not significantly modified between IL-33– or IL-33/DT-treated B6 Foxp3-DTR mice samples. Solid lines indicate direct relationships; dashed lines depict indirect relationships. Yellow color represents predicted upstream regulators. CPM, counts per minute; GSK, glycogen synthase kinase; LTBR, lymphotoxin β receptor; MMP, matrix metalloproteinase; SSC, side scatter; TLR, Toll-like receptor; TNF, tumor necrosis factor.