PRO-INFLAMMATORY ROLE FOR NF-kB IN CHILDHOOD OSA SYNDROME http://dx.doi.org/10.5665/sleep.3236

A Pro-Inflammatory Role for Nuclear Factor Kappa B in Childhood Obstructive Sleep Apnea Syndrome Lee P. Israel, MSc1,2; Daniel Benharoch, MD3; Jacob Gopas, PhD1,4; Aviv D. Goldbart, MD, MSc2,5,6 1 Department of Microbiology and Immunology, 2Pediatric Pulmonary and Sleep Research Laboratory, 3Department of Pathology, 4Department of Oncology, 5Department of Pediatrics, and 6Sleep-Wake Disorders Center, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel

Study Objectives: Childhood obstructive sleep apnea syndrome (OSAS) is associated with an elevation of inflammatory markers such as C-reactive protein (CRP) that correlates with specific morbidities and subsides following intervention. In adults, OSAS is associated with activation of the transcription factor nuclear factor kappa B (NF-kB). We explored the mechanisms underlying NF-kB activation, based on the hypothesis that specific NF-kB signaling is activated in children with OSAS. Design: Adenoid and tonsillar tissues from children with OSAS and matched controls were immunostained against NF-kB classical (p65 and p50) and alternative (RelB and p52) pathway subunits, and NF-kB-dependent cytokines: interleukin (IL)- 1α, IL-1β, tumor necrosis factor-α, and IL-8). Serum CRP levels were measured in all subjects. NF-kB induction was evaluated by a luciferase-NF-kB reporter assay in L428 cells constitutively expressing NF-kB and in Jurkat cells with inducible NF-kB expression. p65 translocation to the nucleus, reflecting NF-kB activation, was measured in cells expressing fluorescent NF-kB-p65-GFP (green fluorescent protein). Setting: Sleep research laboratory. Patients or Participants: Twenty-five children with OSAS and 24 without OSAS. Interventions: N/A. Measurements and Results: Higher expression of IL-1α and classical NF-kB subunits p65 and p50 was observed in adenoids and tonsils of children with OSAS. Patient serum induced NF-kB activity, as measured by a luciferase-NF-kB reporter assay and by induction of p65 nuclear translocation in cells permanently transfected with GFP-p65 plasmid. IL-1β showed increased epithelial expression in OSAS tissues. Conclusions: Nuclear factor kappa B is locally and systemically activated in children with obstructive sleep apnea syndrome. This observation may motivate the search for new anti-inflammatory strategies for controlling nuclear factor kappa B activation in obstructive sleep apnea syndrome. Keywords: Inflammation, nuclear factor kappa B, sleep disordered breathing Citation: Israel LP; Benharoch D; Gopas J; Goldbart AD. A pro-inflammatory role for nuclear factor kappa B in childhood obstructive sleep apnea syndrome. SLEEP 2013;36(12):1947-1955.

INTRODUCTION Obstructive sleep apnea syndrome (OSAS) is a common disorder in children, with a prevalence of 1-5%.1 It is associated with significant neurobehavioral,2,3 cognitive,4,5 and cardiovascular morbidity.6,7 Adenotonsillectomy (T&A) is the most common treatment for the disorder and is reserved for children with moderate to severe OSAS, as defined by polysomnography.8,9 In adults, this seemingly simple mechanical disorder is commonly associated with nasopharyngeal and oropharyngeal inflammation10 as well as with systemic inflammation. Activation of the transcription factor nuclear factor kappa B (NF-kB), leading to increased expression of downstream genes such as tumor necrosis factor (TNF)-α, constitutes an important pathway linking OSAS with systemic inflammation and end-organ cardiovascular disease.11 NF-kB activity is increased in circulating neutrophils and monocytes, and decreases following conventional mechanical therapy with continuous positive airway pressure in adults.12,13 Recent studies suggest

Submitted for publication January, 2013 Submitted in final revised form June, 2013 Accepted for publication June, 2013 Address correspondence to: Aviv D. Goldbart, MD, MSc, Department of Pediatrics, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Post Office Box 151, Beer Sheva, Israel, 84101; Tel/Fax: 972-8-6244465; E-mail: [email protected] SLEEP, Vol. 36, No. 12, 2013 1947 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

that systemic inflammatory markers such as C-reactive protein (CRP), a known cardiovascular risk marker, are increased in children with OSAS and decreased following T&A.14,15 We previously showed that the lypooxygenase pathway is systemically activated in the upper airways of children with OSAS; activation of this pathway correlates with the severity index of the disease.16,17 Moreover, anti-inflammatory therapies such as nasal steroids and leukotriene modifiers improve respiratory parameters during sleep and reduce adenoid size.18,19 Surprisingly, there are no published data regarding inflammatory changes associated with NF-kB activation in children with OSAS. The NF-kB family of transcription factors plays a pivotal role in the regulation of immune responses, proliferation, apoptosis, and expression of certain viral genes,20 and in childhood chronic inflammatory disorders such as inflammatory bowel disease and asthma.21,22 Therefore, the NF-kB signaling pathway has been extensively targeted for pharmacological intervention. The therapeutic and preventive effects of many natural products may, at least in part, be due to their ability to inhibit NF-kB.23 The two known pathways for NF-kB activation are the canonical (classical) and the non-canonical (alternative) pathways. A functional NF-kB molecule is a homodimer/heterodimer composed of members of the Rel family of proteins, which includes RelA (p65) and p50 in the classical pathway and RelB, cRel, and p52 in the alternative pathway. The major form of NF-kB that is rapidly induced after stimulation is the RelA/ p50 complex. NF-kB is maintained in an inactive form in the cytoplasm by its inhibitor IkB, which binds to NF-kB and masks NF-kB in Children with OSAS—Israel et al

Table 1—Demographic data for patients with OSAS and control subjects Patients (N) Mean age in y F/M (N) BMI (z-score) AHI (events/h TST)

OSAS 25

Non OSAS 24

P-value

5.1 ± 3.2 (range 3.2-8.1)

5.3 ± 3.5 (range 3.1-9.3)

NS

14/11

13/11

NS

0.62 ± 1.04

0.57 ± 1.11

NS

14.1 ± 2.9 (range 10.2-17.1)

0.6 ± 0.2 (range 0.4-0.9)

P < 0.001

AHI, apnea-hypopnea index; BMI, body mass index; NS, not significant; TST, total sleep time.

its nuclear localization signal.24 IkB is phosphorylated by the IkB kinase complex (IKK), resulting in its degradation by the proteosome and the release of NF-kB. Following release from IkB, NF-kB translocates to the nucleus, where it stimulates the transcription of a wide variety of genes, including cytokines, cell adhesion molecules, and acute-phase response proteins. In the alternative pathway, activation of IKKα phosphorylates NF-kB precursors (p100/RelB); the proteosome then processes these precursors into the active p52/RelB heterodimer. Based on the functions of NF-kB in adults with OSAS, we hypothesized that NF-kB signaling is similarly activated in children with OSAS. We therefore investigated the local and systemic involvement of NF-kB as a proinflammatory factor in children with and without OSAS. MATERIALS AND METHODS Patients and Setting The Soroka University Medical Center Ethics Committee approved this study, and informed consent was obtained from the legal caretaker of each participant. Twenty-five children in whom severe OSAS was previously diagnosed (apneahypopnea index ≥ 10/h) based on overnight study with polysomnography were recruited, as well as 24 age-, sex-, and body mass index (BMI)-matched control subjects with normal polysomnography findings (apnea-hypopnea index < 1/h). Exclusion criteria were any history of cardiovascular disorder, allergies, asthma/wheezing, smoking in the immediate family, and familial craniofacial or genetic disorders. A summary of the demographic information is presented in Table 1. BMI z-scores were calculated according to the following website: http://stokes.chop.edu/web/zscore/index.php. All children were Caucasian. All subjects received operations 3.1 ± 1.2 mo, mean + standard deviation (SD), range 2.2-6.1 mo, following polysomnography. Sera were collected from children undergoing T&A in the operating room on the morning of the operation. Following centrifugation, sera were stored at -70°C. In addition, during T&A, a portion of the tissue that was removed was immediately fixed in formalin and paraffin for immunohistochemistry. Control tissue samples were obtained from children who underwent T&A due to recurrent tonsillar infections. These subjects were healthy at the time of the operation, and at least 4 w had passed since the last dose of antibiotics for their previous SLEEP, Vol. 36, No. 12, 2013 1948 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

tonsillar infection. All subjects with OSAS were assessed by an ear, nose, and throat surgeon and an anesthesiologist; if acute infection was present, the patients did not undergo the operation and were excluded from the study. C-Reactive Protein High-sensitivity CRP (HS-CRP) was measured via particleenhanced immunonephelometry using the BN ProSpec system (Newark, DE, USA). All samples were assessed in duplicate and assayed at two dilutions. Data are presented in mg %. Blood draws were performed as described in the previous section. Immunohistochemistry Adenoids and tonsils fixed in formalin and embedded in paraffin were cut using a microtome into 4-μm sections in the pathology department; at least three sections from each sample were evaluated. Immunohistochemical analysis was carried out with the ABC peroxidase complex method using the Vectastain kit from Vector Laboratories (Burlingame, CA, USA). The following primary antibodies were obtained: p65 (Diagnostic Biosystems, Pleasanton, CA, USA); p50, p52, and RelB (Santa Cruz Biotechnology, Santa Cruz, CA, USA); TNF-α (ABCAM, Cambridge, MA, USA); and interleukin (IL)-1α and IL-1β (R&D Systems, North Las Vegas, NV, USA). The pathologist, who was blinded to the source of the samples, scored them as negative or as positive if more than 10% of the cells in the sample stained positive.25 We used Hodgkin’s lymphomaderived lymph nodes as a positive control (Reed-Sternberg cells). Unstained cells or cells with background staining in the biopsy specimens served as internal negative controls for the antibodies used. Cell Culture L428 cells (derived from human Hodgkin’s lymphoma) constitutively express active NF-kB due to the absence of IkB, which we confirmed by immunoblotting. NF-kB is inducible in Jurkat cells (derived from human T cell leukemia). Both cell lines were maintained in Roswell Park Memorial Institute medium. African green monkey kidney-derived COS-7 cells were maintained in Dulbecco’s Modified Eagle’s Medium and were passaged by trypsinization. Media were supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, and 1% penicillin-streptomycin (Biological Industries, Beit Haemek, Israel). Transfection L428 cells were stably transfected by electroporation.26 Jurkat cells were transiently transfected via the liposome method using the Jet-PI kit (Tamar, Israel). The cells were transfected with the luciferase-NF-kB reporter gene containing the consensus sequence derived from the human IL-2 promoter. This reporter construct was provided by Professor M. Aboud from the Department of Microbiology and Immunology, Ben Gurion University of the Negev. COS-7 cells were stably transfected by electroporation with GFP-p65 plasmid, which was a gift from Professor Rainer de Martin, Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria. Stable L428 transfectants were selected with 1,000 μg/mL of G418 (Gibco; Langley,OK) and maintained in 500 μg/mL of G418. NF-kB in Children with OSAS—Israel et al

Stable COS-7 transfectants were selected with 2,000 μg/mL of G418 and maintained in 1,000 μg/mL of G418. Luciferase-NF-kB Reporter Gene Assay L428 cells stably expressing the luciferase-NF-kB reporter gene (1 × 106 cells) were incubated for 2 h in 200 μL of Roswell Park Memorial Institute-1640 medium without fetal bovine serum that contained 10% serum from children with OSAS or matched controls. Jurkat cells (5 × 105 cells) were transiently transfected with the same luciferase-NF-kB reporter plasmid and the Renilla control plasmid (Renilla reniformis; Promega, Madison,WI), at ratio of 1:10 Renilla:NF-kB. Forty-eight h after transfection, the cells were washed, resuspended, and incubated for 2 h in 200 µL of medium lacking fetal bovine serum, with the addition of 10% serum from children with OSAS or matched controls. Cells were then harvested, lysed, and monitored by a luciferase reporter assay kit (Promega) according to the manufacturer’s instructions. Measurements were carried out using a luminometer at 300 nm. Data were normalized to the protein concentration in each L428 cell lysate as measured by the Bradford method (BioRad, Hercules, CA). For Jurkat cell lysates, normalization was determined by the luciferase:Renilla ratio. GFP-p65 Translocation Assay African green monkey kidney-derived COS-7 cells stably expressing the GFP-p65 plasmid were seeded into six-well plates and kept in 700 µL of Dulbecco’s Modified Eagle’s Medium without fetal bovine serum. Serum from children with OSAS or controls (10%) was then added to each well, and the cells were photographed under a fluorescence microscope after 10, 30, and 60 min of incubation. Statistical Analyses Results are presented as means ± SD, unless otherwise stated. All analyses were conducted using statistical software SPPS version 17.0 (Armonk, NY, USA). All numeric data were subjected to statistical analyses with either t tests or two-way analysis of variance procedures for repeated measures, as described by Neuman-Keuls. Post hoc tests were performed as appropriate. A two-tailed P < 0.05 was considered statistically significant. RESULTS NF-kB p65 and p50 Subunits are Overexpressed in the Adenoids and Tonsils of Children with OSAS Adenoids and tonsils from a randomly selected subsample of 10 children in whom severe OSAS has been diagnosed and from 10 matched controls were stained with antibodies against the two most common NF-kB classical subunits, p50 and p65 (RelA). We observed substantial differences between the two groups; compared to the control group, the OSAS group exhibited stronger p50 and p65 cytoplasmic and perinuclear staining in germinal center lymphocytes. p50 and p65 staining was nearly absent in the group without OSAS (Figure 1, top panel). Immunohistochemistry against the alternative NF-kB pathway subunits p52 and RelB was negative in all samples (Figure 1, bottom panel). SLEEP, Vol. 36, No. 12, 2013 1949 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

IL-1α, an Inflammatory Cytokine Induced by NF-kB, is Overexpressed in the Adenoid and Tonsillar Tissue of Children with OSAS The IL-1α and IL-1β inflammatory cytokines are induced by the NF-kB classical signaling pathway. IL-1 production in certain cell types, such as macrophages and lymphocytes, is elevated in response to inflammatory stimuli. We stained for IL-1α and IL-1β using the paraffin blocks shown in Figure 1. IL-1α was substantially overexpressed in the germinal centers of the tonsils and adenoids of the OSAS group versus the group without OSAS (Figure 2A). In contrast, IL-1β staining in both groups was low in the germinal center, whereas IL-1β was highly expressed in epithelia from the OSAS group (Figure 2B). CRP, a Marker for Systemic Inflammation, is Increased in Children with OSAS In order to ascertain the presence of systemic inflammation, we investigated the well-established inflammatory biomarker CRP. In comparison with children with OSAS (25), children without OSAS (24) displayed lower circulating concentrations of CRP at the time of diagnosis (0.45 ± 0.21 mg % versus 0.15 ± 0.10 mg %, respectively; P < 0.01; Figure 3). NF-kB is Activated by Stimulation with Sera from Children with OSAS Based on the knowledge that systemic inflammation is involved in the pathophysiology of OSAS, as represented by increased CRP circulating levels in adults and children, we assessed the systemic involvement of NF-kB. We incubated two cell lines with 10% sera from children with OSAS and matched controls for 2 h, then evaluated NF-kB activity via a luciferase reporter assay. Jurkat cells transiently expressing the luciferase-NF-kB plasmid exhibited a 40% increase in NF-kB activity after incubation with OSAS sera, in comparison with sera from the control group (Figure 4A). L428 cells with constitutive activation of NF-kB and stable expression of the luciferase-NF-kB plasmid displayed a 10% increase in NF-kB activity after OSAS sera incubation compared with sera from children without OSAS (Figure 4B). Activation of NF-kB (p65) by OSAS Sera is Mediated by Translocation of GFP-p65 to the Nucleus In order to confirm the systemic involvement and activation of NF-kB in children with OSAS, we evaluated p65 nuclear translocation following the addition of sera from these children to COS cells stably expressing GFP-p65. p65 translocated from the cytoplasm to the nucleus in these cells after a 1-h incubation with 10% sera from children with OSAS. In contrast, incubation with sera from the non OSAS group did not activate NF-kB, and p65 remained cytoplasmic throughout the experiment (Figure 5). DISCUSSION Here, we present evidence that NF-kB, a well-characterized transcription factor that participates in the initiation and progression of inflammation, is involved in the pathophysiology of OSAS in children. We show that both NF-kB and IL-1α are overexpressed in adenoids and tonsils from patients with OSAS versus age-, sex-, and BMI-matched controls. We also found evidence for systemic activation of NF-kB triggered by NF-kB in Children with OSAS—Israel et al

p65

p50

×100

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×100

×600

GC OSAS

GC

GC

GC

×100

Non OSAS

×600

×100

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GC

GC

GC GC

RelB

p52 ×600

×600

×600

×600

OSAS

Non OSAS

Figure 1—Surgically removed adenoids and tonsils from patients and the controls were immunostained using primary antibodies and the ABC immunoperoxidase method. Antibodies against classical nuclear factor kappa B (NF-kB; p50, p65) and alternative NF-kB (p52, RelB) subunits were used. Increased staining of p50 and p65 in the cytoplasm and perinucleus of lymphocytes of the germinal centers (GC) was observed in tonsillar tissue from patients with OSAS in comparison with diffuse and background staining in the control group (top panels). The tissues stained with antibodies against p52 and RelB showed no sabstantial differences between the two groups (bottom panels).

incubation with serum from patients with OSAS. This disorder causes morbidity that involves mechanisms including intermittent hypoxia (IH) and sleep fragmentation.27,28 However, an increasing number of studies have reported evidence of local and systemic inflammation in OSAS pathophysiology in both adults and children.29 To date, systemic activation of NF-kB has been related only to adult OSAS pathophysiology.12,30 Several investigations have uncovered increased levels of neutrophils in the sputum of patients with OSAS, which correlates with disease severity.31 Exhaled condensate specimens revealed high concentrations of leukotrienes in children in whom OSAS was diagnosed that correlate with polysomnographic and radiological parameters.17 An inflammatory process involving cysteinyl leukotrienes was also present in tonsils SLEEP, Vol. 36, No. 12, 2013 1950 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

from pediatric patients with OSAS,32 and immunohistochemical studies revealed high levels of leukotriene 1 and 2 receptors in the tonsils of patients with OSAS.16 Interestingly, Li and colleagues recently reported the IH-induced DNA binding activity of NF-kB in a monocyte cellular model, whereas both MAPK and NF-kB blockers partially inhibited the IH-induced expression of 5-lypooxygenase and leukotriene A4 hydrolase, suggesting that NF-kB plays a major role in leukotrienedependent activation in patients with OSAS.33 Furthermore, IH increases NF-kB translocation, and the NF-kB inhibitors gliotoxin and parthenolide increase neutrophil apoptosis and decrease IL-8 expression.34 Collectively, these observations demonstrate the essential role played by NF-kB signaling and downstream genes in OSAS. NF-kB in Children with OSAS—Israel et al

Non OSAS

OSAS

A

×100

IL1-α

GC

B

IL1-β

×600

×100

GC

GC

×100

×600

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×400

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×400

E E

E E

Figure 2—Slides prepared from the paraffin blocks shown in Figure 1 were stained with antibodies against interleukin (IL)-1α and IL-1β. A, IL-1α staining was positive in obstructive sleep apnea syndrome (OSAS) tissue compared with control tissue. B, IL-1β showed similar, low levels of staining in lymphocytes from both groups, but exhibited positive epithelial (E) expression in OSAS samples. GC, germinal centers.

SLEEP, Vol. 36, No. 12, 2013 1951 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

0.7

P < 0.01

0.6 0.5

CRP (mg/dl)

Overexpression of NF-kB in the lymphoid tissues of children with OSAS is consistent with our findings; NF-kB may propagate local inflammation due to its ability to induce cell and tissue inflammatory responses through various signaling pathways.26,35-37 We observed substantial overexpression of two classical NF-kB pathway subunits, p65 and p50, in the germinal centers of tonsils and adenoids from children with OSAS. Germinal centers are unique compartments in which B cells undergo affinity maturation upon stimulation by T-dependent antigens. Germinal center formation occurs in secondary lymph organs such as lymph nodes and spleen,38 and is induced by various stimuli. Certain stimuli such as cluster of differentiation 40 and lipopolysaccharide also activate NF-kB signaling pathways.22,39 Both p65 and p50 contribute to germinal center function, which is critical for proper maturation and proliferation of B cells.38 The presence of p65 and p50 in the upper airway lymphoid tissue of patients with OSAS, but not in tissue from controls, is consistent with the activation of the adaptive immune system in childhood OSAS or the involvement of NF-kB in the germinal centers during B cell maturation. We believe that this intriguing observation will open new avenues for understanding the interactions of lymphocyte subpopulations, tonsillar hypertrophy, and eventual obstruction of the upper airway. No substantial differences were observed between the OSAS and the control groups with regard to alternative NF-kB subunits p52 and RelB, suggesting that only the classical NF-kB pathway is involved in this disease. There is ample evidence demonstrating activation of the classical NF-kB pathway by proinflammatory cytokines, chemokines, and bacterial or viral products.39 Our detection of the activation of the classical NF-kB pathway (the alternative pathway serving as an internal control) indicates

0.4 0.3 0.2 0.1 0.0

OSAS

Non OSAS

Figure 3—C-reactive protein (CRP, mg %) decreased significantly after adenotonsillectomy (P < 0.01). Data are presented as mean ± standard deviation.

that specific processes are activated in these organs; these pathways are thus candidates for future interventions. Additionally, we observed over-expression of IL-1α in tissue sections from adenoids and tonsils of patients with OSAS. IL-1 is a potent proinflammatory cytokine that consists of two distinct proteins, IL-1α and IL-1β. IL-1 production and cellular secretion is regulated by NF-kB, and expression of the gene encoding IL-1α is controlled by the classical NF-kB subunit p65.27 Moreover, an active binding site for NF-kB exists in the IL-1α promoter, suggesting that NF-kB also regulates IL-1α production.40 NF-kB in Children with OSAS—Israel et al

B

120

NF-κB luciferase activity (%) in L-428

NF-κB luciferase activity (%) in Jurkat cells

A

100 80 60 40 20

OSAS

Non OSAS

110 100 90 80 70 60 OSA

Non OSAS

Figure 4—Jurkat cells transiently transfected with luciferase-nuclear factor kappa B (NF-kB) and Renilla plasmids and L428 cells stably expressing the luciferase-NF-kB reporter were incubated with 10% serum from children with obstructive sleep apnea syndrome (OSAS) and controls. Luciferase activity was normalized to the efficiency of transfection, as determined by Renilla fluorescence. The results (A and B) are expressed as the relative fluorescence (%) of each sample in comparison to the sample with the highest luciferase activity in the experiment. NF-kB activation was significantly higher in cells incubated in OSAS sera than in control sera (t test, P < 0.0003). Horizontal lines represent median values; the upper and lower box limits indicate the 25th percentile and 75th percentile; whiskers represent the 10th and 90th percentiles.

IL-1 binds to its receptor, IL-1R1, and induces signal transduction resulting in the phosphorylation of its inhibitor IkB and the activation and translocation of NF-kB to the nucleus. IL-1-dependent NF-kB signaling leads to the transcription of a variety of proinflammatory genes, including genes encoding inflammatory cytokines, chemokines, and adhesion molecules.41 The strong cytoplasmic and nuclear staining of IL-1α in adenoids and tonsils of children with OSAS was not seen in children with recurrent infections but without OSAS; NF-kBinduced inflammation in OSAS is consistent with IL-1α tissue expression. Moreover, IL-1 may further induce NF-kB activation due to exposure to stimuli such as hypoxia,42 a dominant component in most patients with OSAS. Interestingly, IL-1α was strongly expressed in epithelial cells in the peripheral layer of the germinal centers of adenoids and tonsils from patients with OSAS. IL-1α has been previously shown to induce the migration of airway epithelial cells via the activation of NF-kB, specifically p65. Airway epithelial cell migration is an important step governing the repair of airway cells following injury in inflammatory conditions such as asthma.43 Airway architecture has been shown to change due to an inflammatory process in the pathophysiology of OSAS.10 It is therefore possible that IL-1α is related to classical NF-kB signaling-induced airway epithelial cell migration and cell repair in the upper airway. IL-1β staining showed a low and similar germinal center staining in both. However, epithelial expression of IL-1β in the OSAS group is higher in comparison with the non OSAS group. IL-1β production in the epithelial tissue of inflamed tonsils is of interest because cellular mediators of inflammation are in close contact with epithelial cells, and are thus influenced by the local environment formed by these cells. This microenvironment includes cytokines (such as IL-1β) produced by epithelial cells, which mediate the cellular communication leading to cellular differentiation, maturation, and the regulation of cell function.44,45 SLEEP, Vol. 36, No. 12, 2013 1952 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

We also investigated the systemic role of NF-kB activation in the pathophysiology of OSAS. We found that sera from children with OSAS activated NF-kB in two cell lines in vitro, versus sera from children without OSAS. Systemic inflammation was previously detected in pediatric OSAS, as demonstrated by an elevation of HS-CRP levels.14,15 Furthermore, increases in the plasma concentrations of leukotriene B4 and cysteinyl leukotrienes decreased to normal values following T&A.46 Adult patients with OSAS suffer from several cardiovascular and cerebro-vascular morbidities.47-49 The link to NF-kB clarifies OSAS pathophysiology, because NF-kB activation is related to some of these morbidities.24 NF-kB activation was elevated in both circulating neutrophils and monocytes from patients with OSAS versus control subjects, and decreased after OSAS treatment with continuous positive airway pressure.12,30 High levels of the proinflammatory cytokines IL-6 and IL-8 were previously detected in the circulation of children in whom OSAS was diagnosed,50,51 as well as low levels of the anti-inflammatory cytokine IL-10.50 TNF-α, a classical NF-kBdependent cytokine, was recently reported to be increased in children with OSAS, and its concentration correlated with daytime sleepiness in these young patients.52 All of the aforementioned cytokines are regulated by NF-kB signaling pathways. Various Rel family members bind active sites in the promoters of IL-6, IL-8, and IL-10 to induce or repress their transcription and evoke the cellular inflammatory response.53-55 We detected elevated NF-kB activity after incubation of two cell lines with serum from patients with OSAS: Jurkat cells (inducible NF-kB) and L428 cells (constitutive NF-kB). We therefore suggest that the signaling pathways leading to NF-kB activation in pediatric OSAS are both IkB-dependent (as seen in Jurkat calls) and IkB-independent (as seen in L428 cells; for example, via direct phosphorylation of NF-kB by IKK).56 Our experiments with COS-7 cells emphasized and corroborated the systemic effect of sera from patients with OSAS on NF-kB activation, and implying that these sera may induce NF-kB in Children with OSAS—Israel et al

×400

N

Cos7+TNFα

N

OSAS

N

Non OSAS

N

Control

N

0 min

10 min

30 min

60 min

Figure 5—COS-7 cells were permanently transfected with GFP-p65 plasmid and assessed for nuclear factor kappa B (NF-kB) activity. The cells were incubated with 10% sera from children with obstructive sleep apnea syndrome (OSAS) and controls and photographed under a fluorescence microscope at various times. p65 translocation to the nucleus (N) was observed after 60 min. Incubation with OSAS serum but not with control serum or in the absence of serum led to p65 translocation. Cells incubated with 10 ng/mL tumor necrosis factor-α (positive control) activated NF-kB after 30 min.

signaling in a variety of cells of nonlymphatic origin. It would be of interest to study the effect of such sera in cardiovascular and cerebrovascular tissues to correlate these in vitro findings with patient pathology. To our knowledge, this is the first report of systemic NF-kB activation in pediatric patients with OSAS. Serum-induced NF-kB activation may explain previous observations regarding OSAS inflammatory processes, as well as OSAS-related morbidities. The identification of NF-kB as an important element in the pathophysiology of OSAS underscores the need for specific diagnostic tools and targeted nonsurgical therapies for this relatively common disease. ABBREVIATIONS CRP, C-reactive protein GFP, green fluorescent protein IH, intermittent hypoxia IKK, IkkB kinase IL, interleukin SLEEP, Vol. 36, No. 12, 2013 1953 Downloaded from https://academic.oup.com/sleep/article-abstract/36/12/1947/2709420 by guest on 30 April 2018

NF-kB, nuclear factor kappa B OSAS, obstructive sleep apnea syndrome T&A, adenotonsillectomy ACKNOWLEDGMENTS Dr. Gopas and Dr. Goldbart are equal contributors to this work. They designed the study, analyzed the data, and critically revised the manuscript for important intellectual content. Ms. Israel was responsible for acquisition of data, performance of the experiments, data analysis, and drafting the manuscript. Dr. Benharoch analyzed the immunohistochemical data. The authors thank Dr. Volevich for technical assistance with samples collection and Ms. Mejrovsky for performing the immunohistochemistry. DISCLOSURE STATEMENT This was not an industry supported study. Dr. Goldbart was supported by the Israel Science Foundation (ISF) 753/11. The authors have indicated no financial conflicts of interest. NF-kB in Children with OSAS—Israel et al

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