Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12118
NF-jB Activation and Severity of Gastritis in Helicobacter pylori-Infected Children and Adults rie Segers,¶ Carine Deprez,¶ Francßoise Mascart†,** Patrick Bontems,*,† Ezra Aksoy,†,‡ Alain Burette,§ Vale and Samy Cadranel* Libre de Bruxelles, Av JJ Crocq 15, 1020 *Paediatric Gastroenterology-Hepatology, Queen Fabiola Children’s University Hospital, Universite Brussels, Belgium, †Laboratory of Vaccinology and Mucosal Immunity, Universit e Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium, ‡ Centre for Cell Signalling, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK, §Department of Gastroenterology, Nouvelle Clinique de la Basilique, Rue Pangaert 37, 1083 Brussels, Belgium, ¶Department of Pathology, University Hospital Brugmann, Universit e Libre de Bruxelles, Place Van Gehuchten 4, 1020 Brussels, Belgium, **Immunobiology Clinic, Academic Hospital Erasme, Libre de Bruxelles, Route de Lennik 808, Brussels, Belgium Universite
Keywords Helicobacter pylori, NF-jB activation, gastroduodenal ulcer, children, gastritis. Reprint requests to: Patrick Bontems, Paediatric Gastroenterology-Hepatology, Queen Fabiola Libre de Children’s University Hospital, Universite Bruxelles, Av JJ Crocq 15, 1020 Brussels, Belgium. E-mail: [email protected]
Abstract Background: In contrast to adults, Helicobacter pylori gastritis in children is reported as milder and ulcer disease as uncommon, but unequivocal data are lacking. Objectives: To compare the frequency of gastro-duodenal ulcers in children and adults as well as the proportion of Helicobacter pylori infection in these patients and to study the effect of chronological age on NF-jB activation and on severity of gastritis. Design: Patients referred in one pediatric and one adult facility for upper GI endoscopy were included. Gastric biopsies were obtained in consecutive Helicobacter pylori-infected patients and age-matched negative controls for immunohistochemistry and electrophoresis mobility shift assay. Three age groups were defined: younger than 8 years, 8–17 years, and adults. Results: Peptic ulcer disease was less frequent in children and less frequently associated with Helicobacter pylori infection. When comparing infected subjects to controls, densities of neutrophils and CD20 cells in the lamina propria increased in all age groups, CD3 cells increasing only in patients older than 8 years and CD8 cells only in adults. NF-jB-p65-positive cells were also increased only in infected adults as well as NF-jB-binding activity. A positive correlation was found between age and densities of neutrophils and CD3, but not of CD8 or CD20 cells. Conclusion: Peptic ulcer disease was less frequent in children and less frequently caused by Helicobacter pylori infection. The different clinical outcome of the infection in children can be the consequence of the lower mucosal immune response.
Helicobacter pylori (H. pylori) is a gram-negative microorganism that colonizes the mucus coat overlying the gastric epithelium and causes chronic/active gastritis, peptic ulcer disease (PUD), gastric mucosal atrophy, intestinal metaplasia, gastric adenocarcinoma, and gastric lymphoma [1]. Although the prevalence of infection varies geographically and is declining over time in developed countries, the organism still infects approximately one half of the world’s population [2]. Progression from gastritis toward more severe disease states appears to be influenced by both bacterial determinants and host immune response [3,4].
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 157–167
Immune response against H. pylori involves innate components represented mainly by neutrophils and adaptive immunity involving systemic and mucosal H. pylori-specific antibody production as well as a complex mix of Th1, Th17, and Treg immune responses [5–9]. Interactions of H. pylori strains and bacterial compounds with diverse host epithelial and cytoplasmic signaling pathways such as Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain (NLRs) is followed by the activation of NF-jB, a transcription factor regulating activation of genes responding to
157
NF-jB and severity of H. pylori gastritis
immune or inflammatory signals [8–16]. These immune responses fail to eradicate H. pylori infection and certainly contribute to disease pathogenesis, in part due to tissue remodeling trough inflammation and activation of matrix metalloproteinase [17–22]. Although NF-jB has a key role in mediating gastric mucosal inflammation caused by H. pylori, its activation has not been studied yet in infected children. The systemic humoral response to H. pylori infection is weaker and variable in children [23,24]. In addition, a lower IFN-c secretion in the stomach and a lower infiltration by IFN-c-secreting cells characterize the mucosal immune response to H. pylori in children compared with infected adults [6,7]. Moreover, regulatory T-cell response to H. pylori infection also predominates in children instead of Th17 cell [25–27]. These evidences suggest that a weaker immune response could protect the child from more severe gastroduodenal damages due to the infection. A lesser inflammatory cell mucosal infiltration has been reported in children, but there are still controversies especially concerning the lack or reduced infiltration of the gastric mucosa by polymorphonuclear cells in H. pyloriinfected children [28]. Indeed, most studies are not really comparative because adult patients were not included (the reference to adult patients is only historical). Moreover, three comparative studies that used a semiquantitative scoring of gastritis (the Update Sydney system [29]) give conflicting results [25,30,31]. Using the same score in a pilot study, we also failed to demonstrate a difference in the severity of gastritis between children and adults (P. Bontems, A. NeumanOva, P. Heimann, J.M. Devaster, V. Segers, C. Deprez, S. Cadranel, unpublished observations). In the present study, we compared the frequency of gastric and duodenal ulcer disease between children and adults and the proportion of lesions related to H. pylori infection as well as the cellular density of immune cells in the gastric mucosa using immunohistochemical staining and a morphometric method and the activation of NF-jB by the same method and by electrophoresis mobility shift assay (EMSA).
Patients and Methods Frequency of Gastric and Duodenal Ulcers The source of data was two different endoscopy facilities, one for children and one for adults, located very closely in Brussels. In our routine practice, gastric biopsies for histologic analysis and microbiologic culture were systematically taken from children and adults whenever not contraindicated. All patients undergoing
158
Bontems et al.
an upper GI endoscopy between January 2007 and December 2008 were enrolled to compare the frequency of gastric and duodenal ulcers and the proportion of ulcers associated with H. pylori infection. Only ulcers with a diameter of 5 mm or larger were considered. The following items were recorded: the presence of gastric or duodenal ulcers, H. pylori status, age, and gender. At least two biopsies were taken from the antrum and two more from the fundus for histology and for microbiologic culture. The biopsies were processed according to the previously described methods [32]. Briefly, sections from formalin-fixed, paraffin-embedded biopsies were stained with hematoxylin/eosin to detect histologic features of inflammation (in accordance with the Updated Sydney system [29]) and with Giemsa to detect H. pylori-like microorganisms. Biopsy specimens for culture were dipped with sterile cotton in a semisolid agar transport medium (Cultiplast, LP Italiana SPA, Milan, Italy). Less than 4 hours later, the samples were grounded at 10000 rpm for 15 second with an electric homogenizer (Ultraturrax, Staufen, Germany) and then inoculated onto selective Columbia blood agar and incubated under microaerophilic (5% O2 – 10% CO2 – 85% N2) conditions at 37 °C for up to 7 days. H. pylori infection was confirmed when culture and histology were both positives. Discrepancies between histology and culture were resolved using an additional diagnostic test (urease test or 13C-urea breath test). H. pylori culture in our departments was concordant with histology in 98% [32].
Cellular Density of Immune Cells in the Antral Mucosa Another database was constructed with all the patients undergoing an upper GI endoscopy during 1 year except those presenting with erosive esophagitis, gastric, or duodenal ulcers. Those with inflammatory bowel disease, celiac disease, immunologic disorders as well as those treated during the preceding 4 weeks with antibiotics, proton-pump inhibitors, nonsteroidal anti-inflammatory drugs, steroids, or under immunosuppressive drugs were also excluded. From this database, 30 consecutive H. pylori-infected patients and 30 negative controls were selected in the proportion of 10 in each age group (
NF-jB Activation and Severity of Gastritis in Helicobacter pylori-Infected Children and Adults rie Segers,¶ Carine Deprez,¶ Francßoise Mascart†,** Patrick Bontems,*,† Ezra Aksoy,†,‡ Alain Burette,§ Vale and Samy Cadranel* Libre de Bruxelles, Av JJ Crocq 15, 1020 *Paediatric Gastroenterology-Hepatology, Queen Fabiola Children’s University Hospital, Universite Brussels, Belgium, †Laboratory of Vaccinology and Mucosal Immunity, Universit e Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium, ‡ Centre for Cell Signalling, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK, §Department of Gastroenterology, Nouvelle Clinique de la Basilique, Rue Pangaert 37, 1083 Brussels, Belgium, ¶Department of Pathology, University Hospital Brugmann, Universit e Libre de Bruxelles, Place Van Gehuchten 4, 1020 Brussels, Belgium, **Immunobiology Clinic, Academic Hospital Erasme, Libre de Bruxelles, Route de Lennik 808, Brussels, Belgium Universite
Keywords Helicobacter pylori, NF-jB activation, gastroduodenal ulcer, children, gastritis. Reprint requests to: Patrick Bontems, Paediatric Gastroenterology-Hepatology, Queen Fabiola Libre de Children’s University Hospital, Universite Bruxelles, Av JJ Crocq 15, 1020 Brussels, Belgium. E-mail: [email protected]
Abstract Background: In contrast to adults, Helicobacter pylori gastritis in children is reported as milder and ulcer disease as uncommon, but unequivocal data are lacking. Objectives: To compare the frequency of gastro-duodenal ulcers in children and adults as well as the proportion of Helicobacter pylori infection in these patients and to study the effect of chronological age on NF-jB activation and on severity of gastritis. Design: Patients referred in one pediatric and one adult facility for upper GI endoscopy were included. Gastric biopsies were obtained in consecutive Helicobacter pylori-infected patients and age-matched negative controls for immunohistochemistry and electrophoresis mobility shift assay. Three age groups were defined: younger than 8 years, 8–17 years, and adults. Results: Peptic ulcer disease was less frequent in children and less frequently associated with Helicobacter pylori infection. When comparing infected subjects to controls, densities of neutrophils and CD20 cells in the lamina propria increased in all age groups, CD3 cells increasing only in patients older than 8 years and CD8 cells only in adults. NF-jB-p65-positive cells were also increased only in infected adults as well as NF-jB-binding activity. A positive correlation was found between age and densities of neutrophils and CD3, but not of CD8 or CD20 cells. Conclusion: Peptic ulcer disease was less frequent in children and less frequently caused by Helicobacter pylori infection. The different clinical outcome of the infection in children can be the consequence of the lower mucosal immune response.
Helicobacter pylori (H. pylori) is a gram-negative microorganism that colonizes the mucus coat overlying the gastric epithelium and causes chronic/active gastritis, peptic ulcer disease (PUD), gastric mucosal atrophy, intestinal metaplasia, gastric adenocarcinoma, and gastric lymphoma [1]. Although the prevalence of infection varies geographically and is declining over time in developed countries, the organism still infects approximately one half of the world’s population [2]. Progression from gastritis toward more severe disease states appears to be influenced by both bacterial determinants and host immune response [3,4].
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 157–167
Immune response against H. pylori involves innate components represented mainly by neutrophils and adaptive immunity involving systemic and mucosal H. pylori-specific antibody production as well as a complex mix of Th1, Th17, and Treg immune responses [5–9]. Interactions of H. pylori strains and bacterial compounds with diverse host epithelial and cytoplasmic signaling pathways such as Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain (NLRs) is followed by the activation of NF-jB, a transcription factor regulating activation of genes responding to
157
NF-jB and severity of H. pylori gastritis
immune or inflammatory signals [8–16]. These immune responses fail to eradicate H. pylori infection and certainly contribute to disease pathogenesis, in part due to tissue remodeling trough inflammation and activation of matrix metalloproteinase [17–22]. Although NF-jB has a key role in mediating gastric mucosal inflammation caused by H. pylori, its activation has not been studied yet in infected children. The systemic humoral response to H. pylori infection is weaker and variable in children [23,24]. In addition, a lower IFN-c secretion in the stomach and a lower infiltration by IFN-c-secreting cells characterize the mucosal immune response to H. pylori in children compared with infected adults [6,7]. Moreover, regulatory T-cell response to H. pylori infection also predominates in children instead of Th17 cell [25–27]. These evidences suggest that a weaker immune response could protect the child from more severe gastroduodenal damages due to the infection. A lesser inflammatory cell mucosal infiltration has been reported in children, but there are still controversies especially concerning the lack or reduced infiltration of the gastric mucosa by polymorphonuclear cells in H. pyloriinfected children [28]. Indeed, most studies are not really comparative because adult patients were not included (the reference to adult patients is only historical). Moreover, three comparative studies that used a semiquantitative scoring of gastritis (the Update Sydney system [29]) give conflicting results [25,30,31]. Using the same score in a pilot study, we also failed to demonstrate a difference in the severity of gastritis between children and adults (P. Bontems, A. NeumanOva, P. Heimann, J.M. Devaster, V. Segers, C. Deprez, S. Cadranel, unpublished observations). In the present study, we compared the frequency of gastric and duodenal ulcer disease between children and adults and the proportion of lesions related to H. pylori infection as well as the cellular density of immune cells in the gastric mucosa using immunohistochemical staining and a morphometric method and the activation of NF-jB by the same method and by electrophoresis mobility shift assay (EMSA).
Patients and Methods Frequency of Gastric and Duodenal Ulcers The source of data was two different endoscopy facilities, one for children and one for adults, located very closely in Brussels. In our routine practice, gastric biopsies for histologic analysis and microbiologic culture were systematically taken from children and adults whenever not contraindicated. All patients undergoing
158
Bontems et al.
an upper GI endoscopy between January 2007 and December 2008 were enrolled to compare the frequency of gastric and duodenal ulcers and the proportion of ulcers associated with H. pylori infection. Only ulcers with a diameter of 5 mm or larger were considered. The following items were recorded: the presence of gastric or duodenal ulcers, H. pylori status, age, and gender. At least two biopsies were taken from the antrum and two more from the fundus for histology and for microbiologic culture. The biopsies were processed according to the previously described methods [32]. Briefly, sections from formalin-fixed, paraffin-embedded biopsies were stained with hematoxylin/eosin to detect histologic features of inflammation (in accordance with the Updated Sydney system [29]) and with Giemsa to detect H. pylori-like microorganisms. Biopsy specimens for culture were dipped with sterile cotton in a semisolid agar transport medium (Cultiplast, LP Italiana SPA, Milan, Italy). Less than 4 hours later, the samples were grounded at 10000 rpm for 15 second with an electric homogenizer (Ultraturrax, Staufen, Germany) and then inoculated onto selective Columbia blood agar and incubated under microaerophilic (5% O2 – 10% CO2 – 85% N2) conditions at 37 °C for up to 7 days. H. pylori infection was confirmed when culture and histology were both positives. Discrepancies between histology and culture were resolved using an additional diagnostic test (urease test or 13C-urea breath test). H. pylori culture in our departments was concordant with histology in 98% [32].
Cellular Density of Immune Cells in the Antral Mucosa Another database was constructed with all the patients undergoing an upper GI endoscopy during 1 year except those presenting with erosive esophagitis, gastric, or duodenal ulcers. Those with inflammatory bowel disease, celiac disease, immunologic disorders as well as those treated during the preceding 4 weeks with antibiotics, proton-pump inhibitors, nonsteroidal anti-inflammatory drugs, steroids, or under immunosuppressive drugs were also excluded. From this database, 30 consecutive H. pylori-infected patients and 30 negative controls were selected in the proportion of 10 in each age group (
