Toll-like Receptors in Allergic Rhinitis

University dissertation from Department of Otorhinolaryngology, Institute of Clinical Sciences, Malmö University Hospital, Lund University, Sweden

Abstract: Allergic rhinitis is an inflammatory disorder with the characteristic symptoms itching, sneezing, secretion and blockage upon allergen contact. In addition to the local inflammation in the nose, there is a systemic component that influences peripheral blood, bone marrow and lungs. The immune system is conventionally divided into innate and acquired (adaptive) immunity. The former is important in the early phase of the defense against previously unknown intruders, since there is a delay of 4-7 days for a full function of the adaptive immunity. Repeated encounters with the same antigen will result in a faster and more effective response of the adaptive immunity. Development of allergic airway inflammation is traditionally related to T helper type 2 (Th2) lymphocytes and their production of inflammatory cytokines as a part of the adaptive immunity, but recent data suggest that the innate immunity might also have a role in this process. Toll-like receptors (TLRs) have emerged as key receptors of the innate immune system but information about their role in allergy and airway inflammation is so far limited. The aims of the present study were to investigate the expression of TLR2, TLR3, TLR4 and TLR9 in the nose, peripheral blood and bone marrow, and to find out if pollen encounter affects the local and systemic expression of these proteins. The effects of nasal provocation with lipopolysaccharide (LPS), a ligand to TLR4, were investigated and the functional activity of TLR3 in eosinophils was explored in a series of in vitro experiments. In the six studies included in this thesis, a total of 111 patients with allergic rhinitis and 55 healthy volunteers were investigated. Samples of nasal mucosa, nasal lavage fluid, peripheral blood and bone marrow were examined using enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry and flow cytometry. TLR2, TLR3, TLR4 and TLR9 were found in the nasal epithelium and in inflammatory cells in the submucosa, and TLR2, TLR4 and TLR9 were expressed in neutrophils in nasal lavage fluid. In bone marrow, TLR2, TLR3, TLR4 and TLR9 were found in neutrophils, eosinophils, monocytes and immature granulocytes. These TLRs also appeared in peripheral blood, except for the absence of TLR3 in neutrophils. In addition, TLR9 was seen in basophils and lymphocytes. Generally, the leukocyte expression of TLRs was higher in nasal lavage fluid compared to bone marrow, where it in turn was higher than in peripheral blood. Stimulation of isolated eosinophils with the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)), resulted in an enhanced expression of CD11b and release of interleukin (IL)-8, involving the intracellular pathways of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-?B). Nasal provocation with the TLR4 ligand LPS resulted in leukocyte recruitment and increase in IL-6 and albumin in nasal lavage fluid after 6 hours. In addition, it caused a reduction in the tissue expression and the nasal lavage fluid levels of the anti-inflammatory mediator uteroglobin. Nasal pollen challenge in allergic patients resulted in an increased amount of neutrophils in nasal lavage fluid after one hour and an increased expression of TLR2, TLR3 and TLR4 in the nasal epithelium after 24 hours. Patients with seasonal allergic rhinitis had an activated expression of TLR4 in neutrophils, eosinophils and monocytes in peripheral blood during pollen season. Similarly, an activated expression of TLR4 was found in immature granulocytes and neutrophils in bone marrow and in neutrophils in nasal lavage fluid. The expression of TLR3 in eosinophils decreased in peripheral blood and bone marrow during pollen season, whereas the expression of TLR9 was not affected by allergen exposure. The present findings that pollen-induced upper airway inflammation affects the expression of TLRs locally and systemically, support the idea of a role for the innate immune system during symptomatic allergic rhinitis.

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