KSHV can successfully infect human monocytes and macrophages in vitro
and in vivo
). We infected THP-1 cells with rKSHV.219, expressing green fluorescent protein (GFP) driven by the CMV promoter, red fluorescent protein (RFP) under a lytic viral promoter, and the puromycin resistance gene (40
). Seventy-two hours postinfection, cells were added to puromycin-containing selection medium to maintain KSHV infection and to achieve a 100% KSHV-infected monocytic cell line as monitored by GFP expression (A).
Fig 1 KSHV infection in THP-1 cells. (A) Fluorescence microscopy of GFP expression in KSHV-THP-1 cells compared to that in uninfected THP-1 control cells. (B) Immunofluorescence staining of KSHV LANA expression in KSHV-THP-1 cells compared to that in uninfected (more ...)
To confirm KSHV infection of THP-1 cells, we performed immunofluorescence assays on KSHV-infected and uninfected THP-1 cells for LANA protein expression (B). Briefly, KSHV-THP-1 or THP-1 cells suspended in PBS were spotted on slides, air dried, and fixed with paraformaldehyde. Cells were stained with an antibody directed against LANA followed by a TRITC-conjugated secondary antibody. Cells were also stained with DAPI to demarcate the nucleus. As can be seen in B, uninfected THP-1 cells did not show any LANA staining, while the KSHV-THP-1 cells showed characteristic nuclear speckled staining for LANA protein (19
In order to determine the profile of KSHV viral genes expressed in the THP-1 monocytic cell line, qPCR was performed on KSHV-THP-1 cells and uninfected THP-1 cells. qPCR primer pairs were designed for each KSHV ORF as previously described (11
). Equal amounts of total poly(A) mRNA from THP-1 and KSHV-THP-1 cells were used as starting material. A shows cycle threshold (CT
) values for each KSHV gene versus the relative log fold gene expression in KSHV-THP-1 cells compared to that in uninfected THP-1 cells. The LANA gene and several other classic latent genes, including vCyclin and vIRF-3 genes, were detected at high levels in the KSHV-THP-1 cells. Note that cellular gapdh and hprt, the positive controls, were present at a ratio of 1, i.e., at equal expression levels in infected and uninfected cells. B shows the density distributions, i.e., the number of genes with a given expression level above that for uninfected cells. Fewer than 10 transcripts were expressed at levels significantly greater than 100 times that of the uninfected control cells (B, dotted line); the remainder of genes were undetectable or present at low levels (<100-fold of level for mock-infected cells). The difference between these two groups of genes was significant to a P
value of ≤2.4 × 10−9
, with a 95% confidence interval (95% CI) between the means of expression of 104.99
. Relative expression levels are depicted by heat maps comparing KSHV-THP-1 and THP-1 cells (C). Lytic transcripts detected at high to moderate expression levels likely reflect the low number of cells spontaneously reactivating, similar to results for other KSHV-infected cell lines in culture (33
). Importantly, KSHV-THP-1 cells were capable of reactivation after treatment with 20 ng/ml TPA (A), as evidenced by measuring viral IL-6 mRNA levels after 48 h and 72 h compared to GAPDH mRNA levels as the endogenous control. No signal was obtained in the absence of reverse transcription (−RT) or absence of template (NTC). Lytic viral protein expression of KSHV K8.1 could also be detected in the TPA-reactivated cells (B).
Fig 2 KSHV gene expression profile of KSHV-THP-1 cells. (A) Analysis of log KSHV gene expression versus qPCR cycle threshold (CT) value. Dotted line represents limit of detection for viral genes. (B) Density distribution (on the vertical axis) of relative log (more ...)
Fig 3 TPA treatment of KSHV-THP-1 cells results in lytic replication. (A) Reactivation of KSHV-THP-1 cells. PCR analysis of KSHV lytic gene for vIL-6 at 48 h and 72 h after TPA treatment. (B) KSHV-THP-1 or BCBL-1 control cells (1 × 106) were treated (more ...)
Primary infection of dendritic cells and macrophages with KSHV has been shown to downregulate DC-SIGN, and in B cells KSHV infection decreased MHC I (31
). Hence, we investigated whether latently infected monocytes show downregulation of monocyte activation markers. We found that surface expression of CD86 was reduced from approximately 31% in THP-1 cells to 4% for KSHV-THP-1 cells (average fold change, 5.02 ± 2.4) (). Additionally, we observed that surface expression of CD83 was downregulated from 12% to 1.20% in KSHV-THP-1 cells compared with that in uninfected control cells (average fold change, 5.1 ± 4.0). Since KSHV-infected monocytes are prevalent in KS lesions, reduced expression of costimulatory molecules on the surface of antigen-presenting cells, such as monocytes, during latency may dampen host immune responses against KSHV-infected cells.
Fig 4 KSHV-THP-1 cells exhibit reduced CD86 and CD83 costimulatory molecule expression. THP-1 (A) or KSHV-THP-1 (B) cells were incubated with isotype control, CD86, or CD83 antibody, followed by anti-mouse allophycocyanin-conjugated secondary antibody. Cell (more ...)
In order to confirm coreceptor downregulation, we performed reverse transcription-qPCR (RT-qPCR) for these markers as well as additional costimulatory markers CD80 and CD1a (). We found that several genes associated with macrophage/dendritic cell activation were downregulated compared to levels in uninfected THP-1 control cells (A). By densitometry, we found that genes for costimulatory molecules CD80, CD86, CD1a, and CD83 were downregulated 2.9-, 8.1-, 2.7-, and 4.1-fold, respectively. The downregulation of CD86 and CD80 basal protein expression levels in KSHV-THP-1 cells was confirmed by Western blot analysis (B).
Fig 5 Latent KSHV inhibits expression of costimulatory molecules. (A) qPCR analysis for expression of CD86, CD80, CD83, and CD1a in KSHV-THP-1 cells (K) compared to that in uninfected THP-1 control cells (T). (B) Expression of CD86 and CD80 was determined by (more ...)
There is a heterogeneous level of CD86 coreceptor expression on THP-1 cells. Our findings may allow for the alternative possibility that KSHV selectively enters cells with low CD86 expression and that KSHV-infected latent THP-1 cells display a low CD86 expression due to this fact. To rule out this possibility, THP-1 cells were stained for CD86, and the cells that expressed the highest levels of CD86 coreceptor were isolated using fluorescence-activated cell sorting (FACS). This population is denoted as CD86hi THP-1 (A). Sorted CD86hi THP-1 cells were then infected with KSHV and analyzed by flow cytometry for GFP expression (B). CD86hi THP-1 cells were 68.6% GFP positive 24 h postinfection, suggesting that KSHV can enter the fraction of THP-1 cells with the highest level of CD86 surface expression prior to establishing latency. Hence, it is more likely that CD86 is downregulated by KSHV and that this effect is not an artifact of KSHV preferentially entering THP-1 cells that express the smallest amount of CD86 on their surface.
Fig 6 KSHV does not require low surface coreceptor expression to infect THP-1 monocytes. (A) THP-1 cells (1 × 107) were stained with CD86 antibody and immediately analyzed by fluorescence-activated cell sorting (FACS) to separate THP-1 cells with the (more ...)
We also investigated cytokine expression in KSHV-THP-1 cells compared to that in THP-1 controls by Luminex multianalyte analysis. THP-1 or KSHV-THP-1 cells were incubated for 24 h and cell-free supernatants collected for analysis. Overall, we analyzed the expression of 15 proinflammatory cytokines, and for the most part, expression was unchanged (data not shown). However, a significant difference in tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) was observed (A). TNF-α and IL-1β expression levels were 7.73 ± 0.56 pg/ml and 4.90 ± 0.59 pg/ml, respectively, in THP-1 cells, and these cytokines were undetectable in KSHV-THP-1 cells. In order to confirm that these cytokines were downregulated at the transcription level, RT-PCR analysis was performed. B shows that transcription of both genes is suppressed in KSHV-infected THP-1 cells. TNF-α and IL-β are involved in upregulating the transcription of genes involved in inflammation, hematopoiesis, and immune responses, including costimulatory molecules, and therefore their downregulation may contribute to inhibition of adaptive immunity.
Fig 7 Expression of select cytokines in KSHV-THP-1 cells. (A) TNF-α and IL-1β expression in THP-1 compared to KSHV-THP-1 cells was determined by Luminex multianalyte assay. Units shown are average pg/ml. (B) KSHV-THP-1 (K) or THP-1 (T) cells (more ...)