Single-cell investigation of macrophage activation and plasticity: Focus on STAT1 and STAT6 signalling

Abstract

Phenotypic and functional heterogeneity is a hallmark of macrophage biology. Macrophages are inherently plastic able to adapt their transcriptional programme to a dynamic microenvironment in order to perform opposing functions. Pro-inflammatory/M1 and anti-inflammatory/M2 activation are regarded as two mutually excluded polarisation programmes, however, macrophages expressing mixed (M1 and M2) and intermediate phenotypes are reported within the same environment both in vitro and in vivo. Here, we use quantitative single-cell approaches including live-cell time-lapse imaging and single molecule Fluorescent In Situ Hybridisation (smFISH) to understand the role the Signal Transduces and Activators of Transcription (STATs), in particular STAT1 and STAT6 signalling dynamics in regulation of macrophage activation and plasticity. Using lentiviral transduction, we constructed and validated a dual STAT1 and STAT6 reporter macrophage cell line to study their temporal regulation in response to M1 and M2 signals, IFN-γ and IL-4, respectively. Quantification of stimuli-induced STAT1/6 nuclear translocations across different polarising conditions demonstrates large plasticity of the STAT signalling system. However, the pre-treatment with IFN-γ moderately but significantly potentiated the amplitude of STAT6 activation to subsequent IL-4 treatment, while the pre-treatment with IL-4 moderately but significantly inhibited the IFN-γ-mediated STAT1 response. Downstream of STAT1/6 activation, we observe large variability in mRNA expression of the pro-typical M1/2 markers Nos2 and Arg1, respectively, with only a small subset of cells that produce mRNA output. Notably, pre-treatment resulted in altered fractions of responding cells consistently with changes of STAT signalling dynamics. Additionally, we reveal a dual role of IFNγ-STAT1 signalling, where dynamic inputs lead to complete desensitization of STAT1 responses to repetitive IFN-γ treatment. Our findings suggest a presence of dose-dependent molecular threshold rendering cells with “signal memory” of past IFNγ-STAT1 activation, potentially acting as a homeostatic mechanism to protect against out-of-control inflammation. Last but not least, we show a prominent role of IFNγ-STAT1 signalling in the de novo upregulation of Cd274 mRNA and PD-L1 protein expression in macrophages within a physiological and cancer-disease context. Overall, our single-cell analyses demonstrate plasticity and signal memory within the STAT1 and STAT6 signalling system in the context of macrophage activation and polarisation.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dominik Ruckerl (Supervisor) & Pawel Paszek (Supervisor)