In vitro γ-ray-induced inflammatory response is dominated by culturing conditions rather than radiation exposures

Abstract

The inflammatory pathway has a pivotal role in regulating the fate and functions of cells after a wide range of stimuli, including ionizing radiation. However, the molecular mechanisms governing such responses have not been completely elucidated yet. In particular, the complex activation dynamics of the Nuclear transcription Factor kB (NF-kB), the key molecule governing the inflammatory pathway, still lacks a complete characterization. In this work we focused on the activation dynamics of the NF-kB (subunit p65) pathway following different stimuli. Quantitative measurements of NF-kB were performed and results interpreted within a systems theory approach, based on the negative feedback loop feature of this pathway. Time-series data of nuclear NF-kB concentration showed no evidence of γ-ray induced activation of the pathway for doses up to 5 Gy but highlighted important transient effects of common environmental stress (e.g. CO2, temperature) and laboratory procedures, e.g. replacing the culture medium, which dominate the in vitro inflammatory response.

Figure 1. Simplified view of the NF-kB pathway.

Figure 2. Temporal dynamics of nuclear NF-kB concentration in control samples following a change of culture media at t = 0 h.

Time-series data for the NF-kB concentration (arbitrary units [a.u.]) from sham treated samples (0 Gy) from experiments with, respectively, 50 cGy (a) and 5 Gy (b) irradiated samples, with the best fit curves obtained with Eq. 4. In panel (c) all data from sham samples (0 Gy and no LPS pre-treatment) are shown together, with a common normalization and best fit curve obtained with Eq.4. Reduced chi-squared (“χ²/dof”, i.e. chi squared divided by the number of degrees of freedom) values for the fit are reported in each panel.

Figure 3. Temporal dynamics of nuclear NF-kB concentration in samples irradiated at different doses of γ-rays 45 minutes after the change of culture media at t = 0 h.

Time-series data for the NF-kB concentration (arbitrary units) in samples exposed to 50 cGy (a), 1 (b), 2 (c) and 5 Gy (d) of γ-rays compared to the corresponding controls. Each experimental point is the average over three different replicas, and the error bar represents the standard deviation. In each panel, solid line is the best fit to all sham datasets, taken from Fig. 2c.

Figure 4. Temporal dynamics of nuclear NF-kB concentration in samples not affected by the replacement of culture media and exposed to low doses of γ-rays.

(a) NF-kB concentrations (arbitrary units) for non-irradiated samples, with (red) and without (blue) a change of culture media at t = 0 h. Panels (b) and (c) show respectively data for the irradiations at 25 and 50 cGy and corresponding controls, without any change of media prior to the exposure. Each experimental point is the average over three different replicas, and the error bar represents the standard deviation.

Figure 5. Effects of the LPS pre-treatment on nuclear NF-kB concentration and on NF-kB recruitment to the nuclear compartment following a change of culture media at t = 0 h.

(a) NF-kB concentrations (arbitrary units) for samples subjected to a change of culture media at t = 0 h, with (red) and without (blue) a LPS pre-treatment (4 hours at 5 ng/ml). Each experimental point is the average over three different replicas, and the error bar represents the standard deviation. The ratio of nuclear versus cytoplasmic localization of NF-kB (R ratio of Eq. 1) is plotted against time in panels (b) and (c), respectively without and with a change of culture media occurring at t = 0 h, and for three different initial LPS concentrations during 4 hours of pre-treatment: control-PBS(blue), 5 ng/ml (red), 50 ng/ml (yellow). Error bars represent the Standar Error of the Mean from 10 different fields of fluorescent images. Lines are guide for the eye.