title: Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages.
abstract: 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.
authors.affiliation: a Department of Laboratory Medicine ; Clinical Microbiology; Karolinska Institutet ; Huddinge, Stockholm , Sweden.|b International Centre for Diarrheal Disease Research; Bangladesh ; Mohakhali, Dhaka , Bangladesh.
authors.forename: Rokeya Sultana
authors.initials: RS
authors.lastname: Rekha
authors.affiliation: c Center for Infectious Medicine ; Department of Medicine; Huddinge; Karolinska Institutet ; Stockholm , Sweden.
authors.forename: S S V Jagadeeswara
authors.initials: SS
authors.lastname: Rao Muvva
authors.affiliation: d Department of Medical Biochemistry and Biophysics ; Karolinska Institutet ; Stockholm , Sweden.
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.affiliation: b International Centre for Diarrheal Disease Research; Bangladesh ; Mohakhali, Dhaka , Bangladesh.
authors.forename: Rubhana
authors.initials: R
authors.lastname: Raqib
authors.affiliation: a Department of Laboratory Medicine ; Clinical Microbiology; Karolinska Institutet ; Huddinge, Stockholm , Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Bergman
authors.affiliation: c Center for Infectious Medicine ; Department of Medicine; Huddinge; Karolinska Institutet ; Stockholm , Sweden.
authors.forename: Susanna
authors.initials: S
authors.lastname: Brighenti
authors.affiliation: e Biomedical Center; University of Iceland ; Reykjavik , Iceland.
authors.forename: Gudmundur H
authors.initials: GH
authors.lastname: Gudmundsson
authors.affiliation: a Department of Laboratory Medicine ; Clinical Microbiology; Karolinska Institutet ; Huddinge, Stockholm , Sweden.
authors.forename: Birgitta
authors.initials: B
authors.lastname: Agerberth
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D042541:Intracellular Space / Q000382:microbiology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000166:cytology* / Q000187:drug effects / Q000254:growth & development; D010654:Phenylbutyrates / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism; D014807:Vitamin D / Q000494:pharmacology; D054804:Cathelicidins
chemical_list: D000263:Adenylate Kinase; D023181:Antimicrobial Cationic Peptides; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D002118:Calcium; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D010654:Phenylbutyrates; D019869:Phosphatidylinositol 3-Kinases; D012333:RNA, Messenger; D058486:Receptors, Purinergic P2X7; D014807:Vitamin D; D054804:Cathelicidins; C502778:ATG5 protein, human; C491997:BECN1 protein, human; C477012:MAP1LC3A protein, human; C546468:P2RX7 protein, human; C075773:4-phenylbutyric acid
keywords: P2RX7; antimicrobial peptides; cathelicidin; innate immunity; tuberculosis; vitamin D