In this study, we have explored the roles of HA and HA binding proteins in human asthma. HA has been shown to accumulate in the BAL of asthmatic patients and may correlate with persistence of asthma symptoms33, 34
. Our data show that asthmatic airway fibroblasts produce constitutively higher concentrations of lower molecular weight HA with increased HAS2 gene expression compared to airway fibroblasts from normal controls. In addition, alveolar macrophages from asthmatic patients exhibit altered expression of HA binding proteins as compared to normal subjects, with a decrease in cell surface CD44 and increases in TLR2 and TLR4 expression. This translated into increased responsiveness of asthmatic alveolar macrophages to HA and LPS stimulation as demonstrated by increased IL-8 production. The reduction of CD44 on asthmatic alveolar macrophages could lead to impaired clearance of HA which in turn could promote persistent inflammation and potentially asthma symptoms.
Our data show that asthmatic airway fibroblasts constistutively release more HA than normal fibroblasts. In addition, asthmatic fibroblasts release relatively lower molecular weight HA than normal fibroblasts. The significant increase in HAS2 expression suggests that asthmatic fibroblasts acquired an activated phenotype with increased HA production. 48
In vitro experiments with cultured fibroblasts or COS1 cells suggested that HAS1 and HAS3 generated HA with broad size distributions (molecular masses of 2 × 105
to ~ 2 × 106
Da), whereas HAS2 generated HA with a broad but extremely large size (average molecular mass of > 2 × 106
. Subsequent studies suggested that all three HAS enzymes drive the biosynthesis and release of high molecular mass HA (1 × 106
. Thus, elevated expression of HAS2 may be responsible for increased HA production. HA degrading enzymes HYAL1 and HYAL2 expression was not different between asthmatic and normal fibroblasts. Hyaluronidases usually hydrolyze the hexosaminidic β(1–4) linkages in HA and release HA fragments. Although hyaluronidase from Streptococcus pneumoniae hydrolyzes HA to release disaccharide D-glucuronic acid-N-acetyl-D-glucosamine, the vertebrate hyaluronidases generate a various range of HA oligomers 51
. A recent study reported that asthmatic airway smooth muscle cells showed reduced HA secretion with decreased HAS-1 and HAS2 expression and increased expression of HYAL1 52
. Several reports have demonstrated that HA concentration is elevated in BAL of asthma patients 32–34
and the concentration of HA in BAL was positively correlated with the severity of asthma 34
. Human lung fibroblasts are an important source of HA production 23–25
, in addition to other cell types such as epithelial cells 53
. Furthermore, fibroblasts isolated with our protocol are α smooth muscle actin negative 37
. The reduced HA release by airway smooth muscle cells suggested that smooth muscle cells do not contribute to the HA accumulation in BAL and lung tissue in asthma patients.
Importantly, HA is susceptible to degradation by excessive reactive oxygen species during inflammation. Studies have shown that reactive oxygen species contribute to HA breakdown during lung injury 41–43
and that human lung fibroblasts express NOX4 45, 54
. A recent report has shown that S
-nitrosoglutathione reductase activity and inducible nitric oxide synthase were upregulated in BAL of asthma patients 36
. Our results indicated that NOX4 expression was elevated in asthmatic fibroblasts and might contribute to the production of low molecular weight HA in culture medium of fibroblasts from asthma patients. To our knowledge, we are the first to demonstrate increased NOX4 expression in asthmatic fibroblasts. Collectively, these data suggest that the increase in HA fragments in asthmatic fibroblasts are likely due to enhanced NOX4 expression and oxidative depolymerization rather that digestion by hyaluronidases.
Toll-like receptors are a major component of the innate immune system and have been studied in asthma 17
. Studies with murine asthma models have shown that TLR4 18, 20, 21
as well TLR2 are required for regulating Th2 immune responses in antigen-sensitized mice 55
. Toll like receptors are expressed by all effector cells of innate immunity including epithelial cells, mucosal mast cells and dendritic cells 56
and are important for pathogen recognition. In this study we have found that both TLR2 and TLR4 on human alveolar macrophage from asthmatic patients are upregulated with functional consequences of enhanced cytokine production in response to ligand. Blockade of TLR4 inhibited HA fragment-stimulated expression of IL-8 in asthmatic alveolar macrophages. Future studies will examine the role of TLR2 as well as other inflammatory cells such as eosinophils. This observation has potential implications for the role of infection in the pathobiology of chronic asthma and provides some insights into the potential mechanisms to explain spirals of symptoms that develop in asthmatic patients with airway infections.
Previous work from our group has shown that CD44 is required for HA clearance and the resolution of non-infectious lung inflammation in mice 7
. We have also shown that CD44 also is a negative regulator of TLR signaling. Alveolar macrophages from CD44 null mice produce increased amount of inflammatory chemokines in response to LPS 47
. Loss of CD44 on alveolar macrophages from asthmatic patients may not only impair HA clearance from asthmatic lungs but also enhance TLR signaling, thus promoting persistent inflammation.
Collectively, these data suggest that HA homeostasis is deranged in asthma and may provide some insights into connections between HA accumulation, macrophage function, and fibroblast activation in asthmatic lung. Asthmatic airway fibroblasts produce increased concentrations of low molecular weight HA. Decreased CD44 expression commensurate with increases in TLR2 and TLR4 on asthmatic macrophages may enhance the response to HA and TLR ligand LPS. Impaired HA clearance by decreased CD44 expression on macrophages leading to persistent inflammation in asthmatic lung. This previously unrecognized inflammatory regulatory pathway in asthma could lead to targeted approaches in selected patients experiencing severe and persistent asthma. Targeting the pro-inflammatory cycle involving fibroblasts, macrophages and matrix turnover may present a novel therapeutic strategy in the treatment of asthma.