The effect of hypoxia on mononuclear phagocyte phenotype and activity

Abstract

Monoclonal antibody (mAbs) immunotherapy is considered the main component of cancer treatment, alongside surgery, radiation and chemotherapy. However, a significant fraction of patients fail to respond or develop resistance to treatment. The effectiveness of mAbs has been linked to the expression of Fc gamma receptors (FcγRs) on immune effector cells. The expression of the inhibitory FcγRIIb is detrimental for the efficacy of direct-targeting antibodies, whereas it is required for the function of certain immunomodulatory antibodies. Therefore, understanding the regulation of FcγRIIb expression on effector cells is important to improve different types of mAb therapy.
Previous result have indicated a role for hypoxia in the regulation of FcγRIIb on the surface of monocytes. However, it is unclear how hypoxia regulates expression of FcγRIIb. Therefore the direct influence of hypoxia on FcγRIIb expression on mononuclear phagocytes was investigated here. Gene set enrichment analysis (GSEA) determined that there was enrichment of the hypoxia gene set in monocytes cultured in the presence of dimethyloxalylglycine (DMOG), a hypoxia-inducible factors (HIFs) stabilising agent, compared to untreated monocytes. Furthermore, upstream regulator analysis predicted that hypoxia-related factors, like HIFs and c-Jun were activated in these cells.
The influence of targets identified by upstream regulator analysis (c-Jun, HIF1α and HIF2α) on FcγRIIb expression on macrophages was investigated using small molecule inhibitors. However, the inhibitors did not provide definite data, due to the nature of the inhibitors, their impact on cell viability and the sometimes contrasting effects of inhibitors on the same target. This led to the development of a small interfering RNA (siRNA) transfection method for human monocyte-derived macrophages (hMDMs). Knock-down of HIF2α in M0 and M2 polarised hMDMs prevented the upregulation of protein levels, as well as surface levels of FcγRIIb in response to chemical (DMOG) or physiological (0.1% O2) hypoxia. This effect was even greater when HIF1α and HIF2α were simultaneously knocked-down. The reduction of FcγRIIb surface levels also led to an increase in the activating:inhibitory FcγR ratio on M0 and M2 hMDMs as well as to an increase in functionality of these cells under hypoxic conditions, as measured by antibody-dependent cellular phagocytosis.
In order to be able to translate these results to an in vivo system, a novel mouse model expressing human FcγRs (hFcγRs) was characterised. It was shown that these mice had a similar expression pattern of hFcγRs compared to humans, and that the mice responded appropriately when treated with B-cell and Treg depletion antibodies. In addition, tumours originating from C57BL/6 mice could successfully be engrafted into to these mice and these tumours contained hypoxic regions.
Overall, it has been shown that FcγRIIb in mononuclear phagocytes is a common target of HIF1α and HIF2α, but is preferentially regulated by HIF2α in response to hypoxia. Furthermore, a novel mouse model was identified that can be used in the future to investigate the influence intra-tumoral hypoxia on the efficacy of mAb therapy against solid tumours.

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Identifiers

ORCID for Kri Tanja Janneke Muller: orcid.org/0000-0001-5674-6125

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

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