PurposeTo determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.MethodsUsing sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.ResultsHIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.ConclusionNovel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

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IntroductionBacterial pneumonia remains a frequent and serious complication in asymptomatic HIV ϩ persons, despite relatively preserved peripheral blood CD4 ϩ T-lymphocyte counts 1 and use of highly active antiretroviral therapy (HAART) with undetectable plasma viral loads. 2 These persons have up to 25-fold greater risk of bacterial pneumonia than their healthy cohorts. 3 However, the mechanism contributing to this increased risk is not well understood. Toll-like receptor 4 (TLR4) represents a critical pattern recognition receptor in the innate immune host cell response to bacterial infection. Functional deficiency or genetic deletion of TLR 4 increases susceptibility to Haemophilus influenza, Streptococcus pneumoniae, and Klebsiella pneumoniae respiratory tract infections in murine models. 4,5 Another indication of its importance is that TLR4 polymorphisms are associated with increased susceptibility to lung infection. 6 Our laboratory has reported impaired TLR4-mediated tumor necrosis factor ␣ (TNF␣) release in alveolar macrophages from asymptomatic HIV ϩ persons at increased clinical risk of bacterial pneumonia. 7 Increased susceptibility may be in part related to reduced alveolar macrophage extracellular signalregulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase phosphorylation attributed to elevated MAP kinase phosphatase 1 (MKP-1) activity, 7 and constitutive phosphoinositol-3 kinase (PI3K) activation and heightened PI3K signaling in response to TLR4 activation. 8 These data suggest that the host cell proinflammatory cytokine may be suboptimal in the lungs of HIV ϩ persons and may in part contribute to increased bacterial pneumonia susceptibility and pathogenesis.TLR4 is the most studied of the TLR family of innate receptors. 9 It is a unique member of the TLR family of mammalian receptors in that TLR4 is capable of both adaptor molecule myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent signaling. 10-12 TLR4-mediated MyD88-dependent recognition of bacterial cell wall occurs at the cell surface, 11,13 does not require receptor internalization, 11 involves interleukin-1 receptorassociated kinase (IRAK) phosphorylation, and activation of TNF receptor-associated factor 6 (TRAF6), nuclear factor-B (NF-B), and MAP kinases (such as ERK1/2, p38, and Jun kinase), with the subsequent release of proinflammatory cytokines such as TNF␣ 14 that may contribute to an effective host defense response to bacteria. In contrast, TLR4-mediated MyD88-independent signaling requires receptor internalization and is mediated through Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-␤ (IFN␤; TRIF), 11 involves activation of IFN regulatory factor 3 (IRF3) and STAT1 15 with release of type-1 IFNs, 16 IL-10, 15 RANTES 15 and may be involved in antiviral host defense. 17 Recent work in our laboratory has shown impaired TLR4-mediated signaling response in alveolar macrophages from asymptomatic HIV ϩ persons at high clinical risk of bacterial pneumonia. 7,8 However, whe...