To cite this article: Krysko O, Holtappels G, Zhang N, Kubica M, Deswarte K, Derycke L, Claeys S, Hammad H, Brusselle GG, Vandenabeele P, Krysko DV, Bachert C. Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis. Allergy 2011; 66: 396–403.
Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by biased Th2 inflammation and CRS without nasal polyps (CRSsNP) by a Th1 immune response. Colonization by Staphylococcus aureus is increased in CRSwNP. We aimed to determine macrophage phenotypes in nasal mucosa of CRSwNP and CRSsNP and to examine phagocytosis of S. aureus in these pathologies.
Methods: Macrophage phenotyping was performed by immunohistochemical staining on nasal mucosa sections from 28 patients; in addition flow cytometry analysis was performed. Tissue homogenate protein levels of IFN-γ, IL-5, IL-6, IL-1β, TGF-β, eosinophil cationic protein (ECP) and total IgE were analyzed and correlated with macrophage subtypes. Phagocytosis of S. aureus was analyzed by flow cytometry. Survival of S. aureus in Thp1 cells in the presence of polarizing cytokines was studied in vitro.
Results: By immunohistochemical analysis more M2 macrophages were present in CRSwNP than in CRSsNP. This also was positively correlated with increased levels of IL-5, ECP and locally produced IgE and decreased levels of IL-6, IL-1β and IFN-γ. FACS analysis of dissociated nasal tissue confirmed the presence of increased numbers of M2 macrophages (CD206+HLADR+CD14+CD11c+CD20−) in CRSwNP as compared to controls, while the number of M1 macrophages (CD206−HLADR+CD14+CD11cintCD16−CD20−) was not different. Phagocytosis of S. aureus by human tissue derived macrophages was reduced in CRSwNP as compared to macrophages from the control inferior turbinates.
Conclusions: Decreased phagocytosis of S. aureus and an M2 activation phenotype in CRSwNP could potentially contribute to persistence of chronic inflammation in CRSwNP.
Chronic inflammatory disease of the upper airways, which is also known as chronic rhinosinusitis (CRS), was reported in 9.9% of the European population, with a range of 6.7–19.7% among different countries (1). However, CRS is not one disease but a group of pathologies characterized by distinct expression of innate immune markers, a specific remodeling pattern, and an inflammatory T cell activation profile (2, 3). CRS without nasal polyps (CRSsNP) exhibits a predominant Th1 cytokine profile with increased levels of IFN-γ and TGF-β, while CRSwNP is characterized by a Th2- or a Th1/Th17-skewed eosinophilic or neutrophilic inflammation and low TGF-β (4).
Evidence shows that Staphylococcus aureus can modulate the course of CRSwNP by producing various enterotoxins. These toxins stimulate T cells by their superantigenic activities and stimulate release of cytokines, including IL-4, IL-5, IL-13 and eotaxin, which lead to a Th2 biased inflammation (5). CRSwNP is characterized by high rates of S. aureus colonization as compared to controls (63.6%vs 33.3%) (6). The mechanisms of this increased bacterial colonization in CRS are not clear and it is not known whether it is due to a defect in the defense system in CRSwNP that might involve the epithelial barrier and phagocytes. Mucosal macrophages are located at the interface with the external environment, depending on the factors the macrophages encounter, macrophages become polarized into a classically activated pro-inflammatory (M1) phenotype or into an alternatively activated (M2) phenotype (7–9). M1 macrophages express high levels of pro-inflammatory cytokines, such as IL-1β, IL-12, IL-23 and tumor necrosis factor (TNF), as well as high levels of effector molecules, including nitric oxide. M1 macrophages participate in the induction of a Th1 response and prevent pathogen persistence (10).
M2 macrophages express elevated levels of non-opsonic receptors (e.g. mannose receptor, scavenger receptor-1, CD163, Trem-2) (11). Moreover, M2 polarization of macrophages has been linked to the persistence of allergic disease and asthma (10).
M1 macrophages are microbicidal and pro-inflammatory, while M2 macrophages are immunosuppressive and may support intracellular survival of bacteria and viruses (12). Several aspects of innate defenses were analyzed in CRS patients (3, 13), but the phenotype of macrophages in the nasal tissue of CRS patients is not known, and it is also unclear whether there is any alteration in macrophage function in nasal tissue of CRS patients. Therefore, the aim of this study was to characterize macrophage subsets and phagocytosis of S. aureus in nasal tissue of patients with CRS.
Materials and methods
Patients were recruited from the Department of Otorhinolaryngology of Ghent University Hospital. The local Ethics Committee of Ghent University Hospital approved the study, and informed consent was obtained from all participants before including them in the study. Nasal mucosa tissue was obtained from patients undergoing functional endoscopic sinus surgery.
For macrophage immunohistochemical staining and quantification, cryosections from 28 different patients were obtained and stained with anti-macrophage mannose receptor (BD Pharmingen, Erembodegem, Belgium), CD14, CD68, or CD163 (DAKO, Heverlee, Belgium).
Analysis of cytokine and IgE levels
Fresh human tissue was snap frozen in liquid nitrogen. Levels of cytokines and IgE in tissue homogenates were measured as previously described (5). Samples were assayed for IFN-γ, IL-5, IL-6 and IL-1β by using Luminex (R&D Systems, Oxon, UK). TGF-β was measured by using commercially available ELISA. Eosinophil cationic protein (ECP), total IgE and S. aureus enterotoxin specific IgE (SAE-IgE) were measured using the Unicap system (Phadia, Uppsala, Sweden).
FACS analysis of single cell suspensions of tissue samples
Human nasal tissue was disrupted by Tissue Gentle MACS Dissociator (Miltenyi Biotec, Utrecht, the Netherlands) and single cell suspension of nasal tissue was prepared by enzymatic digestion of tissue fragments with 1 mg/ml collagenase type II (Worthington, Lakewood, NJ, USA) and 0.1 μg/ml DNAse I (Roche Applied Sciences, Vilvoorde, Belgium) for 45 min at 37°C. Antibodies used were anti-human CD206, HLA-DR, CD45, CD11c, CD16, CD14, CD20 and CD3 (BD Biosciences, Erembodegem, Belgium).
Phagocytosis assay and bacterial survival
The phagocytosis assay was performed with live S. aureus strain RN 6390 expressing green fluorescent protein (GFP) according to (14). Bacterial survival rates were determined using the human Thp-1 cell line differentiated with 50 nM phorbol myristate acetate (PMA) and β-mercapto-ethanol (Sigma-Aldrich, Bornem, Belgium) for 72 h and cocultured with bacteria at multiplicity of infection (MOI) 1 : 25 for 45 min.
The data were analyzed using graphpad prism-4 software (San Diego, CA, USA), and statistical comparison was performed using the Kruskal–Wallis test.
Patient selection and profiles
Patients were diagnosed with CRSwNP or CRSsNP according to European position paper on rhinosinusitis and nasal polyps. Patients with different endoscopic scores were included in the study. Aspirin intolerance was diagnosed based on patient’s history. Sinonasal samples from 28 patients were included in this study. The patients were divided into three non-overlapping groups according to the diagnosis. CRSsNP (42.25 ± 6.020 N = 8) and CRSwNP (44.53 ± 4.861 N = 15), but the controls were younger (23.25 ± 3.250 years N = 5). Of the 15 patients with CRSwNP, five had a history of asthma and three a history of aspirin sensitive disease. One of the five control patients and one of the eight CRSsNP patients had asthma. Atopy was confirmed in six out of fifteen CRSwNP patients, three out of seven CRSsNP patients, and one out of five control patients by a skin prick test and by an ImmunoCAP Specific IgE blood test. None of the patients received oral or topical corticosteroids or antibiotics within the 4 week preceding surgery.
Characterization of macrophage phenotype in the nasal tissue by immunohistochemistry
The CRSwNP patients showed significantly more alternatively activated macrophages labeled with CD163 or CD206 than controls or CRSsNP (Figs 1 and 2). In control tissue most CD163+ and CD206+ cells were localized subepithelially, while in CRSwNP they were rather spread over the nasal polyp tissue. Semiquantitative analysis of histological staining performed by two independent observers blinded to the clinical data showed significantly more CD163+ and CD206+ cells in CRSwNP than in CRSsNP. CD68+ cells were also significantly increased in CRSwNP tissue. The numbers of CD14+ cells in all experimental groups were not different (Fig. 2).
Analysis of macrophage phenotypes in the nasal tissue by fluorescence-activated cell sorting (FACS)
To confirm the immunohistochemical data, we performed FACS analysis on nasal tissue isolated from control inferior turbinate and CRSwNP. In agreement with what we observed in tissue sections of CRSwNP, there were significantly more M2 macrophages (CD206+HLADR+CD14+CD11c+CD20−) in the nasal polyp tissue than in control inferior turbinates (3.98 ± 1.5 vs 0.83 ± 0.31, P = 0.0357) (Fig. 3C). The numbers of M1 macrophages that were CD206−HLADR+CD14+CD11cintCD16−CD20− in the two groups of patients were not significantly different (Fig. 3C).
Correlation of alternatively activated macrophages and cytokine profile of the nasal tissue
To determine whether different inflammatory milieus could affect the activation status of macrophages, we measured Th1 and Th2 cytokines in the nasal tissue homogenates. In accordance with the previous results, we observed higher concentrations of IL-5, ECP and total IgE in CRSwNP than in controls and CRSsNP (Fig. 4). There was no difference in IFN-γ and TGF-β protein levels between the groups. Then the group of CRSwNP was split in two subgroups based on the tissue homogenate levels of IL-5 protein. The patients with CRSwNP with undetectable levels of IL-5 were defined as CRSwNP IL5− (mean levels of IL-5 8.68 ± 0.57 pg/ml), and those who had more than 10 pg/ml as the CRSwNP IL5+ group (mean levels of IL-5 1145 ± 237.4 pg/ml).
We found that the CRSwNP IL-5− group had significantly lower levels of IFN-γ, IL-1β, IL-17, IL-6 and TGF-β than the CRSwNP IL5+ group (Fig. 4). SAE-IgE was detected in tissue homogenates in three out of nine CRSwNP IL5+ patients. No SAE-IgE was detected in the sera of controls or CRSsNP and CRSwNP IL5− patients. We looked for a difference in macrophage profile between Th-2 biased and Th-1 biased CRSwNP. Remarkably, CRSwNP IL5+ patients had significantly more CD206+ cells than CRSwNP IL5−, CRSsNP and controls (Fig. 4), but no significant difference was observed between CRSwNP IL5−, CRSsNP and control patients. CD163+ cells were significantly more numerous only in the CRSwNP IL5+ group. Interestingly, the M2 marker CD206 was significantly higher in the CRSwNP IL5+ group than in the CRSwNP IL5−group. There was a tendency to increased CD163 numbers in CRSwNP IL-5+ as compared to CRSwNP IL-5−, but this tendency was only on the borderline of significance (P = 0.0515). The number of CD206+ cells in the tissue was significantly correlated with IL-5 levels, (R = 0.65, P = 0.001) and total IgE levels (R = 0.63, P = 0.002). The numbers of CD163+ cells were also significantly correlated with IL-5 levels (R = 0.72, P = 0.0002) and total IgE levels (R = 0.70, P = 0.0005). However, we could not find any correlations between the number of CD206+ or CD163+ cells and the levels of other cytokines in tissue homogenates. Chronic rhinosinusitis with nasal polyps patients with asthma or aspirin exacerbated respiratory disease (AERD) are known to have more severe chronic Th2 driven eosinophilic inflammation of the airways with high levels of IL-5, total IgE and eosinophilia (15). Eight of the sixteen CRSwNP patients had either asthma alone (n = 5), asthma and AERD (n = 3) or AERD alone (n = 1). Indeed, compared to controls, these patients with comorbidity had significantly higher levels of IL-5 (9.97 ± 0.06 vs 465.4 ± 194.6, P = 0.038) and ECP (517.5 ± 118.5 vs 7670 ± 2396, P = 0.007). They also had significantly more CD206 macrophages than CRSwNP without comorbidity, CRSsNP and controls.
Phagocytosis of S. aureus by tissue macrophages is impaired in CRSwNP
We co-incubated GFP-expressing S. aureus with tissue derived macrophages for 30, 60 or 90 min and then analyzed the phagocytic capacity of CD206+ positive cells by FACS Canto II. The phagocytic index was significantly lower in the CRSwNP group than in controls at all time points of co-incubation (Fig. 5A). No conclusions on the correlation between severity of nasal polyposis and efficiency of phagocytosis could be made, because only the samples from patients with severe IL-5 positive disease could be used in this assay due to the amounts harvested.
IL-5 supports intracellular survival of S. aureus
We also tested whether IL-5 has a direct effect on survival of S. aureus following their engulfment by macrophages. We observed a tendency to increased intracellular survival of S. aureus in PMA – differentiated Thp-1 cells pre-treated with 50 ng/ml of hrIL-5 (1–100 ng/ml) (Fig. 5B). Survival of Thp-1 cells and their phagocytic capacity was not affected (data not shown).
We analyzed the phenotype of macrophages in CRS with and without nasal polyps. Immunohistochemical analysis of macrophages in sections of nasal mucosa showed significantly increased numbers of alternatively activated (M2) macrophages, characterized by abundant expression of nonopsonic receptors, such as the macrophage mannose receptor (CD206, MMR) and CD163 receptor in CRSwNP than in CRSsNP and control nasal mucosa. FACS data confirmed that M2 macrophages were increased in CRSwNP compared to controls, while the number of M1 macrophages was not different. These data are in agreement with previously reported elevated expression of macrophage mannose receptor in the nasal mucosa of CRSwNP (3, 13). The role of macrophages in the primary response to pathogens, coordination of the innate and adaptive immune responses, and tissue repair has been well established. The extracellular milieu affects the phenotype of macrophages and their activation state (8, 16). Classically activated macrophages (M1) are efficient producers of effector molecules (reactive oxygen and nitrogen intermediates) and pro-inflammatory cytokines (IL-1β, TNF, IL-6, IL-12 and IL-23) (10). These macrophages have an enhanced ability to eliminate intracellular pathogens. In contrast, alternatively activated macrophages (M2) express high levels of scavenger, mannose and galactose type receptors (10) and their arginine metabolism is shifted to production of ornithine and polyamines (17). Alternatively activated macrophages are immunosuppressive, they propagate Th2 polarized immune responses, and they contribute to the recruitment of polarized Th2 cells and eosinophils (9, 17). Increased expression of fibronectin on M2 macrophages mediates adhesion of different bacterial strains to these cells (18).
The increased numbers of alternatively activated macrophages in our study positively correlated with markers of a Th2 biased immune response, i.e. higher levels of IL-5 and ECP. A significant correlation was found between the number of CD206+ and CD163+ cells in the nasal mucosa and the IL-5 levels in nasal tissue homogenates of CRSwNP patients (P < 0.001). The level of ECP was also significantly correlated with the number of CD206+, but not with CD163+ cells. In line with our findings, a higher percentage of monocytes prone to alternative activation and expressing CD163, CCL-18 and IL-1RA has been reported in atopic dermatitis and atopic rhinoconjunctivitis (19). In agreement with earlier observations in asthmatic patients (20), we observed that CRSwNP patients who have asthma or AERD co-morbidity had significantly more CD163+ and CD206+ cells in their nasal tissue than CRSwNP patients without these comorbidities. The increased numbers of alternatively activated macrophages in CRSwNP could be explained by increased level of IL-33 (21), which is upregulated in asthma and CRSwNP (21, 22). IL-33 shifts the phenotype of alveolar macrophages toward an M2 phenotype (22). Another possible explanation for the presence of alternatively activated macrophages in the nasal mucosa of CRSwNP is the interaction of macrophages with apoptotic cells, which are reported to be present in large numbers in CRSwNP (23). The engulfment of apoptotic cells by macrophages was also shown to induce up-regulation of IL-10, IL-1ra, CCL-18, CD14, TGF-β, CCL-16 and mannose receptor (12), which are features of alternatively activated macrophages.
Several mediators that might link alternative activation of macrophages to the chronic inflammatory profile have been found in the nasal mucosa of CRSwNP patients. The presence of local IgE has been demonstrated to initiate the alternative activation program in macrophages in vitro (24). The ligation of FceRI by IgE on monocytes resulted in the production of alternatively activated macrophages with high levels of expression of mannose receptor (24). Interestingly, in our study the increase in the number of alternatively activated macrophages was positively correlated with high mucosal IgE production. As we described before, we confirmed that CRSwNP were characterized by higher local IgE concentration and the presence of IgE specific for S. aureus enterotoxins as compared to controls (6). Staphylococcal protein A (SpA) and staphylococcal enterotoxin A (SEA) were shown to promote the proliferation of B cells producing IgM, IgG and IgE (25). In summary, these data suggest that bacterial factors could cause production of local IgE and in this way contribute to the biased M2 polarization of the macrophages.
Compared to the general population, patients with CRSwNP have higher rates of S. aureus colonization in the nasal mucosa. As many as 63.6% of patients with CRSwNP show nasal carriage of S. aureus and 50% of CRSwNP patients develop local IgE to SAEs (3). In contrast, CRSsNP patients have levels of S. aureus colonization comparable to those in healthy subjects. In addition, intramucosal S. aureus was detected in greater numbers in CRSwNP patients with AERD than in controls and CRSsNP (6, 26). We hypothesize that the increased presence of intramucosal S. aureus in CRSwNP could be due to a deficit in the local immune defense and involving deficient phagocytosis of S. aureus. Therefore, in the present study we analyzed the phagocytic capacity of macrophages in nasal tissue from CRS patients and inferior turbinate control tissue using live GFP-expressing S. aureus. We found a significantly lower percentage of phagocytosis of S. aureus by CD206+ cells in CRSwNP as compared to control inferior turbinate tissue. The clearance of non-opsonized bacteria by macrophages depends on their innate immune receptors for detection, binding and internalization of pathogens (27) It has also been shown in the mice that alternatively activated macrophages (IL-4 pre-treated) were less capable of bacterial phagocytosis due to inhibition of PI3K activity (28). Therefore, we propose that the increased presence of S. aureus in CRSwNP can be partly explained by inefficiency of the phagocytic system in the sinomucosal tissue of these patients. In line with our study, defects in phagocytosis of bacteria and apoptotic cells and increased bacterial colonization have been described for several lower airway pathologies, including asthma (29), cystic fibrosis and chronic obstructive pulmonary disease (30). However, the mechanism of this deficient phagocytosis in CRSwNP still needs further investigation.
It has been reported that M2 macrophages produce TGF-β and other mediators. However, we could not find a link between the presence of M2 macrophages and concentrations of TGF-β in tissue. TGF-β was decreased in a group of CRSwNP, whereas in CRSsNP there was extensive tissue fibrosis accompanied by increased TGF-β signaling (31). These observations do not provide support for a role of alternatively activated macrophages in tissue fibrosis in CRS. However, macrophages are just one of many sources for TGF-β, thus may not alter the total amount of production to a meaningful extent.
Macrophages could be a reservoir of living bacteria in the tissue and could support persistence of infections (14). The cytokine milieu is one of the factors that can modulate S. aureus survival. Evidence has been provided for facilitated bacterial proliferation in the presence of exaggerated inflammatory cytokines, including IL-6, IL-1β and TNF (32). However, in opposite to those, IL-4 was shown to inhibit bacterial clearance during S. aureus infection and to promote septic arthritis in mice (33). Macrophages can eliminate intracellular staphylococci more efficiently if they are pre-stimulated with IFN-γ (14), whereas a Th2-polarized response has a negative impact on the innate immune host response to bacteria (34).
Since increased colonization with S. aureus in CRSwNP (35) was accompanied by high levels of IL-5, we questioned whether the augmented levels of IL-5 could contribute to the increased colonization by S. aureus. To answer this question, we analyzed the effect of IL-5 on intracellular S. aureus survival by using a human monocytic cell line (Thp-1 cells). Our data demonstrated that pretreatment of Thp-1 macrophages with IL-5 increases S. aureus survival.
In conclusion, we here demonstrate for the first time the presence of alternatively activated macrophages (M2) and deficient phagocytosis of S. aureus in CRSwNP. These data suggest that a defect in the phagocytic system in CRSwNP might contribute to the increased colonization by S. aureus in this condition. This observation supports the hypothesis that the cytokine milieu in the nasal mucosa could modify macrophage polarization and alter efficiency of the host defense mechanisms.
Professor C. von Eiff, University Hospital of Münster, Germany, is acknowledged for providing RN6390 S. aureus. This project was supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen – Projects 3G.0489. 08 to C.B., G.0642.10N to C.B and O.K., and 3G072810 to D.V.K.) and by the Interuniversity Attraction Poles programme (IUAP) – Belgian state – Belgian Science Policy P6/35. D.V.K. is a postdoctoral fellow paid by fellowship from FWO-Vlaanderen. We thank Dr. A. Bredan for editing the manuscript.