An alternate explanation could be the interaction of HS with other cytokines. For example, heparan sulfate is known to bind IL-8 and might also influence T cell polarization in this way. Additionally, heparin and heparan sulfates are known to interact with IL-7, a cytokine structurally similar to TSLP, and were confirmed in one study to play a role in lymphoid cell binding by initiating binding to the surface. In that study, removing HS from the surface or adding heparin to the media significantly reduced IL-7 binding to cell surfaces. Further, the competition of heparin with cell-surface heparan sulfate for cytokine binding is the suspected reason for the curbed cytokine storm after application of low-molecular-weight heparin (LMWH) during SARS-COV-2 infection. IL-7 and IL-8 are only two examples of the wide range of heparin-and HS-binding proteins. Based on the obtained results, we became interested whether the polyanions also interact with other pro-inflammatory mediators relevant for type 2 inflammation and atopic diseases. Our own recent research has shown that FN occurs at elevated levels in the plasma of children with atopic dermatitis and can also trigger an atopic-like inflammation in human-based 3D bronchial tissue models. We therefore proceeded to investigate the affinity of the polyanions for FN. For the analysis, we established a competitive assay based on the polyglycerol anions' ability to disrupt the binding of FN to immobilized heparin (Figure ). We chose this assay setup instead of a direct binding assay due to the FN degradation that we had observed after attempting its covalent immobilization. Based on the relative binding values, we calculated the respective half-maximal inhibitory concentrations (IC50). The results in Table show that lPGS10 and dPGS10 strongly bind FN, yielding an IC50 in the picomolar range (170 pM for dPGS10 and 280 pM for lPGS10). A reduction in the degree of sulfation from 90 to 40% led to a dramatic increase of the IC50 by more than 3 orders of magnitude to 960 nM, highlighting the need for high functionalization to realize strong electrostatic interactions. Again, in contrast to sulfation, the carboxylated compounds lPGC10 and dPGC10 showed no target binding up to 10 µM. Fibronectin interaction likely relies on binding of heparin to binding sites of type III domains, which are known to display several lysine and arginine amino acids that can form salt bridges with polyanions. To expand our understanding of the evident inhibition activity into more physiological conditions, we next investigated the effect of the strongest inhibitor, dPGS10, on a human-based 3D inflamed bronchial epithelial tissue model. The decision to use a human-cell-based model instead of animal models was driven by the missing cross-reactivity between human and murine TSLP (only a 43 % homology) and TSLPR (35 %). Originally developed following the discovery of elevated FN levels in atopic infant plasma, the model we used was designed to prove whether FN induces an inflammatory phenotype and contributes to the atopic march. Although disease progression in humans typically takes years, a significant FN contribution to inflammation was observed in the model after a three-week cultivation. Here, the inflammation was triggered by adding 1 µg/mL of human plasma FN, leading to increased TSLP expression (Figure ) as well as a significant increase of IL-6 (Figure ). dPGS10 led to a dose-dependent reduction of TSLP expression at 10 µg/mL, as well as a significant reduction of IL-6 in the supernatant at 100 µg/mL. This effect can be explained by the strong binding of dPGS to FN, which likely reduces FN's interaction with cell-surface integrins, and the FN-mediated NFκBp65 activation. It is noteworthy that the concentrations necessary to mediate anti-inflammatory effects in cellbased assays are significantly higher than the IC50 observed in the simplified binding assays. This disparity might result from nonspecific binding of dPGS10 to other proteins and to the collagen matrix of the bronchial epithelium model.