Rekha Rokeya Sultana, Rao Muvva S S V Jagadeeswara, Wan Min, Raqib Rubhana, Bergman Peter, Brighenti Susanna, Gudmundsson Gudmundur H, Agerberth Birgitta
a Department of Laboratory Medicine ; Clinical Microbiology; Karolinska Institutet ; Huddinge, Stockholm , Sweden.
b International Centre for Diarrheal Disease Research; Bangladesh ; Mohakhali, Dhaka , Bangladesh.
Autophagy. 2015;11(9):1688-99. doi: 10.1080/15548627.2015.1075110.
LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca(2+), and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.
LL-37是一种cathelicidin家族的人类抗菌肽（AMP），具有多种活性，包括作为人类巨噬细胞中维生素D诱导自噬的介质，从而导致细胞内杀灭结核分枝杆菌（Mtb）。在之前一项针对健康志愿者的试验中，我们已经表明，4-苯基丁酸（PBA）也能诱导LL-37表达，它可以进一步增强LL-37的表达以及随后的Mtb杀灭作用。在此，我们探究PBA和LL-37诱导自噬以及细胞内杀灭Mtb背后的潜在机制。巨噬细胞感染Mtb会在mRNA和蛋白质水平下调CAMP转录本和LL-37肽以及某些自噬相关基因（BECN1和ATG5）的表达。此外，未检测到原代巨噬细胞和THP-1细胞中LC3-II的激活。PBA和维生素D3的活性形式（1,25[OH]2D3）单独或特别是联合使用时，能够克服Mtb诱导的LL-37表达抑制。值得注意的是，用PBA刺激巨噬细胞可重新激活自噬，并促进LL-37和LC3-II在自噬体中的共定位。重要的是，当LL-37表达沉默时，PBA处理未能在感染Mtb的THP-1细胞中诱导自噬。然而，当向LL-37敲低细胞补充合成的LL-37时，PBA诱导的自噬得以恢复。有趣的是，我们发现LL-37诱导的自噬是通过P2RX7受体介导的，随后胞质游离Ca(2+)增加，以及AMPK和PtdIns3K途径的激活。总之，这些结果表明PBA作为自噬诱导剂具有一种新活性，这种活性依赖于LL-37，并促进人类巨噬细胞中细胞内杀灭Mtb。
