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

Abstract

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.

Fig. 1. KSHV infects macrophages and skews their phenotype towards an M2 phenotype.

a K-bZIP expression in KSHV- and mock-infected macrophages was evaluated after 24 h of infection by western blot and b the percentage of K-bZIP expressing cells was assessed by IFA. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of K-bZIP/β-actin; c LANA, ORF50 and K8.1 mRNA were evaluated by qRT-PCR. The amount of target mRNA was normalised towards the β-actin gene and analysed by comparing mock and KSHV-infected macrophages. Data are plotted in histograms and standard deviation (SD)is also reported. *p-value < 0.05. d Cell viability of mock or KSHV-infected macrophages was studied by trypan blue exclusion assay. Mean plus SD of three independent experiments is reported. *p-value < 0.05; e Morphology of M0, KSHV-infected, M1, M2 and UV-KSHV-treated macrophages was observed utilising an optical microscope (×40 magnification); f FACS analysis of CD86 and CD163 expression of M0, KSHV-infected, M1, M2 and UV-KSHV-treated macrophages. A representative experiment is shown, and the mean of fluorescence intensity is indicated. Grey peaks represent the isotype controls. g Histograms representing the mean plus SD of CD86 and CD163 MFI (Mean fluorescence Intensity) are also reported. *p-value < 0.05.

Fig. 2. KSHV infection promotes the release of pro-tumorigenic cytokines.

a IL-10, VEGF, IL-8, IL-6 and IFN-γ released by KSHV- and mock-infected macrophages were measured by Luminex assay. Mean plus SD of three different experiments is reported. *p-value < 0.05; b Morphology of KSHV-infected HUVEC plus supernatant of KSHV-infected macrophages (sup KSHV-MΦ), KSHV-infected HUVEC, Mock HUVEC, Mock HUVEC treated with supernatant of KSHV-infected macrophages (sup KSHV-MΦ) or treated with supernatant of UV-KSHV-exposed macrophages (sup UV-KSHV-MΦ) was observed utilising an optical microscope (×40 magnification); c Snail expression in mock-, KSHV-infected and KSHV-infected HUVEC plus sup KSHV-MΦ was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of snail/β-actin. *p-value < 0.05.

Fig. 3. KSHV infection activates STAT3 and STAT6 in macrophages.

a STAT6, b STAT3 and c STAT1 activation in M1, M2 and KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of pSTAT/STAT and STAT/ β-ACT. For pSTAT6 the short exposure was used for densitometric analysis. *p-value < 0.05 was calculated between KSHV-infected macrophages and M1 polarised cells.

Fig. 4. KSHV activates UPR and up-regulates PD-L1 on KSHV- infected macrophages.

a Ire1α and XBP1s expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis; b ATF4, CHOP and BIP expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/β-ACT. *p-value < 0.05. c PD-L1 expression on mock- and KSHV-infected macrophages was evaluated by FACS analysis. A representative experiment is shown, and the mean of fluorescence intensity is indicated. Grey peaks represent the isotype controls. d Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are also reported. *p-value < 0.05.

Fig. 5. The Ire1α/XBP1 axis interferes with the pro-tumorigenic cytokines release and PD-L1 expression of KSHV-infected macrophages.

a IL-10, VEGF, IL-8, IL-6 and IFN-γ released by mock-, KSHV-infected macrophages and 4μ8c- (Ire1α inhibitor) or GSK 2606414 (GSK)- (PERK inhibitor) pre-treated KSHV-infected macrophages were measured by Luminex assay. Mean plus SD of three different experiments is reported. *p-value < 0.05; b and c PD-L1 expression on mock-, KSHV-infected macrophages and 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages was evaluated by FACS analysis. Grey peaks represent the isotype controls. Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are reported. *p-value < 0.05 and a representative experiment is shown, and the mean of fluorescence intensity is indicated; d expression of K-bZIP in untreated or 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages; e ATF4 expression in mock-, KSHV-infected and GSK 2606414 (GSK)-pre-treated KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/ β-ACT. *p-value < 0.05.