Azithromycin Reduces Exaggerated Cytokine ... - ATS Journals

Azithromycin Reduces Exaggerated Cytokine Production by M1 Alveolar Macrophages in Cystic Fibrosis ˆ le2, Patrick Lebecque3, Sandra Lo Re2, Magali Meyer1, Franc xois Huaux2*, Ximena Gavilanes1, Sybille van den Bru 2 4 1 1 Dominique Lison , Bob Scholte , Pierre Wallemacq , and Teresinha Leal * 1

Department of Clinical Chemistry, 2Unit of Industrial Toxicology and Occupational Medicine, and 3Division of Paediatric Pulmonology, Universite´ Catholique de Louvain, Brussels, Belgium; and 4Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands

Macrophages phagocyte pathogenic microorganisms and orchestrate immune responses by producing a variety of inflammatory mediators. The cystic fibrosis (CF) transmembrane conductance regulator chloride channel has been reported to be of pivotal importance for macrophage functions. The exact phenotype and role of macrophages in CF is still unknown. Alveolar and peritoneal macrophages were monitored in CF mice homozygous for the F508 del mutation and in wild-type control animals. Classical (M1) and alternative (M2) macrophage polarization and responses to LPS from Pseudomonas aeruginosa were investigated, and the effect of azithromycin was examined in both cell populations. We show that alveolar macrophage counts were 1.7-fold higher in CF as compared with wild-type mice. The macrophage-related chemokine, chemokine C-C motif ligand (CCL)-2, was found to be at least 10-fold more abundant in the alveolar space of mutant mice. Cell count and CCL-2 protein levels were also increased in the peritoneal cavity of CF mice. Both M1 and M2 macrophage polarization were significantly enhanced in alveolar and peritoneal cells from F508del-CF mice as compared with control animals. LPS-stimulated expression of proinflammatory mediators, such as nitric oxide synthase-2, IL-1b, and CCL-2, was increased, whereas anti-inflammatory IL-10 expression was decreased in CF macrophages. Azithromycin, added to cell cultures at 1 mg/liter, significantly reduced proinflammatory cytokine expression (IL-1b, CCL-2, TNF-a) in M1-induced CF and wild-type alveolar macrophages. Our findings indicate that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical and alternative activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages. Keywords: cystic fibrosis; cystic fibrosis transmembrane conductance regulator; inflammation; macrophages; azithromycin

Cystic fibrosis (CF) is the most common fatal genetic disorder affecting the Caucasian population. The disease is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, resulting in malfunctioning or reduction of the CFTR protein. CFTR protein is expressed at the apical membrane of epithelial cells, where it regulates cAMP-dependent chloride transport. Although many organs are affected in

(Received in original form April 19, 2008 and in final form January 12, 2009) * These authors contributed equally to this article. This work was supported in part by Fonds de la Recherche Scientifique (FNRS) Me´dicale and Actions de Recherche Concerte´es, Communaute´ Francxaise de Belgique, Direction de la Recherche Scientifique. F.H. is a Research Associate with the FNRS, Belgium. Cftrtm1eur breeding pairs were provided with support of the European Economic Community European Coordination Action for Research in Cystic Fibrosis program grant EU FP6 LSHM-CT-2005-018932. Correspondence and requests for reprints should be addressed to Teresinha Leal, M.D., Ph.D., Clinical Chemistry, Universite´ catholique de Louvain, Ave Hippocrate 10, Brussels, Belgium. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 41. pp 590–602, 2009 Originally Published in Press as DOI: 10.1165/rcmb.2008-0155OC on February 24, 2009 Internet address: www.atsjournals.org

CLINICAL RELEVANCE The exact phenotype and role of macrophages in cystic fibrosis (CF) is unknown. Azithromycin exerts beneficial effects in CF, but its precise mechanism of action remains unclear. We show here that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical M1 and alternative M2 activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages.

CF, pulmonary disease is the major cause of morbidity and mortality. The lung disease is characterized by a vicious cycle of airway obstruction, neutrophil-dominated inflammation, and infection, culminating in colonization with Pseudomonas aeruginosa. CF airway inflammation has long been considered as a consequence of chronic bacterial infection. However, it has been recently suggested that lung inflammation could represent a primary event due to the genetic defect. In vitro studies performed in airway epithelial cells have described a spontaneous increased production of proinflammatory cytokines (IL-8, TNF-a) and an exaggerated inflammatory response to bacterial stimuli (1–4). Furthermore, it has been shown, in an elegant experimental model of human fetal tracheal grafts in mice with severe combined immunodeficiency, that CF airways display a proinflammatory status even in the absence of any detectable infection, and that the response to P. aeruginosa challenge is earlier and exacerbated in CF (5). These findings led to the suggestion that inflammation in CF may be intrinsically dysregulated. However, the precise cellular and biochemical events initiating and regulating spontaneous inflammation in CF remain unclear. Limiting the effects of the inflammatory process seems to be very important in slowing the decline of lung function in CF. Beneficial effects of anti-inflammatory therapies have been reported in several clinical trials, but therapeutic strategies combining anti-inflammatory properties with an acceptable profile of adverse effects are still needed (6). The macrolide antibiotic azithromycin has been used to treat patients with CF, with significant clinical benefits characterized by improvement in lung function, reduction in pulmonary exacerbations, and fewer courses of antibiotic use, without serious adverse effects (7–11). However, apart from its antibactericidal actions, the precise mechanism of action of azithromycin remains unclear. Our previous study (12), performed in F508del homozygous mice (13), indicates that azithromycin exerts relevant antiinflammatory properties. We have shown that the F508del-CF mouse model (13) displays spontaneous and exaggerated LPSinduced macrophage infiltration in the airways. Both spontaneous and induced inflammatory responses were attenuated after azithromycin treatment (12).

Meyer, Huaux, Gavilanes, et al.: Azithromycin and Macrophage Polarization in CF

Macrophages are an important component of the innate immune defense, and play a key role in the initiation and orchestration of inflammatory responses, particularly in the lungs. It is well known that, in response to environmental signals, macrophages have the ability to develop different phenotypes with distinct physiological activities (14, 15). The classical M1 macrophage activation, which can be induced by stimulation with IFN-g and LPS, is associated with increased proinflammatory cytokine production, and is involved in cytotoxicity and microbicidal activity. Alternatively, M2 macrophage activation, which can be induced by IL-4 and IL-13 stimulation, is involved in inflammatory responses and adaptative type I immunity, and promotes angiogenesis, tissue remodeling, and repair. These functional differences are reflected in the expression of markers that can be monitored at mRNA, protein, or biochemical levels. We hypothesized that CF macrophages express a proinflammatory phenotype, and that azithromycin modulates macrophage biology. We examined the expression of different markers of M1/M2 macrophage polarization in freshly collected, noncultured and primary-cultured macrophages isolated from either the bronchoalveolar space or the peritoneal cavity of F508delCF and normal homozygous mice (13). We also investigated the degree of responsiveness of CF and wild-type macrophages to LPS-induced inflammation, and whether azithromycin modulates macrophage cell phenotype and inflammatory responses.

MATERIALS AND METHODS Animal Model Adult female CF mice homozygous for the F508del mutation in the 129/FVB outbred background (13) and their wild-type homozygous normal littermates were housed in specific pathogen-free conditions, with free access to food and water. To prevent CF intestinal obstruction, Movicol (55.24 g/L; Norgine, Heverlee, Belgium) was administered in drinking demineralized and acidified water. The genotype of each animal was checked at 21 days of age using Taqman quantitative PCR multiplex analysis of tail clip DNA. Primers and minor groove binder (MGB) probes designed for allele-specific PCR using Primer Express Software (Applied Biosystems, Foster City, CA) were as follows: forward primer, 59-TTTCTTGGATTATGCCGGGTA-39; reverse primer, 59-TTGGCAAGCTTTGACAACACT-39; wild-type–specific probe, 59-FAM-AAACACCAAAGATGATATT-MGB-39; mutantspecific probe, 59-VIC-AACACCAATAATATTTTC-MGB-39. These studies and procedures were approved by the local ethics committee for animal welfare and conformed to the European Community regulations for animal use in research (CEE no. 86/609).

Cell Sampling and Culture Primary cultures of resident peritoneal and alveolar macrophages were established from peritoneal cavity lavage (PCL) and bronchoalveolar lavage (BAL) of the bronchoalveolar space from the same group of mice. Animals were killed by subcutaneous injection of 24 mg sodium pentobarbital (Certa, Braine-l’Alleud, Belgium). The peritoneal cavity was first lavaged with 10 ml of sterile NaCl 0.9%. PCL was collected from wild-type or F508del-CF mice, pooled, and centrifuged (280 3 g, 10 min, 48C). BAL was then performed by cannulating the trachea and lavaging the lungs four times with 1 ml of sterile 0.9% NaCl. This step was repeated with three additional volumes (1 ml) of sterile saline. The 4 ml from all lavage fractions were pooled for each animal group and centrifuged (280 3 g, 10 min, 48C). Cell pellets were resuspended in Hanks’ balanced salt solution medium (Invitrogen, Paisley, UK) to determine total and differential cell counts, which were performed on cytocentrifuge preparations and stained with Diff Quick (Dade, Brussels, Belgium). Supernatants were stored at 2208C for further biochemical analyses. Cell pellets collected by an additional centrifugation (280 3 g, 10 min, 48C) were resuspended in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS (Invitrogen), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin (Invitrogen). Cells were seeded at 378C in an atmosphere

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of 5% CO2 in 96-well culture plates at 105 macrophages/well. After cell adherence (2 h), the supernatants containing nonadherent cells were removed by aspiration.

Bacteriology Lavage samples were plated onto Columbia agar base with 5% sheep blood, a polyvalent nonselective medium. Sabaroud agar medium (Becton Dickinson, Franklin Lakes, NJ) was used to select for yeast and fungi. MacConkey’s agar medium was used as a selective Gramnegative bacterial growth medium.

Induction of M1 and M2 Macrophage Polarization Adherent cells, reaching macrophage purity higher than 95% (16), were washed with 200 ml Hanks’ balanced salt solution and then stimulated to induce M1 or M2 macrophage phenotype. Induction of M1 macrophage polarization was performed by a combined exposure of 100 ng/ml LPS from P. aeruginosa (Sigma Chemical Co., St. Louis, MO) and 100 ng/ml (843 IU/ml) of recombinant mouse IFN-g (R&D Systems, Minneapolis, MN). Induction of M2 polarization was performed by a combined exposure of 10 ng/ml IL-4 and 10 ng/ml IL-13 (R&D Systems). Duration of the stimulation phase for both M1 and M2 phenotypes was 6 hours. Cell culture responses to LPS alone (100 ng/ml, 3 or 12 h) were also assessed. Time points based on maximal mRNA transcript expression of the different markers of cultured macrophages were selected. As markers of M1 macrophage activation, we monitored mRNA expression of the inducible nitric oxide synthase (NOS)-2, IL-1b, and TNF-a, biochemical levels of NO production, and TNF-a protein levels. As markers of M2 macrophage activation, we monitored mRNA expression of arginase-1 (Arg-1), found in inflammatory zone (FIZZ)-1, and Ym1-2, as well as arginase enzyme activity. The balance between pro- and anti-inflammatory cytokine production after LPS stimulation was assessed by monitoring, at mRNA or protein levels, TNF-a, chemokine C-C motif ligand (CCL)-2, IL-1b, and NO, and IL-10, respectively.

Azithromycin Treatment Azithromycin (Pfizer, Brussels, Belgium) was added to cell cultures obtained from the peritoneal cavity or the bronchoalveolar space. Azithromycin, varying from 1 to 100 mg/liter, was added at the adherence phase.

RNA Extraction and mRNA Quantification RNA from alveolar or peritoneal macrophages was extracted with TrizolReagent (Invitrogen), according to the manufacturer’s instructions. RNA (122 ng to 1 mg) was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen) with 350-pmol random hexamers (Eurogentec, Seraing, Belgium) in a final volume of 25 ml. Resulting cDNA was then diluted 253 and 103, respectively, for alveolar and peritoneal cells, in sterile distilled water and used as a template in subsequent real-time PCR. Sequences of interest were amplified using the following: forward primers—59-AGA GGG AAA TCG TGC GTG AC-39 (mouse b-actin), 59-CAA GAC AGG GCT CCT TTC AG-39 (mouse Arg-1), 59-CCT GCT GGG ATG ACT GCT ACT-39 (mouse FIZZ-1), 59-CAG CTG GGC TGT ACA AAC CTT-39 (mouse NOS-2), 59-GCC TCT TCT CAT TCC TGC TTG-39 (mouse TNF-a), 59-GAC GGA CCC CAA AAG ATG AAG-39 (mouse IL-1b), 59TGT TCT GGT GAA GGA AAT GCG-39 (mouse Ym1-2); reverse primers—59-CAA TAG TGA TGA CCT GGC CGT-39 (mouse b-actin), 59-GTA GTC AGT CCC TGG CTT ATG G-39 (mouse Arg-1), 59-AGA TCC ACA GGC AAA GCC AC-39 (mouse FIZZ-1), 59-CAT TGG AAG TGA AGC GTT TCG-39 (mouse NOS-2), 59GGC CAT TTG GGA ACT TCT CA-39 (mouse TNF-a), 59-CTC TTC GTT GAT GTG CTG CTG TG-39 (mouse IL-1b), 59-CGT CAA TGA TTC CTG CTC CTG-39 (mouse Ym1-2) (all from Invitrogen). For amplification of mouse IL-10 and mouse CCL-2 sequences, we had to resort to Taqman predesigned primer (Taqman gene expression assay; Applied Biosystems). A relative quantification of mRNA expression was performed on an ABI 7,000 (Applied Biosystems) in the following conditions: 2 minutes at 508C, 10 minutes at 958C (15 s 958C, 1 min 608C) times 40. Six serial 1:10 dilutions of a positive control sample of cDNA were used as standards in each reaction. Standards and samples

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(5 ml) were amplified with 300 nM described primers using Power SYBR Green PCR Master Mix (Applied Biosystems) or with 900 nM of the IL-10 or CCL-2 primers using Taqman Universal Master Mix (Applied Biosystems) in a total volume of 25 ml. PCR product specificity was checked by taking a dissociation curve and by agarose gel electrophoresis. Results were calculated as a ratio of the different gene expression to the expression of the reference housekeeping gene, b-actin. RT-PCR analyses were performed in triplicate.

Cytokine Assays Mouse CCL-2 (Quantikine mouse JE/MCP-1; R&D Systems), IL-10, and TNF-a (BD Pharmigen, San Diego, CA) concentrations were measured in BAL and PCL fluid or in supernatants obtained from 96-well culture plates using a standard sandwich ELISA following the manufacturer’s protocols.

Determination of Arginase Activity Arginase activity was measured in lysates obtained from cultured cells, as described elsewhere (17). At 24 hours after stimulation, cultured macrophages were lysed with 80 ml Triton X-100 (0.1%). After 30 minutes of shaking, arginase was activated by adding 100 ml Tris-HCl (25 mM) and 35 ml MnCl2 (10 mM), and incubating for 10 minutes at 568C. L-arginine hydrolysis was conducted by incubating cell lysates with 100 ml L-arginine (0.5 M, pH 9.7) at 378C for 1 hour. The reaction was stopped with 800 ml H2SO4 (96%)/H3PO4 (85%)/H2O (1:3:7). The produced urea was quantified at 540 nm after addition of 40 ml a-isonitrosopropiophenone (9%, dissolved in 100% ethanol), followed by heating at 1008C for 20 minutes. One unit of enzyme is defined as the amount that catalyzes the formation of 1 mmol urea/min.

Determination of NO Production NO production was monitored in the extracellular medium of cells cultured for 24 hours by assessing nitrite levels using Griess reagent. Briefly, 50 ml cell-free supernatants were added to 100 ml of Griess reagent (5% H3PO4, 1% sulfanilamide, 0.1% N-1-naphtyl-ethylendiamine). Absorbance was measured at 540 nm. Different concentrations of sodium nitrite were used to construct a standard curve. Biochemical analyses were performed in triplicate.

Flow Cytometry Analysis Fluorescent labeling of BAL and PCL macrophages, obtained from F508del-CF and wild-type mice, was undertaken upon resuspension in Hanks’ medium for flow cytometry with 3% decomplemented FBS (Invitrogen) and 10 mM NaN3 (Merck, Darmstad, Germany). Fc receptors were blocked with anti-CD16/32 (clone 2.4G2; BD Biosciences). Cells were stained using antibodies specific for IFN-gR1 (clone 2E2) and Toll-like receptor (TLR)-4 (clone UT41), obtained from eBioscience (San Diego, CA), and IL-4Ra (clone mIL4R-M1) and CD11b (clone M1/70) from BD Biosciences. Samples were fixed in a 1.25% paraformaldehyde solution in PBS for at least 1 hour, acquired on a FACSCalibur (BD Biosciences), and analyzed using CellQuest software (BD Biosciences). Analysis of cell populations was undertaken with appropriate gating according to side and forward scatters to exclude dead cells.

Statistical Analysis Statistical analysis was performed by using JMP software (SAS Institute, Cary, NC). Before statistical analysis, all biochemical data were log transformed and normality of distribution was checked. Between-group comparisons of parametric data were evaluated using one-way ANOVA after checking, by applying the Satterthwaite correction, that variances of populations were homogeneous. Post hoc comparisons were performed by using Student’s t test or TukeyKramer honestly significant difference test, for two or more than two x levels, respectively. To address concerns on potential outliers, a Grubb’s discordance test was applied, as needed. Geometric means are expressed as the antilog of the arithmetic means of log-transformed values. To better illustrate levels of comparison for each individual variable, log-transformed data are plotted in box plots as 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles, and corresponding P values are indicated. Arithmetic means (6SEM) are used for expressing

absolute or relative frequency of normally distributed data. Statistical significance was considered at a P value less than 0.05.

RESULTS Ubiquitous Macrophage Accumulation in CF Mice

Repeated bacteriological examination of lavage samples obtained from CF and wild-type mice showed no known pathogenic infectious agents cultured in polyvalent media, and zero growth detected in selective media for yeast and fungus and for Gram-negative bacteria (data not shown). However, an accumulation of macrophages, along with other cell populations, was found in either PCL or BAL performed in the same naive, nonstimulated animals. As illustrated in Figure 1A, alveolar macrophage counts were found to be 1.7-fold higher in BAL samples obtained from F508del-CF mice compared with those samples obtained from wild-type mice. Macrophage cell number in PCL was 2.3-fold higher in CF compared with wild-type mice (Figures 1A and 1B). The number of neutrophils and lymphocytes collected in PCL was also found to be increased in CF compared with wild-type mice (Figure 1B). To further strengthen our data on macrophage accumulation in CF mice, we monitored, in PCL and BAL fluids, the concentration levels of CCL-2, an essential macrophage-chemoattractant factor. As illustrated in Figure 1C, CCL-2 levels were 14-fold higher in BAL fluid from F508del-CF compared with wild-type mice. The cytokine levels were also significantly increased in the CF peritoneal fluid (Figure 1D). These data indicate that F508delCF mice display a ubiquitous and spontaneous macrophage infiltration associated with increased levels of a macrophage chemoattractant protein, CCL-2. CF Macrophages Display a Marked M1 and M2 Immune Activation Pattern

To investigate whether the ubiquitously increased macrophage infiltration in CF mice is related to a particular immune phenotype, we determined whether CF macrophages harvested from bronchoalveolar and peritoneal cavities displayed a polarized classical (M1) or alternative (M2) phenotype. We initially monitored M1 and M2 phenotype in noncultured conditions. As macrophages represent around 95% of the cells collected from BAL (Figure 1A), experiments in freshly collected cells in noncultured conditions were considered in this work as representative of the macrophage status of the bronchoalveolar space. Among the M1 markers, the expression of NOS-2 in freshly isolated alveolar macrophages was sixfold higher in F508del-CF than in wild-type mice. Indeed, geometric means of NOS-2 mRNA expression relative to b-actin reached 0.006 3 1023 in F508del-CF and 0.001 3 1023 in wild-type mice, respectively (P 5 0.03; n 5 8 for each group). IL-1b and TNF-a mRNA expression were not significantly increased (data not shown). Arg-1 mRNA levels, used as representative of M2 marker, averaged 0.001 in F508del-CF and 0.004 in wild-type mice, respectively (P 5 0.18; n 5 8 for each group), and those levels for Ym1-2 were quite similar in both groups (1.74 and 1.62 for CF and wild-type, respectively; P 5 0.74; n 5 8 for each group). FIZZ-1 expression showed undetectable levels in BAL cells from CF and wild-type mice. We then monitored the alveolar macrophage phenotype in cultured conditions (Figure 2). Under M1 stimulation (LPS plus IFN-g), CF alveolar macrophages displayed an increased expression of NOS-2 and IL-1b (Figure 2A). The increased NOS-2 mRNA expression correlated with NO production, the geometric means of which reached 14.0 mM in F508del-CF and 7.0 mM in wild-type mice, respectively (P 5 0.007; n 5 4 for


593 Figure 1. Differential cell counts in (A) bronchoalveolar lavage (BAL) and (B) peritoneal cavity lavage (PCL) in F508delCF (cystic fibrosis [CF]) and wild-type (WT) mice. Values of cell counts are arithmetic means (6SEM) for six experiments in each group of mice. Chemokine C-C motif ligand (CCL)-2 levels (pg/ml) in BAL fluid (C) and PCL fluid (D) are expressed as geometric means (tables) or plotted as log-transformed values for eight mice per group. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal line across the panel represents the mean of the whole study population. *P , 0.001; †P , 0.005 for comparison of the mean value versus that obtained in the wildtype group of mice.

each group). mRNA TNF-a expression correlated with TNF-a protein concentrations, which reached 14,698 pg/ml in CF cultures and 8,331 pg/ml in wild-type cultures (P 5 0. 11; n 5 4 for each group). In nonstimulated conditions, NO production from cultured alveolar macrophages was also increased, reaching 3.0 mM in F508del-CF and 1.8 mM in wild-type mice, respectively (P 5 0.05; n 5 4 for each group). Under M2 stimulation (IL-4 plus IL-13), a significant increase (P 5 0.02) was observed for Arg-1 mRNA expression (Figure 2) in CF as compared with wild-type cells. Ym1-2 and FIZZ-1 mRNA expression were not significantly different between the two groups of mice (Figure 2). The M1/M2 phenotype of peritoneal macrophages was then investigated. As granulocytes and lymphocytes globally represent around 30% of cells collected from PCL (Figure 1B), experiments in freshly collected cells in noncultured conditions could not be representative of the macrophage status of the peritoneal space. Therefore, experiments with peritoneal macrophages were only performed in cultured conditions after 95% cell purification by adherence into plastic dishes (16). Under stimulated conditions, the M1 cell polarization pattern (Figure 2) was confirmed, particularly for IL-1b, which showed significantly higher mRNA expression in CF compared with wild-type mice. NOS-2 mRNA expression showed an opposite trend (P 5 0.06) as compared with the increased levels observed in alveolar macrophages. Arg-1, Ym1-2, and FIZZ-1 expression showed an increased trend, but no significant effect in stimulated CF peritoneal macrophages as compared with wildtype cells. Determination of arginase activity in cell lysates obtained from stimulated, cultured peritoneal cells reached geometric means of 0.03 mmol urea/min in CF and 0.02 mmol urea/min in wild-type cells, respectively (P 5 0.01; n 5 4 for each group). In nonstimulated conditions, arginase activity

was also increased in CF peritoneal macrophages (0.02 mmol urea/min in CF versus 0.006 in wild-type cells, respectively; P 5 0.01; n 5 4 for each group). Taken together, these findings indicate that F508del-CFTR mutation modulates expression of macrophage markers, resulting in a polarization into M1 and, to a lesser extent, M2 phenotype. Stimulated CF Macrophages Displayed an Exaggerated Proinflammatory and a Reduced Anti-Inflammatory Immune Response

The ability of macrophages to produce pro- and anti-inflammatory cytokines can be used to further characterize macrophage phenotype. The response to LPS was examined in cultured alveolar and peritoneal macrophages harvested from CF and wild-type animals. Overall, we observed an exaggerated response of proinflammatory markers monitored, with the exception of TNF-a (Figures 3D and 3H). Indeed, after stimulation by LPS alone, the mRNA expression of NOS-2, IL-1b and CCL-2 was higher in CF than in wild-type mice (Figure 3). However, in the absence of LPS stimulation, all markers, except CCL-2 (Figure 3G) and TNF-a (Figure 3H) in peritoneal macrophages, were found to be similar in CF and wild-type cells. CCL-2 mRNA levels were 6.8-fold higher in nonstimulated peritoneal macrophages obtained from CF mice as compared with those cells obtained from wild-type mice (Figure 3G). In contrast, TNF-a expression levels were reduced in CF compared with wild-type mice (P 5 0.01; Figure 3H). In contrast to the increased LPS-induced production of proinflammatory markers, CF alveolar and peritoneal macrophages showed a lower responsiveness of anti-inflammatory markers (Figure 4). Indeed, the mRNA expression of IL-10

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Figure 2. mRNA expression of M1 and M2 markers of cultured alveolar or peritoneal cells purified from F508del-CF and WT mice. Nitric oxide synthase (NOS)-2, IL-1b, and TNF-a, used as M1 markers, were monitored under 6-hour stimulation with a combined exposure of 100 ng/ml IFN-g and 100 ng/ml LPS. Arginase (Arg)-1, Ym1-2, and found in inflammatory zone (FIZZ)-1, used as M2 markers, were monitored under 6-hour stimulation with a combined exposure of 10 ng/ml IL-4 and 10 ng/ml IL-13. Data normalized to the expression of b-actin illustrate one of three representative, independent experiments. Each parameter was measured in triplicate. Data are expressed as geometric means (tables) or plotted as logtransformed values for three to four individual cell cultures. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal lines across the panels represent the mean of the whole study population. *P , 0.05; ‡P , 0.001 for comparison of the mean value obtained in CF versus that obtained in the WT group of mice. n.d., not detected.

(Figure 4) was found to be decreased in LPS-stimulated peritoneal macrophages from CF compared with wild-type mice. Similar observations were obtained while assessing protein levels (Figure 4). These findings suggest that F508del-CFTR mutation modulates responsiveness of macrophages to inflammatory stimuli, such as LPS, by enhancing the expression of proinflammatory mediators and reducing the expression of anti-inflammatory mediators, regardless of the cell origin.

(6SEM) of the specific fluorescence labeling relative to CD11b1 cells. Significantly lower density of all three specific receptors was observed in CF compared with wild-type peritoneal (Figure 5) and alveolar macrophages (data not shown). These findings indicate that the differential and exaggerated responses observed between CF and wild-type cells should not be dependent on the density of receptors expressed at their cell surfaces.

Density of Specific Cell Surface Receptors in Macrophages

To investigate whether azithromycin modulates macrophage polarization, we cultured peritoneal and alveolar macrophages with the macrolide under conditions in which cells are differentiated toward M1 or M2 polarization. To select the optimal dose of azithromycin, purified peritoneal macrophages obtained from wild-type mice were first exposed to different azithromycin concentrations, varying from 1 to 100 mg/liter. NO production and arginase activity were

To investigate whether the differential responses that we observed between the two cell genotypes are dependent on the density of specific cell surface receptors involved in these responses, we monitored the number of CD11b1 alveolar and peritoneal macrophages expressing receptors to LPS (TLR-4), to IL-4 and IL-13 (IL-4Ra), and to IFN-g (IFN-gR1) at their cell surfaces. Results are expressed as the arithmetic means

Effect of Azithromycin on Macrophage Immune Responsiveness


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b Figure 3. mRNA expression of NOS-2, IL-1b, CCL-2, and TNF-a in alveolar and peritoneal macrophages cultured in the presence or in the absence of stimulation with 100 ng/ml LPS. Responses are illustrated 3 hours after LPS exposure for TNF-a and CCL2, and 12 hours after LPS exposure for NOS-2 and IL-1b. Data normalized to the expression of b-actin illustrate one of three representative, independent experiments. Each parameter was measured in triplicate. Data are expressed as geometric means (tables) or plotted as log-transformed values for three to four individual cell cultures. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal lines across the panels represent the mean of the whole study population. *P , 0.05; †P , 0.01 for comparison of the mean value obtained in F508del-CF versus that obtained in the corresponding WT group of mice.

monitored as representative markers of M1 and M2 macrophage polarization, respectively. In the absence of azithromycin, NO levels in the extracellular cultured media were manifold higher after M1 differentiation by LPS plus IFN-g as compared with nonstimulated peritoneal macrophages (Figure 6A). In the absence of M1 stimulation, azithromycin did not significantly influence NO production. However, after M1 activation, NO production was significantly lower in the presence of azithromycin, with a more marked effect being observed at 1 mg/liter azithromycin (Figure 6A). In the absence of azithromycin, M2 activation (IL-4 plus IL-13) increased arginase activity by twofold in lysates from cultured peritoneal cells (Figure 6B). In the absence of M2 stimulation, azithromycin increased arginase activity in a dosedependent manner. The combination of azithromycin treatment and M2 activation globally produced a twofold increase of the enzyme activity. These data indicate that azithromycin regulates the inflammatory process in peritoneal macrophages by inhibiting M1 polarization and shifting cell polarization toward the alternatively activated M2 phenotype. We next tested whether azithromycin, at a selected dose of 1 mg/liter, has any effect on the expression of macrophage polarization markers in stimulated CF cells. Although NOS-2 mRNA expression was unmodified (data not shown), the expression of proinflammatory cytokines under M1 activation (LPS plus IFN-g) was decreased by azithromycin in both F508 del-CF and wild-type mice (Figure 7). Indeed, treatment with 1 mg/liter azithromycin significantly reduced IL-1b, CCL-2, and TNF-a in alveolar macrophages from F508del-CF and wild-type mice, with more marked effects in the CF group (Figure 7). The anti-inflammatory effect seemed to be limited to alveolar macrophages as, at the same dose, azithromycin was unable to influence IL-1b, CCL-2, or TNF-a expression in M1-activated peritoneal macrophages. The expression of M2 markers (Arg-1, FIZZ-1, Ym1-2) and that of anti-inflammatory IL-10 cytokine was not modified by azithromycin (data not shown). Azithromycin (1 mg/liter) treatment did not modify proinflammatory cytokine (IL-1b, CCL-2, and TNF-a) production in alveolar macrophages stimulated with LPS alone (data not shown). These results provide evidence that azithromycin regulates the inflammatory process by interfering with proinflammatory cytokine production in stimulated macrophages. Furthermore, the anti-inflammatory effect of azithromycin seems to be limited to airway macrophages, and was only detected in the presence of the M1 inflammatory phenotype.

DISCUSSION The present study was designed to characterize the phenotype of macrophages in CF and to explore the hypothesis that azithromycin modulates macrophage activation and responses. Apart from their involvement in clearing pathogens, macrophages may play a cardinal role in the orchestration of inflammatory responses. Macrophages control switches of the immune system by securing the balance between pro- and anti-inflammatory reactions. The identification of distinct macrophage subpopulations has been put forward to explain contrasting functions of

these key immune cells in different pathologies (14, 15, 18). Depending on the activating stimuli, macrophages can polarize into different subsets: classically (M1) or alternatively (M2) activated mononuclear phagocytes. It has been recognized for many years that M1 macrophages play pivotal roles in the triggering of inflammatory reactions by producing, under adequate stimulation, a large number of mediators, such as cytokines, chemokines, and eicosanoides. These inflammatory factors are known to be able to initiate and orchestrate cellular and molecular responses, and to stimulate resident cells, such as endothelial and epithelial cells (18, 19). M2 has been used as a generic name for various forms of activation other than the classical M1 form of macrophage activation. M2 cells are generally involved in T helper (Th) 2 responses, have immunomodulating function, and promote tissue remodeling and repair after an inflammatory reaction. The biology of macrophages in human and experimental CF is unknown, and the expression of CFTR in macrophages is still debated (20–24). Unfortunately, comparison of studies is difficult because of differences in genotypes studied, experimental protocols used, and origin of cells examined (peripheral blood, bone marrow–derived cells, or mature cells isolated from alveolar or peritoneal cavity) (20–24). It has been recently demonstrated that CFTR is expressed in alveolar macrophages, and that the functional protein is essential for the production of an acidic environment in the phagolysosome, which contributes to macrophage bactericidal activity (21). According to Di and colleagues (21), this disrupted activity in CF macrophages could explain the bacterial colonization of CF lungs. However, another group has not supported this proposed mechanism; that group concluded that phagosomal acidification in alveolar macrophages is not dependent on CFTR channel activity (23). We have previously shown, in naive F508del-CF mice, a spontaneous inflammation characterized by increased accumulation of macrophages and neutrophils in the absence of any detectable bacterial or fungal infection. The inflammation was confirmed histologically, and was combined with up-regulation of a proinflammatory mediator, macrophage inflammatory protein (MIP)-2, a key chemokine in the recruitment of neutrophils, functionally equivalent to the human IL-8. In the present work, we focused our attention on the macrophage cell content in BAL and PCL; we observed, in the absence of any detectable infection, a marked increase of CCL-2 in both fluids from CF mice. The macrophage infiltration that we observed in BAL and PCL, along with infiltration of other cell populations (lymphocytes and neutrophils), supports the finding that the spontaneous inflammation in this CF mouse model is not only confined to the airway compartment, but is a systemic and complex process involving multiple cell types and pathways. Macrophage accumulation in our CF mutant mice is in agreement with that observed during the fetal development in human CF airways (25), and is likely to be related to the elevated CCL-2 protein content. Indeed, CCL-2 protein levels were found to be more than 10-fold higher in the alveolar space of CF mice, and were also increased in the peritoneal cavity. This chemokine, formerly named monocyte chemoattractant protein (MCP)-1, is


597

Figure 4. mRNA and protein expression of IL-10 of alveolar or peritoneal macrophages cultured in the absence or in the presence of stimulation with 100 ng/ml LPS for 3 or 12 hours, respectively. Data normalized to the expression of b-actin illustrate one of three representative, independent experiments. Each parameter was measured in triplicate. Data are expressed as geometric means (tables) or plotted as log-transformed values for three to four individual cell cultures. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal lines across the panels represent the mean of the whole study population. *P , 0.05; †P , 0.01 for comparison of the mean value obtained in F508del-CF versus that obtained in the corresponding WT group of mice. n.d., not detected.

involved in a great variety of lung inflammatory disorders, such as allergic asthma, idiopathic pulmonary fibrosis, and bronchiolitis obliterans syndrome (26). Allergic pulmonary aspergillosis, a frequent syndrome in patients with CF, is also associated with increased CCL-2 pulmonary levels (27, 28). The possible contribution of chemokines involved in macrophage recruitment, such as MCP-1/CCL-2, MIP-1a, MIP-3a, and MIP-1b, to CF lung disease has been recently suggested in a few preliminary clinical studies (29, 30). Indeed, macrophage-related chemokine levels were found to be increased in lungs (29) and serum (30) of young patients with CF. Circulating CCL-2 levels were reported to be higher in patients carrying two mutations associated with a pathological sweat test and pancreatic insufficiency (F508del, W1282X, G542X, N1303K, S549R) than in compound heterozygous patients who carried one mutation

known to cause mild disease with borderline sweat test and pancreatic sufficiency (31). Interestingly, CCL-2 gene polymorphism has been pointed out as an accurate predictor of severity of inflammatory pancreatic disease. Indeed, patients with severe acute pancreatitis have a greater proportion of CCL-2 –2518 A/G allele than control subjects (32). We therefore postulate that CCL-2 could represent a genetic pulmonary modifier in CF. Future investigations are needed to explore this hypothesis. The origin of CCL-2 production was not determined in the present work, but a possible source is epithelial cells. A possible explanation for the manifold higher content of CCL-2 protein levels, particularly higher in CF BAL samples, and the nonsignificant mRNA expression level in alveolar nonstimulated cells is that epithelial cells from the airways could contribute to the in vivo CCL-2 airway overproduction. As a matter of fact, it

598


Figure 5. FACS analysis of Toll-like receptor (TLR)-4 (A), IL-4Ra (B), and IFN-gR (IFNR) (C ) expression in peritoneal lavage of F508del-CF and WT mice. FACS plots are gated on CD11b1 cells. Percentage of CD11b1 cells (D) expressing TLR-4, IL-4Ra and IFN-gR1 receptors. Values are arithmetic means (6SEM) for four animals in each group of mice. Data illustrate one of two representative, independent experiments. *P , 0.05; ‡P , 0.01 for comparison of the mean value obtained in CF versus that obtained in the corresponding WT group of mice.

has been reported that alveolar epithelial cells represent an important source of CCL-2 production (33, 34). Our data show for the first time that CF alveolar and peritoneal macrophages express an M1 polarization profile, denoting that immune functions of CF macrophages are in a proinflammatory status. Indeed, when macrophages isolated from CF mice were stimulated with validated inducers, the expression of M1 activation markers was exacerbated. This effect was identified in both alveolar and peritoneal macro-

phages, even though the relative degree of expression of each marker differed between the two cell populations. Our data support the notion that the differential and exaggerated cytokine responses that we observed in CF macrophages is unlikely to be dependent on the level of expression of receptors at their cell surfaces. Expression of TLR-4, the receptor responsible for the LPS-mediated immune response, has been reported to be increased in airway neutrophils (35), but reduced in bronchial epithelial cells from patients with CF (36). To the best of our knowledge, expression of specific cell surface receptors, including pattern recognition factors, has not been characterized in CF macrophages. As the expression of different specific macrophage receptors is reduced, this observation suggests that intrinsic alterations in the regulation of cell signaling pathways may be involved in the pro- and anti-inflammatory imbalance in CF. Our data indicate that the expression of NOS-2, the inducible isoform of the enzyme catalyzing NO production from the semiessential amino acid, L-arginine, and oxygen, is increased in M1-stimulated alveolar macrophage cells from CF. Conflicting data on NOS-2 expression and NO production have been reported in CF. Indeed, nonaffected (37) or even decreased (38) levels of NOS-2 expression and NO production have been reported in CF airways despite airway inflammation. However, NO production has been more recently recognized as a marker of distal lung inflammation in CF (39). The expression of NOS-2 is known to be dependent on transcription factors, such as NF-kB, activated by proinflammatory cytokines, including IL-1b and TNF-a (40). Our data on the exacerbated expression of proinflammatory mediators, particularly IL-1b, are in keeping with those from clinical studies (41, 42) in which it has been shown that lung macrophages obtained from patients with CF overexpressed proinflammatory cytokines. In agreement with our previously published in vivo data (12), TNF-a was not significantly higher in CF cells. Our study shows that CF macrophages also display an exaggerated response to M2 stimuli, denoting that immunomodulating and remodeling processes seem also to be activated in CF cells. Arg-1, the inducible enzyme that metabolizes L-arginine, is recognized as one of the most specific and validated M2 markers. Biochemical and mRNA determination of arginase supported the M2 polarization in CF macrophages. Interestingly, increased sputum arginase activity has been described in patients with CF (43). Ym1-2, a closely related soluble chitinase-like lectin, and FIZZ-1, a resistin-like molecule, are both recognized as new in vitro markers for M2activated macrophages (15). No corresponding biochemical or protein post-transcriptional biomarkers are known for these new transcripts. In our study, Ym1-2 expression was relatively higher in alveolar than in peritoneal macrophages, whereas FIZZ-1 expression was relatively higher in peritoneal macrophages, with undetectable levels in alveolar cells; however, no significant difference of the two M2 markers was observed between CF and wild-type cells. The M2 activation of macrophages that we observed in our experiments might contribute to remodeling processes and fibrotic changes that occur in the CF lung. Of note, several gene variants of transforming growth factor-b have been reported to affect severity of lung disease in patients with CF (44). Indeed, transforming growth factor-b is a multifunctional peptide involved in proliferation and differentiation in many cell types, but it also plays a critical role in inflammation, fibrosis, and the remodeling process (45). The underlying reasons for higher susceptibility of CF macrophages to M1 and M2 stimuli is not clear, but it could be postulated that the CFTR dysfunction primarily influences inflammatory reactions, or may alter ionic balance at the macrophage microenvironment. Accordingly, con-


599

Figure 6. Effect of 0–100 mg/liter azithromycin (AZM) on NO production and arginase activity in cultured peritoneal macrophages. (A) NO production (mM) was assessed in the presence or in the absence of a combined exposure of 100 ng/ml IFN-g and 100 ng/ml LPS for 48 hours. (B) Arginase activity (mmol urea/min) was assessed in the presence or in the absence of a combined exposure of 10 ng/ml IL-4 and 10 ng/ml IL-13. Data illustrate one of three representative, independent experiments. Each parameter was measured in triplicate. Data are expressed as geometric means (tables) or plotted as log-transformed values for three to four individual cell cultures. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal lines across the panels represent the mean of the whole study population. Levels not connected by same letter are significantly different.

centrations of mono- and divalent ions, such as sodium and calcium, are known to be essential for the development of adequate immune macrophage responses (22, 46, 47). Our data on the evidence of an exaggerated response to LPS of alveolar and peritoneal macrophages from CF mice are in agreement with our previously published data (12), and with those in which human alveolar macrophages, the CFTR of which was suppressed by siRNA, greatly enhance their expression of neutrophilic chemokines after exposure to P. aeruginosa (48). Additionally, down-regulation of IL-10, an anti-inflammatory cytokine, may also be responsible for excessive inflammatory responses in CF (39, 49–51). Taken together, these findings indicate that activated CF lung and peritoneal macrophages may

amplify the inflammatory response to pathogens by both overproducing proinflammatory mediators and underproducing antiinflammatory mediators. In our prior in vivo study (12), we demonstrated that azithromycin exerts some anti-inflammatory effects. Indeed, long-term (4-wk), low-dose (10 mg/kg/day), orally administered azithromycin attenuated lung macrophage infiltration and LPSinduced pulmonary cytokine release in F508del-CF mice and normal homozygous mice. We observed here that azithromycin alters peritoneal macrophage phenotype by inhibiting classical M1 polarization and shifting the cell polarization toward M2 phenotype. This observation is in keeping with a recent study conducted in a mouse macrophage cell line, J774, showing that

600


Figure 7. Effect of 1 mg/liter AZM on IL-1b, CCL-2, and TNF-a mRNA expression in cultured alveolar macrophages. M1 stimulation was performed by 6-hour exposure of cell cultures to 100 ng/ml of LPS plus 100 ng/ml IFN-g. M2 stimulation was performed by 6-hour exposure of cell cultures to 10 ng/ml IL-4 and 10 ng/ml IL-13. Data normalized to the expression of b-actin illustrate one of three representative, independent experiments. Each parameter was measured in triplicate. Data are expressed as geometric means (tables) or plotted as log-transformed values for three to four individual cell cultures. Horizontal lines of box plots illustrate the 0.5th, 25th, 50th (median), 75th, and 99.5th percentiles of the variable. The horizontal lines across the panels represent the mean of the whole study population. *P , 0.01; †P , 0.001; ‡P , 0.001 for comparison of the mean value measured in the presence of AZM versus that obtained in the absence of the macrolide.

overnight exposure to 15–30 mM azithromycin, a dose approximately 10-fold higher than that used in our work, increased arginase activity and reduced NOS-2 expression (52). Here, we also showed that the macrolide reduces IL-1b, CCL-2, and TNF-a mRNA expression by M1-polarized alveolar macrophages. The finding that azithromycin controls the overexpres-

sion of a macrophage-related chemokine (CCL-2) in CF alveolar macrophages may be of particular interest. Apart from its powerful proinflammatory effects, it has been reported that the cytokine is able to promote increased paracellular permeability and disrupted tight junctions in human vascular endothelium cells (53). Interestingly, it has been reported that


azithromycin could counteract these mechanisms. Indeed, azithromycin has been shown to be able to increase transepithelial electrical resistance and induce processing of tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A in human airway epithelia in culture (54). Finally, our results clearly show that CF macrophages are ubiquitously accumulated, and that these cells represent a newly relevant cell population in the apparently constitutive and systemic inflammation in CF. The enhanced number of alveolar and peritoneal macrophages in CF mice is associated with an at least 10-fold higher level of CCL-2, an essential macrophage chemoattractant factor. We also show that CF macrophages are polarized toward classical (M1) and alternative (M2) activation status, denoting that immune functions of CF macrophages are in a proinflammatory status, and that fibrotic and remodeling processes are activated in CF cells. Azithromycin regulates the inflammatory process by inhibiting M1 polarization and shifting macrophage polarization toward a preferential, M2, antiinflammatory status. These findings contribute to shed some new light on the underlying mechanisms of the beneficial in vivo effects of azithromycin in CF. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The authors thank Yousof Yakoub and Francine Uwambayinema for their excellent technical assistance, and are grateful to Jean Cumps for his assistance with statistical analysis.

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