Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Vet Surg. 2021 May;50(4):858-871. doi: 10.1111/vsu.13628. Epub 2021 Apr 2.
To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro.
Controlled laboratory study.
Equine bone-marrow-derived MSCs (three horses).
MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 10 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37.
MSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 10 cells/ml, 2 h, 10 μg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p < .0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05).
TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction.
In vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.
评估 Toll 样受体和核苷酸结合寡聚化结构域（NLR）配体刺激马间充质基质细胞（MSCs）对体外抗菌和免疫调节特性的影响。
对照实验室研究。
马骨髓来源的 MSCs（3 匹马）。
用 TLR（聚肌苷酸：聚胞苷酸[pIC]和脂多糖[LPS]）和 NLR 激动剂（γ-d-Glu-mDAP [IE-DAP]）刺激 MSCs 2 小时，24 小时后将 MSC 以 1×10 个细胞/孔铺板。收集 MSC 条件培养基（MSC-CM），并评估抗菌肽 cathelicidin/LL-37 的产生、对多药耐药浮游和生物膜金黄色葡萄球菌的杀菌作用以及中性粒细胞吞噬作用。通过平板计数细菌和计算活菌菌落、读取培养物吸光度以及使用共聚焦显微镜成像进行活/死染色来测量细菌生长。在对激活刺激进行初步比较后，进一步优化 TLR3 激动剂 pIC 方案（激活和铺板时的细胞密度、培养时间、%血清）以提高杀菌活性和白细胞介素-8（IL-8）、单核细胞趋化蛋白-1（MCP-1）和 cathelicidin/LL37 的分泌。
pIC（p =.004）和 IE-DAP（p =.03）刺激 MSCs 可促进杀菌活性增加，这表现为浮游菌落活菌计数减少。pIC 刺激（2×10 个细胞/ml，2 小时，10 μg/ml）进一步抑制生物膜形成（p =.001），增强中性粒细胞对细菌的吞噬作用（p =.009），增加 MCP-1 的分泌（p < .0001），并增强 cathelicidin/LL-37 的产生，当培养基中血清浓度降低至 1%（p =.01）和 2.5%（p =.05）时，这种作用变得明显。
这里测试的 TLR-3 pIC MSCs 激活最有效地增强了抗菌和细胞因子反应，这受血清减少的影响。
在此处测试的体外 TLR-3 激活马 MSCs 可能是一种提高 MSCs 抗菌特性以治疗抗生素耐药感染的策略。
