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

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.Author summary: Hyperthermia has been considered a promising strategy to sensitize cancer cells to treatment. As such, it might potentially increase treatment efficacy while reducing negative side effects. So far, this potential has not been fully realized. One of the major obstacles is a lack of quantitative understanding of crosstalk mechanisms involved in the heat shock and other cellular stress-factor systems. In this work, we used experimental population-level and live-cell imaging approaches to characterize the NF-κB signaling system response to physiologically high temperature. We then iteratively applied mathematical modeling of single cells to infer the underlying regulatory mechanisms. We showed experimentally that elevated temperature attenuated cytokine-induced NF-κB system responses, resulting in complex and unintuitive single cell behavior. Our analyses suggested that these responses are consistent with stochastic cell-intrinsic heat-shock-mediated regulation of the NF-κB system via the IKK signaling module. Considering the oncogenic associations of the NF-κB pathway, the crosstalk mechanisms described in this work are important for the understanding of chemo- and radio-resistance to treatment. However, more but similar, quantitative studies are required to ultimately change currently used therapy protocols.