These data demonstrate the production of HIV-derived miRNAs by human macrophages in vitro, including vmiR-TAR as previously reported in addition to novel HIV-derived vmiR88 and vmiR99. Novel HIV vmiR88 and vmiR99 are produced by HIV-infected human macrophage cell lines, human alveolar macrophages following in vitro HIV infection, and by alveolar macrophages from (-)-Tetramisole asymptomatic HIV-infected persons with advanced HIV infection, especially following PMA stimulation. Full-length sequences of vmiR88 and vmiR99 expressed in infected cells were confirmed. Exosome preparations harbored full-length vmiR88 as well as longer variants of vmiR88 and vmiR99 bearing 39 extensions of viral sequence. Furthermore, incubation of uninfected recipient macrophages with exogenous vmiR88 or vmiR99 stimulate a pathway in macrophages that elicits TNFa release. The mechanism of these pro-inflammatory miRNAs was not due to the role of miRNA in targeted gene silencing by RNA interference. Instead, the HIV derived miRNAs directly stimulated a signaling pathway in macrophages resulting in TNFa release, a process that was Citiolone dependent partly on G+U base composition of the miRNA, and partly on macrophage TLR8 expression. Using a flow cytometry based fluorescence resonance energy transfer assay, we demonstrated binding of ssRNA40 to human TLR8. Furthermore, TNFa release was inhibited by antagomir88 and antagomir99 even with partial or little complementarity to the ssRNA ligand, suggesting that these antagomirs may function more strongly as receptor antagonists relative to their intended function as ligand antagonists. Finally, novel HIV vmiRNAs are detected in sera of HIV-infected persons, and associated with exosomal fraction. Biological significance is suggested by the finding that exosomes from serum of an HIV-infected aviremic person as well as exosomes from HIV-infected U1 macrophages elicit a pro-inflammatory response by human macrophages, whereas exosomes from healthy serum and from uninfected parental macrophages did not stimulate TNFa release.These data support a potential role for novel HIV-derived vmiRNAs from macrophages as contributing to chronic immune activation in HIV-infected persons.