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

Abstract

These authors contributed equally to this work.Keywords: antimicrobial peptides, cathelicidin, innate immunity, P2RX7, tuberculosis, vitamin D Abbreviations: 1,25(OH) 2 D 3 , 1,25-dihydroxy vitamin D 3 ; AMPK, adenosine monophosphate-activated protein kinase; AMPs, antimicrobial peptides; ATG, autophagy related; BECN1, Beclin 1, autophagy related; CAMP, cathelicidin antimicrobial peptide; CFUs, colony-forming units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MDMs, monocyte-derived macrophages; Mtb, Mycobacterium tuberculosis; P2RX7, purinergic receptor P2X, ligand gated ion channel, 7; PBA, 4-phenylbutyrate; PBS, phosphate-buffered saline; PtdIns3K, phosphatidylinositol 3-kinase; RNA18S/18S rRNA, RNA 18S ribosomal; SQSTM1, sequestosome 1; TB, tuberculosis 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 D 3 (1,25[OH] 2 D 3 ), 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 2C , 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.

Role of endoplasmic reticulum stress in drug‐induced toxicity

“…2012; Rekha et al. 2015). Thus, protection afforded by 4‐BPA might not be fully related to ER stress defense.…”

Potent Inducers of Endogenous Antimicrobial Peptides for Host Directed Therapy of Infections

“…In addition, LL-37 was shown to work in synergy with the antibiotic azithromycin towards multi-drug resistant gram negative bacteria11. Recently it has been demonstrated that autophagy is activated by LL-3712. The expression of AMPs is downregulated by specific pathogens13.…”

Circulating cathelicidin LL-37 in adult patients with pulmonary infectious diseases

“…This results in the suppression of neutrophil apoptosis [8], which extends the life-span of neutrophils and increases phagocytosis. Recently, it was shown that cathelicidin LL-37 up-regulates the autophagy-related gene expression in macrophages and induces autophagosome formation to promote killing of intracellular bacteria [9]. There is also information that LL-37 can influence the course of adaptive immune response [2].…”