BACKGROUND AND PURPOSEThere is current interest in vitamin D as a potential anti-inflammatory treatment for chronic inflammatory lung disease, including cystic fibrosis (CF). Vitamin D transcriptionally up-regulates the anti-inflammatory gene DUSP1, which partly controls production of the inflammatory chemokine IL-8. IL-8 is overabundant in CF airways, potentially due to hyperinflammatory responses of CF macrophages. We tested the ability of vitamin D metabolites to down-regulate IL-8 production in CF macrophages. EXPERIMENTAL APPROACHCF and healthy monocyte-derived macrophages (MDM) were treated with two vitamin D metabolites, 25-hydroxyvitamin D3 (25OHD3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or paricalcitol, synthetic analogue of 1,25(OH)2D3. 25OHD3 was tested at doses of 25-150 nM, whereas 1,25(OH)2D3 and paricalcitol at doses of up to 100 nM. IL-8 was stimulated by bacterial virulence factors. As potential anti-inflammatory mechanism of vitamin D metabolites, we assessed up-regulation of DUSP1. KEY RESULTSMDM from patients with CF and some healthy donors showed excessive production of stimulated IL-8, highlighting their hyperinflammatory phenotype. Vitamin D metabolites down-regulated stimulated IL-8 only in those hyperinflammatory MDM, and only when used at high doses (>100 nM for 25OHD3, or >1 nM for 1,25(OH)2D3 and paricalcitol). The magnitude of IL-8 down-regulation by vitamin D metabolites or paricalcitol was moderate (∼30% vs. >70% by low-dose dexamethasone). Transcriptional up-regulation of DUSP1 by vitamin D metabolites was seen in all tested MDM, regardless of IL-8 down-regulation.

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Pharmacological stimulation of the antiviral cytokine IFN-β in the airways may help to counter deleterious virus-induced exacerbations in chronic inflammatory lung diseases (asthma, chronic obstructive pulmonary disease, or cystic fibrosis). Polyinosinic-polycytidylic acid [poly(I:C)] is a known inducer of IFN-β but also costimulates an inflammatory response. The latter response is undesirable given the pre-existing airway inflammation in these diseases. The objective of our study was to identify conditions for poly(I:C) to selectively upregulate IFN-β in airway epithelial cells without a concomitant inflammatory response. The inflammatory response was gauged by production of the chemokine IL-8. Using cell lines and primary airway epithelial cells (both submerged and well-differentiated), we observed that pure poly(I:C) stimulated IFN-β mainly through the TLR3/TRIF pathway and IL-8 through an unidentified pathway. The magnitude of the IL-8 response stimulated by pure poly(I:C) matched or even exceeded that of IFN-β. Furthermore, this IL-8 response could not be pharmacologically downregulated without affecting IFN-β. In contrast, we show that stimulation of the RIG-I/MAVS pathway, such as when poly(I:C) is delivered intracellularly in a complex with liposomes or via nucleofection, selectively stimulates IFN-β with low IL-8 costimulation. The magnitude of IFN-β stimulation by liposome-encapsulated poly(I:C) is markedly diminished in well-differentiated cells. In conclusion, it is feasible to augment IFN-β production in airway epithelial cells without excessive costimulation of IL-8 if the RIG-I/MAVS pathway is stimulated, such as via liposomal delivery of poly(I:C). Better cytoplasmic delivery vehicles are needed to efficiently stimulate this pathway in well-differentiated cells.

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