Does airway colonization cause systemic inflammation in bronchiectasis ?

Son dönemde yapılan çalışmalar kronik obstrüktif akciğer hastalığı ve astım gibi kronik hava yolu hastalıklarında eşlik eden bir sistemik inflamatuvar yanıtın varlığını göstermiştir. Hava yollarında kronik bakteriyel kolonizasyon ve buna ikincil hava yolu inflamasyonu ve ilerleyici hava yolu hasarı ile karakterize bir hastalık olan bronşektazide, sistemik inflamatuvar yanıtın var olup olmadığı bilinmemektedir. Bu çalışmada bronşektazide hava yolundaki bakteriyel kolonizasyon ve sistemik inflamasyon arasındaki ilişkinin değerlendirilmesi amaçlanmıştır. Klinik olarak stabil olan bronşektazi hastaları (n= 50) ile yaş ve cinsiyete göre eşleştirilmiş kontrol grubunda (n= 30) serum inflamasyon parametreleri [beyaz küre sayısı, eritrosit sedimentasyon hızı, C-reaktif protein (CRP), fibrinojen, interlökin-8, tümör nekroz faktörü-α ve leptin] değerlendirilmiştir. Ayrıca, bronşektazili hasta grubunda hava yolu bakteriyel kolonizasyonuna göre alt analiz yapılmıştır. Bronşektazili hasta ve kontrol grupları arasında serum inflamasyon parametreleri seviyeleri açısından fark saptanmamıştır. Ancak bronşektazili hasta grubu hava yolu bakteriyel kolonizasyonuna göre değerlendirildiğinde median (çeyrekler arası aralıkÇAA) beyaz küre sayısı, CRP ve fibrinojen seviyeleri hava yolu kolonizasyonu olan hastalarda (n= 14) olmayanlara göre belirgin derecede yüksek olarak saptanmıştır [sırasıyla 8.2 (6.4-9.5) ve 6.4 (5.8-7.7) x 103/mm3, 0.91 (0.45-1.29) ve 0.42 (0.30-0.77) mg/dL, 433.5 (390.3-490.3) ve 392.0 (327.0-416.0) mg/dL; p< 0.05]. Bu çalışmada tüm bronşektazi grubunda, kontrol hastaları ile karşılaştırıldığında, sistemik inflamatuvar parametrelerde artış saptanmamıştır. Ancak hastalar hava yolunda bakteriyel kolonizasyon varlığına göre değerlendirildiklerinde kolonize hastalarda beyaz küre, CRP ve fibrinojen seviyelerinin yüksek olduğu gözlenmiştir. Bu bulgular stabil bronşektazi hastalarında gelişen kronik hava yolu kolonizasyonunun sistemik inflamasyona neden olabileceğini desteklemekedir.

Bronchiectasis, a pathological entity characterized by permanent and abnormal dilatation of one or more bronchi, is a common airway disease in developing countries due to increased prevalence of tuberculosis and other respiratory infections (1)(2)(3)(4).The airway pathology observed in bronchiectasis is not a stationary process due to the ongoing local inflammatory response (4,5).The inflammatory response triggered by microbial infection in bronchiectatic areas causes the release of proteolytic enzymes and oxygen radicals from neutrophils.This leads to impairment in mucociliary activity and retaining of secretions, which in turn creates an environment suitable for recurrent bacterial infections and colonization.The repetitive bouts of infection and inflammation create a vicious cycle, which in end cause progressive damage in the airways (4)(5)(6).
In this study our objective was to determine the levels of systemic inflammatory markers in clinically stable bronchiectasis patients and identify its relationship with airway colonization.

Study Population
We included a consecutive series of patients with an established or new diagnosis of bronchiectasis confirmed by high-resolution computerized tomography (HRCT).The study was performed in a university hospital between January and December 2005.We only included clinically stable patients."Clinically stable" was defined as 1) absence of any change in respiratory symptoms (increased dyspnea, increased sputum production, change in sputum color), 2) absence of any new respiratory symptoms like cough, dyspnea, hemoptysis, 3) absence of any change in medications, and 4) absence of hospital admission, within six weeks prior to the study.Patients with exacerbations (defined as clinical worsening with increased symptoms like cough, dyspnea, sputum production and increased sputum volume), diagnosis of a systemic disease which may be associated with bronchiectasis (COPD, bronchial asthma, cystic fibrosis, sarcoidosis, interstitial fibrosis, allergic bronchopulmonary aspergillosis) and concurrent use of any medications that may influence systemic inflammation (immunosupressive drugs or systemic corticosteroids) were excluded from the study.A consecutive series of healthy volunteers without any evident airway disease were enrolled into the study as the control group.The study was approved by the local institutional review board and a written informed consent was obtained from all study participants.

Data Collection
Baseline demographic and clinical [age, gender, height, weight, body mass index (BMI), smoking history] data were collected from all study participants.Bronchiectasis group was also reviewed for the type and duration of symptoms, time of diagnosis, possible risk factors for bronchiectasis, and ongoing treatments for bronchiectasis.
HRCT scans of the bronchiectasis patients were evaluated independently by two radiologists and a pulmonologist.The extent of bronchiectasis was evaluated for Ergan Arsava B, Çöplü L.

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Tüberküloz ve Toraks Dergisi 2011; 59(4): 340-347 on in bronchiectasis.Although bronchiectasis was initially considered a stationary process, chronic bacterial colonization causes airway inflammation and progressive airway damage.The aim of this study was to determine the level of systemic inflammation in bronchiectasis patients and identify its relationship with colonization.White blood cell (WBC) count, erythrocyte sedimentation rate, serum C-reactive protein (CRP), plasma fibrinogen, interleukin-8, tumor necrosis factor-α and leptin levels were determined in clinically stable bronchiectasis patients (n= 50), and age-and sex-matched controls.Bronchiectasis patients were also analyzed according to colonization in sputum samples.There was no significant difference between bronchiectasis and control groups with respect to inflammatory markers but median (interquartile range-IQR) WBC count, CRP and fibrinogen levels were significantly higher in colonized patients (n= 14) when compared to non-colonized patients [8.2 (6.4-9.5) vs. 6.4 (5.8-7.7)x 10 3 /mm 3 , 0.91 (0.45-1.29) vs. 0.42 (0.30-0.77) mg/dL, 433. 5 (390.3-490.3)vs. 392.0(327.0-416.0)mg/dL, respectively; p< 0.05].There was no evidence supporting the presence of systemic inflammation in the overall bronchiectasis group when compared to controls.However, elevated WBC count, CRP and fibrinogen levels in patients with colonization suggest the presence of a systemic inflammatory response in clinically stable bronchiectasis patients with colonization.Key Words: Bronchiectasis, C-reactive protein, colonization, fibrinogen, systemic inflammation.each lobe individually; lingula was considered as a separate lobe (16).

Lung Function
Forced expiratory volume in one second (FEV 1 ), forced vital capacity (FVC), FEV 1 /FVC ratio were measured by spirometer (Spirolab II, Medical International Research-MIR, Italy) using standard protocols.Lung function was expressed as both actual and percentage of reference values (17).

Laboratory Analysis
White blood cell (WBC) count, erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), immunoglobulin G, A, M and E, and plasma fibrinogen levels were determined in all subjects.Venous blood samples were centrifuged at 3000 rpm and plasma was collected to be stored at -70°C.IL-8 and TNF-α levels were determined by enzyme linked immunosorbent assay (ELISA) and leptin levels were determined by solid phase enzyme amplified sensitivity immunoassay (BioSource International Inc, California, USA).All samples were tested twice and the mean value was used in further analyses.

Microbiological Analysis
Every patient was asked to give sputum samples for quantitative culture at least twice.The quality of specimen was screened by Gram stain; specimens with > 25 leukocytes and < 10 epithelial cells per examination field (x100 magnification) were considered as acceptable.Samples were diluted with an equal volume of 0.9% NaCl solution and vortex mixed for five minutes.Once homogenised, the samples were left for five minutes at room temperature and then ten-fold serial dilutions of sputum samples were plated on blood and haemophilus test medium agars (BD diagnostic system).The cultures were evaluated for growth after 24 and 48 hours.Negative bacterial cultures were discarded after five days.Growth with potentially pathogen microorganisms (Haemophilus influenzae and parainfluenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, enterobacteria) were considered as positive culture growth and colonization was defined as the presence microorganism growth with ≥ 1 x 10 5 colony-forming units per milliliter (18)(19)(20).

Statistical Analysis
All numerical variables are expressed as median (interquartile range, IQR).Continuous variables were compared by Mann-Whitney U test and categorical variables were compared by Chi-Square test.Spearman's correlation test was used to assess the relationship between two continuous variables.A two-tailed p value of < 0.05 was considered significant.All statistical analyses were performed using SPSS 14.0.
The underlying etiologies considered to be associated with the development of bronchiectasis were as follows: idiopathic (48%), previous tuberculosis infection (26%), post-pneumonic (22%) and primary ciliary dyskinesia (4%).All patients had normal immunoglobulin levels.According to HRCT findings, the median (IQR) number of lobes involved was 2 (1-4).The patients were followed with a diagnosis of bronchiectasis for a median (IQR) duration of 1.5 (0.0-7.5) years (range: 0-25 years) and none of the patients were on long term prophylactic antibiotic therapy.
When compared to controls, bronchiectasis patients had a lower prevalence of active smoking (8% vs. 40%, p< 0.01).The control group, as expected, had better results in pulmonary function tests.No significant difference was present between patients and controls with respect to levels of inflammatory markers (Table 1).Plasma concentrations of TNF-α remained below the lower detection limit (1.7 pg/dL) in all patients and controls.There was no statistically significant difference for inflammatory markers between patients who were and were not receiving inhaled corticosteroid therapy in the bronchiectasis group.Inflammatory marker levels were similar between smoker and nonsmoker controls.

DISCUSSION
Permanent dilatation of the airways and impairment of mucociliary clearance result in bacterial colonization in patients with bronchiectasis.Chronic airway infection, in turn, triggers an intense local inflammatory response (12).There is limited data about the presence of a systemic inflammatory component in addition to this local response during the clinically stable period in bronchiectasis (15).In this study, we sought to identify the presence of systemic inflammation in clinically stable bronchiectasis.We measured systemic markers of inflammation in a consecutive series of patients with bronchiectasis and controls.We were not able to find any evidence suggestive of systemic inflammation in the overall bronchiectasis cohort when compared to controls.On the other hand, when markers of systemic inflammation were compared within the bronchiectasis subgroups, we found evidence for elevated WBC count, CRP and fibrinogen levels in patients with bacterial colonization.
It is known that colonization with pathogenic microorganisms in the airways trigger a cascade of events resulting in local airway inflammation (9,12).A previous Ergan Arsava B, Çöplü L.

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Tüberküloz ve Toraks Dergisi 2011; 59(4): 340-347   study has shown that the inflammation is limited to the airways in patients with bronchiectasis and this inflammation is more intense in colonized patients (9).This inflammation might in turn flood into the systemic circulation and lead to a systemic inflammatory response, as suggested by elevated WBC count, CRP and fibrinogen levels in colonized patients in our study.This finding raises the question of whether systemic inflammation in bronchiectasis during clinically stable period is dependent on bronchiectasis severity, which is closely related to colonization.Wilson et al. showed elevated levels of systemic inflammatory markers (WBC count, ESR and CRP) in bronchiectasis; these inflammatory parameters were significantly correlated with HRCT scores (15).In a study done among patients with diseases that cause end-stage respiratory failure, bronchiectasis patients had higher CRP levels with respect to other diseases that cause respiratory failure (21).CRP levels were also independently associated with mortality in this study (21).In addition, it has been shown that systemic inflammation, as reflected by elevated ESR and CRP levels, is related with rapid decline in FEV 1 in patients with bronchiectasis (20).All these data point out to the presence of a systemic inflammatory component in bronchiectasis; this component is probably related to disease severity, and therefore systemic inflammation might only be restricted to severe bronchiectasis cases in whom bacterial colonization is expected.The absence of any significant difference with respect to systemic inflammatory markers between the patient and control groups in our study might be explained by the relatively mild disease severity of our bronchiectasis cohort, as shown by the pulmonary function tests and HRCT findings.

Table 2. Baseline characteristics and results of laboratory tests in bronchiectasis patients
The association between systemic inflammation and disease severity is also supported by the findings observed in the P. aeruginosa group of this study.The most commonly accused microorganism with disease severity and worse clinical outcome in bronchiectasis is P. aeruginosa.The presence of this microorganism during stable period is related with more extensive disease, severe airflow limitation and rapid decline in lung function (20,(22)(23)(24)(25). Consistently, in this study, patients with P. aeruginosa colonization had more impairment in pulmonary function tests and more diffuse disease on HRCT.All inflammatory markers in the P. aeruginosa group were higher when compared to other groups; however these differences did not reach statistical significance except for WBC count and fibrinogen levels.We believe that the small number of patients has considerably contributed to the statistical non-significance in this subgroup analysis.Nonetheless, these fin-dings point out to a potential role of colonization with certain microorganisms, particularly P. aeruginosa, in the initiation of low-grade systemic inflammation in bronchiectasis.
We found elevated levels of fibrinogen in colonized patients.The only study in the English literature looking for fibrinogen levels in bronchiectasis reported a significant relationship between fibrinogen levels and patient activity score in St. George Respiratory Questionnaire (26).Other studies, analyzing fibrinogen in respiratory diseases have mostly been performed in COPD patients.High fibrinogen levels were seen both during clinically stable periods and exacerbations of COPD (27).The relationship between fibrinogen and bacterial growth was not evaluated in this study; however elevation of fibrinogen during exacerbations was related with sputum purulence, which might be an indirect sign of bacterial infection.In a previous study performed in clinically stable COPD patients, subjects colonized with potentially pathogenic microorganisms had increased levels of fibrinogen (28).Groenewegen et al. reported elevated fibrinogen level as an independent risk factor for recurrent COPD exacerbations (29).We think that these findings reported for COPD are also valid for bronchiectasis patients and fibrinogen is elevated in bronchiectasis patients with airway colonization.
Although the levels of common systemic inflammatory markers (WBC count, CRP and fibrinogen) were increased in bronchiectasis patients with colonization, we were not able to find a difference in levels of TNF-α and IL-8 between study groups.This finding is similar to the literature, in which serum levels of these cytokines were reported within normal limits, despite elevations in the bronchiectatic airways (9).One previous study has reported elevated plasma TNF-α levels in bronchiectasis patients with severe disease and respiratory failure, supporting the probable relationship between systemic inflammation and disease severity (30).
Some limitations of our study merit consideration.First, the study population was relatively small; we were not able to perform detailed statistical analyses in subgroups stratified for colonization or disease severity.Second, the controls and bronchiectasis patients were not entirely matched; smoking prevalence was unexpectedly higher in the control group (active smoking 40%).This was possibly a reflection of high smoking prevalence in our country (31).Smoking is associated with inflammation and previous studies have shown elevated levels of systemic inflammatory markers, such as WBC count, CRP and fibrinogen, in active smokers (32).This is also true for leptin, which was found to increase in asymptomatic smokers (33).All these data raise the possibility that the high smoking prevalence among controls, might have masked the ongoing systemic inflammation in bronchiectasis patients of our cohort.Third, about half of the patients (52%) in the bronchiectasis group were on inhaled corticosteroid therapy, which potentially might have suppressed the inflammatory response (34).Previous studies have shown that inhaled corticosteroids suppress airway inflammation in bronchiectasis (7).Similarly, Sin et al. showed that inhaled corticosteroid may have systemic effects and can decrease serum CRP levels in COPD patients (35).
In conclusion, bronchiectasis is a disease with definite local inflammation.We were not able to show any evidence for systemic inflammation in the overall bronchiectasis cohort with respect to controls.However, there were findings suggestive of systemic inflammation in patients with colonization.We believe that colonization with pathogenic microorganisms in bronchiectasis might trigger a cascade of events leading to systemic inflammation, which in turn might contribute to disease progression.Further studies are needed to clarify the presence of systemic inflammation in bronchiectasis patients with different degrees of disease severity and colonization.

1 :
sputum was available in 12 patients.a denote for p< 0.05 when colonized patients were compared to non-colonized patients.b and c denote p< 0.05 when patients who had Pseudomonas aeruginosa colonization were compared to patients with colonization other than P. aeruginosa and to non-colonized patients, respectively.Categorical variables are expressed as n (%) and continuous variables are expressed as median (interquartile range).PA: Pseudomonas aeruginosa.NA: Not applicable, BMI: Body mass index, FEV Forced expiratory volume in one second, FVC: Forced vital capacity, HRCT: High-resolution computerized tomography, ESR: Erythrocyte sedimentation rate, WBC: White blood cell, CRP: C-reactive protein, IL-8: Interleukin-8.