Fig 1.
KSHV preferentially infects monocytes and triggers the monocyte inflammatory transcription program.
PBMCs were infected with rKSV.219 at MOI = 1, fixed at various time points (pre-infection, day 1, 2, and 4) after infection, and subjected to 10x Genomics Chromium scRNA-seq analysis. (A) UMAP visualizes 9 PBMC clusters (CL1: Classical Monocytes, CL2: Intermediate Monocytes, CL3 and CL4: NK cell subset, CL5: T cell subset, CL6: B cells, CL7: Non Classical Monocytes, CL8: IFN-responsive cells, and CL9: CD8 enriched T cell subset) in a 1 day-post-infection (dpi) sample based on lineage-specific gene expression of KLRD1, CD14, APOBEC3A, VMO1, CD79A, and CD3E genes for NK cells, monocytes, intermediate monocytes, non-classical monocytes, B cells, and T cells, respectively. The detailed list of differentially expressed genes for each cluster is shown in S1 Table. (B) KSHV-encoded K2 (vIL-6) and ORF16 (vBCL2) expression overlapped with CD14 expression at 1 dpi. (C) The kinetic scRNA-seq analysis on pre-infection, 1 dpi, 2 dpi, and 4 dpi revealed that the colocalized expression of KSHV-encoded K2 and ORF16 and the monocyte-related genes CD14, IL3RA (CD123), CD68, and IL6ST (gp130). (D) KSHV infection in CD14+ monocytes triggered the activation and inflammatory response at 1 dpi with the upregulation of CD86, CD80, TNFα, CCL2, IL1b, and IL6. (E) KSHV infection in CD14+ monocytes later upregulated the expression of genes associated with differentiation of M2-like macrophages with suppressive phenotype (MRC1, CD163, IL10, RNASE1, C1QA, and TREM2). Gene expression levels are indicated by heatmap bars. A representative of three similar experiments is shown.
Fig 2.
KSHV infection preferentially triggers STAT activation in monocytes and dendritic cells.
PBMC from healthy donors (n = 6) were infected with vIL-6REV (KSHV with vIL-6 expression) or vIL-6STOP (KSHV without vIL-6 expression) (MOI = 1), or mock-infected (PBS). At 1 dpi, cells were fixed and subjected to CyTOF analysis. The expression levels of 10 signaling molecules were evaluated. Data combine two experiments (n = 3 each). (A) tSNE for dimension reduction analysis was applied for each infection group after 1000 events from 6 samples in each group were concatenated. Immune cell subsets were clustered based on each lineage’s marker expression (Right panel). (B) The phenotypic changes induced in three immune cell populations corresponding to CD4+CD3+, CD19+CD20-, and CD123+CD14+ clusters by infection were identified by comparison between vIL-6STOP, vIL-6REV, and the mock-infected (PBS) control group. (C) The activation status of 10 signaling molecules among plasmacytoid dendritic cells (pDC) and monocytic cell subsets, including CD123+CD11c+ cells, myeloid (m)DC, CD14loCD16+ non-classical monocytes, CD14+CD16+ intermediate monocytes, and CD14+CD16- classical monocytes are shown in heat maps. Each subset was identified based on the lineage cell surface marker expression with gating strategies shown in S6 Fig. (D) The fold change in the CyTOF signal intensity of STAT1 and pSTAT3 for CD123+CD11c+ cells (TOP panel) and CD14+CD16+ cells (Bottom panel) in infected groups compared to mock-infected (PBS) control group are shown. p values shown are by ratio paired t-test. p<0.05: statistically significant. (E) Individual percentages of pDCs, CD14+CD16+, and CD123+CD11c+ cells among DC gate for each PBMC sample (n = 6) are compared between vIL-6REV- and vIL-6STOP- infected groups, and the uninfected group (PBS). p values shown are by ratio paired t-test. p<0.05: statistically significant.
Fig 3.
KSHV preferentially infects and expands CD14+ monocytes with an inflammatory response in a manner dependent on vIL-6.
Monocytes were isolated negatively from PBMCs (n = 6) using magnetic beads-based negative enrichment and infected with vIL-6STOP or vIL-6REV rKSHV (MOI = 1). (A) The frequencies of live cells at 2 dpi by live/dead staining are shown. (B) Representative FACS profiles of infected monocytes analyzed by flow cytometry for Ki67, CD16, and PD-L1 expressing cells. (C) The frequencies for Ki67+, CD16+, and PD-L1+ cells are shown. Data represent two similar experiments. (D) Representative images of cell blasts in the indicated treatment conditions (x100). (E) Representative FACS profiles of each sample for pSTAT1 and pSTAT3 intracellular staining with the percentage of gated pSTAT1- and pSTAT3- expressing cells are shown. Data represent two similar experiments. p values shown are by ANOVA with a paired comparison with Tukey’s multiple comparison test. p < 0.05: statistically significant.
Fig 4.
Differential gene expression analysis between wild-type KSHV and vIL-6STOP KSHV infected monocytes.
(A) A schematic illustration of the KSHV infection experiment (Left). Monocytes were infected with vIL-6REV, vIL-6STOP, or mock-infected (PBS). At 7 dpi, cells were harvested for RNA-seq analysis (n = 2/group). Principal component analysis (PCA) of RNA-seq data from independent biological replicates is shown (Right). The number in parenthesis indicates the percentage of total variance explained by each PC. Colored dots represent individual samples infected with mock (PBS-treated), vIL-6REV, and vIL-6STOP. (B) Volcano plots for differentially expressed genes (log2 FC threshold = 1, p-value threshold = 0.05) between vIL-6REV and vIL-6STOP (Top), vIL-6 REV and mock (Middle) and vIL-6STOP and mock (Bottom). The log2 FC indicates the mean expression for each. The functional enrichment analysis of upregulated genes (C) and downregulated genes (D) in vIL-6REV- compared to vIL-6STOP-infected monocytes. DAVID functional enrichment analysis was performed, and the analysis results of the top 5 enriched pathways are shown (Right). The representative results of Gene Set Enrichment Analysis (GSEA) are shown (Left). (E) Heatmap of representative genes from enriched gene sets for upregulated IFN-inducible genes (C) or downregulated MHC-II genes (D) in vIL-6REV- compared to vIL-6STOP-infected monocytes. (F) Putative transcription factors that are responsible for differentially expressed genes between vIL-6 REV and vIL-6 STOP KSHV infection. The bar charts indicate p values for enrichment. The following parameters were used; Organism: Homo sapiens (hg38), Experiment type: ChiP (TFs and others), Cell type Class: Blood, and Threshold for significance: 50.
Fig 5.
KSHV infection promotes macrophage differentiation with an immunosuppressive phenotype in a manner dependent on vIL-6 expression.
Monocytes were infected with vIL-6REV or vIL-6STOP (MOI = 1), uninfected (PBS) and cultured for 7–14 days, or treated with vIL-6 (200 ng/ml in serum-free medium) for 9 days. Each sample was analyzed by flow cytometry with isotype control staining as a control. (A) The average frequency of macrophages in each culture (n = 3) is based on representative FSC-H and SSC-H profiles for uninfected (PBS) and vIL-6REV- and vIL-6STOP-infected samples. The macrophage gate was further analyzed for HLA-DR and CD16 expression to confirm typical macrophage phenotype. A representative overlay for the treated sample with unstained control (blue) and stained with antibodies for HLA-DR and CD16 (red). Data combine four independent experiments with different donor samples. Dots represent the average % of biological replicates (n = 2–3). (B) The average geometric mean fluorescent intensity (MFI) of HLA-DR for each macrophage sample with representative overlays is shown. Data combine four independent experiments with different donor samples. Dots represent the average HLA-DR gMFI of biological replicates (n = 2–3). (C) Representative FACS plots of live monocytes one week after vIL-6 treatment and the average frequencies (n = 3) are shown. (D) The average gMFI of HLA-DR for each macrophage sample in (C) with representative overlays are shown. Data represent two similar experiments. (E) An experimental design of T cell proliferation assay with macrophages (Top) and representative histograms of T cells (gated based on FSC-A and SSC-A) for CSFE are shown. T cells without or with anti-CD3/CD28 tetramer stimulation are used as controls. (F) The average frequencies of the CFSE-negative population determined as in (E) for each sample are shown (n = 3). p values shown are by ANOVA with a paired comparison with Tukey’s multiple comparison test. p < 0.05: statistically significant. Data represent two similar experiments.
Fig 6.
vIL-6 protein expression is important to sustain KSHV inducible gene expression.
(A) iSLK cell lines were infected with vIL-6 REV or vIL-6 STOP rKSHV. The proportion of GFP-positive cells was determined by FACS analysis at 1 dpi. (B) Total RNA was isolated from vIL-6 REV or vIL-6 STOP-infected iSLK cells at 0, 1, 7, 14 dpi. LANA, K8.1, vIL-6, and PAN mRNA expression normalized with β-actin were determined in triplicates by qRT-PCR. Each mRNA expression was then normalized by the viral genome copy. p values shown are by unpaired Student’s t-test. (C) Monocytes were infected with vIL-6-REV or vIL-6 STOP rKSHV, and vIL-6 and PAN mRNA expression levels normalized to β-actin were measured in triplicates by qRT-PCR at 1 and 7 dpi. p values shown are by ANOVA with a paired comparison with Tukey’s multiple comparison test. p < 0.05: statistically significant. Data represent two similar experiments.