Polyomavirus JC infection inhibits differentiation of oligodendrocyte progenitor cells

Abstract

Reactivation of the human polyomavirus JC (JCV) in the CNS results in a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). The lytic destruction of oligodendrocytes, which occurs at the terminal stage of the viral infection cycle, is considered a critical factor in the development of demyelination and the pathogenesis of PML. However, knowledge is limited about interaction of JCV with oligodendrocytes and its impact on the denudation of axons at the early stage of viral reactivation and prior to the destruction of the infected cells. We have developed an in vitro neuroprogenitor cell culture using human fetal brain that can be differentiated to the oligodendrocyte lineage to investigate interactions of JCV with its host cells. Results show that infection with JCV delays oligodendrocyte maturation as shown by reduced levels of oligodendrocytic markers, including myelin basic protein, proteolipid protein, and platelet-derived growth factor receptor-α. Furthermore, replication of JCV in these cells caused substantial dysregulation of several chemokines, including CCL5/RANTES, GRO, CXCL1/GROα, CXCL16, CXCL8/IL-8, CXCL5/ENA-78, and CXCL10/IP-10, all of which play a role in cell growth and differentiation.

Fig. 1

Preparation of human neural progenitor cells (hNPC) and their differentiation into human oligodendrocyte progenitor cells (hOPC) and human oligodendrocytes (hOL). A: Schematic representation of the preparation of hNPC from human fetal brain tissue (hfB, embryonic age 16 weeks) based on protocols of Espinosa-Jeffrey at al. (2009), with slight modifications. After dissociation of brain tissue, cells were plated onto nontissue-culture-grade Petri dishes coated with anti-PSA-NCAM antibody. Unattached cells were cultured in neural stem cell medium (NSCM) either as two- or as three-dimensional hNPC cultures. The hNPC were then gradually switched mixed with glial defined medium plus growth factors (GDM⁺) to differentiate them into hOPC and then to glial defined medium minus growth factors (GDM) to differentiate them into hOL. B: The hNPC stage of cultures was ascertained by immunolabeling of hNPC plated on slides with antibodies specific to nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. C: Immunolabeling of hOPC for expression of nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. D: Immunolabeling of hOL cultures for expression of GalC, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. Average percentages of cells expressing specific markers were quantified in at least 15 fields of view (~700–800 cells total). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 2

JCV infection of undifferentiated hNPC and hNPC induced to differentiate into hOL. A: Cultures of hNPC on poly-D-lysinecoated dishes and slides in two sets were left uninfected or were infected with the Mad-1 strain of JC virus for 7 days, and CM was collected, total protein lysates were prepared, and cells on slides were fixed. Immunocytochemistry was performed for βIII-tubulin, GFAP, GalC, and the viral proteins VP1 and agnoprotein. Expression of JCV T-antigen, VP1, and agnoprotein was analyzed by Western blot assay. Viral DNA content in CM was determined by qPCR. B: Cultures of hNPC were grown in NSCM for 3 days, and then differentiation of hNPC into the OL lineage was initiated. Cells were kept in GDM⁺ with growth factors for 4 days, and then culture medium was switched to the GDM without growth factors. Two days later, cells were uninfected or infected with the Mad-1 strain of JC virus. At day 7 of JCV infection (9 days in GDM without growth factors), CM was collected, protein lysates were prepared, and cells on slides were fixed. Immunocytochemistry was performed for βIII-tubulin, GFAP, GalC, and the viral proteins VP1 and agnoprotein. Expression of JCV T-antigen, VP1, and agnoprotein was analyzed by Western blot assay. Viral DNA content in CM was determined by qPCR. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 3

Effect of JCV infection on the expression of OL lineage-specific markers in hNPC undergoing OL lineage progression. hNPC were induced to differentiate into the oligodendroglial lineage and infected or uninfected with JCV as described in the legend to Figure 2. Seven days following infection (9 days in GDM), RNA was isolated, and the levels of mRNAs for the oligodendroglial lineage markers Nkx2.2, Olig1, Olig2, and PDGFRα (A); MBP and PLP (B); and Oct4B, Nanog, and 5HT2AR (C) were measured by qRT-PCR. All values in the histograms are normalized relative to hOPC cultures that were neither induced to differentiate nor infected with JCV (1.0). Finally, expression of A2B5 was examined in these cells by immunocytochemistry (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 4

Effect of JCV infection on growth/survival and secretion of specific cytokines by hNPC undergoing OL lineage progression. hNPC were induced to differentiate into the oligodendroglial lineage and infected or uninfected with JCV as described in the legend to Figure 2. A: Seven days following infection (9 days in GDM), the number of viable cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assay. CM from JCV-infected and uninfected hOL was subjected to immunoprecipitation with anti-VP1 antibody to deplete viral particles and for treatment of differentiating hOPC. Cell viability was measured by MTT assay (right). B: A chemokine array assay was performed using CM from the JCV-infected or uninfected cells. Chemokines that were significantly upregulated by JCV infection are indicated by colored boxes. C: Expression of chemokines that were significantly upregulated by JCV infection in the chemokine array assay was quantitated by densitometry: CCL5/RANTES, GRO, CXCL1/GROα, CXCL16, CXCL8/IL-8, CXCL5/ENA-78, and CXCL10/IP-10. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 5

Effect of JCV infection on the expression of specific cytokines/chemokines in hNPC undergoing OL lineage progression. Chemokine mRNA expression levels in the hOL cultures were measured by qRT-PCR using the same RNA preparations that were analyzed for Figure 3. The levels of mRNAs for CXCL1/GROα, CXCL8/IL-8, CXCL5/ENA-78, CXCL6/GCP-2, and the CXC cytokine receptor CXCR2 are shown. ENA78, GCP-2, GROα, and IL-8 cytokines are shown, along with the mRNA for the CXC cytokine receptor CXCR2.