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Temperature Regulates NF-Κb Dynamics and Function Through Timing of A20 Transcription

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Temperature Regulates NF-Κb Dynamics and Function Through Timing of A20 Transcription Temperature regulates NF-κB dynamics and function through timing of A20 transcription C. V. Harpera,1, D. J. Woodcockb,c,1, C. Lama, M. Garcia-Albornozd, A. Adamsona, L. Ashalla, W. Rowea, P. Downtona, L. Schmidta, S. Weste, D. G. Spillera, D. A. Randb,c,2, and M. R. H. Whitea,2 aSystems Microscopy Centre, Division of Molecular and Cellular Function, School of Biology, Faculty of Biology, Medicine and Health, Manchester Academic Health Sciences Centre, University of Manchester, M13 9PT Manchester, United Kingdom; bMathematics Institute, University of Warwick, CV4 7AL Coventry, United Kingdom; cZeeman Institute for Systems Biology and Infectious Epidemiology Research, University of Warwick, CV4 7AL Coventry, United Kingdom; dDivision of Evolution and Genomic Sciences, School of Biology, Faculty of Biology, Medicine and Health, Manchester Academic Health Sciences Centre, University of Manchester, M13 9PT Manchester, United Kingdom; and eInstitute of Integrative Biology, University of Liverpool, L69 7ZB United Kingdom Edited by Ronald N. Germain, National Institutes of Health, Bethesda, MD, and approved April 27, 2018 (received for review March 7, 2018) NF-κB signaling plays a pivotal role in control of the inflammatory by measuring both the oscillatory dynamics and the downstream gene response. We investigated how the dynamics and function of NF-κB expression response. were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase Results in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor To investigate the effect of temperature on NF-κB dynamics, we Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling used time-lapse live-cell microscopy to study cytoplasmic to nu- suggested that this temperature sensitivity might be due to an clear oscillations of NF-κB across the physiological temper- A20-dependent mechanism, and A20 silencing removed the sensi- ature range, 34 °C to 40 °C. Bacterial artificial chromosomes tivity to increased temperature. The timing of the early response of expressing human RelA and IκBα fused to fluorescent proteins a key set of NF-κB target genes showed strong temperature depen- were generated to enable detection of both the dynamics of dence. The cytokine-induced expression of many (but not all) later RelA translocations and IκBα degradation and resynthesis, genes was insensitive to temperature change (suggesting that they allowing expression at more physiological levels. The RelA and might be functionally temperature-compensated). Moreover, a set IκB dynamics were observed to have regular opposite phases α SYSTEMS BIOLOGY of temperature- and TNF -regulated genes were implicated in NF- (Fig. 1 A and B). A rapid temperature shift from 37 °C to 40 °C κ – B cross-talk with key cell-fate controlling pathways. In conclusion, caused an immediate decrease in period of RelA translocation from κ NF- B dynamics and target gene expression are modulated by tem- ∼100 min to ∼74mininTNFα-treated (10 ng/mL), transiently perature and can accurately transmit multidimensional information to control inflammation. Significance NF-κB | temperature | A20 | transcription | dynamics Inflammation is often accompanied by temperature change, κ but little is known about the role of temperature in the in- he nuclear factor kappa B (NF- B) signaling pathway is a key flammatory response. We show that physiologically relevant Tmediator of the immune system, which regulates cell fate as temperature changes significantly perturb NF-κB dynamics well as stress and inflammatory responses. It responds to diverse following TNFα stimulation in single cells. Using experimenta- stimuli, including viral and bacterial pathogens, free radicals, tion informed by mathematical modeling, we found that these cytokines, and growth factors (1), and can process information changes were mediated, at least in part, through the key > from multiple input signals to regulate the transcription of 500 feedback gene TNFAIP3/A20. Curtailing A20 expression re- genes (1, 2). In response to persistent cytokine signaling, the NF-κB moved temperature sensitivity across the fever range (37 °C to transcription factor complex (typically RelA/p65:p50) can oscillate 40 °C). Gene expression was generally unaffected between repeatedly between the cytoplasm and nucleus with a period of these temperatures, although a select set of NF-κB−regulated ∼100 min (3–5). This is regulated by a set of negative feedback genes was up-regulated at early time points. These genes were loops, notably via the genes expressing IκBα,IκBe,andA20pro- predominantly involved in inflammation, signaling, and cell teins. The dynamics, and particularly frequency, of NF-κB oscilla- fate. The cellular response to inflammation may therefore be tions may be one component that regulates the pattern of target mechanistically and functionally regulated by temperature. gene expression and the physiological response (3, 6–8). Changes in temperature alter the rates of all biological– Author contributions: C.V.H., D.J.W., D.A.R., and M.R.H.W. designed research; C.V.H., D.J.W., C.L., L.A., P.D., L.S., S.W., and D.A.R. performed research; D.J.W. and A.A. contrib- chemical reactions. The ability of mammals to regulate tem- uted new reagents/analytic tools; C.V.H., D.J.W., M.G.-A., and W.R. analyzed data; C.V.H., perature is an important aspect of survival. In humans, there are D.J.W., D.G.S., D.A.R., and M.R.H.W. wrote the paper; D.G.S. managed microscopy facility natural physiological temperature changes involved in day−night and assisted with microscopy experiments; D.A.R. jointly directed project with a focus on (36.4 °C to 37.5 °C) (9), menstrual (∼0.5 °C increase after ovu- modeling and bioinformatics; and M.R.H.W. initiated and jointly directed project with a focus on experimental work. lation) (10), and (more variable) seasonal temperature cycles, as The authors declare no conflict of interest. well as between peripheral tissues (∼34 °C) and core body temperature (∼37 °C). Temperature also increases with fever This article is a PNAS Direct Submission. (typically 37 °C to 40 °C). The direct functional consequences of This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). these temperature changes are largely unclear, but there has Data deposition: Gene expression data reported in this paper have been deposited in the been a growing body of evidence that the efficacy of the immune ArrayExpress database, https://www.ebi.ac.uk/arrayexpress (accession no. E-MTAB-5158). response may be modulated by temperature (11, 12). 1C.V.H. and D.J.W. contributed equally to the work. Despite the long-established associations between tempera- 2To whom correspondence may be addressed. Email: [email protected] or mike. ture, fever, and inflammation, as well as between inflammation [email protected]. κ and NF- B signaling, it remains unknown how temperature af- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fects the NF-κB system. We investigated how temperature 1073/pnas.1803609115/-/DCSupplemental. change in the physiological range affects the NF-κB system response, www.pnas.org/cgi/doi/10.1073/pnas.1803609115 PNAS Latest Articles | 1of7 Downloaded by guest on September 26, 2021 transfected SK-N-AS cells (Fig. 1 C and D). TNFα treatment at those parameters that had the greatest effect on period (Fig. 2C). different (constant) incubation temperatures (34 °C, 37 °C, and Parameters controlling the key inflammatory regulator A20 40 °C) showed pronounced modification of the oscillation period (TNFAIP3) were observed to have the largest effect on the pe- (Fig. 1 E–G). In both cell lines and primary cells, there were signif- riod (Fig. 2C). icant (approximately linear) decreases in period from 34 °C to 37 °C The control coefficients used to quantify this sensitivity were to 40 °C (Fig. 1 H–J),andalsodownto28°C(SI Appendix,Fig.S1). used to predict the change in period per degree of temperature The Q10 temperature coefficient [a measure of the temperature change (SI Appendix, SI Methods 1). This analysis was then sensitivity of a process due to an increase of 10°, it is calculated as compared with the experimental data (Fig. 2D). This showed a = 10/(T2 − T1) Q10 (R2/R1) , where R1 and R2 are the oscillation fre- greater experimental change in period than could be predicted quencies at temperatures T1 and T2, respectively; temperatures are ∼ by the original model, indicating that it was necessary to modify given in kelvin] was measured as being between 2.3 and 3.1 in the the structure of the model. Due to the observed sensitivity of the different experimental systems. These data suggest that NF-κBos- original model to parameters controlling A20, we investigated cillation frequency is very temperature sensitive, in contrast with the effect of temperature on TNFα-stimulated A20 expression, temperature-compensated processes like the circadian clock (13). We analyzed an established deterministic mathematical model using time-course microarray experiments. These data showed a of the NF-κB system (3) by incorporating temperature de- significant greater than twofold increase in A20 expression at pendence of the rate parameters using the Arrhenius relation 30 min at 40 °C (SI Appendix, Fig. S2), compared with 37 °C. No (Fig. 2A and SI Appendix, SI Methods 1), informed by previous significant differences were observed at subsequent time points, approaches for circadian clocks (14). This analysis predicted a suggesting that that the ratio of transcription rate to degrada- decrease in period as temperature increased (Fig. 2B), in tion rate was largely unchanged at these later times. These data agreement with the overall experimental trend (Fig. 1). Sensi- implied that changes in these rates could not reasonably account tivity analysis (SI Appendix, SI Methods 1) was used to identify for the change in period.
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