In the present investigator-initiated study we evaluated the inflammatory profile in expectorated sputum of patients with bronchiectasis participating in the BAT trial and treated with maintenance AZM or placebo for one year.13 Our most remarkable finding was the fact that markers of airway inflammation remained stable or even increased during long-term macrolide treatment, suggesting that the clinically beneficial effect of macrolide treatment may not, or not as much, be driven by an anti-inflammatory effect as generally assumed. However, our results contrast with a recently published observational study whereby NETs were identified as a key marker of treatment response in bronchiectasis.12 In this study, lower concentrations of NET proteins were found after one year of maintenance AZM, especially in patients with non-eosinophilic asthma and in patients with PA infection. NETs were also identified as a key marker of disease severity.12 In our study, the inflammatory profile was examined at baseline in relation to the severity of the disease based on components of the BSI.2 Similar results were found, whereby an increase in disease severity based on a FEV1 of < 50% predicted as well as an increase in exacerbation rate in the year prior to the start of maintenance treatment were reflected by an upregulation of the pro-inflammatory markers at baseline.
In contrast to previous studies, the patients colonized with PA in our analysis showed no inflammatory upregulation.2,25,26 Moreover, levels of VEGF, IL-8 and MMP-9 were significant lower as compared to non-PA patients, probably due to the diversity of PA strains in bronchiectasis patients.27 But, it is also possible that long-term colonization with PA would lead to a more chronic inflammation and, to a lesser extent, active inflammation. In our analysis, only 10 patients were colonized with PA, and in addition, only 16 patients with HI. Due to the small number of samples, the results must be interpreted by caution. Surprisingly, the presence of HI in our sputum samples at baseline showed a significant upregulation of the inflammatory markers, suggesting a more marked inflammation in these patients, with probably even more clinical signs of active inflammation. In our study, patients with HI had more exacerbations in the year prior to the study as compared to patients colonized with PA. Both in COPD and bronchiectasis patients, HI is related to an increase in inflammation with higher levels of IL-6, IL-8, IL-1β and MPO and is an independent predictor for future exacerbations.28,29 These higher levels of inflammation were also found in our analyses, as compared to both non-HI and PA colonization.
Our analysis showed that during an exacerbation the inflammatory response increased, with an excessive and significant increase in patients treated with placebo as compared to a non-significant increase in the AZM-treated patients. This may suggest that there is indeed a dampening effect on the inflammatory response with macrolide treatment, but exclusively during exacerbations and, at least in the current study not picked up during stable state. In addition, this finding of reduced upregulation of inflammatory markers during an exacerbation may be driving the clinical finding of a marked reduction in the number of exacerbations in the active treatment group during the BAT trial.13 However, for this sub analysis the sample size was low, with a total of 29 sputum cultures collected during the exacerbation.
Prior to this study, we hypothesized that the inflammatory profile at baseline might be predictive of the effect of macrolide treatment, with higher levels of inflammation predicting better outcome, because of its supposed anti-inflammatory mode of action. However, in the current study, upregulation of no specific inflammatory marker at baseline was predictive for the treatment effect of AZM on exacerbations, FEV1% of predicted or QoL based on the SGRQ. Instead, lower values of (neutrophilic) inflammation expressed as based on VEGF, IL-8, IL-1α and MMP-9 were significantly associated with an increased improvement in FEV1% of predicted during AZM maintenance treatment. This may reflect reduced disease severity at baseline with a higher tendency to regenerate and improve during treatment. However, an association between downregulation of these specific inflammatory markers was not for other outcome measures such as exacerbation frequency and QoL, so the importance of this finding remains unsure.
Contrary to what is generally believed, the current study failed to show an attenuation of the inflammatory response in bronchiectasis patients with AZM treatment. A previous published systematic review of Zimmermann et al18 described an overall decrease in inflammatory markers in both sputum and serum samples of patients with various kinds of respiratory tract infections/inflammation, and skin and eye infections treated with macrolides. However, AZM treatment was more frequently associated with no influence on the immunological markers as compared to the other macrolides. In contrast to these results, a review of Huckle et al30 included 12 RCT’s of patients with stable COPD and described that prophylactic use of AZM (as compared to non-macrolides) is of benefit in reducing exacerbation frequency with reduced levels of a wide range of inflammatory markers in sputum. The effect of macrolide maintenance treatment on the inflammatory markers in the heterogeneous group of bronchiectasis patients has been studied in detail in a limited number of previous studies. Conflicting results were found, with a decrease of concentrations of IL-8, NE, and MMP-9 after treatment with clarithromycin (for 3 months) or roxithromycin (for 6 months) in two small open label studies.31,32 And in addition, one cohort study in patients with bronchiectasis and PA infection treated with maintenance AZM for one year found an decrease in NET concentrations.12 However, in one other RCT included 20 patients, treatment with low- dose erythromycin for 2 months did not effected inflammatory markers as IL-1α, IL-8, and TNF-α.33 Difference in treatment doses and duration of maintenance treatment of macrolides could be an explanation for these discordant results. Additionally, although we have measured a wide array of inflammatory markers, the effect of macrolide treatment on the immune system shows high complexity and is not fully understood yet. Therefore, one could argue that some effects may have been missed due underrepresentation of certain types of markers. However, this appears not very likely when considering the extensive panel, with markers representing different immunomodulatory pathways.18 In light of the above; other factors likely contribute to the observed clinical benefit of macrolides treatment in bronchiectasis. A previous study showed that AZM attenuated IL-8 without attenuating neutrophilic inflammation, which is suggestive for the inflammatory response due to viral infections too.34
This is to our knowledge the first study investigating the effect of macrolide treatment on airway inflammation during exacerbations. Interestingly, during an exacerbation AZM treatment appeared to have a dampening effect on the upregulated inflammatory response during an exacerbation, as compared to placebo. However, due to the small number of samples collected during an exacerbation, these results should be interpreted by caution. Nevertheless, this agrees very well with the most important clinical effect of long-term macrolide treatment, which is a marked reduction of the exacerbation frequency.13–15
In our study the inflammatory profile was related to the severity of the disease expressed as an increase in number of exacerbations and a FEV1 < 50% of predicted. This is in concordance with previous studies, mentioning upregulation of inflammatory markers (IL-8, IL-13, and NET proteins) as correlated to functional measurements of disease severity and an increase in exacerbations.8,9,12 In our study mainly neutrophilic inflammation was found with a significant upregulation of MPO, ECP, IP-10, TNF-α, IL-21,VEGF, MMP-9 in relation to the severity of the disease, however higher levels of ECP suggested also an eosinophilic component. These results are in line with earlier findings suggesting that in bronchiectasis patients the inflammatory profiles are dominated by neutrophilic inflammation, but there is also a role for eosinophilic inflammation, and other innate immune mediators.4,10,12
The current study is unique and provides interesting and new information on the effect of AZM on the sputum inflammatory markers during maintenance treatment, in a very well described patient population in a randomized controlled trial. However, there are some limitations to mention. From the total 83 patients conducted the BAT trial13, a total of 391 sputa were collected during the study, from a maximum of 60 patients per visit (73%). During AZM treatment, the availability of sputum samples gradually decreased in contrast to the placebo-treated patients. Only the patients with persistent respiratory symptoms and probably a reduced response on AZM could still expectorate sputum at the end of the study. Second, mainly for logistical reasons, only a small number of sputum samples were collected during an exacerbation. And therefore, our subgroup analysis of the inflammatory profile during an exacerbation in the AZM-treated patients as compared to the placebo-treated patients must be interpreted carefully. In addition, the inflammatory markers were measured in spontaneously expectorated sputum, and not in bronchial alveolar lavage fluid, causing possible oral contamination and represent not exactly the inflammatory profile of the lower respiratory tract.
In conclusion, in this study, we investigated the longitudinal effect of macrolide maintenance treatment on airway inflammation in bronchiectasis patients during stable state disease and exacerbations and inflammation in relation to disease severity. We found that disease severity was related to a higher mainly neutrophilic inflammatory response at baseline, with a significant upregulation in patients especially colonized by HI and not in patients with PA. Maintenance AZM treatment did not attenuate the inflammatory response as compared to placebo, but a dampening effect on the immune response during exacerbation was seen in AZM-treated patients, which may be responsible for the observed clinical benefit of macrolide maintenance treatment.
For a better understanding of the pathways through which macrolides exert their effect in bronchiectasis, more research is needed. This may need to be more aimed at understanding an anti-bacterial or anti-viral effect and type of anti-inflammatory effect. Also, our observation of reduced upregulation of the inflammatory response during an exacerbation warrants further research.