Abstract
Toll-like receptor 3 (TLR3) activation by viral infections plays a key role in promoting inflammatory immune responses that contribute to pulmonary fibrosis in chronic inflammatory respiratory diseases. Vitamin D3 has been shown to be beneficial to patients with asthma and chronic obstructive pulmonary disease (COPD) through its anti-inflammatory and anti-fibrotic properties. Smooth muscle cells are one of the major contributors to airway remodeling in asthma and COPD. We therefore aimed to investigate the effect of vitamin D3 treatment on viral-induced TLR3 responses in Bronchial Smooth Muscle Cells (BSMCs) as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Primary BSMCs from patients with asthma (n=4), COPD (n=4), and healthy control subjects (n=6) were treated with polyinosinic: polycytidylic acid (polyI:C), TLR3 agonist in the presence or absence of vitamin D3 (1,25D3). Here we report the mRNA expression and protein levels of pro-inflammatory and pro-fibrotic markers (IL-6, IFN-β1, CCL2/MCP-1, fibronectin 1 and type I collagen) among BSMCs groups: asthma, COPD, and healthy controls. We show that at the baseline, prior to polyI:C stimulation, asthma and COPD BSMCs presented increased pro-inflammatory and pro-fibrotic state compared to healthy control subjects, as measured by quantitative PCR and immunoassays (ELISA/Flow Cytometry. Ligation of TLR3 by polyI:C in BSMCs was associated with increased TLR3 mRNA expression, and 1,25D3 treatment significantly reduced its expression. In addition, 1,25D3 decreased the expression of IL-6, IFN-β1, CCL2, FN1 and COL1A1 induced by polyI:C in BSMCs. The regulatory effect of 1,25D3 treatment on polyI:C-stimulated BSMCs was further confirmed at protein levels. Our findings suggest that vitamin D3 attenuates TLR3 agonist-induced inflammatory and fibrotic responses in BSMCs and support the clinical relevance of vitamin D3 supplementation in patients with viral infections having chronic respiratory diseases, such as asthma and COPD.
Highlights
Pulmonary fibrosis is a complex process that involves activation of several pattern recognition receptors (PRRs) and an interplay of many cell types that contribute directly or indirectly to airway remodeling
The mRNA expression of vitamin D receptors (VDRs) was only slightly increased in polycytidylic acid (polyI):C-stimulated Bronchial Smooth Muscle Cells (BSMCs) and this effect was significantly increased by the addition of 1,25D3 (0.866 ± 0.23fold increase, p = 0.01 in asthma (Figure 1C) and 1.6 ± 0.73-fold increase in chronic obstructive pulmonary disease (COPD), p < 0.05) (Figure 1D)
Abnormal immune responses in viral infections may worsen acute lung injury, increasing airflow obstruction or pulmonary fibrosis, and pathological conditions already existent in patients with asthma or COPD
Summary
Pulmonary fibrosis is a complex process that involves activation of several pattern recognition receptors (PRRs) and an interplay of many cell types that contribute directly or indirectly to airway remodeling. The chemokine (CXC motif) ligand 10 (CXCL10) and the chemokine (CC motif) ligand 2 (CCL2), encoding monocyte chemoattractant protein 1 (MCP-1), are potent inflammatory mediators involved in inflammatory immune cell migration. These pro-inflammatory mediators have been recently described in the context of viral infections [13] and activates T helper 1 (Th1) cell-mediated immune responses. Increases in pro-inflammatory mediators in viral infections, in turn, may further activate signaling pathways involved in fibrosis [14], characterized by excessive deposition of ECM proteins, mostly fibronectin 1 (FN1) and type I collagen (COL1A1) [15]
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