Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB

Abstract

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1β-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

The mannose receptor (CD206) identifies a population of colonic macrophages in health and inflammatory bowel disease

“…5 A), or in the proportions of CD206 − and CD206 + macrophages in colonic biopsies from patients with IBD. Other investigators have reported increased proportions of less-mature monocyte-derived cells in inflamed samples from patients with IBD 17 . The difference between our data and other reports is unlikely to reflect differences in our patient population, as all samples designated as ‘active’ (CDa and UCa) were obtained from visibly-inflamed regions of the colon.…”

“…Much progress has already been made in identifying DC populations in the human intestine 12 – 17 , therefore, we focused on intestinal macrophage populations. Recent detailed descriptions of murine MNP populations have enabled clearer identification of these cells in samples from human tissue, including skin 18 , liver, lung, blood 19 and lymph nodes 20 .…”

CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody

“…Cell-penetrating peptides (CPPs) induce cellular uptake in a receptor-independent manner. Since their discovery in the 90s [ 24 , 25 ] they have been employed to mediate uptake of oligonucleotides [ 26 ], peptides [ 27 ], antibodies [ 28 , 29 ] and nanoparticles [ 30 , 31 ]. Therefore, CPPs are also good candidates to enhance cellular uptake and tissue retention of tumor targeting agents.…”

Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

“…NF-kB is a pleiotropic transcription factor that increases proinflammatory cytokine production, and regulation of its activation is therefore considered one of the approaches to reduce the risk of PTB [ 33 , 34 , 35 ]. Novel therapeutics that reduce inflammation by inhibiting NF-kB using anti-inflammatory drugs [ 36 , 37 , 38 ], cell-penetrating peptides, small molecule inhibitors [ 33 , 39 , 40 ], nonspecific inhibitors [ 41 , 42 , 43 , 44 ], and flavonoids are now being tested [ 45 , 46 ]. However, very few have made it to clinical trials so far and none are in use clinically, likely due to several issues including, but not limited to, the mode of delivery, placental permeability, teratogenicity, and transgenerational effects.…”