title: Phenylbutyrate Is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection, Synergistically with 25-Hydroxy-Vitamin D3.
abstract: Adjunctive vitamin D treatment for pulmonary tuberculosis enhances resolution of inflammation but has modest effects on bacterial clearance. Sodium 4-phenylbutyrate (PBA) is in clinical use for a range of conditions and has been shown to synergise with vitamin D metabolites to upregulate cathelicidin antimicrobial peptide (CAMP) expression. We investigated whether clinically attainable plasma concentrations of PBA (0.4-4 mM) directly affect Mycobacterium tuberculosis (Mtb) growth and human macrophage and PBMC response to infection. We also tested the ability of PBA to enhance the immunomodulatory actions of the vitamin D metabolite 25(OH)D3 during infection and synergistically inhibit intracellular Mtb growth. PBA inhibited Mtb growth in broth with an MIC99 of 1 mM, which was reduced to 0.25 mM by lowering pH. During human macrophage infection, PBA treatment restricted Mtb uptake, phagocytic receptor expression and intracellular growth in a dose-dependent manner. PBA independently regulated CCL chemokine secretion and induced expression of the antimicrobial LTF (lactoferrin), the anti-inflammatory PROC (protein C) and multiple genes within the NLRP3 inflammasome pathway. PBA co-treatment with 25(OH)D3 synergistically modulated expression of numerous vitamin D-response genes, including CAMP, CYP24A1, CXCL10 and IL-37. This synergistic effect was dependent on MAPK signalling, while the effect of PBA on LTF, PROC and NLRP3 was MAPK-independent. During PBA and 25(OH)D3 co-treatment of human macrophages, in the absence of exogenous proteinase 3 (PR3) to activate cathelicidin, Mtb growth restriction was dominated by the effect of PBA, while the addition of PR3 enhanced growth restriction by 25(OH)D3 and PBA co-treatment. This suggests that PBA augments vitamin D-mediated cathelicidin-dependent Mtb growth restriction by human macrophages and independently induces antimicrobial and anti-inflammatory action. Therefore through both host-directed and bacterial-directed mechanisms PBA and vitamin D may prove an effective combinatorial adjunct therapy for tuberculosis to both resolve immunopathology and enhance bacterial clearance.
authors.affiliation: Clinical Infectious Diseases Research Initiative, Institute of Infectious Disease & Molecular Medicine, University of Cape Town, South Africa; Division of Mycobacterial Research, MRC National Institute of Medical Research, London, United Kingdom.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Coussens
authors.affiliation: Clinical Infectious Diseases Research Initiative, Institute of Infectious Disease & Molecular Medicine, University of Cape Town, South Africa; Department of Medicine, Imperial College London, London, United Kingdom; The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wilkinson
authors.affiliation: Division of Mycobacterial Research, MRC National Institute of Medical Research, London, United Kingdom; Blizard Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Martineau
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002112:Calcifediol / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000469:parasitology*; D009169:Mycobacterium tuberculosis; D010654:Phenylbutyrates / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014376:Tuberculosis
chemical_list: D000900:Anti-Bacterial Agents; D010654:Phenylbutyrates; C075773:4-phenylbutyric acid; D002112:Calcifediol