Pattern-recognition receptors in airway inflammation

Abstract

Airway inflammation is a defining feature of allergic rhinitis and asthma. Bacterial and viral infections are known to cause exacerbations of both diseases, but knowledge about the mechanisms involved is limited. The pattern-recognition receptors (PRRs) comprise several receptor families which recognize microbial structures or host-derived danger signals, and trigger an immune response. The Toll-like receptors (TLRs) are the best characterized set of receptors, but another family of PRRs, the Nod-like receptors (NLRs), has recently been described. The TLRs and NLRs are found both in leukocytes and structural cells throughout the airways and are becoming increasingly implicated in airway inflammation. This thesis characterizes the presence and functional response of various members of the TLR and NLR families.

Lipopolysaccharide (LPS) is found in the cell wall of Gram-negative bacteria and exerts its effects through TLR4. In the first three papers nasal LPS challenge was used as a method to study neutrophil inflammatory responses in the nose.

In the first paper we explored if the upper airways could be used as a model for inflammatory events in the lung. LPS-induced neutrophil inflammation in the nose was inhibited and the results compared with findings in a study of similar design on the lower airways. The nasal model was found to mimic the responses seen in the lower airway study, with no signs of systemic activation or adverse effects. This suggests the nasal LPS model to be a safe and convenient method for studying neutrophilic airway inflammation.

The nasal model was subsequently used in the second paper to analyze the role of macrophage inflammatory protein (MIP)-1α in LPS-induced local inflammation. The LPS challenge resulted in a neutrophil-mediated secretion of MIP-1α, dependent on the nuclear factor (NF)-κB, protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK) pathways. LPS also delayed neutrophil apoptosis in vitro, suggesting that the secretion of MIP-1α may be boosted by a prolonged neutrophil survival. This indicates that MIP- 1α plays a role in neutrophilic airway disease.

In the third paper we investigated whether symptomatic allergic rhinitis affected the expression of TLR4 in nasal lavage, blood and bone marrow. Provocation with LPS in a milieu of allergic inflammation, caused by allergen challenge, resulted in a release of cytokines like interleukin (IL)-4, IL-5, IL-10, IL-13, Interferon (IFN)-γ and tumor necrosis factor (TNF)-α. No such cytokine release was seen with either allergen or LPS alone, nor when LPS preceded allergen. The systemic up-regulation of TLR4 seen during on-going allergic rhinitis might contribute to the presently seen increase in LPS response. It is therefore tempting to extrapolate these findings to a clinical situation in which a local infection can cause exacerbation or aggravation of allergic symptoms. The systemic up-regulation of TLR4 seen during symptomatic allergic rhinitis might contribute to the observed increase in response when LPS was applied following allergen challenge. The results strengthen the suggestion that a local infection may exacerbate allergic inflammation.

The fourth study aimed to visualize the effects of allergen on two major populations of dendritic cells, the myeloid (mDCs) and the plasmacytoid dendritic cells (pDCs), in the nose of patients with allergic rhinitis. Allergen challenge increased the number of pDCsin the nasal sub-epithelium. In vitro studies of pDCs revealed that they could be activated by TNF-α, IL-4 and CpG stimulation, and that TNF-α caused a higher activation among atopic than non-atopic subjects. The data support the notion of mDCs and pDCs as distinct populations with different roles in the allergic process. Further, it suggests that the pDCs observed upon allergen challenge might be of an activated phenotype and play a role in the course of allergic rhinitis.

The fifth study focused on the effects of TLR7 stimulation on airway smooth muscle contraction. Guinea pig tracheas were stimulated with the TLR7 agonists R837 and R848 for three days, and the contractile response along with the underlying signal pathways were investigated in a myograph model. TLR7 stimulation was found to reduce airway smooth muscle contraction in an epithelium-independent manner, dependent on p38 MAPK and NF-κB pathways. This indicates that TLR7 stimulation can be part of a protective mechanism against virus infection and that TLR7 deficiency might be a cause of airway disease. Further, it suggests that TLR7 ligands might be a future option for treatment of airway hyperresponsiveness.

The sixth study characterized the expression and function of NLRs in neutrophils, with focus on Nucleotide-binding oligomerization domain (NOD)1, NOD2 and NACHTLRR- PYD binding protein (NLRP)3. An expression of NOD2 and NLRP3 protein and mRNA was found in isolated neutrophils. NOD2 activation resulted in IL-8 secretion and a change in neutrophil phenotype, while activation of NLRP3 caused secretion of IL-1β. Both receptors caused an increased neutrophil migration. The findings might reflect a previously unknown pathway for activation of these cells.