TLR5 Activation Exacerbates Airway Inflammation in Asthma

Abstract

Introduction: Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms.

Methods: We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma.

Results: We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma.

Conclusion: Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

Keywords: Airway inflammation; Asthma; Flagellin; TLR5.

Figure 1.. Overview of the research presented in this paper.

Initially we analyzed house dust from homes of asthma patients for TLR5 biological activity. We then used an ovalbumin model of allergic airway inflammation, with or without additional TLR5 activation via flagellin, to evaluate the role of TLR5 activation ion established asthma. Lastly, we translated our findings in human health insights by analyzing the symptoms of asthma patients with or without functional TLR5 (based on self-reported data).

Figure 2.. FLA is present in house dust from asthma patients’ homes.

FLA activity was assessed by measuring IL-8 in the supernatant of HEK293 cells transfected with either empty vector or human TLR5 and exposed to house dust extract from 24 homes of asthma patients. Because house dust has several impurities which may induce IL-8, the difference in IL-8 levels between empty vector and TLR5-transfected cells is depicted as indicator of specific FLA activity.

Figure 3.. FLA exacerbates allergic airway inflammation in the mouse model.

A). Airway resistance in response to 100 mg/ml aerosolized methacholine, measured by flexiVent, in C57BL/6J mice after sensitization with OVA, and exposure to OVA alone, FLA alone, or OVA+FLA, in comparison to unchallenged mice. Only OVA+FLA exposure leads to significant increase in airway responsiveness in this (relatively resistant) mouse strain. B). FLA + Ovalbumin exposure increases total cells, neutrophils and eosinophils in the lung lavage fluid.

Figure 4.. FLA/OVA co-exposure increases cytokine and chemokine levels in mouse lung compared to OVA or FLA exposure alone.

*p<0.5, **p<0.01, ***p<0.001, ANOVA with Holm-Sidak post-hoc correction.

Figure 5.. FLA/OVA co-exposure generates a unique gene expression signature.

A). Principal component analysis. B). Supervised Hierarchical Clustering of group expression means using the 276 genes significantly elevated in the (FLA vs FLA/OVA) and (OVA vs FLA/OVA) comparisons. Various shades of yellow indicate higher, and blue lower, relative expression. C)Venn diagram of gene activation groups, comparing unique genes between FLA/OVA to FLA and FLA/OVA to OVA comparisons. . D) Gene enrichment score for relevant pathways comparing OVA vs. OVA/FLA exposure (the pattern was very similar in the FLA vs OVA/FLA comparison).