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Minocycline modulates NF?B phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

ABSTRACT: Mesenchymal 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.Modulation 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.Minocycline leads to the phosphorylation of transcriptional nuclear factor-?B (NF?B), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NF?B 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.Minocycline modulates the NF?B 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.

SUBMITTER: Guerra AD

PROVIDER: S-EPMC5521110 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Guerra Alberto Daniel AD Rose Warren E WE Hematti Peiman P Kao W John WJ

Stem cell research & therapy 20170721 1

<h4>Background</h4>Mesenchymal 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 MSC ...[more]

PMID: 28732530

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Project description:Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties including an anti-inflammatory cytokine profile and the promotion of angiogenesis via expression of growth factors in pre-clinical models. MSCs encapsulated in poly(ethylene glycol) diacrylate (PEGdA) and thiolated gelatin poly(ethylene glycol) (Gel-PEG-Cys) crosslinked hydrogels have led to controlled cellular presentation at wound sites with favorable wound healing outcomes. However, the therapeutic potential of MSC-loaded hydrogels may be limited by non-specific protein adsorption on the delivery matrix that could facilitate the initial adhesion of microorganisms and subsequent virulent biofilm formation. Antimicrobials loaded concurrently in the hydrogels with MSCs could reduce microbial bioburden and promote healing, but the antimicrobial effect on the MSC wound healing capacity and the antibacterial efficacy of the hydrogels is unknown. We demonstrate that minocycline specifically induces a favorable change in MSC migration capacity, proliferation, gene expression, extracellular matrix (ECM) attachment, and adhesion molecule and growth factor release with subsequent increased angiogenesis. We then demonstrate that hydrogels loaded with MSCs, minocycline, vancomycin, and linezolid can significantly decrease bacterial bioburden. Our study suggests that minocycline can serve as a dual mechanism for the regenerative capacity of MSCs and the reduction of bioburden in triple antimicrobial-loaded hydrogels.Wound healing is a complex biological process that can be hindered by bacterial infection, excessive inflammation, and inadequate microvasculature. In this study, we develop a new formulation of poly(ethylene glycol) diacrylate and thiolated gelatin poly(ethylene glycol) crosslinked hydrogels loaded with minocycline, vancomycin, linezolid, and mesenchymal stromal/stem cells that induces a favorable wound healing phenotype in mesenchymal stromal/stem cells and prevents bacterial bioburden on the hydrogel. This combinatorial approach to biomaterial development has the potential to impact wound healing for contaminated full thickness cutaneous wounds.

Project description:A promising alternative to antibiotics for treatment of Staphylococcus aureus infections is photodynamic inactivation (PDI), which employs a photosensitizer (PS) that produces cytotoxic reactive oxygen species (ROS) when exposed to molecular oxygen and antimicrobial blue light in the spectrum of 400-470 nm. Although the precise mechanistic basis of PDI has not been defined, the formation of ROS and free radicals that oxidize a number of cellular targets, including membrane lipids, damage to proteins and nucleic acids result in inactivation of essential cellular functions and subsequent cell death. Because PDI is non-selective and affects multiple cellular targets, development of resistance or tolerance to PDI has been considered to be unlikely and attempts to induce S. aureus resistance or tolerance upon repeated sub-lethal doses of PDI have not succeeded. However, multiple aspects of PDI suggest that development of tolerance is highly probable, in particular when PDI is used for treatment of infections where the environment at the infection site prevents penetration of PDI at a level sufficient to cause death of all bacteria and with tolerant phenotypes emerging from the surviving bacteria. In this study, we sought to identify the mechanisms that contribute to PDI tolerance in S. aureus. S. aureus HG003 and the isogenic HG003DmutSL strain with defects in DNA mismatch repair were used to evaluate the response of S. aureus and the roles of DNA mismatch repair and gene regulatory networks to repeated sublethal doses of PDI. Global transcriptome and genome analyses were used in an agnostic approach to identify the underlying transcriptional responses and genetic adaptations that occur as a result of repeated PDI and contribute to PDI tolerance. Our results reveal multiple metabolic, transport and cell wall biogenesis pathways that contribute to PDI tolerance, and a S. aureus regulatory gene likely responsible for the adaptive transcriptional response to PDI.

Dataset Information

Minocycline modulates NF?B phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

Publications

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

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