Toll-like Receptors in Airway Inflammation in vitro and in vivo

During evolution a variety of solutions has been developed by different living
organisms in order to protect itself from invading potential pathogens and particles.
Protection of the host is essential for its survival and involves efficient recognition and
elimination of potential pathogens. The recognition is often mediated by pathogen
recognition receptors (PRRs). Activation of PRRs is mainly driven by exogenous
pathogen associated molecular patterns (PAMPs) or endogenous danger associated
molecular patterns (DAMPs) and result in secretion of multiple pro-inflammatory
cytokines. Toll-like receptors (TLRs) are members of the PRR family and the
importance of these receptors during pro-inflammatory conditions is addressed in the
present thesis, both in vitro and in vivo.
In Paper I we showed that farmers and smokers, two groups that are continuously
exposed to organic material through daily work at the farm or through tobacco smoke,
have an ongoing inflammation in the respiratory tract. It was shown that chronically
exposed subjects develop an adaptation to the effects of acute exposure to inhaled
organic material. Further, it was demonstrated that exposure in the swine barn was a
much stronger pro-inflammatory stimulus than inhaled pure lipopolysaccharide (LPS),
in vivo.
In Paper II, the gene expression of TLR2 on primary bronchial epithelial cells was
demonstrated. This expression was synergistically enhanced by co-stimulation with
pro-inflammatory stimuli and glucocorticosteroids. Dust obtained from the swine barn
was a more potent pro-inflammatory stimulus than pure LPS or pure peptidoglycan
(PGN), in vitro, as already shown in vivo. The secreted pro-inflammatory cytokines
from the epithelial cells were diminished by blocking of the TLR2 and TLR4 with
monoclonal antibodies, indicating that the pro-inflammatory stimulation was at least
partly dependent on TLR2 and TLR4.
In paper III we found that TLR2 on blood neutrophils was down-regulated by proinflammatory
stimuli, whereas the expression TLR4 and CD14 were unaffected by the
pro-inflammatory stimulation, in vitro. The expression of TLR4 and CD14 were
increased by the presence of epithelial cells, irrespective of stimulation. Moreover, we
showed synergistically enhanced secretion of CXCL8 (IL-8) and sCD14 during coculture
compared to single culture condition and a strong positive correlation between
CXCL8 and sCD14 in LPS-stimulated co-cultured cells. These findings strongly
suggest an active bidirectional cross-talk between alveolar epithelial cells and
neutrophils.
In paper IV we confirmed what we already had shown in vitro, that TLR2 was down
regulated by pro-inflammatory conditions on neutrophils, this time in vivo. We also
showed the presence of soluble TLR2 (sTLR2) in BAL and sputum and that this
expression was altered in COPD compared to healthy subjects. Moreover, CD14
expression on sputum neutrophils was enhanced compared with blood neutrophils and
that the gene expression of CD14 on alveolar macrophages in BAL-fluid was increased
in smokers compared with non-smokers. These findings indicate that PRRs expression
2
is altered by smoking per se, but also that the disease, COPD, contributes. This is likely
of importance in COPD patho-physiology, in particular for exacerbations, which often
are caused by microorganisms.
Overall, these studies have shown the involvement of PRRs in several immunological
active cell types during pro-inflammatory conditions. A better understanding of the
mechanisms behind PRRs regulation and outcome would potentially benefit drug
development and in the end many patients with inflammatory diseases