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 (0.4-4mM) 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 1mM, which was reduced to
0.25mM 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.