Cathelicidin

The Clinical Respiratory Journal

ORIGINAL ARTICLE

Cathelicidin (LL-37) and human b2-defensin levels of children with post-infectious bronchiolitis obliterans Ahmet Hakan Gedik1, Erkan Cakir1, Yasemin Gokdemir2, Zeynep Seda Uyan3, Abdurrahim Kocyigit4, Emel Torun5, Bulent Karadag2, Refika Ersu2 and Fazilet Karakoc2 1 Division of Pediatric Pulmonology, Bezmialem Vakif University, Istanbul, Turkey 2 Division of Pediatric Pulmonology, Marmara University, Istanbul, Turkey 3 Division of Pediatric Pulmonology, Kocaeli University, Istanbul, Turkey 4 Department of Biochemistry, Bezmialem Vakif Univeristy, Istanbul, Turkey 5 Department of Pediatrics, Bezmialem Vakif University, Istanbul, Turkey

Abstract Introduction: The antimicrobial peptides (AMPs) human b-defensins and cathelicidin (LL-37) are key factors in innate and adaptive immune responses of the respiratory tract and play an important role in many respiratory diseases. No data are available in the literature about the levels of these AMPs in paediatric patients with post-infectious bronchiolitis obliterans (BO). This study aimed to determine human b-defensin 2 (hBD2) and LL-37 levels and compare between post-infectious BO patients and the control group. Method: The patients diagnosed with post-infectious BO between September 2012 and 2013 and age- and-gender matched healthy controls were enrolled in this multi-center study. Serum hBD2 and LL37 levels were determined with the enzyme-linked immunosorbent assay method. Results: Sixty-three post-infectious BO patients and 65 healthy children (median age 73 6 55 and 78.74 6 36.32 months, respectively) were enrolled in the study. The mean of hBD2 levels in patients and the control group were 1.06 6 0.24 and 0.67 6 0.72 ng/mL, respectively (P < 0.001). The mean of LL-37 levels in patients and the control group were 72.13 6 29.06 and 50.10 6 21.98 ng/mL, respectively (P < 0.001). No correlation was found between these AMPs levels and chronological age, age at the time of diagnosis, gender, Z-scores of weight and length, hospitalization numbers, the disease history before diagnosis and 25-OH D vitamin levels. Conclusion: This is the first study to demonstrate the higher levels of serum hBD2 and LL-37 levels in paediatric post-infectious BO patients. These AMPs may have important roles in the immune systems and pathogenesis of these patients. Please cite this paper as: Gedik AH, Cakir E, Gokdemir Y, Uyan ZS, Kocyigit A, Torun E, Karadag B, Ersu R and Karakoc F. Cathelicidin (LL-37) and human b2defensin levels of children with post-infectious bronchiolitis obliterans. Clin Respir J 2015; 00: 000–000. DOI: 10.1111/crj.12331.

Key words antimicrobial peptide–bronchiolitis obliterans–cathelicidin–child–defensin Correspondence Ahmet Hakan Gedik, MD, Bezmialem Vakif University, Faculty of Medicine, Adnan Menderes Bulvarı (Vatan Cad.) Fatih Istanbul/ Turkey 34093 Tel: 1902124531700 Fax: 1902126217580 email: [email protected] Received: 03 September 2014 Revision requested: 21 April 2015 Accepted: 14 May 2015 DOI:10.1111/crj.12331 Authorship and contributorship Gedik AH and Cakir E designed and performed the study, wrote the initial manuscript. Gokdemir Y, Uyan SD and Torun E collected the data and took responsibility for the content of the manuscript. Kocyigit A performed the biochemistrical tests and analysed the data. Karadag B, Ersu R and Karakoc F contributed important reagents, reviewed and revised the manuscript and approved the final manuscript as submitted. Ethics The local ethic committee of Bezmialem Vakif University approved our present study, and informed consent was obtained from overall patients. This study meets the ethical guidelines. Funding source No funding was secured for this study. Conflict of interest The other authors have no conflicts of interest to disclose.

The Clinical Respiratory Journal (2015) • ISSN 1752-6981 C 2015 John Wiley & Sons Ltd V

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hBD2 and LL-37 in paediatric bronchiolitis obliterans

Introduction Antimicrobial peptides (AMPs) are cationic peptides that form essential elements of the innate immune system, which is particularly important in the respiratory tract because of continual exposure to particulates and pathogens entering through inhalation or other contact with the nasal or oral surface. The AMPs human b-defensins (hBDs) and cathelicidin (LL-37) are key factors in innate and adaptive immune responses of the respiratory tract and play an important role in many respiratory diseases (1). Tracheal antimicrobial peptide (TAP) produced by bovine respiratory epithelium was first described by Diamond et al. (2). TAP was shown to be a homologue to human b-defensin 2 (hBD2), which is secreted from epithelial cells in the lungs (1, 3, 4). LL-37 was first identified in rabbit granulocytes in 1991. LL-37 is produced by neutrophils, macrophages and various epithelial cells including respiratory cells (5, 6). LL-37 and hBD2 have similar features, directly inhibit the pathogens and modulate the innate and adaptive immune responses (3). The roles of AMPs in the pathogenesis of various respiratory diseases have been evaluated in several studies (1, 3, 7). Although post-infectious BO is one of the important aetiologies for chronic obstructive lung disease in children in developing countries, data are scarce about the pathogenesis of post-infectious BO. Although the role of AMPs in post-transplantation bronchiolitis obliterans (BO) pathogenesis has been studied (8–10), similar studies are lacking for postinfectious BO. In this study, we aimed to evaluate the level of AMPs in patients with post-infectious BO compared to ageand gender-matched healthy children. We also aimed to investigate the relationship between AMPs levels and the clinical parameters and vitamin D levels in the same group of patients.

Materials and methods Study design and patients This multi-center and cross-sectional study included 63 children who were diagnosed as post-infectious BO between 2012 and 2013 in the study centres, the Pediatric Pulmonology Departments of Bezmialem Vakif University, Marmara University, and Kocaeli University. The following well-defined criteria were used for the inclusion of a post-infectious BO diagnosis (11, 12): (1) Requiring hospitalization, persistent obstructive respiratory symptoms such as wheezing, crackles, 2

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tachypnea, hypoxemia for at least 60 days after the initial illness following a diagnosis of acute bronchiolitis or severe pneumonia in a previously healthy child, (2) Computerized tomography findings of mosaic pattern, vascular attenuation and expiratory air trapping, (3) Exclusion of other diagnosis such as congenital and acquired immunodeficiency, bronchopulmonary dysplasia, a-1-antitripsin deficiency, cystic fibrosis, congenital heart disease and pulmonary tuberculosis. A standardized data extraction form was used to obtain demographic characteristics, including date of birth, gender, age at diagnosis, weight and length Z scores and respiratory symptoms from medical records. Also, 25-OH vitamin D levels were measured and noted. The control group consisted of age- and gendermatched children who presented to Bezmialem Vakif University paediatric outpatient clinic for medical conditions other than infectious and inflammatory diseases. In both groups, there was no history of immune suppressive drug use or immune deficiency, or other chronic diseases.

Blood sampling and biochemistrical evaluation The blood samples, 5 mL taken from each patient, were centrifuged at the 3000 rpm for 10 min and the serum was quickly stored at 2808C. The concentrations of hBD2 and human LL-37 in the serum were measured by enzyme-linked immunosorbent assay (ELISA) kit, according to protocols provided by the manufacturers (Human DEF b2 ELISA kit; cat no.: 20782; lot no.: 140118103; Beijing, China, Human LL-37 Hycult Biotechnology; cat no.: HK321; lot no.: 14518K1213-N; R Microplate Uden; the Netherlands). Multiskan FCV Photometer (Thermo Scientific; USA) was used for reading at 450 nm. All results were expressed in ng/mL.

Statistical analysis Statistical Package for the Social Science (SPSS) 15.0 version was used for analysis. All data were registered to SPSS and evaluated. The numerical parameters were described with the mean, median and standard deviation; distributions of the categorical measurements were determined by frequencies and percentages. The one-sample Kolmogorov–Smirnov Test was used to evaluate the distributions of the levels of hBD2 and LL-37. Because of the high range of distributions, Mann–Whitney test was used to compare the levels between patients and the control group. Spearman’s


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Table 1. The demographic characteristics of the patients and controls Characteristics Gender n (%) Female Male Age (month) mean 6 SD (min–max) Age at the time of diagnosis (month) mean 6 SD (min–max) Z-scores of weight mean 6 SD Z-scores of height mean 6 SD Number of hospitalization before diagnosis mean 6 SD (min–max) Type of infection n (%) Viral Bacterial Unidentified

Patients (n 5 63)

Controls (n 5 65)

P

21 (33) 42 (67) 73.11 6 55.39 (8–192) 43.12 6 34.57 (4–146) 20.56 6 2.40 20.78 6 1.97 3.36 6 2.69 (1–12)

29 (44.6) 36 (55.4) 78.74 6 36.32 (12–192) –

0.19* 0.5†

20.01 6 1.31 20.28 6 1.3 –

0.18† 0.091† –

35 (55.6) 13 (20.6) 15 (23.8)

–

–

–

*chi-squared test. †student t-test.

correlation and chi-squared tests were used to evaluate the correlations between other characteristics and hBD2 and LL-37 levels.

Ethics committee approval Signed informed consent forms were obtained from the families. Ethical approval was obtained from the Bezmialem Vakif University Local Research Ethics Committee (DN. 2013/469).

Results The demographic characteristics of the children with post-infectious BO and controls were shown in Table 1. Age and gender distributions were not statistically different between the two groups. The most com-

Figure 1. The serum human b2-defensin levels of the patients and controls.


mon symptoms of the patients were cough (n 5 45, 71.4%), wheezing (n 5 41, 65%), sputum (n 5 35, 55.5%), dyspnea and tachypnea (n 5 5, 7.9%). The physical examination findings were wheezing (n 5 35, 55.5%), prolonged expiration (n 5 23, 36.5%), crackles (n 5 20, 31.7%) and rhonchus (n 5 12, 19%). BO patients had significantly higher hBD2 levels compared to controls (1.06 6 0.24 and 0.67 6 0.72 ng/ mL, respectively, P < 0.001; Fig. 1). The mean of LL-37 levels in patients and the control group were 72.13 6 29.06 and 50.10 6 21.98 ng/mL, respectively. A statistically significant difference was found between the two groups (P < 0.001; Fig. 2). No correlation was found between hBD2 and LL-37 levels and age, age at the time of diagnosis, gender,

Figure 2. The serum cathelicidin (LL-37) levels of the patients and controls.

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Z-scores of weight and length, hospitalization numbers, the disease history before diagnosis and 25-OH D vitamin levels.

Discussion AMPs hBDs and LL-37 are important factors in the immune response of the respiratory tract and play an important role in many respiratory diseases (1, 3, 7). To our knowledge, this is the first study evaluating the role of AMPs in patients with post-infectious BO. In this study, levels of hBD2 and LL-37 were found to be higher in children with post-infectious BO compared to healthy controls. Several studies have evaluated the role of AMPs in different respiratory diseases and shown complex relationship. In cystic fibrosis patients, there appeared to be impaired activity, impaired production, and accelerated breakdown of hBDs, which may have a role in reduced antibacterial activity, and LL-37 levels have also been shown to be high in these patients (13, 14). In diffuse panbronchiolitis, which is a progressive inflammatory disease, Hiratsuka et al. (15) showed high levels of hBD2. Bacterial pneumonia, pulmonary tuberculosis and sepsis were also found to be associated with elevated hBDs and LL-37 levels in the lung fluids and blood (3, 7). AMPs may affect the course of respiratory disease via different mechanisms. In addition to their direct antimicrobial and antiviral activities, hBDs and LL-37 stimulate the recruitment of inflammatory cells to the mucosa in different respiratory diseases (3). Defensins can be chemotactic for dendritic cells, stimulate mast cells, and hBD2 can activate dendritic cells via binding to toll-like receptors (16). LL-37 stimulates chemotaxis of neutrophils, monocytes and T cells (17). LL-37 also down-regulates the monocyte inflammatory cytokine production and has a role in the generation of chemokines and IL-10 (18). So it is clear that these peptides have a role in modulating further innate and adaptive immune responses in inflammation of diseases (1, 3). Inflammation and fibrosis of the terminal and respiratory bronchioles are also the main characteristics of the pathogenesis of BO. BO is an uncommon and severe form of chronic obstructive lung disease that usually develops after lower respiratory tract infections in children and is known as postinfectious BO. Stevens–Johnson syndrome, lung and bone marrow transplantations and connective tissue disorders are the other underlying aetiologies in patients with BO (11, 12). To date, only a few studies have evaluated the role of AMPs in patients with post-transplant BO (8–10). 4

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Anderson et al. hypothesized that these elements of the pulmonary innate response play an etiological role in post-transplant BO. They found high levels of LL-37 and a-defensins, but normal hBD2 levels (9). Ross et al. and Triveedhi et al. (8, 10) showed high levels of hBD2. In these studies, the authors concluded that hBD2 production leads to an increase in cellular activation resulting in increased production of inflammatory cytokines, increase the initiation and propagation of an adaptive immune system, affect epithelial repair because of being mitogenic for fibroblast and increasing collagen type I vs III expression, and resulting epithelial cell proliferation and remodelling that contribute towards the development of post-transplant BO. In addition, no relation was shown between LL37 and hBD2 levels and prognosis of BO patients in these studies. We also found high levels of both hBD2 and LL-37 in paediatric post-infectious BO similar to patients with post-transplantation BO. The significantly higher levels of these peptides may trigger excessive inflammatory response and play an important role in the pathogenesis of lung damage in patients with post-infectious BO.

Conclusion This is the first study demonstrating high levels of serum hBD2 and LL-37 levels in paediatric postinfectious BO patients. These AMPs may have important roles in immune systems and the pathogenesis of these patients. Further studies are required in order to evaluate the role of AMPs levels in patients with postinfectious BO.

Acknowledgments The remaining authors have no financial relationships relevant to this article to disclose.

References 1. Tecle T, Tripathi S, Hartshorn KL. Review: defensins and cathelicidins in lung immunity. Innate Immun. 2010;16: 151–9. 2. Diamond G, Zasloff M, Eck H, Brasseur M, Maloy WL, Bevins CL. Tracheal antimicrobial peptide, cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc Natl Acad Sci USA. 1991; 88: 3952–6. 3. Doss M, White MR, Tecle T, Hartshorn KL. Human defensins and LL-37 in mucosal immunity. J Leukoc Biol. 2010;87: 79–92.


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4. Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson JM. Human beta-defensins 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest. 1998; 102: 874–80. 5. Bals R, Wang X, Zasloff M, Wilson JM. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA. 1998;95: 9541–6. 6. Larrick JW, Morgan JG, Pallings I, Hirata M, Yen MH. Complementary DNA sequence of rabbit CAP18-a unique lipopolysaccharide binding protein. Biochem Biophys Res Commun. 1991;179: 170–5. 7. Torun E, Cakir E, Aktas EC, Gedik AH, Deniz G. Intracellular cytokine and cathelicidin secretion from monocytes and neutrophils in childhood tuberculosis. Pediatr Infect Dis J. 2014;33: 224–6. 8. Ross DJ, Cole AM, Yoshioka D, et al. Increased bronchoalveolar lavage human beta-defensin type 2 in bronchiolitis obliterans syndrome after lung transplantation. Transplantation. 2004;78: 1222–4. 9. Anderson RL, Hiemstra PS, Ward C, Forrest IA, Murphy D, Proud D, Lordan J, Corris PA, Fischer AJ. Antimicrobial peptides in lung transplant recipients with bronchiolitis obliterans syndrome. Eur Respir J. 2008;32: 670–7. 10. Tiriveedhi V, Banan B, Deepti S, Nataraju A, Hachem R, Trulock E, Alexander PG, Thalachallour M. Role of defensins in the pathogenesis of lung allograft rejection. Hum Immunol. 2014;75: 370–7. 11. Champs NS, Lasmar LM, Camargos PA, Marquet C, Fischer DB, Mocelin HT. Post-infectious bronchiolitis obliterans in children. J Pediatr (Rio J). 2011;87: 187–98.



12. Fischer GB, Sarria EE, Mattiello R, Mocelin HT, Castro-Rodriguez JA. Post-infectious bronchiolitis obliterans in children. Paediatr Respir Rev. 2010;11: 233–9. 13. Chen CI, Schaller-Bals S, Paul KP, Wahn U, Bals R. Beta-defensin and LL-37 in bronchoalveolar lavage fluid of patients with cystic fibrosis. J Cyst Fibros. 2004;3: 45–50. 14. Taggart CC, Greene CM, Smith SG, Levine RL, McCray PB Jr, O’Neill S, McElvaney NG. Inactivation of human betadefensins 2 and 3 by elastolytic cathepsins. J Immunol. 2003;171: 931–7. 15. Hiratsuka T, Mukae H, Liboshi H, Ashitani J, Nabeshima K, Minematsu T, Chino N, Ihi T, Kohno S, Nakazato M. Increased concentrations of human betadefensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis. Thorax 2003;58: 425–30. 16. Biragyn A, Ruffini PA, Leifer CA, et al. Toll-like receptor 4dependent activation of dendritic cells by beta-defensins 2. Science. 2002;298: 1025–9. 17. De Yang, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, Oppenheim JJ, Chertov O. LL-37-the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL 1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med. 2000; 192: 1069–74. 18. Mookherjee N, Lippert DN, Hamill P, et al. Intracellular receptor for human host defense peptide LL-37 in monocytes. J Immunol. 2009;183: 2688–96.

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