Quantitative analysis reveals crosstalk mechanisms of heat shock-induced attenuation of NF-κB signaling at the single cell level

Abstract

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.

Heat shock response pathways regulate stimulus-specificity and sensitivity of NF-κB signalling to temperature stress

“…Previously we characterised HS-mediated inhibition of the TNFα-dependent NF-κB signalling (Kardynska et al, 2018). Here we sought to investigate the long-term modulation and recovery of NF-κB responses following HS.…”

“…HS exposure may lead to heterogenous NF-κB responses in single cells, which can be masked in the population level analyses (Kardynska et al, 2018). Therefore, we developed a MCF7 cell line stably expressing p65-EGFP suitable for quantitative single time-lapse microscopy analyses.…”

“…TNFα acts through its cognate receptor to activate IKK, and a number of mechanisms have been proposed to understand how HSR affect this process (Kardynska et al, 2018;Sheppard et al, 2014). In the first attempt to investigate these mechanisms, we utilised IL1β cytokine, which is known to activate IKK via signal transduction pathway parallel to that of TNFα ( Fig.…”

“…We previously showed that exposure of human osteosarcoma cells to 1h 43°C HS resulted in depletion of soluble IKKα and IKKβ levels, effectively limiting the amount of IKK (and thus NF-κB) that can be activated by the cytokine stimulation (Kardynska et al, 2018). The cellular adaptation to HS requires restoration of IKK signalling.…”

“…However, a better understanding of the temperature effect on cellular signalling, in general, and crosstalk between HSR and NF-κB systems, in particular, is required for better efficacy. The NF-κB system can adapt to physiological (<40°C) temperatures (Harper et al, 2018), but exposure to higher temperatures (>40°C) results in the attenuation of the NF-κB signalling and function (Esquivel-Velazquez et al, 2015;Janus et al, 2015;Kardynska et al, 2018;Wong et al, 1997b). This involves IKK denaturation (Kardynska et al, 2018;Lee et al, 2004;Pittet et al, 2005), with inhibition of IκB degradation, NF-κB phosphorylation and translocation as well as target gene expression (Ayad et al, 1998;Cooper et al, 2010;Feinstein et al, 1997;Furuta et al, 2004;Janus et al, 2015;Wong et al, 1997a, b).…”