Toll-like receptors (TLRs) are key components of the innate immune
system involved in surveillance and early detection of infection, as well as
coordination of the subsequent adaptive immune response. Pulmonary immune
responses are initiated by TLRs expressed on airway epithelial cells. In this
thesis, primary normal human bronchial epithelial (NHBE) cells, immortalized
human bronchial epithelial (HBE) cells, a glandular lung adenocarcinoma cell line
(Calu-3) and an alveolar epithelial adenocarcinoma cell line expressing NF-κB
(A549/NF-κB-luc) were analyzed for expression of mRNAs for TLRs, coreceptors,
adaptor proteins and non-TLR pathogen recognition receptors (PRRs).
Quantitative RT-PCR analysis in Calu-3 and NHBE/HBE cells revealed mRNA
expression of only TLRs 1-6, with TLR4/TLR6 having the lowest abundance and
TLR3 exhibiting the highest level of expression. Conversely, in A549/NF-κB-luc
cells mRNA expression of all TLRs 1-10 was detectable; with TLR5 mRNA
expressed at the highest level.
Poly i/c, a synthetic viral dsRNA analogue, elicited the greatest effect on expression of TLR subtypes. The effect was far more pronounced in NHBE/HBE
cells as compared to Calu-3 and A549-NF-κB-luc cell lines. Poly i/c increased
TLR2 mRNA expression by over sixty-fold and TLR3 by eleven-fold in NHBE
cells, whereas TLR5 was significantly reduced. TLR2 protein was also enhanced
by Poly i/c, but not by the TLR2 ligand, PAM3CSK4. Additionally, Poly i/c
enhanced mRNA expression of adaptor molecules (MyD88, TIRAP, and TRIF) and co-receptors (Dectin-1, CD14) involved in TLR2-signaling. In contrast,
mRNA and protein expression of co-receptor CD36 was significantly reduced by
Poly i/c. Overall, Poly i/c activation of NHBE cells differentially regulated
expression of multiple TLRs, non-TLR receptors, adaptor proteins and coreceptors.
ELISA analysis of apical and basolateral solutions from Poly i/cstimulated
NHBE monolayers revealed significantly higher levels of IL-6 and GMCSF.
After priming with Poly i/c, an increase in IL-6 secretion was observed in cells stimulated with PAM3CSK4 and with Alternaria extract, a fungal allergen
known to signal partly through TLR2. However, IL-6 secretion was not stimulated
by other TLR2 ligands, zymosan or lipothechoic acid (LTA). Pretreatment with
anti-TLR2 blocking antibody inhibited the PAM3CSK4-induced increase in IL-6
secretion after Poly i/c exposure. Up-regulation of TLR2 following exposure to
dsRNA enhanced functional responses of the airway epithelium to certain
(PAM3CSK4), but not all (zymosan, LTA) TLR2 ligands. In TLR3-deficient cells,
induction of TLR2 mRNA expression by Poly i/c was reduced by thirteen-fold,
indicating that Poly i/c-dependent TLR2 induction was TLR3-mediated.
Furthermore, Poly i/c-dependent IL-6 secretion was abrogated by 83% in TLR3
knock-down cells. Following priming of NHBE cells with Poly i/c and
subsequently with TLR5 ligand flagellin, no reduction in IL-6, GM-CSF, RANTES
and IFN-β transcripts was observed. Similarly, IL-6 and GM-CSF protein
secretion did not match Poly i/c-dependant down-regulation of TLR5. Using A549/NFκB-luc cells and wild-type (flagellated) and flagella-mutated Burkholderia
(B.) cenocepacia, intact flagella were found to be critical for NF-κB activation and
enhanced secretion of IL-8.
To elucidate the in vivo actions of viral infection on the magnitude of TLR2
expression, and subsequent innate immune response to TLR2 activation, the
effects of Poly i/c challenge on the inflammatory response of mouse airways to a
selective TLR2 ligand (PAM3CSK4) and a common fungal allergen (Alternaria
alternata) were investigated. Poly i/c enhanced mRNA and protein expression of TLR2 in the lung. It also elicited a marked induction of Th1 cytokines. However,
priming with Poly i/c abrogated Alternaria-mediated Th2 cytokine production.
Inflammatory cell recruitment into the airways, predominantly with neutrophils,
was also observed. Taken together, both the in vitro and the in vivo studies show
that activation of TLR3 by Poly i/c alters the inflammatory response of the
airways to other microbial ligands, thereby modulating the severity of
inflammation. This effect is partly due to changes in the expression of other TLR
receptors and their associated co-receptors and adaptor proteins.
University of Minesota Ph.D. dissertation. June 2010. Major: Comparative and Molecular Biosciences. Advisor: Scott M. O’Grady. 1 computer file (PDF); xvi, 201 pages.
Toll-like receptor interactions and their contribution to airway inflammation..
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