Abstract
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi’s sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Using scRNA-seq, we demonstrate that KSHV preferentially infects CD14+ monocytes, sustains viral lytic replication through the viral interleukin-6 (vIL-6), which activates STAT1 and 3, and induces an inflammatory gene expression program. To study the role of vIL-6 in monocytes upon KSHV infection, we generated recombinant KSHV with premature stop codon (vIL-6(-)) and its revertant viruses (vIL-6(+)). Infection of the recombinant viruses shows that both vIL-6(+) and vIL-6(-) KSHV infection induced indistinguishable host anti-viral response with STAT1 and 3 activations in monocytes; however, vIL-6(+), but not vIL-6(-), KSHV infection promoted the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL-6(+) KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL-6(-) KSHV infection or uninfected control. Notably, a viral nuclear long noncoding RNA (PAN RNA), which is required for sustaining KSHV gene expression, was substantially reduced in infected primary monocytes upon vIL-6(-) KSHV infection. These results highlight the critical role of vIL-6 in sustaining KSHV transcription in primary monocytes. Our findings also imply a clever strategy in which KSHV utilizes vIL-6 to secure its viral pool by expanding infected monocytes via differentiating into longer-lived dysfunctional macrophages. This mechanism may facilitate KSHV to escape from host immune surveillance and to support a lifelong infection.
Author summary
Kaposi’s sarcoma-associated virus (KSHV) is the causative agent of highly inflammatory diseases that include Kaposi’s sarcoma and KSHV-inflammatory cytokine syndrome (KICS). Macrophages are important immune cells that regulate inflammation by stimulating both innate and adaptive immune systems. A small fraction of monocytes differentiates into macrophages to acquire a longer life span and migrate to residential areas. Deregulation of macrophage functions weakens host immune defense mechanisms, allowing secondary infections resulting in prolonged inflammation. Here, we demonstrate that KSHV infection to monocytes facilitates their transition into macrophages; however, those infected macrophages have impaired immune stimulatory function. Such tradition depends on the expression of KSHV vIL-6, a virally encoded IL-6 homolog. Prolonged vIL-6 stimulation in culture also induced a similar phenotype in monocytes. These results suggest that continuous stimulation by KSHV-derived vIL-6 deregulates host macrophage functions. This mechanism may be associated with hyperinflammatory phenotypes seen in KSHV-associated diseases.
Introduction
A virus is an infectious agent that can only replicate within a living host organism. Because of this dependence, viruses have evolved mechanisms to exploit normal cell functions to escape host immune surveillance for their survival advantage. This exploitation is sometimes associated with prolonged damage to the host, leading to pathologic processes and diseases caused by the viral infection [1].
Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV), or human gamma herpesvirus 8 (HHV-8), is an oncogenic double-stranded DNA virus that establishes a lifelong latent infection [2]. KSHV is the etiologic agent of Kaposi’s sarcoma and is associated with two lymphoproliferative disorders: multicentric Castleman’s disease (MCD) and HIV-associated primary effusion lymphoma (PEL). KSHV-inflammatory cytokine syndrome (KICS) may also represent a prodromic form of KSHV-MCD, which exhibits elevated KSHV viral loads and circulating inflammatory cytokines including IL-6, IL-10, and a KSHV-encoded IL-6 homolog (vIL-6) [3–8]. These highly inflammatory diseases are devastating and a leading cause of cancer deaths in people living with HIV. Therefore, understanding the mechanism of KSHV infection and its association with inflammatory disease development is crucial for finding a cure for these diseases.
Natural transmission of KSHV most likely occurs through salivary and sexual transmission or during transplantation of KSHV-positive organs into a naïve recipient, although initial KSHV infection is typically asymptomatic [2,9]. In experimental settings, KSHV has been shown to infect various types of cell lines and primary cells, such as epithelial cells and immune cells that include B cells, monocytes, and dendritic cells through binding to specific cell surface receptors such as Siglec DC-SIGN [10–14]. However, it remains unclear as to whether KSHV may strategically infect a particular cell type among PBMC. The mechanisms by which KSHV facilitates a lifelong infection by increasing viral reservoirs and impacts the host immune system are also not entirely clear.
KSHV-encoded viral interleukin-6 (vIL-6) is a homolog of human interleukin-6, which is encoded by KSHV ORF-K2 and is highly expressed during the lytic replication cycle [15]. Viral IL-6 is also expressed at physiologically functional levels in latently infected cells [16] and is detectable in the sera and/or tumor tissues of patients with KS, PEL, and MCD [17]. Viral IL-6 enhances cell proliferation, endothelial cell migration, and angiogenesis, leading to tumorigenesis, and has been suggested to be a driver of KICS [18,19]. In addition, vIL-6 transgenic mice developed IL-6-dependent MCD-like disease [20] and supported tumor metastasis in a murine xenograft model [21]. Mechanistically, vIL-6 directly binds to the gp130 subunit of the IL-6 receptor without the need for the IL-6 receptor α, and actives the JAK/STAT pathway to induce STAT3 phosphorylation and acetylation [8,22,23]. In addition, vIL-6 activates the AKT pathway to promote numerous oncogenic phenotypes [18,19,24,25]. STAT3 activation by vIL-6 also increases the VEGF expression through the downregulating caveolin 1 [8] and promotes angiogenesis, suggesting that vIL-6 plays an important role in tumorigenesis through STAT activation. Given that the prototypical human IL-6 plays a critical role in immune regulation and inflammation, vIL-6 is thought to play a pivotal role in inflammatory KSHV diseases. Here, we reveal the role of vIL-6 in the regulation of monocytes by utilizing recombinant KSHV and de novo infection to the peripheral blood mononuclear cells.
Results and discussion
KSHV preferentially infects CD14+ monocytes among PBMC, triggering an inflammatory response and macrophage differentiation
To study cell type-specific KSHV infection, we employed a single cell (sc)RNA-seq analysis approach. Recombinant KSHV (rKSHV.219) virions were purified by two serial ultracentrifugations from the culture supernatant of the iSLK.219 cell line, an inducible recombinant KSHV producer cell. Peripheral blood mononuclear cells (PBMCs) were infected with rKSHV.219 at MOI = 1, fixed at various time points (day 0, 1, 2, and 4) after infection, and subjected to scRNA-seq analysis. KSHV infection and lytic replication in single cells were then monitored by the expression of all KSHV genes.
As shown in Fig 1A, unsupervised Uniform Manifold Approximation and Projection (UMAP) for dimension reduction analysis identified 9 clusters among peripheral blood mononuclear cells (PBMCs). The detailed list of differentially expressed genes for each cluster is shown in S1 Table. Each of the clusters was then associated with known immune cell subsets based on corresponding lineage-specific gene expression. Thus, KLRD1(CD94), CD14, APOBEC3A, VMO1, CD79A, and CD3E genes were used as guiding markers for the classification of NK cells, monocytes, intermediate monocytes, non-classical monocytes, B cells, and T cells, respectively, based on the immune cell data available from the Human Protein Atlas [26] (S1A Fig). All four types of immune cells were present during 4-day infection period, and monocyte cell population was slightly decreased at day 4. (S1B Fig)…
