KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression.

BACKGROUND: Kaposi's Sarcoma Herpesvirus (KSHV) is a gammaherpesvirus strongly linked to human cancer. The virus is also able to induce immune suppression, effect that contributes to onset/progression of the viral-associated malignancies. As KSHV may infect macrophages and these cells abundantly infiltrate Kaposi's sarcoma lesions, in this study we investigated whether KSHV-infection could affect macrophage polarisation to promote tumorigenesis. METHODS: FACS analysis was used to detect macrophage markers and PD-L1 expression. KSHV infection and the molecular pathways activated were investigated by western blot analysis and by qRT-PCR while cytokine release was assessed by Multi-analyte Kit. RESULTS: We found that KSHV infection reduced macrophage survival and skewed their polarisation towards M2 like/TAM cells, based on the expression of CD163, on the activation of STAT3 and STAT6 pathways and the release of pro-tumorigenic cytokines such as IL-10, VEGF, IL-6 and IL-8. We also found that KSHV triggered Ire1 α-XBP1 axis activation in infected macrophages to increase the release of pro-tumorigenic cytokines and to up-regulate PD-L1 surface expression. CONCLUSIONS: The findings that KSHV infection of macrophages skews their polarisation towards M2/TAM and that activate Ire1 α-XBP1 to increase the release of pro-tumorigenic cytokines and the expression of PD-L1, suggest that manipulation of UPR could be exploited to prevent or improve the treatment of KSHV-associated malignancies.

HHV-6B reduces autophagy and induces ER stress in primary monocytes impairing their survival and differentiation into dendritic cells.

HHV-6A and HHV-6B are ubiquitous human betaherpesviruses sharing more than 80% homology. HHV-6B is the most common cause of encephalitis in transplant patients and its primary infection may cause the exanthema subitum and febrile seizures in infants. HHV-6A and HHV-6B are able to infect several immune cell types such as T cells, monocytes and dendritic cells (DCs). In this study we found that HHV-6 B derived from patients affected by exanthema subitum impaired monocyte differentiation into DCs, as the infected cells acquired less CD1a DC marker and retained more CD14 monocyte marker. In agreement with the previous finding that HHV-6B dysregulated autophagy and induced endoplasmic reticulum (ER) stress in cells in which it replicated, here we found that these effects occurred also in differentiating monocytes and that ER stress relief, by using the chemical chaperone sodium 4-phenylbutirate (PBA), partially restored DC formation. This suggests that the induction of ER stress, likely exacerbated by autophagy inhibition, could contribute to the immune suppression induced by HHV-6B derived from exanthema subitem patients.

Kaposi Sarcoma Herpes Virus (KSHV) infection inhibits macrophage formation and survival by counteracting Macrophage Colony-Stimulating Factor (M-CSF)-induced increase of Reactive Oxygen Species (ROS), c-Jun N-terminal kinase (JNK) phosphorylation and autophagy.

Kaposi Sarcoma Herpes Virus (KSHV) is an oncovirus belonging to the human gammaherpesvirus family, able to infect several immune cell types including B cells, dendritic cells (DCs) and monocytes. In this study, we found that KSHV infection of monocytes counteracted the Reactive Oxygen Species (ROS) increase induced by Macrophage Colony-Stimulating Factor (M-CSF), prevented c-Jun N-terminal kinase (JNK) and B-cell lymphoma-2 (Bcl-2) phosphorylation and inhibited autophagy, leading to an impairment of cell survival and differentiation into macrophages. We also show that, to further dysregulate immune response in monocytes, KSHV reduced the production of pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNF α) while increased the release of the immune suppressive cytokine Interleukin-10 (IL-10). These results unveils new strategies put in place by KSHV to induce immune suppression and to persist into the infected host.

Mutant p53, Stabilized by Its Interplay with HSP90, Activates a Positive Feed-Back Loop Between NRF2 and p62 that Induces Chemo-Resistance to Apigenin in Pancreatic Cancer Cells.

Pancreatic cancer is one of the most aggressive cancers whose prognosis is worsened by the poor response to the current chemotherapies. In this study, we investigated the cytotoxic effect of Apigenin, against two pancreatic cell lines, namely Panc1 and PaCa44, harboring different p53 mutations. Apigenin is a flavonoid widely distributed in nature that displays anti-inflammatory and anticancer properties against a variety of cancers. Here we observed that Apigenin exerted a stronger cytotoxic effect against Panc1 cell line in comparison to PaCa44. Searching for mechanisms responsible for such different effect, we found that the higher cytotoxicity of Apigenin correlated with induction of higher level of intracellular ROS, reduction of mutant (mut) p53 and HSP90 expression and mTORC1 inhibition. Interestingly, we found that mutp53 was stabilized by its interplay with HSP90 and activates a positive feed-back loop between NRF2 and p62, up-regulating the antioxidant response and reducing the cytotoxicity of Apigenin. These results suggest that targeting the molecules involved in the mTOR-HSP90-mutp53-p62-NRF2-antioxidant response axis could help to overcome the chemo-resistance of pancreatic cancer to Apigenin.

EBV up-regulates PD-L1 on the surface of primary monocytes by increasing ROS and activating TLR signaling and STAT3.

Programmed death ligand 1 (PD-L1) (also called B7-H1) is a membrane immune-modulatory protein whose overexpression on the surface of tumor cells as well as APCs impairs T-cell-mediated killing. Viruses that establish chronic infections have developed a number of strategies to escape from immune recognition including the up-regulation of PD-L1. This study shows for the first time that the human oncovirus EBV infects human primary monocytes using HLA-DR and induced a strong up-regulation of PD-L1 expression on their surface. Searching for the underlying mechanism/s leading to this immune suppressive effect, we found that EBV activated TLR signaling, increased intracellular ROS, and phosphorylated STAT3. Targeting these molecules partially reverted PD-L1 up-regulation that correlated with an altered cytokine production and a reduction of monocyte cell survival, strongly impairing the antiviral immune response.