Institute of Fundamental Technological Research

Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon-induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-γ stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-α/β1 elicit different level of desensitisation from IFN-γ, where cells refractory to IFN-α/β1 are sensitive to IFN-γ, but not vice versa. We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT network’s ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.

JAK-STAT network, STAT1 kinetics, interferons, pathway desensitisation, mathematical modelling, signal memory, live-cell microscopy

Background

Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Heat Shock (HS) affects the signalling and gene expression responses of the Nuclear Factor κB (NF-κB) transcription factor, a critical regulator of proliferation and inflammation, however, our quantitative understanding of how cells sense and adapt to temperature changes is limited.
Methods

We used live-cell time-lapse microscopy and mathematical modelling to understand the signalling of the NF-κB system in the human MCF7 breast adenocarcinoma cells in response to pro-inflammatory Interleukin 1β (IL1β) and Tumour Necrosis Factor α (TNFα) cytokines, following exposure to a 37–43 °C range of physiological and clinical temperatures.
Results

We show that exposure to 43 °C 1 h HS inhibits the immediate NF-κB signalling response to TNFα and IL1β stimulation although uptake of cytokines is not impaired. Within 4 h after HS treatment IL1β-induced NF-κB responses return to normal levels, but the recovery of the TNFα-induced responses is still affected. Using siRNA knock-down of Heat Shock Factor 1 (HSF1) we show that this stimulus-specificity is conferred via the Inhibitory κB kinase (IKK) signalosome where HSF1-dependent feedback regulates TNFα, but not IL1β-mediated IKK recovery post HS. Furthermore, we demonstrate that through the temperature-dependent denaturation and recovery of IKK, TNFα and IL1β-mediated signalling exhibit different temperature sensitivity and adaptation to repeated HS when exposed to a 37–43 °C temperature range. Specifically, IL1β-mediated NF-κB responses are more robust to temperature changes in comparison to those induced by TNFα treatment.

Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular “all-or-nothing” encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were “analogue”. In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited “all-or-nothing” encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of “all-or-nothing” crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.