The biological response of liquid silk towards inflammatory cells involved in stroke – relevance to its potential use as a regenerative stroke therapy

Background: Neuroinflammatory responses in stroke involve peripheral macrophage infiltration, that induces injury as well as repair, depending upon their activation status. Thus, a promising strategy in treating ischaemic stroke is the modulation of macrophage responses. Silk fibroin is a biocompatible, natural biomaterial currently undergoing consideration as a neuroregenerative stroke therapy1, yet its immunomodulatory effects are not known. Here, we examine these immunomodulatory effects and hypothesise that liquid silk fibroin is anti-inflammatory in response to LPS-induced inflammatory responses that as similar to those associated with stroke2.

Methods: RAW264.7 macrophages were treated with liquid silk fibroin (Bombyx mori, 0.01, 0.1, and 1 mg/mL), either alone or before LPS (200 ng/mL), in serum-free medium (24 h). Cell viability (MTT), cytokines (ELISA, cytokine array), nitric oxide (Griess assay), CD86 polarisation (flow cytometry) and surface area/morphology (ImageJ) were quantified.

Results: Liquid silk was not cytotoxic itself, but significantly increased TNF-α, IL-1β and NO release in a dose-dependent manner and significantly exacerbated the LPS-induced increase. Cytokine profiling confirmed enhanced expression of pro-inflammatory mediators (e.g., CCL2, CXCL2, RANTES) at higher concentrations of silk. Flow cytometry revealed a dose-dependent upregulation of CD86 expression, with an increase in its expression at 1 mg/mL of silk alone and when co-applied with LPS. In addition, morphology analysis revealed a significant increase in surface area with higher concentrations of silk.

Conclusions: Liquid silk fibroin is biocompatible but promotes a pro-inflammatory phenotype in macrophages and exacerbates the LPS-induced response. These data reveal that, contrary to our hypothesis, liquid silk alone is pro-inflammatory and exacerbates neuroinflammation associated with stroke.

References
1. Totten JD, Alhadrami HA, Jiffri EH, McMullen CJ, Seib FP, Carswell HVO. Towards clinical translation of 'second-generation' regenerative stroke therapies: hydrogels as game changers? Trends Biotechnol. 2022;40(6):708-720.
2. Hakoupian, M., Ferino, E., Jickling, G.C. et al. Bacterial lipopolysaccharide is associated with stroke. Sci. Rep. 2021;11:6570.