Investigating the influence of hypoxia on cGAS-STING-IFN signalling in macrophages

Abstract

Background: The double-stranded DNA sensor, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and its downstream effector, stimulator of interferon (IFN) genes (STING) contribute to the type I IFN response, critical to anti-cancer immunity and response to immunotherapy. Hypoxia within the tumour microenvironment (TME) promotes immune dysregulation, typically immunosuppression. This project aimed to determine 1) whether hypoxia impairs the cGAS-STING-IFN axis in macrophage models, and 2) whether the cGAS-STING-IFN axis in macrophages is activated by atovaquone, an anti-parasitic drug that reduces hypoxia in solid tumours. Methods: RAW 264.7 and differentiated THP1-Dual cells were cultured in 20% (normoxia) or 0.2% (hypoxia) oxygen for up to 72 hours. ISG54 and NFкB activity were interrogated with THP1-Dual cell reporter assays. Real-time quantitative polymerase chain reaction assessed effects on selected cGAS-STING-IFN induced transcripts. Enzyme linked immunosorbent assay determined CXCL10 concentrations in supernatant. Additionally, differentiated THP1-Dual cells were treated with atovaquone (10 μM) for 48 hours. Results: Hypoxic THP1-Dual cells demonstrated significantly decreased ISG54 reporter levels, while the NFкB reporter was unaffected. Under hypoxia, transcript levels in THP1-Dual cells were significantly decreased for CXCL10, IFIT1, TNF, and IFIT2, increased for IL6 and IL10, and unchanged for IFIT3, IFNB1, and CGAS. Comparatively, hypoxic RAW 264.7 cells exhibited significantly downregulated Cgas, Cxcl10, and Tnf, upregulated Ifit3, and unchanged Ifnb1 and Ifit1. Both cell lines demonstrated significantly reduced CXCL10 secretion under hypoxia. Transfection with double-stranded DNA appeared to overcome most of hypoxia’s effects, although hypoxic downregulation of Cgas in RAW 264.7 cells was maintained. Atovaquone did not alter transcripts downstream of cGAS-STING. Conclusions: Prolonged hypoxia impaired the macrophage-mediated cGAS-STING-IFN axis, decreasing CXCL10 secretion. Hypoxia did not affect the expression of CGAS in THP1-Dual cells, whereas its expression was significantly suppressed in hypoxic RAW 264.7 cells. Expression of IFNB1 was unchanged by hypoxia, suggesting that suppression of the pathway occurs downstream of IFN-β. Finally, atovaquone did not modulate the cGAS-STING-IFN axis in the human macrophage model studied, but still has the potential to remodel the tumour immune landscape and enhance immunotherapy in vivo, by decreasing hypoxia.