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Human cytomegalovirus stimulates monocyte-to-macrophage differentiation via the temporal regulation of caspase 3.

ABSTRACT: Monocytes are primary targets for human cytomegalovirus (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Biologically, monocytes have a short life span of 48 h in the circulation, a period of time during which monocytes must make a cell fate decision on whether to undergo apoptosis or differentiate into a macrophage. We have previously shown that HCMV infection stimulates monocyte-to-macrophage differentiation; however, the mechanism(s) by which HCMV-infected monocytes simultaneously navigate the 48-h "viability gate" and undergo macrophagic differentiation has remained elusive. Studies have demonstrated that the level of caspase 3 and 8 activities in monocytes may mediate the delicate balance between apoptosis and macrophage colony-stimulating factor (M-CSF)-induced myeloid differentiation. Here, we show that HCMV infection, unlike M-CSF treatment, does not induce caspase 8 activity to promote myeloid differentiation. However, HCMV infection does induce a temporal activation of caspase 3, with only a low level of active caspase 3 being observed after the 48-h viability checkpoint. Consistent with the role of a time-dependent activation of caspase 3 in promoting myeloid differentiation, the inhibition of caspase 3 blocked HCMV-induced monocyte-to-macrophage differentiation. Temporal transcriptome and functional analyses identified heat shock protein 27 (HSP27) and Mcl-1, two known regulators of caspase 3 activation, as being upregulated prior to the 48-h viability gate following HCMV infection. Using small interfering RNAs (siRNAs), we demonstrate that HCMV targets the rapid induction of HSP27 and Mcl-1, which cooperatively function to precisely control caspase 3 activity in order to allow for HCMV-infected monocytes to successfully traverse the 48-h cell fate decision checkpoint and commence macrophage maturation. Overall, this study highlights a unique regulatory mechanism employed by HCMV to tightly modulate the caspase 3 activity needed to promote myeloid differentiation, a key process in the viral dissemination and persistence strategy.

SUBMITTER: Chan G

PROVIDER: S-EPMC3457328 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Human cytomegalovirus stimulates monocyte-to-macrophage differentiation via the temporal regulation of caspase 3.

Chan Gary G Nogalski Maciej T MT Yurochko Andrew D AD

Journal of virology 20120725 19

Monocytes are primary targets for human cytomegalovirus (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Biologically, monocytes have a short life span of 48 h in the circulation, a period of time during which monocytes must make a cell fate decision on whether to undergo apoptosis or differentiate into a macrophage. We have previously shown that HCMV infection stimulates monocyte-to-macrophage differentiation; however, the mechanism(s) by which HC ...[more]

PMID: 22837201

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Project description:Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection.Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence in in vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.

Project description:BackgroundDysregulation of monocyte-macrophage differentiation is a hallmark of vascular and metabolic diseases and associated with persistent low grade inflammation. Plasmalogens represent ether lipids that play a role in diabesity and previous data show diminished plasmalogen levels in obese subjects. We therefore analyzed transcriptomic and lipidomic changes during monocyte-macrophage differentiation in vitro using a bioinformatic approach.MethodsElutriated monocytes from 13 healthy donors were differentiated in vitro to macrophages using rhM-CSF under serum-free conditions. Samples were taken on days 0, 1, 4 and 5 and analyzed for their lipidomic and transcriptomic profiles.ResultsGene expression analysis showed strong regulation of lipidome-related transcripts. Enzymes involved in fatty acid desaturation and elongation were increasingly expressed, peroxisomal and ER stress related genes were induced. Total plasmalogen levels remained unchanged, while the PE plasmalogen species pattern became more similar to circulating granulocytes, showing decreases in PUFA and increases in MUFA. A partial least squares discriminant analysis (PLS/DA) revealed that PE plasmalogens discriminate the stage of monocyte-derived macrophage differentiation. Partial correlation analysis could predict novel potential key nodes including DOCK1, PDK4, GNPTAB and FAM126A that might be involved in regulating lipid and especially plasmalogen homeostasis during differentiation. An in silico transcription analysis of lipid related regulation revealed known motifs such as PPAR-gamma and KLF4 as well as novel candidates such as NFY, RNF96 and Zinc-finger proteins.ConclusionMonocyte to macrophage differentiation goes along with profound changes in the lipid-related transcriptome. This leads to an induction of fatty-acid desaturation and elongation. In their PE-plasmalogen profile macrophages become more similar to granulocytes than monocytes, indicating terminal phagocytic differentiation. Therefore PE plasmalogens may represent potential biomarkers for cell activation. For the underlying transcriptional network we were able to predict a range of novel central key nodes and underlying transcription factors using a bioinformatic approach.

Dataset Information

Human cytomegalovirus stimulates monocyte-to-macrophage differentiation via the temporal regulation of caspase 3.

Publications

Human cytomegalovirus stimulates monocyte-to-macrophage differentiation via the temporal regulation of caspase 3.

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