Cellular and Molecular Biology

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: KSHV infects macrophages and skews their phenotype towards an M2 phenotype.
Fig. 2: KSHV infection promotes the release of pro-tumorigenic cytokines.
Fig. 3: KSHV infection activates STAT3 and STAT6 in macrophages.
Fig. 4: KSHV activates UPR and up-regulates PD-L1 on KSHV- infected macrophages.
Fig. 5: The Ire1α/XBP1 axis interferes with the pro-tumorigenic cytokines release and PD-L1 expression of KSHV-infected macrophages.

References

  1. 1.

    Dittmer, D. P. & Damania, B. Kaposi sarcoma-associated herpesvirus: immunobiology, oncogenesis, and therapy. J. Clin. Invest 126, 3165–3175 (2016).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Cirone, M., Lucania, G., Bergamo, P., Trivedi, P., Frati, L. & Faggioni, A. Human herpesvirus 8 (HHV-8) inhibits monocyte differentiation into dendritic cells and impairs their immunostimulatory activity. Immunol. Lett. 113, 40–46 (2007).

    CAS  PubMed  Google Scholar 

  3. 3.

    Santarelli, R., Granato, M., Pentassuglia, G., Lacconi, V., Gilardini Montani, M. S., Gonnella, R. et al. KSHV reduces autophagy in THP-1 cells and in differentiating monocytes by decreasing CAST/calpastatin and ATG5 expression. Autophagy 12, 2311–2325 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Cirone, M., Di Renzo, L., Lotti, L. V., Conte, V., Trivedi, P., Santarelli, R. et al. Activation of dendritic cells by tumor cell death. Oncoimmunology 1, 1218–1219 (2012).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Gilardini Montani, M. S., Falcinelli, L., Santarelli, R., Romeo, M. A., Granato, M., Faggioni, A. et al. 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. Int J. Biochem Cell Biol. 114, 105560 (2019).

    CAS  PubMed  Google Scholar 

  6. 6.

    Santarelli, R., Gonnella, R., Di Giovenale, G., Cuomo, L., Capobianchi, A., Granato, M. et al. STAT3 activation by KSHV correlates with IL-10, IL-6 and IL-23 release and an autophagic block in dendritic cells. Sci. Rep. 4, 4241 (2014).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Bhaskaran, N., Ghosh, S. K., Yu, X., Qin, S., Weinberg, A., Pandiyan, P. et al. Kaposi’s sarcoma-associated herpesvirus infection promotes differentiation and polarization of monocytes into tumor-associated macrophages. Cell Cycle 16, 1611–1621 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A. & Locati, M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25, 677–686 (2004).

    CAS  PubMed  Google Scholar 

  9. 9.

    Yang, L. & Zhang, Y. Tumor-associated macrophages: from basic research to clinical application. J. Hematol. Oncol. 10, 58 (2017).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Olson, O. C. & Joyce, J. A. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat. Rev. Cancer 15, 712–729 (2015).

    CAS  PubMed  Google Scholar 

  11. 11.

    Gregory, S. M., Wang, L., West, J. A., Dittmer, D. P. & Damania, B. Latent Kaposi’s sarcoma-associated herpesvirus infection of monocytes downregulates expression of adaptive immune response costimulatory receptors and proinflammatory cytokines. J. Virol. 86, 3916–3923 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wang, C., Zhu, C., Wei, F., Zhang, L., Mo, X., Feng, Y. et al. Constitutive activation of interleukin-13/STAT6 contributes to Kaposi’s sarcoma-associated herpesvirus-related primary effusion lymphoma cell proliferation and survival. J. Virol. 89, 10416–10426 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Cheng, Z., Zhang, D., Gong, B., Wang, P. & Liu, F. CD163 as a novel target gene of STAT3 is a potential therapeutic target for gastric cancer. Oncotarget 8, 87244–87262 (2017).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Gong, M., Zhuo, X. & Ma, A. STAT6 upregulation promotes M2 macrophage polarization to suppress atherosclerosis. Med Sci. Monit. Basic Res. 23, 240–249 (2017).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Rahal, O. M., Wolfe, A. R., Mandal, P. K., Larson, R., Tin, S., Jimenez, C. et al. Blocking Interleukin (IL)4- and IL13-mediated phosphorylation of STAT6 (Tyr641) decreases M2 polarization of macrophages and protects against macrophage-mediated radioresistance of inflammatory breast cancer. Int J. Radiat. Oncol. Biol. Phys. 100, 1034–1043 (2018).

    CAS  PubMed  Google Scholar 

  16. 16.

    Yan, D., Wang, H. W., Bowman, R. L. & Joyce, J. A. STAT3 and STAT6 signaling pathways synergize to promote cathepsin secretion from macrophages via IRE1alpha activation. Cell Rep. 16, 2914–2927 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Zhang, L. & Wang, A. Virus-induced ER stress and the unfolded protein response. Front. Plant Sci. 3, 293 (2012).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Smith, J. A. Regulation of cytokine production by the unfolded protein response; implications for infection and autoimmunity. Front. Immunol. 9, 422 (2018).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Corazzari, M., Gagliardi, M., Fimia, G. M. & Piacentini, M. Endoplasmic reticulum stress, unfolded protein response, and cancer cell fate. Front Oncol. 7, 78 (2017).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Condamine, T., Kumar, V., Ramachandran, I. R., Youn, J. I., Celis, E., Finnberg, N. et al. ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R-mediated apoptosis. J. Clin. Invest. 124, 2626–2639 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Osorio, F., Tavernier, S. J., Hoffmann, E., Saeys, Y., Martens, L., Vetters, J. et al. The unfolded-protein-response sensor IRE-1alpha regulates the function of CD8alpha+ dendritic cells. Nat. Immunol. 15, 248–257 (2014).

    CAS  PubMed  Google Scholar 

  22. 22.

    Cubillos-Ruiz, J. R., Silberman, P. C., Rutkowski, M. R., Chopra, S., Perales-Puchalt, A., Song, M. et al. ER stress sensor XBP1 controls anti-tumor immunity by disrupting dendritic cell homeostasis. Cell 161, 1527–1538 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Martinon, F., Chen, X., Lee, A. H. & Glimcher, L. H. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nat. Immunol. 11, 411–418 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Chang, L. C., Chen, T. P., Kuo, W. K. & Hua, C. C. The protein expression of PDL1 is highly correlated with those of eIF2alpha and ATF4 in lung cancer. Dis. Markers 2018, 5068701 (2018).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Host, K. M., Jacobs, S. R., West, J. A., Zhang, Z., Costantini, L. M., Stopford, C. M. et al. Kaposi’s sarcoma-associated herpesvirus increases PD-L1 and proinflammatory cytokine expression in human monocytes. mBio. https://doi.org/10.1128/mBio.00917-17 (2017).

  26. 26.

    Gilardini Montani, M. S., Santarelli, R., Granato, M., Gonnella, R., Torrisi, M. R., Faggioni, A. et al. EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation. Autophagy 15, 652–667 (2019).

    CAS  PubMed  Google Scholar 

  27. 27.

    Granato, M., Gilardini Montani, M. S., Filardi, M., Faggioni, A. & Cirone, M. Capsaicin triggers immunogenic PEL cell death, stimulates DCs and reverts PEL-induced immune suppression. Oncotarget 6, 29543–29554 (2015).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Gilardini Montani, M. S., Granato, M., Cuomo, L., Valia, S., Di Renzo, L., D’Orazi, G. et al. High glucose and hyperglycemic sera from type 2 diabetic patients impair DC differentiation by inducing ROS and activating Wnt/beta-catenin and p38 MAPK. Biochim Biophys. Acta 1862, 805–813 (2016).

    CAS  PubMed  Google Scholar 

  29. 29.

    Granato, M., Gilardini Montani, M. S., Romeo, M. A., Santarelli, R., Gonnella, R., D’Orazi, G. et al. Metformin triggers apoptosis in PEL cells and alters bortezomib-induced unfolded protein response increasing its cytotoxicity and inhibiting KSHV lytic cycle activation. Cell Signal 40, 239–247 (2017).

    CAS  PubMed  Google Scholar 

  30. 30.

    Jaguin, M., Houlbert, N., Fardel, O. & Lecureur, V. Polarization profiles of human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin. Cell Immunol. 281, 51–61 (2013).

    CAS  PubMed  Google Scholar 

  31. 31.

    Carmeliet, P. VEGF as a key mediator of angiogenesis in cancer. Oncology 69(Suppl. 3), 4–10 (2005).

    CAS  PubMed  Google Scholar 

  32. 32.

    Zheng, T., Ma, G., Tang, M., Li, Z., Xu, R. IL-8 secreted from M2 macrophages promoted prostate tumorigenesis via STAT3/MALAT1 pathway. Int J Mol Sci. https://doi.org/10.3390/ijms20010098 (2018).

  33. 33.

    Samaniego, F., Young, D., Grimes, C., Prospero, V., Christofidou-Solomidou, M., DeLisser, H. M. et al. Vascular endothelial growth factor and Kaposi’s sarcoma cells in human skin grafts. Cell Growth Differ. 13, 387–395 (2002).

    CAS  PubMed  Google Scholar 

  34. 34.

    Masood, R., Cai, J., Tulpule, A., Zheng, T., Hamilton, A., Sharma, S. et al. Interleukin 8 is an autocrine growth factor and a surrogate marker for Kaposi’s sarcoma. Clin. Cancer Res. 7, 2693–2702 (2001).

    CAS  PubMed  Google Scholar 

  35. 35.

    Darwich, L., Coma, G., Pena, R., Bellido, R., Blanco, E. J., Este, J. A. et al. Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18. Immunology 126, 386–393 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Wu, X., Lahiri, A., Haines, G. K. 3rd, Flavell, R. A. & Abraham, C. NOD2 regulates CXCR3-dependent CD8+ T cell accumulation in intestinal tissues with acute injury. J. Immunol. 192, 3409–3418 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Wang, L., Wakisaka, N., Tomlinson, C. C., DeWire, S. M., Krall, S., Pagano, J. S. et al. The Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) K1 protein induces expression of angiogenic and invasion factors. Cancer Res. 64, 2774–2781 (2004).

    CAS  PubMed  Google Scholar 

  38. 38.

    Gasperini, P., Espigol-Frigole, G., McCormick, P. J., Salvucci, O., Maric, D., Uldrick, T. S. et al. Kaposi sarcoma herpesvirus promotes endothelial-to-mesenchymal transition through Notch-dependent signaling. Cancer Res 72, 1157–1169 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Oh, J., Riek, A. E., Weng, S., Petty, M., Kim, D., Colonna, M. et al. Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J. Biol. Chem. 287, 11629–11641 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Soto-Pantoja, D. R., Wilson, A. S., Clear, K. Y., Westwood, B., Triozzi, P. L. & Cook, K. L. Unfolded protein response signaling impacts macrophage polarity to modulate breast cancer cell clearance and melanoma immune checkpoint therapy responsiveness. Oncotarget 8, 80545–80559 (2017).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Cook, K. L., Soto-Pantoja, D. R., Clarke, P. A., Cruz, M. I., Zwart, A., Warri, A. et al. Endoplasmic reticulum stress protein GRP78 modulates lipid metabolism to control drug sensitivity and antitumor immunity in breast cancer. Cancer Res. 76, 5657–5670 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Nomura, M., Liu, J., Rovira, I. I., Gonzalez-Hurtado, E., Lee, J., Wolfgang, M. J. et al. Fatty acid oxidation in macrophage polarization. Nat. Immunol. 17, 216–217 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Polizzotto, M. N., Uldrick, T. S., Wang, V., Aleman, K., Wyvill, K. M., Marshall, V. et al. Human and viral interleukin-6 and other cytokines in Kaposi sarcoma herpesvirus-associated multicentric Castleman disease. Blood 122, 4189–4198 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Nagai, K., Ochi, T., Fujiwara, H., An, J., Shirakata, T., Mineno, J. et al. Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity. Blood 119, 368–376 (2012).

    CAS  PubMed  Google Scholar 

  45. 45.

    Su, Y. L., Banerjee, S., White, S. V., Kortylewski, M. STAT3 in tumor-associated myeloid cells: multitasking to disrupt immunity. Int. J. Mol. Sci. https://doi.org/10.3390/ijms19061803. (2018)

  46. 46.

    Johnson, D. E., O’Keefe, R. A. & Grandis, J. R. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol. 15, 234–248 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Liu, Y., Shao, M., Wu, Y., Yan, C., Jiang, S., Liu, J. et al. Role for the endoplasmic reticulum stress sensor IRE1alpha in liver regenerative responses. J. Hepatol. 62, 590–598 (2015).

    CAS  PubMed  Google Scholar 

  48. 48.

    Gilardini Montani, M. S., Santarelli, R., Falcinelli, L., Gonnella, R., Granato, M., Di Renzo, L. et al. EBV up-regulates PD-L1 on the surface of primary monocytes by increasing ROS and activating TLR signaling and STAT3. J. Leukoc. Biol. 104, 821–832 (2018).

    CAS  PubMed  Google Scholar 

  49. 49.

    Salmaninejad, A., Zamani, M. R., Shabgah, A. G., Hosseini, S., Mollaeim, F., Hosseini, N. et al. Behcet’s disease: an immunogenetic perspective. J. Cell. Physiol. 234, 8055–8074 (2019).

    CAS  PubMed  Google Scholar 

  50. 50.

    Porcheray, F., Viaud, S., Rimaniol, A. C., Leone, C., Samah, B., Dereuddre-Bosquet, N. et al. Macrophage activation switching: an asset for the resolution of inflammation. Clin. Exp. Immunol. 142, 481–489 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. G. Girelli (Blood bank, Sapienza University of Rome) and Dr G. Mandarello and Dr R. Gasbarri (Immunohematology and Transfusion Service, Belcolle Hospital, Viterbo) for providing buffy coats. We also thank Alessia Garufi and Micol Di Segni for technical assistance in qRT-PCR experiments.

Author information

Affiliations

Authors

Contributions

M.S.G.M. performed macrophage preparation and FACS analysis. L.F., R.S., N.C., M.S.G.M. performed western blot analysis and analysed the data. M.A.R., M.G., R.G. performed cell culture and Elisa assays. G.D.O. carried out qRT-PCR. M.C., G.D.O. and A.F. discussed the data. M.C. and M.S.G.M. conceived the experiments. M.C. wrote the paper.

Corresponding author

Correspondence to Mara Cirone.

Ethics declarations

Ethics approval and consent to participate

This research involving human subjects has been performed in accordance with the Declaration of Helsinki and has been approved by the ethic committee of Policlinico Umberto I, Rome, Italy (847/19).

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Funding information

This work was supported by grants from Istituto Pasteur Italia-Fondazione Cenci Bolognetti, PRIN 2017 (2017K55HLC) and by the Italian Association for Cancer Research (AIRC) Grant (IG 2019 Id.23040).

Additional information

Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gilardini Montani, M.S., Falcinelli, L., Santarelli, R. et al. 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. Br J Cancer 123, 298–306 (2020). https://doi.org/10.1038/s41416-020-0872-0

Download citation