Background

Melanin is an important virulence factor for Sporothrix globosa, the causative agent of sporotrichosis, a subcutaneous mycosis that occurs worldwide. Although previous research suggests that melanin is involved in the pathogenesis of sporotrichosis, little is known about its influence on the macrophages that represent the frontline components of innate immunity.

Objectives

To evaluate the effects of melanin on phagocytic activity and the expression of Toll-like receptor (TLR)2 and TLR4 during S. globosa infection of macrophages in vitro.

Methods

To compare phagocytic activity and survival rates, THP-1 macrophages and primary mouse peritoneal macrophages were co-cultured with a wild-type S. globosa strain (Mel+), an albino mutant strain (Mel˗), a tricyclazole-treated Mel + strain (TCZ-Mel+), or melanin ghosts extracted from S. globosa conidia. Reactive oxygen species (ROS), nitric oxide (NO) generation, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed in THP-1 cells infected with S. globosa conidia. Quantitative PCR and western blotting were used to observe the effect of melanin on TLR2 and TLR4 expression. Knockdown of TLR2/4 expression with small interfering RNA was performed to further verify the role of these receptors during infection.

Results

Macrophages infected with Mel + conidia showed a lower phagocytosis index and a higher survival rate than TCZ-Mel+ and Mel˗ in vitro. After incubation with S. globosa, the release of ROS, NO, TNF-α and IL-6 by THP-1 were decreased in the presence of melanin. Increased mRNA and protein expression of TLR2 and TLR4 occurred upon S. globosa infection in THP-1, whereas the presence of melanin suppressed TLR2 and TLR4. Moreover, TLR2 or TLR4 knockdown showed a trend toward reducing the pernicious effect of S. globosa conidia on THP-1 cells in vitro.

Conclusions

Collectively, our results indicated that melanin inhibits the phagocytosis of S. globosa and guards against macrophage attack by providing protection from oxygen- and nitrogen-derived radicals, as well as suppressing the host pro-inflammatory cytokine response (TNF-α and IL-6). Melanin was also involved in modulating TLR2 and TLR4 receptor expression, weakening the killing efficiency of S. globosa.

中文翻译：

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球形孢子丝菌黑色素影响THP-1巨噬细胞的功能并调节TLR2和TLR4的表达

背景

黑色素是球状孢子丝菌的重要毒力因子，球状孢子丝菌是孢子丝菌病的病原体，孢子丝菌病是一种在世界范围内发生的皮下真菌病。尽管先前的研究表明黑色素参与了孢子丝菌病的发病机制，但对其对代表先天免疫前线成分的巨噬细胞的影响知之甚少。

目标

评估黑色素对体外巨噬细胞S.globosa感染过程中吞噬活性和Toll样受体（TLR）2和TLR4表达的影响。

方法

为了比较吞噬活性和存活率，将 THP-1 巨噬细胞和原代小鼠腹腔巨噬细胞与野生型S. globosa菌株 (Mel+)、白化突变菌株 (Mel˗)、三环唑处理的 Mel + 菌株共培养(TCZ-Mel+) 或从S. globosa分生孢子中提取的黑色素鬼影。活性氧 (ROS)、一氧化氮 (NO) 生成、肿瘤坏死因子 (TNF)-α 和白细胞介素 (IL)-6 在 THP-1 细胞中检测到S.globosa分生孢子。采用定量PCR和蛋白质印迹法观察黑色素对TLR2和TLR4表达的影响。用小干扰 RNA 敲低 TLR2/4 表达以进一步验证这些受体在感染过程中的作用。

结果

Mel + 分生孢子感染的巨噬细胞在体外显示出比 TCZ-Mel+ 和 Mel˗ 更低的吞噬指数和更高的存活率。与S. globosa孵育后，THP-1 释放的 ROS、NO、TNF-α 和 IL-6 在黑色素存在的情况下减少。TLR2 和 TLR4 的 mRNA 和蛋白质表达增加发生在 THP-1 中的S.globosa感染后，而黑色素的存在抑制了 TLR2 和 TLR4。此外，TLR2 或 TLR4 敲低显示出降低球形分生孢子对体外THP-1 细胞的有害影响的趋势。

结论

总的来说，我们的结果表明，抑制黑色素的吞噬S.藻和巨噬细胞对攻击防护装置通过从含氧和氮的衍生的基团提供保护，以及抑制宿主促炎细胞因子应答（TNF-α和IL-6 ）。黑色素还参与调节 TLR2 和 TLR4 受体的表达，削弱了球状链球菌的杀伤效率。