Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds

Abstract

BackgroundThe prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies in humans. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC on the growth of representative gram-negative and gram-positive bacterial species commonly found in skin wounds and (ii) define the mechanisms by which MSC inhibit bacterial growth.MethodsMSC were isolated from the peripheral blood of healthy horses. Gram-negative E. coli and gram-positive S. aureus were cultured in the presence of MSC and MSC conditioned medium (CM), containing all factors secreted by MSC. Bacterial growth was measured by plating bacteria and counting viable colonies or by reading the absorbance of bacterial cultures. Bacterial membrane damage was detected by incorporation of N-phenyl-1-naphthylamine (NPN). Antimicrobial peptide (AMP) gene and protein expression by equine MSC were determined by RT-PCR and Western blot analysis, respectively. Blocking of AMP activity of MSC CM was achieved using AMP-specific antibodies.ResultsWe found that equine MSC and MSC CM inhibit the growth of E. coli and S. aureus, and that MSC CM depolarizes the cell membranes of these bacteria. In addition, we found that equine MSC CM contains AMPs, and blocking these AMPs with antibodies reduces the effects of MSC CM on bacteria.ConclusionsOur results demonstrate that equine MSC inhibit bacterial growth and secrete factors that compromise the membrane integrity of bacteria commonly found in skin wounds. We also identified four specific AMPs produced by equine MSC. The secretion of AMPs may contribute to the value of MSC as a therapy for cutaneous wounds in both horses and humans.

Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells

“…Bacterial death continued to increase for up to 3 hours, at which time 98% of the culture consisted of bacteria with disrupted membrane integrity (Figure B). Thus, MSC CM factors triggered bacterial killing extremely rapidly, consistent with what has been reported previously for AMP‐mediated killing …”

“…Human MSC have been shown to produce multiple AMPs, including the cathelicidin peptide LL‐37, hepcidin, β‐defensin 2, and lipocalin 2 . MSC‐produced AMPs are thought to be one critical component regulating the ability of MSC administered therapeutically to control or eliminate bacterial infections, as explored in multiple animal models …”

Cuantificación de la efectividad de la indolicidina y protegrina-4 por medio de análisis de parámetros cinéticos

“…Pero, este fenómeno no se observa contra E. coli, ya que ambos péptidos muestran la misma efectividad antimicrobiana contra esta bacteria. Posiblemente sea debido al tiempo de exposición de los antimicrobianos contra las bacterias, por ejemplo, Harman et al, (2017) y Sung et al, (2016 reportan una exposición directa de 6 horas entre los AMPs y bacterias patógena, mientras que Miyasaki et al (1997) reportaron que 2 horas de exposición son suficientes para observar un efecto significativo en la viabilidad celular. En este trabajo podemos decir que debido a la concentración celular y péptidos en suspension fue suficiente para eliminar más del 90 % de las bacterias.…”

“…Existen pocos trabajos en la literatura que reporten el uso de parámetros cinéticos para determinar la susceptibilidad de bacterias patógenas a la presencia de péptidos antimicrobianos usando modelos matemáticos y muchos de ellos son solamente descriptivos tales como los reportados por Steinberg et al, (1997); Harman et al, (2017);Fanter et al, (2010); Freire et al, (2015); Bolintineau et al, (2010); Sung et al, (2016). Esto es debido a que posiblemente, los AMPs, en muchos casos, son todavía una promesa terapéutica para reemplazar a los antibióticos, por lo tanto, existen pocos estudios clínicos sistemáticos.…”

“…La viabilidad celular se determinó a través de Unidades Formadoras de Colonias (UFCs), la cual es una técnica ampliamente reportada para determinar la susceptibilidad de las bacterias contra antibióticos y de partículas de plata (Theophel et al, 2014). Recientemente, Harman et al, (2017) utilizaron este método para medir la viabilidad celular cuando se expusieron bacterias a concentraciones de péptidos antimicrobianos; estos autores demostraron que es un método altamente preciso ya que se obtienen prácticamente los mismos valores cuando se compara con las lecturas de absorbancia obtenidas. La factibilidad y rapidez de este método, así como el corto tiempo de incubación y el uso de pocos reactivos utilizados para esta investigación hace que sea posible llevar a cabo un diagnóstico simple de los péptidos propuestos contra cepas patógenas: Escherichia coli, Staphylococcus epidermidis y Salmonella sp.…”