Targeting synergetic endothelial inflammation by inhibiting NFKB and JAK-STAT pathways

Abstract

Multiple systemic vascular inflammatory disorders are associated with endothelial dysfunction and elevated levels of TNFα and IFNγ. Combined TNFα and IFNγ stimulation induces synergetic hyperinflammation in endothelial cells (ECs) through the activation of the NFKB and JAK/STAT pathways. Here, we assess how targeting these pathways affects EC inflammation. Using mass spectrometry based proteomics, we investigate system-wide effects of TNFα- and IFNγ-stimulated Endothelial Colony Forming Cells (ECFCs) in combination with inhibitors targeting NFKB and JAK/STAT pathways. JAK1 inhibitor itacitinib blocked IFNγ-, but not TNFα-induced proteomic responses. IKK2/STAT3 inhibitor TPCA1 attenuated both responses. Most TNFα+IFNγ-induced proteins, such as pyroptosis mediators, chemokines, and Weibel-Palade Body content, were inhibited by both inhibitors, highlighting their synergetic dependency on both pathways. Imaging of Von Willebrand Factor (VWF) revealed an extracellular VWF network induced by combined stimulation, a phenotype which was reverted by both inhibitors. This study provides a preliminary basis for inhibiting endothelial inflammation in vascular inflammatory disorders.

Keywords: Biochemistry; Cell biology; Proteomics.

Graphical abstract

Figure 1

IKK2/STAT3 and JAK1 inhibition affects endothelial inflammation states (A) Overview of inhibitor panel with their targets and proteomics workflow. (B) Differentially abundant proteins of inhibitors versus steady state proteome (LFC >1, BH adjusted p-value <0.05, moderated t-test). Mean of N = 3 replicates, concentrations are shown in μM. (C) Facetted principal component analysis (PCA) of samples combined with IKK2/STAT3i (TP), JAK1i (IT) or without inhibitor per stimulus as indicated: steady state (gray), TNFα (green), IFNγ (blue), TNFα+IFNγ (red). Shapes indicate concentrations: 0 μM (circle), 0.1 μM (square), 1 μM (diamond), 10 μM (triangle). (D) Mean z-scores of regulated proteins per stimulus and inhibitors as indicated. TNFα-induced (green, N = 60 proteins), IFNγ-induced (blue, N = 84), Mix-induced (red, N = 208). Horizontal lines indicate average Z score of conditions without inhibitor. (E) LFQ levels of inflammation proteins across inhibitors and concentrations per stimulus. n.d. indicates protein was not detected. Crossbar indicates mean (N = 9 biological replicates for stimuli without inhibitors, and N = 3 for stimuli with inhibitors). Dotted line shows the mean steady sate or stimulated levels without inhibitors. (F) Schematic overview of pathway inhibition for separate stimuli, showing how JAK1i has no effect on TNFα induced response.

Figure 2

Inhibition of synergetic EC inflammation by IKK2/STAT3i and JAK1i (A) Pie chart of Mix-induced proteins (LFC >1, BH adjusted p-value <0.01, moderated t-test) categorized by inhibition: IKK2/STAT3i (TP) (light green), JAK1i (IT) (light blue), either (both TP or IT, light red), or none (gray). (B) Top 6 enriched terms of proteins inhibited by IKK2/STAT3i (green), JAKi (blue) or either inhibitor (red) in KEGG (purple), WikiPathways (dark red), and Reactome (dark purple) databases. (C) Protein interaction network based on the highest confidence (>0.9) StringDB interactions. Processes of interest are indicated in squares. (D) Heatmap of chemokine LFQ levels in the secretome at 4h, 8h, and 24h in conditions as indicated. TNFα and IFNγ are added experimentally. Color indicates conditions, unstimulated (gray), Mix (red), Mix + JAKi (pink), Mix + IKK2/STAT3i (dark red). (E) LFQ levels of chemokines CXCL1 and CXCL10 in conditions as indicated. Crossbar indicates mean (N = 2 biological replicates).

Figure 3

Inhibition of inflammation-induced VWF release (A) LFQ levels of WPB proteins per inhibitor (10 μM) and stimulus as indicated: steady state (gray), TNFα (green), IFNγ (blue), TNFα+IFNγ (red). Crossbar indicates mean (N = 9 biological replicates for stimuli without inhibitors, and N = 3 for stimuli with inhibitors). (B) Quantified WPB count per cell from confocal imaging in conditions as indicated. Boxplot middle line indicates median, upper and lower parts of boxplots indicate the 25th and 75th percentiles (N = 4 independent experiments for unstimulated and mix conditions, N = 2 independent experiments for TNFα and IFNγ stimulated conditions, 3 images were analyzed per experiment). Significant differences are indicated: ∗∗ = adj. p-value <0.01, ∗∗∗ = adj. p-value <0.0001. (one-way-Anova and Tukey post-hoc test). (C) Confocal images of ECs stained with anti-VWF (green) and anti-Ve-cadherin (red) antibodies in conditions as indicated. Scale bar (white) indicates 100 μm for overall image and 25 μm for the detail images. Brightness and contrast were adjusted equally across images, representative image shown (N = 4 independent experiments, 3 images were analyzed per experiment).