Guerra Alberto Daniel, Rose Warren E, Hematti Peiman, Kao W John
School of Pharmacy, Division of Pharmaceutical Sciences, Pharmacy Practice Division, University of Wisconsin-Madison, 777 Highland Avenue, 7123 Rennebohm Hall, Madison, WI, 53705, USA.
School of Medicine and Public Health, Department of Medicine, Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, 1685 Highland Avenue, Madison, WI, 53705, USA.
Stem Cell Res Ther. 2017 Jul 21;8(1):171. doi: 10.1186/s13287-017-0623-1.
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-κB (NFκB), but not c-Jun NH-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.
Minocycline 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.
间充质基质/干细胞（MSCs）因其抗炎、促血管生成甚至抗菌特性而具有促进愈合的特性。我们之前已经表明，米诺环素可增强MSCs的伤口愈合表型，并且封装在含米诺环素的聚乙二醇和明胶基水凝胶中的MSCs对金黄色葡萄球菌（SA）具有抗菌特性。在此，我们研究了米诺环素在MSCs中调节的信号通路，该通路导致其伤口愈合表型增强，并确定用米诺环素预处理MSCs是否对抗菌活性有影响。我们进一步研究了MSCs和载抗生素水凝胶在接种的全层皮肤伤口中的体内抗菌效果。
通过蛋白质免疫印迹、免疫荧光和酶联免疫吸附测定（ELISA）分析米诺环素对MSCs中细胞信号通路的调节作用。用米诺环素预处理的MSCs的抗菌效果通过与SA的直接共培养和Transwell共培养来确定。SA共培养后MSCs的活力通过活/死染色来确定。用共聚焦成像确定用米诺环素预处理的MSCs对SA的内化作用。所有蛋白质和细胞因子分析均通过ELISA进行。在接种SA的Sprague-Dawley大鼠中确定MSCs和载抗生素水凝胶的体内抗菌效果。使用双向方差分析进行多重比较，并采用Bonferroni检验评估，分别使用不成对双尾学生t检验来确定所有具有多个或两个条件的试验的p值。
米诺环素导致转录核因子-κB（NFκB）磷酸化，但不导致c-Jun氨基末端激酶（JNK）或丝裂原活化蛋白激酶（ERK）磷酸化。抑制NFκB激活可阻止米诺环素诱导的血管内皮生长因子（VEGF）分泌增加。用米诺环素预处理MSCs会导致抗菌肽LL-37的产生减少，但通过增加白细胞介素-6（IL-6）的产生和SA内化增强了对SA的抗菌活性。MSCs和载抗生素水凝胶在3天内降低了接种伤口中的SA生物负荷，并加速了上皮再形成。
米诺环素调节MSCs中的NFκB通路，导致IL-6产生增加和SA内化。这种机制可能有助于MSCs和载抗生素水凝胶的体内抗菌效果。
