Fukuyama Kohtaro, Islam Md Aminul, Takagi Michihiro, Ikeda-Ohtsubo Wakako, Kurata Shoichiro, Aso Hisashi, Vignolo Graciela, Villena Julio, Kitazawa Haruki
Food and Feed Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan.
Livestock Immunology Unit, International Education and Research Centre for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan.
Pathogens. 2020 May 25;9(5):410. doi: 10.3390/pathogens9050410.
Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle's environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with -derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes-interleukin 1 alpha (), , monocyte chemotactic protein 1 (), , chemokine (C-X-C motif) ligand 2 (), and were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for CRL2074, which reduced the expression of , , , , and , whereas CRL2084 diminished , , and expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.
牛乳腺炎是乳腺的炎症，会影响牛奶产量的质量和数量。乳腺炎的控制依赖于抗生素治疗的单一或多种组合。由于病原体对抗生素的耐药性不断增加，通过提高宿主免疫力，向乳房内注入乳酸菌（LAB）已被视为治疗和预防牛乳腺炎的一种潜在抗生素替代方法。益生菌作用具有菌株依赖性；因此，必须对候选乳酸菌菌株进行有效评估，以找出最具潜力的菌株。在此，我们研究了最初从饲养场牛的环境中分离出的乳酸菌菌株在体外诱导牛乳腺上皮（BME）细胞中Toll样受体（TLR）触发的炎症反应的能力。将BME细胞分别用乳酸菌菌株预刺激12、24和48小时，然后用脂多糖（LPS）刺激12小时。通过实时定量PCR（RT-qPCR）对选定的免疫基因——白细胞介素1α（IL-1α）、白细胞介素6（IL-6）、单核细胞趋化蛋白1（MCP-1）、白细胞介素8（IL-8）、趋化因子（C-X-C基序）配体2（CXCL2）和肿瘤坏死因子α（TNF-α）的mRNA表达进行定量。结果表明，用一些乳酸菌菌株预处理能够差异调节BME细胞中的LPS炎症反应；然而，发现了菌株依赖性差异。对CRL2074的影响最为显著，它降低了IL-1α、IL-6、MCP-1、IL-8和TNF-α的表达，而CRL2084则降低了IL-6、IL-8和TNF-α的表达。用CRL2074菌株对BME细胞进行预刺激，在LPS刺激后导致TLRs的三种负调节因子的表达上调，包括泛素编辑酶A20（也称为肿瘤坏死因子α诱导蛋白3，TNFAIP3）、单免疫球蛋白IL-1单受体（SIGIRR）和Toll相互作用蛋白（Tollip）。CRL2084预刺激仅上调了Tollip的表达。我们的结果表明，CRL2074菌株对BME细胞中LPS诱导的炎症具有显著的免疫调节能力。由于该菌株通过乳房内注入对牛乳腺炎具有有益作用，可作为体内试验的候选菌株。我们的研究结果还表明，BME细胞免疫测定系统对于体外评估乳酸菌对乳腺炎相关病原体乳房内感染引起的炎症的免疫调节能力可能具有价值。
