Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells

Abstract

BackgroundMesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds.MethodsModulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague–Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student’s t test was used to determine p values for all assays with multiple or two conditions, respectively.ResultsMinocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization.ConclusionsMinocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0623-1) contains supplementary material, which is available to authorized users.

Minocycline impairs TNF-α-induced cell fusion of M13SV1-Cre cells with MDA-MB-435-pFDR1 cells by suppressing NF-κB transcriptional activity and its induction of target-gene expression of fusion-relevant factors

“…Moreover, as shown here and in our previous study [13], ICAM1 expression levels were increased in minocycline + TNF-α-treated MDA-MB-435-pFDR1 cells. This finding is in agreement with data indicating that increased phosphorylation levels of NF-κB concomitant with increased expression of vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) observed in minocycline-treated human mesenchymal stem cells (MSCs) [66, 67]. Even though MMP9 mRNA levels were also increased in MDA-MB-435-pFDR1 cells treated with minocycline and minocycline + TNF-α, only very low MMP9 protein expression could be determined [13].…”

Identification of Functional mimotopes of human Vasorin Ectodomain by Biopanning

“…HepG2 motility were assayed using 12-well transwell plates (Corning) as described before 23 . In brief, 1×10 5 cells were seeded on the upper chamber with a cell-permeable 8.0 μm membrane, and the lower chamber was filled with serum-free DMEM containing the antibodies with or without the peptide-BSA proteins.…”

Mesenchymal Stromal Cells as Potential Antimicrobial for Veterinary Use—A Comprehensive Review

“…A number of preconditioning methods have been employed over the years including hypoxia, serum deprivation, and exposure to antagonistic substances to improve MSCs' ability to differentiate or modulate immune cells. Conditions promoting antimicrobial activity are being examined with exposure to cytokines, target bacteria, bacterial components, vitamins, and antibiotics improving both direct and indirect antimicrobial effects (Gupta et al, 2012;Guerra et al, 2017;Johnson et al, 2017;Cahuascanco et al, 2019;Yagi et al, 2020).…”

“…Notably, cathelicidins across different species can vary their mechanism of action, albeit bacterial membrane disruption, and cell lysis generally occur (Schneider et al, 2016;Scheenstra et al, 2019). In humans, cathelicidin LL-37 has been implicated in the direct bacterial killing effects of MSCs (Guerra et al, 2017;Ren et al, 2019). LL-37 has bactericidal properties as well as the ability to decrease cytokine and endotoxin levels in septic models.…”