in vivo 30: 47-52 (2016)

CpG-Oligodeoxynucleotides in Chronic Rhinosinusitis Cell Culture

RICHARD BIRK, CHRISTOPH ADERHOLD, KARL HÖRMANN, ANGELA WENZEL, BENEDIKT KRAMER, TILL ESCHENHAGEN and J. ULRICH SOMMER

Department of Otorhinolaryngology Head and Neck Surgery, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Abstract. In chronic rhinosinusitis (CRS) an important nasal congestion, reduced olfaction and secondary acute feature is the infiltration of eosinophils, triggered by T-helper exacerbations with an increased bacterial infection of the type 2 cells (TH2). Binding of the CpG oligodeoxynucleotide nasal sinuses. These symptoms have a serious impact on the (CpG-ODN) ligand to toll-like receptor 9 (TLR9) induces a patient’s quality of life. One study has even suggested that shift from a TH2- to a TH1-type response. We evaluated the their quality of life is more severely impaired than patients hypothesis that CpG-ODN could reduce the predominantly with congestive heart failure (4). Due to the unknown TH2-driven response in our cultures. Materials and pathophysiology, CRS treatment is still a challenging Methods: Twenty samples from CRS patients with (CRSwNP) process. Furthermore, we have to distinguish CRS with nasal and without nasal polyposis (CRSsNP) were cultivated. The polyps (CRSwNP) from CRS without nasal polyps expression of interleukin 5 (IL-5), eotaxin 3 and matrix (CRSsNP). A predominant histological feature of CRS is a metalloprotease 9 (MMP-9) were evaluated with and without persistent underlying infiltration by eosinophils (5). This CpG-ODN. Results: Addition of CpG did not influence the accumulation of eosinophils might be caused by an influx of expression of IL-5 and eotaxin-3 DNA. Elevated MMP-9 T-helper type 2 (TH2) cells with a subsequent dominant TH2 expression in cultures from CRSwNP and CRSsNP patients profile (2). The TH2 lymphocytes and eosinophils could be established. Conclusion: CpG does not reduce the release interleukin (IL)-3, IL-5, IL-13 and the pro- attraction of eosinophils since no reduced IL-5 expression inflammatory eotaxin (6-9). These promote the was measured in our cultures. Yet, MMP-9 - an important production, recruitment and activation of eosinophils. IL-5 factor in tissue remodelling - was elevated in cultures from expression was observed in nasal polyps of patients suffering CRS patients. from CRSwNP (10). In CRSsNP, IL-13 is also significantly elevated in sinus lavage from patients with CRS compared Chronic rhinosinusitis (CRS) is one of the most common to non-CRS controls. IL-13 seems to afflict the function of chronic diseases in the United States and Europe. Up to 16% matrix metalloproteases, which remodel the extracellular of the Western population suffer from CRS (1). Currently, matrix (11). For example, addition of IL-13 could induce a CRS is defined by symptoms and endoscopic findings such significant increase in β-catenin expression in eosinophilic as of the nasal and paranasal sinus mucosa, CRS cultures compared to non-eosinophilic cultures (7). that presents for durations of longer than three months (2). Eotaxin, especially eotaxin-3, is supposed to play a pivotal Furthermore, it is associated with mucosal alterations role in the regulation of eosinophilic inflammation and ranging from inflammatory thickening to nasal polyps (3). extracellular matrix breakdown in CRSwNP. Due to the fact Patients complain of thick mucus production and subsequent that MMP-9’s exact role in polyposis still remains unclear (12) and MMP-9 is involved in tissue remodelling (11) this study was also set-up to determine the expression of MMP- 9. Additionally, Lim et al. found elevated expression of Correspondence to: Richard Birk, MD, Department of MMP-9 in CpG stimulated murine macrophages (13). Otorhinolaryngology Head and Neck Surgery, Medical Faculty Compared to bacterial DNA, vertebrate DNA shows an Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D- under-representation of the phosphate-guanosine 68167 Mannheim, Germany. Tel: +49 06213831600, Fax: +49 06213833827, e-mail: [email protected] (CpG) complex. In vertebrate DNA, the cytosine in the CpG motif is mostly methylated compared to bacterial DNA, that Key Words: Chronic rhinosinusitis, CpG-ODN, IL-5, MMP-9, is not methylated (14). The unmethylated CpG dinucleotides eotaxin 3. cause an activation of the innate immune system, via toll-like

0258-851X/2016 $2.00+.40 47 in vivo 30: 47-52 (2016) receptor 9 (TLR9). The activation of the immune system may Statistical analysis. Statistical analysis was performed with SAS be reproduced by the application of synthetic CpG- (SAS/STAT; Version 8, SAS Institute Inc., Cary, NC, USA). p- oligodeoxynucleotides (ODN) (15). TLR9 is expressed by Values were calculated by using the Student’s t-test and Dunnett’s procedure against the untreated control group (21). The level of leukocytes and able to detect CpG motifs in DNA (16). statistical significance was defined to be p<0.05, as no Bonferroni Binding of the CpG-ODN ligand to TLR9 induces a shift from adjustments were necessary. Plotting was done using “R”, an open a TH2- to the TH1-type response in the target tissue (17). It source environment for statistical computing and graphics (22). has been shown that the TH2 dominant inflammatory responses in the lungs could be prevented by the application of Results CpG-ODN (18). Furthermore, eosinophilic inflammation was abrogated in a murine model of asthma by CpG-ODN (19). Regarding the incubation duration, there was no significant In the present study, we tested the hypothesis that CpG- difference in expression of IL-5, eotaxin-3 or MMP-9 after ODN would reduce the predominantly TH2-driven chronic 24, 36, 72 and 96 h. inflammation in our cultures. IL-5. Treatment with CpG did not have any significant effect Materials and Methods on the expression of IL-5. But, IL-5 concentration seemed to be altered when comparing the control group (0.095 pg/ml Patients, tissue collection and cultures. Tissue collection and ±0.027 pg/ml; p=0.097) with the CRSwNP group (0.142 experiments were carried-out in the same manner as already pg/ml ±0.357 pg/ml), and when comparing the CRSsNP outlined in a previous study (20). Cultures were obtained from group (0.073 pg/ml ±0.134 pg/ml; p=0.05) with the patients suffering from CRSwNP or CRSsNP and undergoing CRSwNP group. No significant effect on the expression of functional endoscopic sinus surgery (FESS) at the Department of IL-5 was found when using CpGas or CpGsc as an Otorhinolaryngology at the University Hospital Mannheim, addendum. For details see Figure 1. Germany. Each group consisted of cultures from five different patients. The specimens from the control group were obtained from patients suffering from inverted papilloma during FESS. Written Eotaxin-3. No significant effect on the expression of eotaxin- consent was obtained from all patients and the study was approved 3 in the different groups could be shown by adding CpG to by the Ethics Committee of the Faculty of Medicine, Mannheim, the cultures, regardless of whether it was CpGas or CpGsc University of Heidelberg, Germany. (control group: 4.72 pg/ml±2.343 pg/ml; CRSwNP group: 4.262 pg/ml±1.577 pg/ml; CRSsNP group: 4.405 Test agent. The test agents were oligodeoxynucleotides (Eurofins pg/ml±2.293 pg/ml). For details see Figure 2. MWG Operon, Ebersberg, Germany) containing the following sequence: CpGas: TCC ATG ACG TTC CTG ACG TT and CpGsc: TCC ATG AGC TTC CTG AGT CT (Scramble siRNA). MMP-9. During treatment with CpG, a significant alteration in the MMP-9 expression was observed. These alterations were Incubation with CpG-ODN. ODNs were added in a concentration observed between the control group (6.43 pg/ml±1.78 pg/ml) of 20 μg/ml when the cultures showed at least 70% confluence (day and the CRSwNP group (47.186 pg/ml±48.914 pg/ml; 0) to all samples, except for the “negative control”. IL-5, eotaxin 3 p<0.001), as well as between the control group and the and MMP-9 expression in the supernatants of the cultures were CRSsNP group (33.037 pg/ml±53.244 pg/ml; p<0.001). evaluated after 24, 48, 72 and 98 h. However, between the two CRS groups, no significant effect Cytokine immunoassay. Storage of culture supernatants was was observed (p=0.418). As an addendum, no significant effect performed in sterile test tubes at –20˚C until an ELISA technique on the expression of MMP-9 was shown when using CpGas or (R&D Systems, Wiesbaden, Germany) was used to examine the CpGsc. Figure 3 shows graphs of data. expression of the interleukin, cytokine and proteinase. The system used a solid-phase monoclonal antibody and an enzyme-linked Discussion polyclonal antibody raised against the objectives. The exact products used included the following: DuoSet Human Total MMP9 Elisa/Order No. DY 911/R+D Systems/Minneapolis/USA. BD The innate immune system includes the so-called toll-like- OptEIA Human IL-5 Elisa Set/Order No. 555202/BD Biosciences, receptors (TLRs). The family of TLRs consists of 13 Franklin Lakes, New Jersey, USA. BD OptEIA Human Eotaxin members (TLR1 to TLR13). They recognize various Elisa Set/Order No 555175/BD Biosciences, Franklin Lakes, New structures such as lipoproteins, flagellin, lipopolysaccharide Jersey, USA. and nucleic acids (23). TLR9 mediated recognition of viral According to the manufacturer’s instructions, each ELISA assay and bacterial DNA (in terms of CpG) activates the innate determined IL-5, eotaxin 3 or MMP-9 concentrations in 100 μl of immune system and plays a critical role in adaptive immunity supernatant. The cells were grown in 96-well plates with 12 strips of 8 walls coated with an antibody against either of the proteins of (24). It has already been shown that TLR9 expression is the interest. After 24-96 h of incubation, the expression of markers of same in patients with CRSwNP or CRSsNP and in patients interest in the supernatants was determined (20). without CRS. Due to the fact that TLR9 stimulation results

48 Birk et al: CpG-oligodeoxynucleotides in Chronic Rhinosinusitis Cell Culture

Figure 1. Box-Whisker plot of IL-5 expression. The boxes represent the Figure 2. Box-Whisker plot of eotaxin 3 expression. The boxes represent interquartile range (IQR) with the whiskers extending up to 1.5-times the interquartile range (IQR) with the whiskers extending up to 1.5- the IQR. The median is marked with a solid line. times the IQR. The median is marked with a solid line.

in a strong TH1 response and induces a TH2/TH1 shift (25), we expected that the CpG-ODN addition to our cultures would reduce the predominantly TH2-driven chronic inflammatory state in our cultures. In a murine allergic model it has already been shown that CpG-ODN prevents the development of TH2-mediated eosinophilic inflammation and symptoms of allergic rhinosinusitis (19).

IL-5. The factor concentration did not have a significant effect on the expression of IL-5. Only a trend to an alteration of IL- 5 expression could be seen when comparing the control group with the CRSwNP group and the CRSwNP with the CRSsNP group. The cytokine IL-5, which is also called eosinophil differentiation factor, is a regulation factor of eosinophil growth, activation and expression (26). Eosinophil-mediated diseases are linked to increased levels of IL-5 expression (e.g., asthma, rhinitis and eosinophilic esophagitis) (27). It also plays a critical role in CRS. In CRSwNP, significantly elevated IL-5 levels were reported (28), in accordance to our results, which displayed an elevation tendency. Additionally, high Figure 3. Box-Whisker plot of MMP-9 expression. The boxes represent mRNA and protein levels of IL-5 have been detected in nasal the interquartile range (IQR) with the whiskers extending up to 1.5- polyposis (29). Hussain et al. showed that treatment with CpG times the IQR. The median is marked with a solid line. oligodeoxynucleotides could prevent the development of TH2- mediated eosinophilic inflammation and symptoms in a murine model of allergic rhinosinusitis (19). This has also inflammation, and airway hyper-responsiveness (30, 31). In been shown in other diseases with eosinophilic inflammation. our cultures, we could not confirm these findings, a fact that In murine and primate models of allergen-induced airway could be attributed to the different culture models used (30, hyper-responsiveness, CpG inhibits IL-5, eosinophilic 31). Our results showing a tendency of altered IL-5 levels in

49 in vivo 30: 47-52 (2016)

CRSwNP compared to both the control and CRSsNP groups, remains unclear (12). Elevated expression of MMP-9 has which supports the well-established theory that IL-5 plays a been described in murine macrophages by TLR9 stimulation role only in CRSwNP (32). Another explanation for our results with CpG (13). Therefore, our results confirm the findings might be that the limited surface area of cell cultures— of Lechapt-Zalcman et al. who reported an elevated compared to the sinus mucosa—does not allow for revelation concentration of MMP-9 in inflammatory endothelial cells of alterations in eosinophilopoiesis. in nasal polyps (43). No significant difference between patients with CRSwNP and CRSsNP in MMP-9 expression Eotaxin 3. Eotaxin, also called chemokine (C-C motif) ligand could be found, that is in line with a study by Watelet et al. 26 (CCL26), macrophage inflammatory protein 4-alpha (36). We assume that this is associated with the lack of (MIP-4-alpha), or thymic stroma chemokine-1 (TSC-1), is an difference in expression of TLR9 between controls and eosinophil specific β-chemokine. Shinkai et al. demonstrated patients with CRSwNP or CRSsNP (44). that eotaxin 3 is involved in the accumulation of leukocytes, especially eosinophils. The hypothesis states that eosinophils Conclusion are activated by eotaxin 3 expressed on vascular sites of endothelial cells, extravasate from the bloodstream, and then In conclusion, addition of CpG did not have any influence migrate to the center of the inflamed tissue through the on the expression of IL-5 or the concentration of eotaxin 3 action of eotaxin (33). In eosinophilic inflammatory diseases DNA. However, it affected MMP-9 expression in terms of an such as atopic dermatitis, allergic rhinitis, asthma and elevation in cultures from CRS patients with or without nasal parasitic infections, it is assumed that eotaxin is a key protein polyposis no matter if CpGas or CpGsc was used. Due to in their pathophysiology (34). In our cultures, the addition these results, we assume that CpG does not reduce the of CPG did not influence eotaxin 3 DNA concentration at attraction of eosinophils. Elevated levels of MMP-9 all. This is partly inconsistent with the results from Yao et expression in CRS could not be reduced by the addition of al., where eotaxin 1, 2 and 3 levels were all elevated in CpG to the cultures. Due to the fact that CpG stimulation eosinophilic nasal polyposis (6). Furthermore, Olze et al. increased the percentage of epithelial and polyp cells found a correlation between eotaxins and tissue eosinophilia expressing TLR9, we expected more differences in MMP-9 in CRSwNP (35). A consistent explanation why eotaxins, as expression, but could not confirm that. In allergic key chemotactic factors for eosinophils in eosinophilic nasal rhinosinusitis in BALB/c mice, the addition of CpG during polyposis, were not altered by CPG treatment in our ovalbumin sensitization prevents the development of TH2- experiments, cannot be given by the authors. mediated eosinophilic inflammation and symptoms result in a model of allergic rhinosinusitis (19). Our data could not MMP-9. During the treatment with CpG, significant confirm these reduced IL-5 levels in our cultures. As some alterations in the MMP-9 expression could be evaluated results only show trends towards a change, more studies are when comparing the control group with both the CRSwNP necessary to elevate these effects. group and the CRSsNP group. Metalloproteinases are proteolytic enzymes mainly responsible for the remodelling Conflicts of Interest of the extracellular matrix (ECM). 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