Macrophage Differentiation and Rhinovirus Infection

To generate bone marrow-derived macrophages, mouse bone marrow monocytes were isolated from C C57BL/6J and TLR2−/− (B6.129-Tlr2tm1Kir/J) mice (Jackson Laboratories) and cultured in L929 medium, a source of macrophage colony-stimulating factor, for 7 days ^{49 (link)}. Human primary peripheral blood mononuclear cells (PBMC; Precision for Medicine, Frederick, MD) were cultured in RPMI-1640 media supplemented with 20ng/mL of recombinant human M-CSF. THP-1 human monocytic cells (ATCC) were differentiated with 5 ng/ml of phorbol 12-myristate 13-acetate as described⁵⁰ . Cells were infected with RV-A1B and RV-C15 at a multiplicity of infection (MOI) of 1.0 at 33°C. To study virus entry, human THP-1-derived macrophages and mouse bone marrow-derived macrophages were incubated with RV-A1B or RV-C15 for 1 hour and washed three times with PBS. Viral RNA and CDHR3 mRNA expression were quantified by qPCR (see below) and the presence of RV-C was examined by Western blot and immunofluorescence using anti-EV-D68 vp3. Late endosomes were visualized with anti-EEA1 (Invitrogen, Carlsbad, CA). Images were visualized using a Leica SP5 inverted laser confocal microscope (Buffalo Grove, IL).