10779/crick.12666752.v1 Anna K Coussens Anna K Coussens Robert J Wilkinson Robert J Wilkinson Adrian R Martineau Adrian R Martineau Phenylbutyrate is bacteriostatic against Mycobacterium tuberculosis and regulates the macrophage response to infection, synergistically with 25-hydroxy-vitamin D3 The Francis Crick Institute 2020 Anti-Bacterial Agents Calcifediol Cell Proliferation Drug Synergism Flow Cytometry Humans Macrophages Mycobacterium tuberculosis Phenylbutyrates Reverse Transcriptase Polymerase Chain Reaction Tuberculosis Wilkinson, R U117588499 Virology 0605 Microbiology 1107 Immunology 1108 Medical Microbiology 2020-07-17 10:07:57 Journal contribution https://crick.figshare.com/articles/journal_contribution/Phenylbutyrate_is_bacteriostatic_against_Mycobacterium_tuberculosis_and_regulates_the_macrophage_response_to_infection_synergistically_with_25-hydroxy-vitamin_D3/12666752 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.