A computational analysis of dynamic, multi-organ inflammatory crosstalk induced by endotoxin in mice

Abstract

Bacterial lipopolysaccharide (LPS) induces an acute inflammatory response across multiple organs, primarily via Toll-like receptor 4 (TLR4). We sought to define novel aspects of the complex spatiotemporal dynamics of LPS-induced inflammation using computational modeling, with a special focus on the timing of pathological systemic spillover. An analysis of principal drivers of LPS-induced inflammation in the heart, gut, lung, liver, spleen, and kidney to assess organ-specific dynamics, as well as in the plasma (as an assessment of systemic spillover), was carried out using data on 20 protein-level inflammatory mediators measured over 0-48h in both C57BL/6 and TLR4-null mice. Using a suite of computational techniques, including a time-interval variant of Principal Component Analysis, we confirm key roles for cytokines such as tumor necrosis factor-α and interleukin-17A, define a temporal hierarchy of organ-localized inflammation, and infer the point at which organ-localized inflammation spills over systemically. Thus, by employing a systems biology approach, we obtain a novel perspective on the time- and organ-specific components in the propagation of acute systemic inflammation.

Fig 1. Blunted response to LPS in TLR4^-/- mice.

C57BL/6 and TLR4^-/- mice were injected with LPS (3 mg/kg, i.p.). At different time points (0, 1, 4, 6, 12, 24 and 48 h) upon sacrifice, blood was collected and inflammatory mediators were measured by Luminex as described in Materials and Methods. Figure shows plasma concentrations of (A): TNFα, (B): IL-10, (C): IL-6, (D): IL-17A, and their respective AUCs (E) in both C57BL/6 and TLR4^-/- mice. The significant reduction in damage to parenchymal cells is reflected in lower ALT concentrations in the TLR4^-/- animals (F). Results represent the mean ± SEM from n = 5–8 (C57BL/6) and n = 4 (TLR4^-/-) animals for each experimental group (C57BL/6 vs. TLR4^-/-, analyzed by Two-Way ANOVA as indicated).

Fig 2. Analysis of endotoxemic C57BL/6 mice by Time-Interval PCA (TI-PCA).

Animals (n = 5–8 for each experimental group) were injected with LPS (3 mg/kg, i.p.). At different time points upon sacrifice, the inflammatory mediators in blood and different organs (liver, heart, gut, lung, spleen and kidney) were measured by Luminex as described in Materials and Methods. Identification of the inflammatory mediators contributing to the top 25% variance of the inflammatory response (shown above the red line in the PCA graph) in all organs together during each of the following six time frames: 0-1h, 1-4h, 4-6h, 6-12h, 12-24h, and 24-48h (A) was performed using Time-Interval PCA as described in Materials and Methods. The number of mediators contributing to the top 25% variance of the inflammatory response in each organ as well as in plasma during each time-interval is represented in Panel B.

Fig 3. Analysis of endotoxemic TLR4^-/- mice by Time-Interval PCA (TI-PCA).

Animals (n = 4 for each experimental group) were injected with LPS (3 mg/kg, i.p.). At different time points upon sacrifice, the inflammatory mediators in blood and different organs were measured by Luminex, and identification (A) as well as quantification (B) of the inflammatory mediators contributing to the top 25% variance of the inflammatory response was performed as described in Fig 2 legend.

Fig 4. Total number of mediators contributing to the top 25% variance by Time-Interval PCA (TI-PCA) in C57BL/6 vs. TLR4^-/- mice.

Animals (C57BL/6, n = 5–8 and TLR4^-/-, n = 4 for each experimental group) were injected with LPS (3 mg/kg, i.p.). At different time points upon sacrifice, the inflammatory mediators in blood and different organs (liver, heart, gut, lung, spleen and kidney) were measured by Luminex and Time-Interval PCA was performed as described in Materials and Methods. Figure shows the total number of mediators contributing to the top 25% variance of the inflammatory response in each organ as well as in plasma of C57BL/6 vs. TLR4^-/- mice during the indicated time-intervals.

Fig 5. Pearson’s correlation between concentrations of inflammatory mediators in C57BL/6 and TLR4^-/- mice.

Animals (C57BL/6, n = 5–8 and TLR4^-/-, n = 4 for each experimental group) were injected with LPS (3 mg/kg, i.p.). At different time points upon sacrifice, the inflammatory mediators in blood and different organs (liver, heart, gut, lung, spleen and kidney) were measured by Luminex and Pearson’s correlation was calculated as described in Materials and Methods. Figure shows the 20x20 correlation matrix of mediators' interactions in all organs as well as in plasma for each time-point in C57BL/6 (Panel A) and TLR4^-/- (Panel B) mice. The time-point at which these results suggest the systemic spillover begins to occur in C57BL/6 mice is shown by a red box in Panel A.