This is the first clinical study to examine innate immune responses in the airways of subjects with asthma and bronchiectasis. Neutrophilic asthma was associated with an upregulation of the innate immune response. In particular, we found that neutrophilic asthma was characterised by increased expression of several key innate immune receptors: TLR2, TLR4, CD14 and SP‐A, as well as pro‐inflammatory cytokines IL8 and IL1β. We also found high levels of airway endotoxin in subjects with neutrophilic asthma. Innate immune activation may therefore be a key mechanism in the development of neutrophilic asthma.
Subjects with bronchiectasis were used in this study to provide a positive reference group, and this represents the first report of innate immune markers in bronchiectasis. These subjects were expected to have innate immune activation owing to their chronic bacterial infection and chronic airway inflammation with neutrophils. Persistent bacterial colonisation of the airways in bronchiectasis is caused by impaired mucociliary transport and mucus clearance, and initiates a vicious cycle of inflammation characterised by activated neutrophils and neutrophil proteases.31
On persistent exposure to PAMPs, we hypothesised and confirmed that innate receptors would be stimulated and activated to induce the production of IL8 and an influx of neutrophils. Subjects with bronchiectasis had increased sputum mRNA expression of TLR2 and supernatant SP‐A compared with controls. No difference was observed in TLR4 mRNA expression, suggesting that there may be differential expression of TLRs in bronchiectasis.
Innate immune activation seems to be an important pro‐inflammatory mechanism in neutrophilic asthma. While the pathway leading to eosinophilic asthma is well characterised, the mechanisms of neutrophilic inflammation in asthma are poorly understood. Although neutrophils were thought to be primarily involved in severe asthma,32,33
increased neutrophil levels have also recently been reported in stable asthma.16,34,35
The increased neutrophil levels in those studies were not caused by respiratory tract infections as subjects with a reported lower respiratory tract infection in the preceding month were excluded. In the study by Green et al
subjects with neutrophilic asthma were older, had a later onset of disease and were less atopic than subjects with normal levels of neutrophils. Our study was too small to assess these differences. However, it is important to note that neutrophilic asthma has been described in people with mild, moderate and severe asthma, and it is not merely a feature of severe asthma with fixed air flow obstruction.6,13
Sputum TNFα mRNA levels were also highest in subjects with neutrophilic asthma and bronchiectasis, and TNFα mRNA was significantly correlated with both TLR4 and TLR2 mRNA expressions. Recently, TNFα has been implicated in the upregulation of TLR2 expression in epithelial cells and, therefore, may be an important cytokine in the perpetuation of innate immune activation in the airways.32
Further, a recent randomised controlled trial of a soluble TNFα receptor in people with severe refractory asthma has shown improvements in a number of asthma outcomes including asthma control score and AHR, highlighting the potentially important pathogenic role of TNFα in difficult asthma.33
The inflammatory cell counts in neutrophilic asthma were similar to bronchiectasis and, in addition, both groups had an increased frequency of chronic bacterial colonisation of the airways when compared with the other asthma subtypes. This indicates that neutrophilic asthma and bronchiectasis have a similar pattern of airway inflammation, with evidence of innate immune activation. Sputum endotoxin levels were high in subjects with neutrophilic asthma, which suggests that endotoxin may be the source of PAMPs driving the innate response in this group. The levels were 6–8‐fold higher than those observed in the other asthma subtypes studied. In contrast, to subjects with bronchiectasis, there was increased mRNA expression of both TLR2 and TLR4 in subjects with neutrophilic asthma compared with both eosinophilic and paucigranulocytic asthma. Although TLR4 has been identified as the primary receptor for bacterial LPS, recent studies have shown that TLR2 is also capable of responding to LPS.36
This may explain why both TLR2 and TLR4 were upregulated. Alternatively, other PAMPs or cytokines known to upregulate TLR2 expression may have a role.
LPS signalling is complex and involves a number of accessory proteins including LPS binding protein and CD14. Subjects with neutrophilic asthma had increased mRNA expression of CD14 and TLR4 compared with controls, subjects with eosinophilic and paucigranulocytic asthma and those with bronchiectasis. The reason for this increase in neutrophilic asthma (which was not observed in bronchiectasis) is unclear, but may be due to the higher levels of endotoxin measured in the sputum supernatant, which may be upregulating the expression of LPS signalling proteins. There was also a strong positive correlation between TLR4 mRNA and CD14 mRNA in subjects with asthma, suggesting that the expression of these proteins is co‐regulated.
The roles of gene polymorphisms in determining innate immune responses are conflicting. The TLR4 polymorphism Asp299Gly has been associated with hyporesponsiveness to airway challenge with LPS37
and increased risk of Gram‐negative infection.38
In another study, similar rates of chronic infection were reported between the subjects with a TLR4 polymorphism and wild‐type TLR4, indicating a role for this polymorphism in the risk of acute infection but not in chronic infection.39
The same TLR4 polymorphism has been associated with asthma in Swedish children,40
but no association was found between TLR4 polymorphisms and a diagnosis of asthma in a large adult study.41
A polymorphism in the CD14 promoter has been associated with increased soluble CD14 expression and lower IgE levels in serum,42
whereas polymorphisms of the LPS‐binding protein gene have been associated with increased risk of sepsis in association with male gender.43
Regardless of these conflicting data, it is plausible that a polymorphism in innate immune receptors and associated proteins (CD14 and LPS‐binding protein) may explain the variation in immune and inflammatory responses reported.
SP‐A functions primarily as an opsonin, identifying targets for phagocytosis and binding of pathogens in vitro. In SP‐A null mice, exposure to pathogens, including viruses, results in increased neutrophilia, epithelial injury and persistence of infection compared with control mice, indicating a protective role for surfactant proteins in lung defence.44
In this study, levels of SP‐A were increased in subjects with eosinophilic and neutrophilic asthma compared with controls, indicating host‐defence activity in these subjects. SP‐A did not seem to be a specific marker of innate immune activation as levels were similar between the asthma subtypes. Although levels of SP‐A are increased, it is unknown whether the SP‐A is functional and intact. Neutrophil proteases present in airway fluids degrade SP‐A and reduce its antimicrobial and anti‐inflammatory functions. SP‐A levels are also increased in bronchial lavage fluid from subjects with asthma.45
Further investigation is required to determine whether the integrity of SP‐A from the airways of subjects with asthma is maintained.
In summary, we conclude that persistent activation of the innate immune system in stable asthma results in the production of pro‐inflammatory cytokines which may contribute to the pathogenesis of neutrophilic asthma. This is an important observation that identifies a specific mechanism operating in the neutrophilic subtypes of non‐eosinophilic asthma. This adds to our understanding of the heterogeneity of airway inflammation in asthma. The mechanisms of this persistent activation are unclear but may be related to endotoxin exposure or chronic bacterial colonisation of the lower airways.