Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of <i>Mycobacterium tuberculosis</i> in human macrophages

<div><p>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 <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent <i>Mtb</i>-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 <i>Mtb</i>. <i>Mtb</i> infection of macrophages downregulated the expression of both the <i>CAMP</i> transcript and LL-37 peptide as well as certain autophagy-related genes (<i>BECN1</i> and <i>ATG5</i>) 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 D<sub>3</sub> (1,25[OH]<sub>2</sub>D<sub>3</sub>), separately or particularly in combination, were able to overcome <i>Mtb</i>-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 <i>Mtb</i>-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<sup>2+</sup>, 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 <i>Mtb</i> in human macrophages.</p></div>