Characterization of macrophage activation states in patients with cystic fibrosis

Journal of Cystic Fibrosis 9 (2010) 314 – 322 www.elsevier.com/locate/jcf

Original Article

Characterization of macrophage activation states in patients with cystic fibrosis☆ Brian S. Murphy a,b,⁎, Heather M. Bush c , Vidya Sundareshan a , Christina Davis a , Jennifer Hagadone d , Theodore J. Cory d , Heather Hoy a , Don Hayes Jr. a,b , Michael I. Anstead b , David J. Feola d a

d

Department of Internal Medicine, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA b Department of Pediatrics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA c Department of Biostatistics, 205c College of Public Health, Lexington, KY 40536, USA Department of Pharmacy Practice and Science, University of Kentucky College of Pharmacy, 725 Rose Street, Lexington, KY 40536, USA Received 25 September 2009; received in revised form 19 April 2010; accepted 25 April 2010 Available online 8 June 2010

Abstract Background: Chronic airway inflammation characterizes patients with cystic fibrosis (CF). The role of alternative macrophage activation in this disease course is unknown. Objective: We evaluated markers of alternative and classical macrophage activation in the lungs of patients with CF and evaluated these characteristics in the context of Pseudomonas aeruginosa (PA) infection, immunomodulatory drug therapy and pulmonary function. Methods: Bronchoalveolar lavage or spontaneously expectorated sputum samples were collected from 48 CF patients. Clinical data were related to macrophage surface expression of mannose receptor (MR) (up-regulated in alternatively activated macrophages) and TLR4 (up-regulated in classically activated macrophages). Also, the activity of the alternatively activated macrophage effector molecule arginase was compared among patient groups, and pro- and anti-inflammatory cytokines produced by alternatively and classically activated macrophages were measured. Results: There were significant differences between PA-infected and -uninfected patients in several clinical measurements. PA-infected patients exhibited increased use of azithromycin, up-regulation of MR on CD11b+ cells and increased arginase activity in their lung samples, and had a strong inverse relationship between MR and arginase activity to FEV1. Upon further analysis, PA-infected patients who were treated with azithromycin had the highest arginase activity and the highest number of macrophages that were MR+TLR4−, and both of these markers were inversely related to the FEV1. Conclusions: Our findings suggest an increase in both MR and arginase expression as pulmonary function declines in PA-infected patients with CF. These markers of an alternatively activated macrophage phenotype give cause for future study to define the function of macrophage activation states in the CF lung. © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Pseudomonas; Azithromycin; Pulmonary; Macrophage; Inflammation; Immunomodulation

Abbreviations: AAM, alternatively activated macrophage; AZM, azithromycin; BAL, bronchoalveolar lavage; CAM, classically activated macrophage; CF, cystic fibrosis; ECM, extracellular matrix; FEV1, forced expiratory volume in 1 s; IFNγ, interferon gamma; IL, interleukin; MR, mannose receptor; PA, Pseudomonas aeruginosa; TLR, toll-like receptor; TGF-β, transforming growth factor beta; TNFα, tumor necrosis factor alpha; UKMC, University of Kentucky Medical Center. ☆ Portions of these data have been presented at the 2009 American Thoracic Society meeting in San Diego, California. ⁎ Corresponding author. 800 Rose St., MN 672 Lexington, KY 40536, USA. Tel.: +1 859 323 5999; fax: +1 859 323 8926. E-mail address: [email protected] (B.S. Murphy).

1. Background The disease process in cystic fibrosis (CF) is in large part a result of the chronic host inflammatory response. The airways of these patients are often chronically infected by mucoid Pseudomonas aeruginosa (PA) which proliferates a cycle of airway inflammation, airway remodeling, re-infection, and tissue damage [1–3]. The ongoing response of immune cells to this chronic infection results in progressive lung destruction, increased exacerbations of disease, and increased mortality [4].

1569-1993/$ - see front matter © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2010.04.006

B.S. Murphy et al. / Journal of Cystic Fibrosis 9 (2010) 314–322

Alveolar macrophages have a fundamental role in regulating the innate host response against infection, maintaining homeostasis, and mediating inflammation. Exposure of macrophages to either the Th1 cytokine interferon (IFN)-γ or the Th2 cytokines, IL-4 and IL-13, induces two distinct phenotypes deemed classically (CAM) or alternatively activated macrophages (AAM), respectively [10,11,22,23]. These cells function as part of a comprehensive host defense process that includes initiation of inflammation, clearance of pathogens and extracellular debris, tissue homeostasis, and repair [5]. The combined action of IL-4 and IL-13 provides an elegant strategy for host protection against certain infections, but in other cases (e.g. asthma) the effector functions induced by these cytokines can have pathologic consequences [29]. Recent studies have described the importance of the AAM effector molecule arginase in patients with asthma, chronic obstructive lung disease (COPD), CF, pulmonary hypertension, and interstitial pulmonary fibrosis [30]. AAMs and CAMs have been defined on a continuum of activation states [7,8] with AAMs typically demonstrating an increased expression of mannose receptor (MR) [9], low expression of toll-like receptor 4 (TLR4) [11], and up-regulation of arginase [12] relative to CAMs. The function of AAMs has mostly been defined in the context of infections with pathogens that trigger Th2-type responses and in fibrotic diseases where chronic infections have not been present [13]. Data describing the role of AAMs in response to bacterial infections is scant. In CF patients who are chronically infected with PA, the immune response is triggered and maintained, but this response is ineffective in the clearance of mucoid strains of PA and promotes alveolar wall thickening, airway remodeling, and eventual pulmonary decline [6,13,24,31]. The contribution of AAMs to the process of lung remodeling and fibrosis is just beginning to be evaluated. AAMs are involved in the fine tuning of CAMs and Th1 inflammatory responses by driving Th2 polarization, actively inhibiting CAM-driven inflammatory processes, and mediating repair through up-regulation of the arginase pathway and other proteins including TGFβ [11,13,14]. Arginase has been postulated to be an important mediator of airway remodeling and lung fibrosis in patients with CF [19], and others have described how arginase-controlled production of collagen precursors increases in CF patients in an age-dependent manner [25]. Whether AAMs are helpful (through suppression of dysregulated inflammatory responses) or harmful (through increased fibrosis development) during a chronic infection such as occurs with PA in CF patients remains unclear. We have previously reported that azithromycin (AZM) can polarize macrophages in vitro toward an AAM-like phenotype [20]. Because of this and because of the effective clinical use of AZM as an immunomodulatory agent in CF patients, we are interested in describing the role of AAMs in the pathophysiology of CF. The present study describes the activation status of CD11b+ cells in the airways by characterizing surface marker expression (MR and TLR4), airway cytokine concentrations, and expression of arginase in PA-infected and -uninfected CF patients. We then relate these to the patient's pulmonary function as defined by the forced expiratory volume in 1 s (FEV1). We postulated that PA-infected patients would have a predominant AAM phenotype in their bronchoalveolar lavage (BAL) or sputum and that AZM-

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treated patients would have an increased proportion of markers associated with AAMs than those not treated with AZM. These data will lay a foundation for future studies examining the function of various macrophage phenotypes in the CF lung. 2. Methods 2.1. Population Forty-eight patients with CF participated in this observational study. Subjects were match based on PA status only. The diagnosis of CF had been confirmed by genetic analysis (Ambry Genetics, CA) demonstrating two disease causing mutations and/ or 2 separate sweat chloride tests (N 60 mEq/l) and clinical features consistent with the disease in all subjects. BAL fluid was collected from 17 acutely ill patients undergoing bronchoscopy as part of their medical care at the University of Kentucky Medical Center (UKMC). Sputa were collected from 31 clinically stable CF patients (i.e., no acute exacerbation, upper respiratory infection, or requirement for an antibiotic (other than AZM maintenance) during the 4 weeks prior to collection) who were able to spontaneously expectorate sputa during routine clinic visits at UKMC. Table 1 lists the characteristics of the enrolled patient population. Overall, 27 male and 21 female patients were enrolled between the ages of 1 and 50 years. The cohort was under the age of 25 years with the exception of two participants who were 49 and 50 years old. Spirometry was performed using Knudson criteria [15] on the same visit as the sputum sample collection or within a week before the BAL sample. Serum C-reactive protein and leukocyte count were measured on the same day as the sample was collected. Table 1 Patient characteristics.

N Age, years ⁎ Sex, (% male) FEV1 (% predicted) ⁎ Source of sample ⁎ BAL Sputum C reactive protein ⁎ Peripheral leukocytosis ⁎ Other infection a, ⁎ Systemic azithromycin ⁎ Systemic steroids Inhaled steroids CFTR mutation Homozygous F508del Heterozygous F08del Non-F508del Unknown

Overall

Pseudomonasinfected

NonPseudomonas

48 14 (1–50) 56.2% 65 (13–125)

25 16.5 (8–50) 48% 44.5 (13–83)

23 13 (1–25) 65.2% 89 (39–125)

35.4% 64.6% 0.7 (0.5–16.5) 8.6 (4–21.9) 68.8% 64.6% 16.7% 54.2%

44% 56% 1.7 (0.5–16.5) 10.6 (4.4–21.9) 48% 84% 20% 64%

26.1% 73.9% 0.55 (0.5–1.8) 7.1 (4–14.6) 91.3% 43.5% 13% 43.5%

47.9% 35.4% 8.3% 8.3%

44% 36% 12% 8%

52.2% 34.8% 4.3% 8.7%

Note: continuous variables are described with median and range (min–max) or as % of group. a Other infection includes one or more of the following: Staphylococcus aureus (MRSA and MSSA), H. influenzae, S. maltophilia, S. marcescens, A.baumannii, K. oxytoca, A. xylosoxidans, B. bronchiseptica, and A. fumigatus. ⁎ p b 0.05 between groups.

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Patients were described as receiving AZM, oral steroids or inhaled steroids if they had received the treatment for at least one month prior to the sample collection. AZM doses ranged between 250 mg and 500 mg daily or every other day. Subjects and/or legal guardians provided written informed consent/assent as approved by the Internal Review Board at UKMC. 2.2. Sample processing Two to 5 ml of sputum or BAL fluid was collected and immediately placed on ice. For BAL samples, up to 60 ml of room temperature saline was used for the lavage. A 3–5 ml aliquot of the first lavage was obtained for analyses for the purposes of this study. Sputum samples were processed by digestion in 0.1% diothiothreitol and DNase 30 μg/ml for 30 min [28]. The sputum sample was deemed adequate for further analysis if there were b 10 epithelial cells and N25 white blood cells per lower powered field (10×). Digest supernatants were frozen at −80 °C. Cells were washed thrice to create single cell suspensions and immediately processed. Since each lavage may have had a different diluting effect on the concentration of BAL fluid contents, cytokine concentrations were normalized to total protein concentration to correct for this as has been previously described [16]. Total protein concentrations were assessed using the Micro BCA (bicinchoninic acid) protein assay reagent kit (Pierce Biotechnology). Microbiological analysis of patients' sputa and BAL fluid included the following: 1) a semi-quantitative evaluation on nonselective medium, 2) isolation of respiratory pathogens, and 3) antimicrobial susceptibility testing by single-disk diffusion test (Kirby–Bauer). These studies were performed by the UKMC Clinical Microbiology Laboratory using methods approved by the Clinical and Laboratory Standards Institute. 2.3. Flow cytometry Cells isolated from BAL fluid and sputum were incubated with fluorescently-labeled monoclonal antibodies specific for CD11b, MR, and TLR4. The monocyte marker CD11b is up-regulated on infiltrating monocytes/macrophages and was used to identify this group of cells, and MR and TLR4 were chosen based on their differential levels of expression in AAMs and CAMs, respectively [8–10]. Cells were analyzed by multiparameter flow cytometry using a FACSCalibur Flow Cytometer (BD Biosciences, Mountain View, CA). Greater than 50,000 events/sample were examined. Cells were classified by gating on characteristic size and granularity, thereby eliminating lymphocytes, apoptotic cells, and debris. The remaining fraction positive for CD11b surface expression were then individually assessed for expression of MR and TLR4. 2.4. Arginase activity Arginase activity was assessed via the measurement of the conversion of L-arginine to urea [17]. Briefly, 10 mM MnCl2 in Tris–HCl was incubated with cell lysates and L-arginine overnight. The reaction was terminated by the addition an acid solution. Alpha-isonitrosopropiophenone 9% in ethanol

was added to each tube and the OD was read using a 490 nm filter. Readings were compared to a standard curve of known urea concentration. A unit of arginase activity converts 1 μmol of L-arginine to urea per minute. 2.5. Cytokine assays IL-10, IL-12, TNFα, IL-6, IL-1β, and IL-8 from CD11b+ cells were simultaneously measured in the cell lysates using Cytometric Bead Array kits (BD Pharmingen, San Jose, CA) with a limit of detection of 3.3 pg/ml, 4 pg/ml, 3.7 pg/ml, 2.5 pg/ml, 7.2 pg/ml, and 3.6 pg/ml, respectively [18,21]. Bead populations with distinct fluorescence intensities coated with capture antibodies specific for each cytokine were incubated with detection antibodies along with each sample. Fluorescence intensities were assayed by flow cytometry and compared with a standard curve to determine the concentration. 2.6. Statistical analyses The primary objective was to ascertain whether AAM surface markers and arginase activity correlated with a particular patient profile based upon FEV1 and PA infection status. The secondary objective was to determine if AZM was an important determinant of the predominant macrophage phenotype. Patients were grouped based on PA-infection status. Categorical data were analyzed using the chi-square test of independence or Fisher's exact test where appropriate. Data that failed normality testing were compared using the Mann Whitney Rank Sum Test, otherwise a two-sample t-test was performed. Surface receptor expression and arginase activity were correlated to FEV1 using the Pearson Product Moment Correlation with subsequent forward and backward stepwise multiple regression. Since the data presented in this study was of a hypothesis-generating and exploratory in nature, multiple test adjustments were not performed. Differences were determined to be statistically significant when a p-value of b 0.05 was attained. All analyses were performed using SigmaStat 3.5 (Systat Software, Chicago, IL). 3. Results 3.1. Patient groups Patient characteristics and differences between the PAinfection groups are shown in Table 1. There was no significant difference in the number or males and females between the PAinfection groups (p = 0.3). Gender made no difference when comparing arginase activity, surface receptor expression, and cytokine concentrations between the 2 groups (p N 0.1). The median age of the PA-infected group was significantly higher (p b 0.05), and patients who were above the median age of 14 were more likely to be infected with PA (p b 0.001). However, there were no differences in arginase activity, surface receptor expression or cytokine concentrations between males and females. The groups also did not differ in terms of CFTR gene mutations present (p N 0.5).

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The overall median FEV1 % predicted was 65% with the median FEV1 values of the PA-infected and -uninfected groups differing significantly (44.5% vs. 89%, respectively; p b 0.05). The median FEV1% between males and females (regardless of age, PA status or AZM status) was not significantly different (p = 0.57). As expected, the FEV1 was lower among patients who were older than the median compared to those who were younger (80.2% vs. 52%, respectively; p = 0.001). There was no significant difference in the PA-infection status between patient groups whose samples were obtained by spontaneous expectoration vs. BAL sample (p = 0.32). Serum markers varied significantly between the groups with PAinfected patients having a higher median peripheral leukocyte count and C reactive protein value (p b 0.05). Finally, patients in the two groups received different therapies. Patients infected with PA were more likely to have received AZM N 4 weeks (p b 0.05). Importantly, there was no difference between AZM use between males and females, no difference in AZM use between patients above and below the median age, and the rates of inhaled and oral steroid use were not significantly different between groups (p N 0.3). 3.2. Surface markers, arginase activity and cytokines We then asked whether the prevalent macrophage activation state as measured by surface receptors, cytokine production and arginase activity was different between the groups. While leukocyte differential counts were not performed, the mean percentages ± standard error of CD11b+ cells (infiltrating monocytes) were 35.0% ± 4.3% and 39.5% ± 4.5% for the PA-infected and -uninfected groups, respectively (p = 0.49). Differences in the mean percentages of CD11b+ cells between BAL and sputum samples were not observed (p = 0.98). The median percentage of CD11b+ cells expressing high levels of MR was significantly higher in the PA-infected group than in the PA-uninfected group (32.46% vs. 13.03%, p = 0.014) (Fig. 1A). However, there was no significant difference in the median mean fluorescence intensity (MFI) of MR (p N 0.1). TLR4 expression on CD11b+ cells did not differ between the groups (p N 0.5). Samples from PA-infected patients had higher arginase activity than samples from PAuninfected patients (1.99 × 10− 4 U/mg protein vs. 8.01 × 10− 5 U/ mg protein; p b 0.001) (Fig. 1B). Finally, cytokines were measured in BAL and sputum and normalized to 500 μg of total protein. There were no significant differences between the concentration of TNFα, IL-6, and IL-10 between the groups (p N 0.1). However, PA-infected patients had higher concentrations of IL-12 (16.2 pg/ml vs. 11.5 pg/ml, p = 0.02), IL-1 (398.02 pg/ml vs. 198.7 pg/ml, p = 0.03), and a trend toward higher IL-8 concentrations (5304.2 pg/ml vs. 2910.35 pg/ml, p = 0.07) (Fig. 2). 3.3. Relationship of activation markers to FEV1 The relationship with markers of alternative macrophage activation and disease severity were analyzed utilizing FEV1 as a clinical measurement of a patient's pulmonary status. Correlation coefficients (r) and p-values for each relationship tested are given

Fig. 1. Box and whisker plots of macrophage markers in Pseudomonas-infected and -uninfected patients as measured by (A) percentage of CD11b+ cells with up-regulation of MR expression, and (B) arginase activity. MR expression on CD11b+ cells was determined by flow cytometry. Arginase activity was measured by the fluorimetric measurement of the conversion of L-arginine to urea. p b 0.0001 for differences between median MR expression and arginase activity. Box plots demonstrate medians, range, 5th and 95th percentiles with outliers demonstrated for each.

in Table 2. Patients infected with PA were more likely to have an FEV1 less than the median (OR = 15, CI 3.6–61.9). Overall, there was a significant inverse relationship between the percentage of CD11b+ cells expressing MR and FEV1 (r = −0.55, p = 0.001) (Fig. 3A) and between FEV1 and the degree to which MR was expressed as defined by the MFI (r = −0.304, p = 0.05). Further subset analysis revealed a negative trend in the relationship between MR expression and FEV1 in PA-infected patients (r = −0.407, p = 0.06) and a strong inverse relationship between FEV1 and MR in the PA-uninfected patients (r = −0.662, p = 0.001). This result corresponds with the median MR data above, as infected subjects were primarily those within the lower FEV1 range. There was no significant relationship between expression of TLR4 on CD11b+ cells and FEV1 (p N 0.1). Similarly, we sought to characterize the relationship between arginase expression and disease severity. We observed a strong inverse relationship between arginase activity and FEV1 when analyzing values from all patients (r = −0.483, p = 0.002) (Fig. 3B). This association was strongest in the PA-infected patients (r = −0.567, p = 0.02), and further analysis of this patient subset showed that the relationship between arginase activity and FEV1 was greatest among those whose FEV1 was b 65% predicted (p = 0.003).

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Fig. 2. Comparison of median cytokine expression in cell lysates from patients with CF who were Pseudomonas-infected and -uninfected. Cytokine concentration was determined by a multiplex assay. Box plots demonstrate medians, range, and 5th and 95th percentiles with outliers demonstrated for each cytokine.

TNFα concentrations in the samples also negatively correlated to FEV1 (r = −0.486; p = 0.016). There was a trend in IL-10's inverse relationship to FEV1 (r = −0.36, p = 0.08) and between the concentration of IL-12 and FEV1 (r = −0.524, p = 0.06). However, in subset analyses of the PA-infected group, IL-12 concentration displayed a strong direct relationship to FEV1 (r = 0.474, p = 0.01). The ratio of the anti-inflammatory cytokine IL-10 to the proinflammatory IL-12 was significantly inversely related to FEV1 (r = −0.739, p = 0.038). None of the other cytokine concentrations measured correlated to FEV1 (p N 0.2).

3.4. Comparison of sample source Because of the difference between the source of the samples (BAL vs. sputa) in the groups (p = 0.065), subset analysis of the surface receptors and arginase activity was performed. The percentages of isolated cells that were CD11b positive were compared between sample sources. Mean percentages from BAL and sputum samples that were CD11b positive were not significantly different (37.5 ± 24.3% and 37.8 ± 15.8%, respectively, p-value = 0.975). There was no difference in relationships

Table 2 Relationship of FEV1 to markers. MR+TLR4− expression

Arginase activity

TNFα

Grouped by:

MR expression r

p

r

r

p

r

p

r

p

Overall PA-infected PA-uninfected AZM treated AZM untreated PA+AZM+ PA+AZM− PA− AZM+ PA− AZM−

−0.550 −0.407 0.662 NS −0.391 NS NS NS NS

0.001 0.06 0.001

NS NS NS − 0.482 NS − 0.615 NS NS NS

− 0.483 − 0.567 NS − 0.380 NS − 0.69 NS NS NS

0.002 0.02

− 0.486 NS NS − 0.728 NS NS NS NS NS

0.016

− 0.36 NS NS NS NS NS NS NS NS

0.08

NS = non-significant p-value N 0.1.

0.08

p

0.048 0.039

0.025 b0.001

IL-10

0.004

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and FEV1 in the AZM-treated patients (r = 0.342, p = 0.06) that was not observed in the untreated group (p = 0.16). Among AZM-treated patients, FEV1 and TNFα concentration were inversely related (r = − 0.728, p = 0.004). No other relationships were found between FEV1 and any of the other cytokines. Further subgroup analyses were performed taking into consideration both PA infection and AZM treatment status. The median arginase activity in the sample differed between AZM+PA+ (n = 20) and AZM−PA− (n = 12) groups (p = 0.006) and between AZM+PA− (n = 8) and AZM−PA− (n = 8) groups (p = 0.015). The relationship between arginase activity and FEV1 was found to be greatest among AZM+PA+ subjects (r = − 0.69, p b 0.001) (Fig. 4A). Likewise, AZM+PA+ patients had the highest percentage of CD11b+ MR+TLR4− cells (6.5%, p = 0.009). Among these patients, this same cell population inversely correlated to FEV1 (r = − 0.615, p = 0.039) (Fig. 4B). No significant correlations were found between FEV1 and arginase activity, surface receptor expression, or cytokine expression in any of the other subgroups (p N 0.1). Because of the overlap in AZM and oral steroid use in the groups, we repeated the analysis and excluded AZM-treated patients who also were receiving oral steroids. For the 13 patients treated with AZM and not steroids, there was no significant change of the relationships that are reported above for MR expression and arginase activity (p b 0.02). In the 12 patients who were treated with either inhaled or oral steroids but not AZM, no significant correlations were found between FEV1 and any of the markers measured in this study (p N 0.1). Fig. 3. Relation of FEV1 and (A) surface expression of MR on CD11b+ cells and (B) arginase activity in sample lysates from PA-infected and -uninfected patients. Lines represent the linear regression analysis between the variables for the infected (– – –) and uninfected (—) groups.

between FEV1 and macrophage surface markers, cytokine concentrations or arginase activity when the data was analyzed only in terms of the source (p N 0.4 for all parameters). 3.5. Comparison of AZM and steroid treatment Since our previous work demonstrated that AZM can induce properties of alternative macrophage activation, we were interested in evaluating macrophage activation markers relative to AZM exposure. There was no significant difference between AZM-treated and -untreated patients in regards to the median arginase activity (1.19 × 10− 4 vs. 8.95× 10− 5 U/mg protein, respectively; p N 0.5), surface receptor expression (p N 0.2), or cytokine concentration (p N 0.5). Table 2 shows there were no significant relationships between FEV1 and the percentage of CD11b+ cells that had up-regulated MR expression among either AZM-treated or -untreated subjects (p N 0.05); however, the percentage of CD11b+ MR+TLR4− cells in the AZM-treated group directly related to the FEV1 (r = −0.482, p = 0.048). Arginase was inversely related to FEV1 in the AZM-treated group (r = −0.38, p = 0.025) but not in the AZMuntreated group (p = 0.24). Additionally, there was a trend toward significance in the relationship between TLR4 surface expression

3.6. Infection with a non-PA organism Overall 68.8% of the total patients were infected with at least one other organism other than PA (including Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, Serratia marcescens, Acinetobacter baumannii, Klebsiella oxytoca, Achromobacter xylosoxidans, Bordetella bronchiseptica, and Aspergillus fumigatus). PA-uninfected patients demonstrated a higher prevalence of infection with at least one additional organism in the lungs as compared to the PAinfected group (91.3% vs. 48%, respectively; p b 0.05). The predominant non-PA organism was S. aureus (methicillin resistant (MRSA) and sensitive (MSSA)). Twenty percent of PA-infected patients and 26% of the PA-uninfected patients were infected with MRSA; 20% of the PA-infected patients and 52% of the PA-uninfected patients were infected with MSSA. To evaluate possible interactions between PA and/or any of these other organisms that could affect macrophage activation states, subset analyses were performed of patients who were infected with only PA, those who were infected with PA and another organism, and those who were only infected with a nonPA organism. Infection with organisms other than PA did not significantly affect whether patients had FEV1 measurements above or below the FEV1 median (OR = 0.73, CI = 0.03–18.19). Analyses revealed no significant interactions between any nonPA organism and FEV1 or any of the inflammatory markers evaluated in this project, although power to detect differences

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infection status (p b 0.001), AZM treatment status (p = 0.003), macrophage MR surface expression (p = 0.013), and arginase activity (p = 0.032). Therefore, it can be concluded that even controlling for other factors, MR and arginase activity are still significant. 4. Discussion

Fig. 4. Relation of FEV1 to (A) arginase activity and (B) percentage of CD11b+ cells that were both MR+ and TLR4− among patients who are AZM-treated and PA-infected. Lines represent the linear regression analysis between the variables.

was reduced in these small subsets. Further, co-infection subgroup analyses did not significantly change any of the reported differences or correlations that exist between the PA-infected and PA-uninfected groups.

3.7. Prediction of FEV1 To determine whether characteristics of AAMs were predictive of a patient's overall disease severity, we performed a multiparameter modeling analysis of the impact of all measured variables upon FEV1. Of the factors evaluated, the FEV1 in CF patients enrolled in this study can best be predicted from a linear combination of the patient's age (p b 0.001), PA

This study shows that the BAL fluid and sputum from PAinfected patients with CF contain a population of CD11b+ cells that have an increase in characteristics consistent with an AAM phenotype (i.e., higher percentage of MR expression and high arginase activity). Further, AZM treatment of PA-infected patients was associated with the highest percentage of CD11b+ MR+TLR4− cells and highest arginase activity. This is the first study correlating select characteristics of alternative macrophage activation to pulmonary function and is the first study evaluating the association between these AAM markers and the use of AZM in patients with CF. These data support the hypothesis that AAMs are relevant in the pathophysiology of chronic PA infection in CF patients and relate to our previous description of AZM's ability to polarize the macrophage toward the AAM phenotype [3]. While the role of alveolar macrophages in response to lung pathogens in CF is not completely understood, previous studies have implicated a potential role of the AAM-activation cytokines IL-4 and IL-13 in the disease process. Hartl et al. reported that BAL fluid from patients with CF infected with PA had higher IL-4 and IL-13 concentrations and lower levels of IFNγ compared with uninfected patients, and BAL fluid levels of IL-4 and IL-13 correlated inversely with FEV1 [5]. Additional studies have shown that, in response to PA outer membrane proteins, monocytes from PA-infected CF patients produce more IL-4 and less IFNγ than monocytes from non-PA-infected patients [6]. These studies support the notion that alternative macrophage activation may be prevalent in CF patients with declining pulmonary function. Our work extends these investigations further by examining the impact that this shift has upon the activation state of the macrophage compartment in CF patients. There are also further implications of a relationship between a CD11b+ MR+TLR4− cell and high activity of arginase-1, recognized as one of the most specific AAM markers and an important mediator of airway remodeling and lung fibrosis [19]. However, it remains to be determined whether the increased prevalence of AAMs in PAinfected patients is a beneficial alteration of an immune system that is compensating for a progressive pathology or if this indeed contributes to airway damage and fibrosis. Several possibilities exist to explain the expression of MR and increased arginase activity in patients infected with PA. The IL-4/IFNγ imbalance that exists in CF patients [5] suggests that the cytokine environment in chronic PA-infected lungs drives the macrophage phenotype. Because the main purpose of this project was to characterize the macrophage activation status we did not measure IL-4 and IL-13 concentrations. However, it is possible that increased IL-4/IL-13 in the patients with decreasing FEV1 could have driven the macrophage response toward a prevalence of alternatively activated macrophages.

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Still, there were no significant differences between anti- and pro-inflammatory cytokines between the AZM and PA subgroups that could alone explain these differences. Due to the observational nature of this study, there are several limitations. As patients decline clinically, they are more likely to be infected with PA, and physicians are more likely to start empiric therapies with AZM. Hence, we were unable to infer causality between the macrophage phenotype that predominates in the lung of the CF patient at any point in time to the patient's lung function or to medications such as AZM with which a patient is treated. Additionally, because some samples were collected from sputum and some were collected during BAL, we are unable to completely compare differences that could occur between alveolar macrophages and macrophages in the central airways, respectively. Differences in neutrophil and macrophage counts between sputum and BAL fluid have been described previously [37]; it is possible that by increasing the numbers of patients enrolled in the study or by comparing patients matched for age and disease severity we would detect differences in macrophage phenotypes between the sample sources. It is important to note that we primarily matched patients based on PA-infection status. Our study confirms the results of other studies that have shown high concordance between expectorated sputum and BAL samples in regard to microbial burden [38]. Nonetheless, the subset analyses of AZM exposure and PA infection status suggest that the strongest correlation between AAM markers and decline in pulmonary function are found among patients who are treated with AZM and infected with PA, regardless of the source of the sample. It is difficult in this study to separate the effects of PA and AZM since there is substantial overlap of these patient groups. However, we believe that AZM directly affects macrophage activation states in PA-infected patients for several reasons: 1) quantitative microbiological data revealed no significant difference in PA bacterial burden between groups based on AZM treatment status, indicating that the AAM phenotype is not exclusively an effect of clearance of PA; 2) there were overall correlations between MR and arginase activity to FEV1 that were found to be strongest among PA-infected patients treated with AZM; and 3) subgroup analyses of patients co-infected with nonPA organisms revealed no changes in the differences among inflammatory markers or correlations to lung function. An alternative explanation of the data could be due to AZM's ability to alter bacterial virulence factor production including biofilm proteins [27]. While this is possibly a contributing factor, we believe that AZM directly affects the macrophage based on our previous findings. In our in vitro studies with J774 macrophages, we demonstrate that AZM will polarize cells to an AAM-like phenotype not only when stimulated with PA (unpublished) but also when stimulated with purified LPS [20]. The compelling aspect of this work is that AZM polarizes macrophages to AAM despite exposure to the classical activation stimuli IFNγ and LPS. We believe this is relevant in this current study where patients have continual exposure to PA and its LPS. Our data support the notion that the preference for AAMs in the lungs of PA-infected CF patients is dependent on additional factors other than the exaggerated cytokine response as pro-

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inflammatory and not anti-inflammatory cytokines predominated in the lungs of the PA-infected patients. However, it is possible that the macrophage receptor expression and arginase activity that we observed in these patients is a result of changes in cell signaling pathways. While the effect of CFTR expression on alveolar macrophage's activity is still debated [32,33], it has been reported that CFTR is a negative regulator of NF-κB mediated innate immune response [34]. A possible link between the NF-κB signaling pathway and the control of macrophage activation characteristics has been reported previously [26], but the molecular pathways for induction of different macrophage gene expression programs during PA infection or AZM treatment has not yet been described in any model, regardless of CFTR expression. It should be noted that there were no significant differences among CFTR mutations between the PA or AZMtreatment groups in this current study, leading us to conclude that CFTR expression is not a significant factor in PA or AZM's polarization of the macrophage phenotype. Still, other variations in cell signaling and cell programming could cause alveolar macrophages to differentially express effector molecules, surface receptors, and cytokines associated with AAMs and CAMs. Responses governed by NF-κB represent a plausible explanation for the hyper-responsive and constitutive inflammation seen in the lungs of patients with CF. Since AZM has been shown to blunt this hyper-responsiveness by inhibiting NF-κB signaling [35,36], this may, in part, account for the differences we have observed in the macrophage activation states. Our results indicate that characteristics of alternative macrophage activation predominate in PA-infected patients with CF and that this cell population may be relevant to the remodeling and fibrotic changes that occur during this disease. There are likely complex interactions between several immune cell populations that govern inflammation and defense against bacterial pathogens that predominate in the lungs of patients with CF. Differences in macrophage activation likely play an important role during the disease process, and it remains to be determined whether induction of alternative macrophage activation with drugs such as AZM is beneficial. The potential long-term effect of an increase in AAMs, their effector molecules such as arginase, and their subsequent influence on airway remodeling has only been demonstrated in other lung diseases where chronic infections have not been present [13]. Future studies evaluating long-term use of AZM in PA-infected patients should include evaluation of the contribution that macrophage phenotype alteration has upon disease severity and progression. References [1] Kosorok MR, Jalaluddin M, Farrell PM, Shen G, Colby C, Laxova A, et al. Comprehensive analysis of risk factors for acquisition of Pseudomonas aeruginosa in young children with cystic fibrosis. Ped Pulmonol 1998;26(2): 81–8. [2] Li Z, Kosorok MR, Farrell PB, Laxova A, West S, Green C, et al. Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. JAMA 2005;293(5):581–8. [3] Murphy TF, Brauer AL, Eschberger K, Lobbins P, Grove L, Cai X, et al. Pseudomonas aeruginosa in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177:853–60.

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