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Cunegundes, P. S.

Publications and source records attributed to Cunegundes, P. S..

2 recordsLinked to original sources
bioRxiv2026

Immunomodulatory Effects of Insulin-Derived Fibrils from Infusion Pumps: Role of Phenolic Preservatives in Macrophage Activation

BackgroundProtein fibrillation represents a critical challenge in therapeutic insulin delivery, yet the structural determinants and immunological consequences of insulin-derived fibrils (IDFs) formed in the presence of phenolic preservatives remain poorly characterized. ObjectiveThis study investigated the structural characteristics of IDFs formed with (IDF (+)) and without (IDF (-)) phenolic preservatives and elucidated their differential immunomodulatory mechanisms in bone marrow-derived macrophages (BMDMs). MethodsIDF structural properties were characterized using Thioflavin T fluorescence and nanoparticle tracking analysis (Spectradyne nCS1). BMDMs were treated with serial dilutions of IDF (+), IDF (-), or m-cresol. Cytotoxicity, reactive oxygen species (ROS) production, MIP-1 levels, and expression of signaling pathways were quantified. ResultsStructural analysis revealed similar aggregation states between IDF (+) and IDF (-). However, IDF (+) induced greater cytotoxicity and ROS production than IDF (-), which produced minimal ROS. Both fibrils increased MIP-1 chemokine levels. Additionally, IDF (-) upregulated NRF2 whereas m-cresol downregulated STAT6 compared to control. Together, these results support the existence of distinct mechanisms of macrophage activation and suggest that protein aggregates can directly induce macrophage responses independent of ROS production. ConclusionsInsulin fibrils activate macrophage inflammatory pathways through ROS-independent mechanisms. Phenolic preservatives enhance fibril cytotoxicity and likely ROS production while differentially modulating inflammatory signaling. These findings suggest that strategies to remove or reduce the effects of IDFs in insulin infusion therapy may increase longevity and biocompatibility of these devices.

immunology↗
bioRxiv2026

Insulin preservatives trigger neutrophil extracellular trap formation through SYNE1-mediated nuclear disassembly

Neutrophil extracellular traps (NETs) are critical effector molecules in sterile inflammation, yet the molecular mechanisms by which xenobiotic chemical exposures trigger NETosis remain poorly defined. Here, using phenolic preservatives present in all FDA-approved insulin formulations as a discovery platform, we show that these compounds induce NETosis in primary human neutrophils (34.3 {+/-} 5.0% vs. 2.8 {+/-} 0.9% for preservative-free insulin; p < 0.001) via a mechanism distinct from canonical PKC- and calcium-dependent pathways. Data-independent acquisition mass spectrometry (n = 6 donors) reveals that preservatives prompt coordinated dephosphorylation of SYNE1 (nesprin-1) at Ser8724 and Ser8727 (log2FC = -4.41 and -4.01, respectively; both q-value < 0.0001), disrupting LINC complex-mediated nuclear-cytoskeletal anchoring, through a phosphatase-dependent pathway distinct from canonical PKC- and calcium-dependent NETosis. In a porcine subcutaneous catheter model, preservative-containing formulations drive progressive NET accumulation, neutrophil infiltration, and early fibrotic changes over 7 days, whereas removing preservatives reduces the histological inflammation score by 40% (P < 0.001). These findings establish phenolic preservatives as non-pathogen triggers of NETosis, identify disruption of the SYNE1-LINC complex as the underlying mechanism, and demonstrate that preservative-free formulations lessen device-related inflammation, offering a translatable strategy for safer implantable drug delivery systems.

immunology↗