Long-term HIV-1 infection induces an antiviral state in primary macrophages

Highlights

• Productive HIV-1 infection of macrophages triggers an innate antiviral response.

• HIV-1 infected macrophages activate interferon stimulated genes and cell death.

• HIV-1 infection increases Rig-I and MDA5 expression and p21-mediated cell cycle arrest.

• Infected macrophages become refractory to over infection through SAMHD1 activation.

• Innate immune responses may serve as targets for eradicating HIV-1 infection.

Abstract

HIV-1 infection is thought to impair type I interferon (IFN-I) production in macrophages, a cell type that is also relatively resistant to HIV-1 cytotoxic effects. Here, we show that monocyte differentiation into macrophages by M-CSF led to cell proliferation and susceptibility to HIV-1 infection that induced cell cycle arrest and increased cell death. Established HIV-1 infection of monocyte-derived macrophages induced the upregulation of the pattern recognition receptors MDA5 and Rig-I that serve as virus sensors; production of interferon-β, and transcription of interferon-stimulated genes including CXCL10. Infected macrophages showed increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription and CDK1 downregulation. Additionally, HIV-1 infection limited deoxynucleotide pool by downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1 together with increased cell death. In conclusion, HIV-1 induced an innate antiviral mechanism associated to IFN-I production, interferon stimulated gene activation, and p21-mediated G2/M arrest leading to elevated levels of cell death in monocyte derived macrophages. Upregulation of MDA5 and Rig-I may serve as targets for the development of antiviral strategies leading to the elimination of HIV-1 infected cells.

Introduction

Macrophages represent the most plastic cell of the hematopoietic system, they are present in virtually all tissues and exhibit a great functional diversity (Geissmann et al., 2010). Among their activities, macrophages perform important immunological functions during the innate response to pathogens and the initiation of inflammation but also contribute to the maintenance of tissue homeostasis, tissue repair and cancer pathogenesis (Wynn et al., 2013), and play a critical role in human immunodeficiency virus (HIV) transmission, viral spread and as a viral reservoir. Recently, it has been demonstrated that macrophages originate from two different sources: tissue-resident macrophages derived from embryonic precursors capable of self-maintain by local proliferation (Schulz et al., 2012) or from infiltrating monocyte-derived macrophages (MDM) (Sieweke and Allen, 2013) changing the traditional view that all tissue-resident macrophages derive from circulating monocytes (van Furth and Cohn, 1968). Several studies have shown that newly recruited monocytes proliferate locally under various conditions (Davies et al., 2013) and may also integrate into the resident population of self-renewing macrophages (Franklin et al., 2014, van de Laar et al., 2016), suggesting that tissue-resident macrophages have the same proliferative potential regardless of their source or ontogeny.

Non-stimulated monocytes are refractory to infection by HIV type 1 (HIV-1). Conversely, differentiated macrophages, as well as other myeloid lineage cells, become susceptible to HIV-1 infection after degradation or inactivation of the restriction factor SAMHD1, a triphosphohydrolase enzyme that controls the intracellular level of dNTPs (Ballana and Este, 2015, Hrecka et al., 2011, Laguette et al., 2011, Lahouassa et al., 2012). Macrophages play crucial roles in viral dissemination and pathogenesis (Herbein et al., 2010). As macrophages are relatively resistant to the cytopathic effect of HIV-1 and are able to harbor the virus for long periods of time, they may represent an important element in maintaining immune activation or serving as long-term viral reservoirs (Igarashi et al., 2001). Importantly, it has been suggested that HIV-1 infection does not induce interferon type I (IFN-I) production in macrophages, and evades immune recognition in this cell type (Harman et al., 2015, Rasaiyaah et al., 2013, Yan et al., 2010).

The close relation of HIV-1 and cell cycle regulation became evident with the observation that the HIV-1 Vpr protein induces cell cycle arrest at the G2/M transition (reviewed in (Andersen and Planelles, 2005)). Since then, several studies have shown different mechanisms through which Vpr is able to halt the cell cycle: recently, premature activation of the SLX4 complex, causing incorrect processing of replication forks have been described as inducer of cell cycle arrest at G2/M (Laguette et al., 2014, Lahouassa et al., 2016). Other mechanisms proposed include ATR-CHK1 signaling triggered by replication stress that inhibits the Cdc25C and CDK1:CyclinB1 complex leading to G2/M arrest (Bregnard et al., 2014). In addition, it has also been shown that Vpr can induce the expression of the of cyclin-dependent kinase p21/Waf1/Cip1 (p21) (Vazquez et al., 2005) or the activation of the NF-KB pathway (Liang et al., 2015) known to stimulate p21 (Wuerzberger-Davis et al., 2005) expression, leading to cell cycle arrest at G2/M.

We have identified the CDK6-dependent CDK2 inactivation of the HIV-1 restriction factor SAMHD1 (Baldauf et al., 2012, Ballana and Este, 2015, Laguette et al., 2011) in primary CD4⁺ T cells and macrophages (Pauls et al., 2014b) and the effect of p21 in regulating SAMHD1 function (Pauls et al., 2014c). The activity of SAMHD1 has been reported to trigger cell death in HIV-1 infected CD4⁺ T cells (Doitsh et al., 2010, Monroe et al., 2014) because it may prevent complete viral DNA synthesis that is sensed by the host cell triggering an innate immune response, resulting in caspases activation and apoptosis. Although HIV-1 has been reported to inhibit the secretion of type I interferon and other proinflammatory cytokines (Harman et al., 2011, Laguette et al., 2014), it may stimulate type I IFN production in astrocytes (Na et al., 2011) and could induce interferon-stimulated genes (ISGs) in macrophages and monocytes through Vpr (Zahoor et al., 2014, Zahoor et al., 2015).

Identification and validation of host mechanisms that might be susceptible targets for novel antiviral therapies may provide the basis of therapeutic strategies to eradicate and cure HIV infection (Ballana and Este, 2013). In the present study, we aimed to investigate the link between cell cycle regulation, innate immune activation and cell death induced by HIV-1 in primary human macrophages.