Fig 7. Critical role of IFN-I signaling on resident CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ DCs in the production of initial CCL2 protein for early migration of CD11b⁺Ly-6C^hi monocytes.

(A) Early expression of CCL2 and CCL3 by sorted CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ DCs. Subsets of macrophages (CD11b^hiF4/80^hi, CD11b^hiF4/80^int/lo, CD11b^loF4/80^hi, and CD11b^loF4/80^lo) and DCs (CD11c^hiEpCAM⁺, CD11c^hiEpCAM⁻, CD11c^loEpCAM⁺, and CD11c^loEpCAM⁻) were sorted 12 h after mucosal HSV-1 infection, and CCL2 and CCL3 expression was analyzed with real-time qRT-PCR. (B,C) Regulation of CCL2 and CCL3 production from CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ DCs by IFN-I signaling. CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ DCs producing CCL2 (B) and CCL3 (C) were determined by flow cytometric analysis at 12 h pi. Blue line, HSV-1-infected BL/6; Red line, HSV-1-infected IFNAR KO; Gray line, mock-infected. (D) Confocal microscopic analysis of early CCL2 production by resident CD11b⁺ macrophages and CD11c⁺ DCs. CD11b⁺ macrophages and CD11c⁺ DCs producing CCL2 protein were visualized by confocal microscopy at 12 h pi. CD11b⁺ macrophages and CD11c⁺ DCs producing CCL2 protein are denoted by white arrows. (E,F) Essential role of IFN-I signaling in the production of CCL2 protein from CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ DCs. Vaginal CD11b^hiF4/80^hi macrophages (E) and CD11c^hiEpCAM⁺ DCs (F) isolated from WT and IFNAR KO mice were stimulated with recombinant IFN-α (2,000 and 4,000 IU/ml) for 6 h. The expression and production of CCL2 were determined by real-time qRT-PCR and CBA using stimulated cells and collected culture media, respectively. Data represent the average ± SD of values derived from three individual experiments (n = 4–5). *, p<0.05; **, p<0.01; ***, p<0.001 compared with the level of the indicated group.