FIGURE 4.

Exposure to TAR RNA alters cytokine profiles in primary monocyte-derived macrophages. A, primary human MDMs were differentiated by incubating in 10 ng/ml M-CSF and phorbol 12-myristate 13-acetate for 1 week before incubating 10⁶ cells with 10⁴ copies of either TAR WT or mutated TAR D RNA packaged into DOTAP liposomal transfection reagent or with DOTAP only (control). After 48 h, supernatants were analyzed for the presence of 23 cytokine proteins using human cytokine antibody array I. Cytokines that are highly up-regulated after treatment with TAR are indicated by enclosed rectangles. B, exosomes were first isolated from CEM, OM10.1, and J1.1 cell supernatants using Nanotrap particles as described in Fig. 1B; total RNA was purified and then packaged into DOTAP reagent as in A. The DOTAP (liposomes) containing extracellular RNA, as well as TAR (WT) and mutated TAR (D) RNA, were incubated with MDM for 48 h, and then supernatants were analyzed for 23 cytokines (data not shown) as in A. Only IL-6 and TNF data are shown as bar graphs. MDMs were incubated with DOTAP alone as a control. C, primary macrophages were treated with either DOTAP alone (control) or DOTAP/TAR wild type (10⁴ copies/10 μl/experimental), and supernatants were assayed for the presence of cytokines after 0, 3, 6, and 24 h. Results are presented as mean ± S.D. of at least three independent measurements.