Interplay Between Helicobacter pylori Infection,Interleukin-11,and Leukemia Inhibitory Factor in Gastric Cancer Among Egyptian Patients

Abstract

Helicobacter pylori is a ubiquitous Gram-negative bacterium, that is responsible for gastric mucosal inflammation. It is the most common risk factor for gastric cancer (GC). The current study aimed to investigate the association between interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) levels among H. pylori-infected Egyptian patients with gastritis and GC. One hundred forty-seven patients with gastric lesions were endoscopically biopsied and assessed using rapid urease test and immunohistochemistry. Quantitative real-time polymerase chain reaction was done for the detection of H. pylori load in gastric biopsies and detection of LIF as well as IL-11 relative gene expression. The mean values of H. pylori load, LIF, and IL-11 were significantly elevated in GC patients compared to gastritis group (P < 0.0001). A positive significant correlation was detected between mucosal levels of LIF, IL-11, and H. pylori load in both groups. Both LIF and IL-11 had the same pattern of expression in gastric tissues with different types of gastritis and different types and grades of gastric carcinoma. This report could clarify the molecular events associated with the immune response against H. pylori infection and H. pylori-associated pathology. Therefore, development of immunotherapy strategies against H. pylori-induced cytokines becomes inevitable.

Introduction

H elicobacter pylori is one of the most common human pathogens. Approximately, this ubiquitous pathogen is responsible for infection in 50% of the world population (Al-Moayad and others 2014). Nearly 70% of all gastric cancer (GC) cases are directly attributable to previous H. pylori infection (Kim and others 2011). Among the Middle East countries, Egypt recorded the highest prevalence of H. pylori infection (80%; Al-Moayad and others 2014). Nevertheless, the molecular and cellular events responsible for the development of gastric malignancies due to H. pylori are still poorly defined. Probably, this may be due to the decreased gastric adherence of H. pylori because of the metaplastic changes in gastric cells and diminished acid secretion as a result of the loss of specialized acid-secreting cells from the atrophied gastric glands (Kim and others 2011). Aberrant epithelial proliferation and sustained gastric mucosal inflammation are the main pathophysiological events in gastric mucosa infected with H. pylori. This leads to synthesis and stimulation of some pro-inflammatory mediators such as interleukin (IL)-1β, tumor necrosis factor, IL-2, IL-6, IL-8, and some anti-inflammatory mediators such as IL-4 and IL-10 (Hwang and others 2002; Malfertheiner and others 2012). The upregulation of mRNA of pro-inflammatory cytokines results in inflammation of gastric mucosa, by binding to the receptors on target cells (Sugimoto and others 2010).

IL-11 is one among the IL-6 cytokine family. Expression of IL-11 and IL-11Rα, its receptor, is required for signal transduction, which is mediated by gp130 (Heinrich and others 2003). This expression was found to be associated with proliferation and invasion in gastric and colorectal cancers (Yoshizaki and others 2006; Nakayama and others 2007), indicating that IL-11 plays an important role in cancer pathogenesis. Moreover, IL-11 can influence epithelial homeostasis through decreasing stem cell apoptosis, prolonging survival, and enhancing mitosis (Orazi and others 1996; Potten 1996). Ulcer healing is accelerated by proliferation and angiogenesis mediated by IL-11. Both factors are important in early GC (Wen and others 2002, 2003).

Leukemia inhibitory factor (LIF) also is one among the members of the IL-6 cytokine family. Previous studies on LIF revealed its role in the induction of maturation and differentiation of macrophage and murine M1 myeloid leukemia that leads to suppression of leukemia proliferation; that is why LIF was so named (Wen and others 2002). LIF has various effects that include bone formation, induction of hematopoietic cell proliferation, hormone production, neuron survival, and the formation of acute phase proteins. Also, it is important for embryo development (Wen and others 2003). LIF receptors are widely distributed, and therefore LIF has a broad range of functions on almost all body systems (Yue and others 2015). The high degree of functional redundancy shared by members of this cytokine family is mainly due to sharing a single receptor subunit to exert their effects.

Variable tests have been developed to diagnose H. pylori infection or to confirm treatment. Urea breath test, antigen stool assay, and serology are noninvasive tests, while culture, rapid urease test (RUT), histological detection, and polymerase chain reaction (PCR) are invasive tests (Al-Moayad and others 2014). The aim of this work was to evaluate the effect of H. pylori infection on the expression levels of IL-11 and LIF genes in gastric tissues of Egyptian patients with gastritis and GC.

Patients and Methods

Patients

One hundred forty-seven patients were enrolled in the study. They were recruited from the gastrointestinal tract endoscopy unit, Fayoum University Hospital. The study was approved by the Research Ethics Committee, Faculty of Medicine, Fayoum University. Patients with a history of chronic use of nonsteroidal anti-inflammatory drugs were excluded. Patients were instructed to stop antibiotics for at least 4 weeks and proton pump inhibitor drugs for at least 2 weeks before the study. All enrolled patients signed a written informed consent after detailed explanation of the study.

All patients were subjected to abdominal ultrasonographic examination and upper gastrointestinal endoscopy. Patients with gastric lesions were endoscopically biopsied and assessed by RUT (Quick Urease Test; Biohit Oyj Laippatie, Helsinki, Finland). Gastric biopsies were collected in duplicate; a part was fixed in buffered formalin for histologic examination and DNA extraction, and the other part was stored in RNA lysis extraction buffer containing guanidinium thiocyanate and β-mercaptoethanol for further quantification of LIF and IL-11 gene expression.

Histopathological studies

Biopsies from the gastric mucosa, which were fixed for 24 h in 10% formalin, were dehydrated in xylene and alcohol and embedded in paraffin. Sequential sections (4–5 μm thickness) were stained with hematoxylin and eosin after they were embedded in paraffin for routine histopathological examination. Immunohistochemistry was performed to confirm H. pylori infection by immune-staining with anti-H. pylori antibody (Dako, High Wycombe, United Kingdom). Chronic gastritis was diagnosed by the presence of lymphocytes and an infiltrate of plasma cells in the lamina propria, with or without acute inflammation (polymorphs reflecting active inflammation), and lymphoid aggregates. Gastric adenocarcinoma was histologically graded as I, II, and III (Novelli 2007).

Quantitative real-time PCR for H. pylori ureA

Genomic DNA was isolated from gastric tissue using a DNA extraction kit (QIAamp; Catalog No. 51304; Qiagen, Hilden, Germany). Primer sequence specific for H. pylori ureaseA (ureA) was prepared as forward (5′-CGTTGTCTGCTTGCCTATCA-3′) and reverse (5′-CGGCTCACACTTCCATTTCT-3′; GenBank accession number CP011330). The ureA primer set was used in quantitative real-time PCR (qRT-PCR) for a final volume of 25 μL containing 12.5 μL Syber Green PCR mastermix (Fermentas), 9.5 μL water, 4 pmol of each primer set, and 0.2 μg of DNA under the following conditions: 35 cycles of denaturation at 94°C/15 s, annealing at 60°C/30 s, and extension at 72°C/1 min. Calculation of H. pylori copy number was based on the standard curve generated using 10-fold dilutions of DNA extracted from H. pylori culture control strain varying from 10⁵ to 10¹ copies (Shukla and others 2011).

Real-time reverse transcriptase PCR for LIF and IL-11

Gastric samples from all patients were lysed, and total RNA was extracted and purified with Gene JET Kit (No. K0732; Thermo Fisher Scientific, Germany). LIF primer sequence was synthesized as: forward (5′-TGGTTCTGCACTGGAAACATG-3′) and reverse (5′-GTAATAGAGAATAAAGAGGGCATTGG-3′; GenBank accession number NT_011520.12). Primer sequence for IL-11 was synthesized as: forward (5′-TCTCTCCTGGCGGACACG-3′) and reverse (5′-AATCCAGGTTGTGGTCCCC-3′; GenBank accession number NT_011109.16); and for the housekeeping 18sRNA gene (as an internal control) as: forward (5′-CAGCCACCCGAGATTGAGCA-3′) and reverse (5′-TAGTAGCGACGGGCGGGTGT-3′; GenBank accession number JX132355.1). Twenty nanograms of purified RNA from each sample were applied for reverse transcription with subsequent quantitative PCR amplification with SensiFAST™SYBR^®Hi-ROX One-step Kit (catalog number PI-50217V; Bioline, United Kingdom). Thermal profile was as follows: 45°C for 20 min in 1 cycle (for cDNA synthesis) followed by 10 min at 95°C for reverse transcriptase enzyme inactivation. Forty cycles of PCR amplification were further carried out as: 10 s at 95°C, 30 s at 58°C, and 1 min at 72°C. Changes in the expression of each target gene were normalized relative to the mean cycle threshold (CT) values of the housekeeping gene 18sRNA by the ^ΔCt method. The real-time PCRs were carried out in 48-well-plate Step One real-time PCR systems (Applied Biosystems, Foster City, CA), and results were analyzed using Applied Biosystems software version 2.

Statistical analysis

The collected data were organized, tabulated, and statistically analyzed using SPSS software package version 18 (SPSS, Inc.). For quantitative data, the mean, median, standard deviation, and interquartile range (IQR) were calculated. Kolmogorov–Smirnov (KS) test was performed as a test of normality. If variables were normally distributed, independent t-test was used to compare between the two groups. For non-normally distributed variables, Mann–Whitney U-test was used as a test of significance. Qualitative data were presented as numbers and percentages; chi-square was used as a test of significance. Spearman correlation was run to identify the relation between H. pylori load and LIF and IL-11 mRNA expression levels in gastric mucosa. For the interpretation of results of tests of significance, significance was adopted at P ≤ 0.05.

Results

Demographic features of study subjects

This cross-sectional study was performed on 147 patients; male/female ratio was 61.2%/38.8%. The mean age of all included patients was 51.2 ± 13.9. Based on histopathological examination of gastric mucosal biopsies, the patients were divided into 2 main groups: Group I, patients with gastritis (88 patients); and Group II, patients with GC (59 patients). The mean age was 43.1 ± 11.6 years for gastritis patients and 63.3 ± 5.9 for GC patients. No significant difference was found by sex for both gastritis and GC groups, while age was found to be significantly higher for GC group (P < 0.0001). The main demographic features of the study population are given in Table 1.

Table 1.

Demographic Characterization of All Enrolled Patients

	All patients (N = 147)	Gastritis (N = 88)	Gastric cancer (N = 59)
Age, mean ± SD				P
Age, mean ± SD	51.2 ± 13.9	43.1 ± 11.6	63.3 ± 5.9	<0.0001 ^a,b
Sex, n (%)				P
Male	90 (61.2)	49 (55.7)	41 (69.5)	0.092^c
Female	57 (38.8)	39 (44.3)	18 (30.5)	0.092^c

Independent t-test.

Bold values are significant (p ≤ 0.05).

Chi-square test.

SD, standard deviation.

Histopathological findings of study subjects

According to the semi-quantitative grading of different histopathological features, gastritis patients were divided into 3 groups: (1) acute gastritis group with 63 patients (71.6%), (2) chronic gastritis group with 19 patients (21.6%), and (3) atrophic gastritis group with 6 patients (6.8%). Data are shown in Table 2.

Table 2.

Characterization of Study Subjects According to Histopathological Findings

Group	Findings of biopsy	n	%
Gastritis (N = 88)	Acute gastritis	63	71.6
	Chronic gastritis	19	21.6
	Atrophic gastritis	6	6.8
Gastric cancer (N = 59)	Adenocarcinoma grade I	14	23.7
	Adenocarcinoma grade II	18	30.5
	Adenocarcinoma grade III	24	40.7
	Lymphoma	3	5.1

Similarly, GC patients were divided into 2 groups: (1) adenocarcinoma (56 patients; 94.9%) and (2) lymphoma (3 patients; 5.1%). Based on the dataset for histopathological reporting of GC (Novelli 2007), adenocarcinomas were divided into 3 groups, according to its grade; 14 patients had adenocarcinoma grade I, another 18 patients were of grade II, while 24 patients were of grade III (Table 2).

Endoscopic and laboratory findings of study subjects

The most frequent abnormality seen on endoscopy in gastritis group was antral gastritis in 30 patients (20.4%), while malignant gastric ulcer was the most predominant finding in 24 patients (40.7%) of the GC group (Table 3). Other pathological conditions were also noted among gastritis patients, including congestive gastropathy (21 patients), gastric ulcer (16 patients), diffuse gastritis (12 patients), diffuse gastric hyperemia (6 patients), erosive gastritis (3 patients), bulb duodenitis (3 patients), and gastroesophageal reflux disease (3 patients).

Table 3.

Results of Endoscopy for Gastritis and Gastric Cancer Patients (Some Patients Had More Than One Finding on Endoscopy)

U/E ^a	All patients (N = 147)	Gastritis (N = 88)	Gastric cancer (N = 59)
U/E ^a	n (%)
Antral gastritis	30 (20.4)	30 (34.1)	0 (0.0)
Congestive gastropathy	21 (14.3)	21 (23.9)	0 (0.0)
Gastric ulcer	23 (15.6)	16 (18.2)	7 (11.9)
Diffuse gastritis	12 (8.2)	12 (13.6)	0 (0.0)
Diffuse gastric hyperemia	6 (4.1)	6 (6.8)	0 (0.0)
Erosive gastritis	3 (2.0)	3 (3.4)	0 (0.0)
Bulb duodenitis	3 (2.0)	3 (3.4)	0 (0.0)
GERD	3 (2.0)	3 (3.4)	0 (0.0)
Fungating gastric mass	15 (10.2)	0 (0.0)	15 (25.5)
Gastric fold thickening	6 (4.1)	0 (0.0)	6 (10.2)
Malignant gastric ulcer	24 (16.3)	0 (0.0)	24 (40.7)
Ulcerating gastric mass	7 (4.8)	0 (0.0)	7 (11.9)

Not mutually exclusive.

GERD, gastroesophageal reflux disease; U/E, upper endoscopy.

Other endoscopic findings for the GC group included 15 patients with fungating gastric mass, 7 patients with gastric ulcer, 6 patients with gastric fold thickening, and 7 patients with ulcerating gastric mass (Table 3).

It was observed that there were significant differences between gastritis and GC patients as regards the mean values of hemoglobin, erythrocyte sedimentation rate (ESR) first hour, ESR second hour, and albumin (P < 0.001 for each). No significant difference was observed as regards the mean values of neutrophils (P = 0.548; Table 4).

Table 4.

Comparison Between Laboratory Data in Gastritis and Gastric Cancer Patients

	All patients (N = 147)	Gastritis (N = 88)	Gastric cancer (N = 59)	P
	Mean ± SD			P
Neutrophils	13.57 ± 0.98	13.59 ± 1.09	13.54 ± 0.79	0.548^a
HB	10.95 ± 1.79	11.74 ± 1.54	9.83 ± 1.47	<0.0001 ^a,b
ESR1	19.29 ± 13.88	12.67 ± 4.59	38.00 ± 14.32	<0.0001 ^b,c
ESR2	40.00 ± 27.39	19.93 ± 5.65	67.90 ± 20.15	<0.0001 ^b,c
ALB	3.58 ± 0.71	3.97 ± 0.56	3.01 ± 0.50	<0.0001 ^a,b

Independent t-test.

Bold values are significant (p ≤ 0.05).

Mann–Whitney U-test.

ALB, albumin; ESR, erythrocyte sedimentation rate; HB, hemoglobin; SD, standard deviation.

Observing the difference in H. pylori load in gastric tissues of gastritis and GC patients

All patients were proved to be H. pylori-infected by one or more positive tests (RUT, immunohistochemistry, or qRT-PCR). According to qRT-PCR, H. pylori load (median [IQR]) was 24.8 (6.5–67.8) thousand copies in gastritis patients and 107 (99.5–111.1) thousand copies in GC patients. This difference in mean values of H. pylori load between both groups was significant (P < 0.0001; Fig. 1).

FIG. 1.

Box-plot (median value, lower and upper quartile) analysis of Helicobacter pylori load, and LIF and IL-11 expression levels among gastritis and gastric cancer patients. (A) H. pylori load, (B) LIF expression levels, (C) IL-11 expression levels. H. pylori load and LIF and IL-11 expression levels were analyzed for gastritis group (n = 88) and GC group (n = 59) using RT-qPCR. The asterisks indicate significant difference (P ≤ 0.0001) by Mann–Whitney U-test. GC, gastric carcinoma; IL-11, interleukin-11; LIF, leukemia inhibitory factor; RT-qPCR, reverse transcriptase quantitative PCR.

Studying the association of H. pylori load with histopathological features of gastric mucosal biopsies

By comparing the H. pylori load between different histopathological findings of gastritis patients, we found that acute gastritis patients had the highest load of 37.2 (6.5–67.8) thousand copies. However, this value was not significantly different from chronic or atrophic gastritis (Table 5).

Table 5.

Difference in Helicobacter pylori Load, Leukemia Inhibitory Factor, and Interleukin-11 Expression Levels According to Histopathological Findings

	H. pylori load^**	LIF expression level	IL-11 expression level
Findings of biopsy		Median (IQR)
Gastritis (N = 88)
Acute gastritis (N = 63)	37.2 (6.5–67.8)	0.69 (0.55–0.84)	0.15 (0.14–0.15)
Chronic gastritis (N = 19)	33.3(7.2–79.3)	0.52 (0.46–0.88)	0.14 (0.12–0.26)
Atrophic gastritis (N = 6)	24.3 (6521.00–56221.00)	0.54 (0.24–0.89)	0.13 (0.00–0.31)
P	0.625	0.878	0.864
Gastric cancer (N = 59)
Adenocarcinoma grade I (N = 14)	110.2(97.5–120.7)	1.47 (1.26–1.68)	0.57 (0.51–0.64)
Adenocarcinoma grade II (N = 18)	102.0 (98.0–104.7)	1.49 (1.42–2.53)	0.49 (0.48–1.53)
Adenocarcinoma grade III (N = 24)	108.5 (103.1–126.6)	2.21 (1.75–2.72)	1.09 (0.61–1.68)
Lymphoma (N = 3)	70.4 (69.5–71.3)	0.95 (0.87–1.03)	0.36 (0.33–0.39)
P	≤0.0001 ^*	≤0.0001 ^*	≤0.0001 ^*
	0.011 ^* ^,a, 0.003 ^* ^,c	<0.0001 ^* ^,b, 0.003 ^* ^,c	<0.0001 ^* ^,b, 0.003 ^* ^,c
	0.0001 ^* ^,d, 0.002 ^* ^,e	0.006 ^* ^,d, 0.002 ^* ^,e	0.006 ^* ^,d, 0.002 ^* ^,e
	0.001 ^* ^,f	0.001 ^* ^,f	0.001 ^* ^,f

Adenocarcinoma grade I versus adenocarcinoma grade II.

Adenocarcinoma grade I versus adenocarcinoma grade III.

Adenocarcinoma grade I versus lymphoma.

Adenocarcinoma grade II versus adenocarcinoma grade III.

Adenocarcinoma grade II versus lymphoma.

Adenocarcinoma grade III versus lymphoma.

Bold values are significant (p ≤ 0.05 is significant by Mann-Whitney U-test).

Values in thousands.

IQR, interquartile range; LIF, leukemia inhibitory factor.

As regards H. pylori load in different histopathological types of GC, gastric lymphoma had the lowest level of 70.4 (69.5–71.3) thousand copies. This low load was significantly different from grades I, II, and III of adenocarcinoma group (P = 0.003, 0.002, 0.001, respectively; Table 5).

Studying the association of H. pylori load with different upper endoscopic findings

By studying the difference in H. pylori load according to endoscopic findings of gastritis patients, the highest load was in the gastric ulcer group, 67.8 (38.5–79.3) thousand copies. This difference was significant compared to H. pylori load in other gastritis types: antral gastritis, congestive gastropathy, and diffuse gastritis (P = 0.006, <0.0001, and 0.006, respectively). However, H. pylori load in GC had the highest value in gastric fold thickening, 115.6 (109.0–122.2). This was significantly higher than other endoscopic findings in GC (Table 6).

Table 6.

Difference in Helicobacter pylori Load, Leukemia Inhibitory Factor, and Interleukin-11 Expression Levels According to Endoscopic Findings

U/E	H. pylori load^**	LIF expression level	IL-11 expression level
U/E	Median (IQR)
Gastritis (N = 88)
Antral gastritis and other findings (N = 30)	17.8 (7.2–74.2)	0.55 (0.46–0.96)	0.16 (0.12–0.39)
Congestive gastropathy and hyperemia (N = 27)	30.0 (2.2–56.2)	0.29 (0.24–0.84)	0.00 (0.00–0.31)
Diffuse gastritis (N = 15)	24.8 (24.3–56.2)	0.64 (0.45–0.89)	0.13 (0.12–0.14)
Gastric ulcer (N = 16)	67.8 (38.5–79.3)	0.84 (0.60–0.88)	0.21 (0.14–0.26)
P	≤0.0001 ^*	0.062	0.014 ^*
	0.014 ^* ^,a, 0.006 ^* ^,c	0.006 ^* ^,e	0.011 ^* ^,a
	0.004 ^* ^,d, <0.0001 ^* ^,e		<0.0001 ^* ^,f
	0.006 ^* ^,f		<0.0001 ^* ^,f
Gastric cancer (N = 59)
Fungating gastric mass (N = 15)	104.3 (99.5–104.6)	1.51 (1.49–2.53)	0.51 (0.49–1.53)
Gastric fold thickening (N = 6)	115.6 (109.0–122.2)	2.83 (2.81–2.84)	1.743 (1.741–1.744)
Gastric ulcer (N = 7)	111.1 (111.1–149.2)	1.67 (1.67–1.69)	0.64 (0.63–0.64)
Malignant gastric ulcer and ulcerating mass (N = 31)	101.6(98.0–109.3)	1.67 (1.39–1.97)	0.57 (0.51–0.68)
P	≤0.0001 ^*	0.001 ^*	0.001 ^*
	<0.0001 ^* ^,g, <0.0001 ^* ^,h	<0.0001 ^* ^,g	<0.0001 ^* ^,g
	<0.0001 ^* ^,g, <0.0001 ^* ^,h	0.002 ^* ^,j	0.001 ^* ^,j
	0.048 ^* ^,k	<0.0001 ^* ^,k	<0.0001 ^* ^,k
	0.001 ^* ^,l	<0.0001 ^* ^,k	<0.0001 ^* ^,k

Antral gastritis versus congestive gastropathy.

Antral gastritis versus diffuse gastritis.

Antral gastritis versus gastric ulcer.

Congestive gastropathy versus diffuse gastritis.

Congestive gastropathy versus gastric ulcer.

Diffuse gastritis versus gastric ulcer.

Fungating gastric mass versus gastric fold thickening.

Fungating gastric mass versus gastric ulcer.

Fungating gastric mass versus malignant gastric ulcer.

Gastric fold thickening versus gastric ulcer.

Gastric fold thickening versus malignant gastric ulcer.

Gastric ulcer versus malignant gastric ulcer.

Bold values are significant (p ≤ 0.05 is significant by Mann-Whitney U-test).

Values in thousands.

IQR, interquartile range; LIF, leukemia inhibitory factor.

LIF expression levels in gastric tissues among gastritis and GC patients

LIF expression level was 0.55 (0.29–0.89) in gastritis patients and 1.68 (1.42–2.44) in GC patients. This difference between both groups was significant (P < 0.0001).

Studying the association of LIF expression levels with histopathological features of gastric mucosal biopsies

Acute gastritis had the highest LIF expression level of 0.69 (0.55–0.84) among other histopathological findings in gastritis patients; however, this was not significantly different (Table 5). For the GC group, adenocarcinoma grade III had the highest LIF expression level of 2.21 (1.75–2.72) and gastric lymphoma had the lowest level of 0.95 (0.87–1.03). Both values were significantly different from the LIF expression level in other histopathological findings (Table 5).

Studying the association of LIF expression levels with different upper endoscopic findings

By studying the difference in LIF expression levels according to endoscopic findings in gastritis group, the highest level of 0.84 (0.60–0.88) was found for gastric ulcer, while the lowest level of 0.29 (0.24–0.84) was found for congestive gastropathy. The difference between both endoscopic findings was significant (P = 0.006; Table 6).

Gastric fold thickening was the endoscopic finding with the highest LIF expression level of 2.83 (2.81–2.84) among other endoscopic findings of the GC group. The difference between this LIF expression level and its level among other GC endoscopic findings, namely, fungating gastric mass, gastric ulcer, and malignant gastric ulcer, was statistically significant (P = <0.0001, 0.002, and <0.0001, respectively; Table 6).

IL-11 expression levels in gastric tissues of gastritis and GC patients

IL-11 expression level was 0.14 (0.00–0.27) in gastritis patients and 0.63 (0.49–1.49) in GC patients. This difference between both groups was significant (P < 0.0001).

Studying the association of IL-11 expression levels with histopathological features of gastric mucosal biopsies

Similar to LIF expression level, IL-11 expression levels were not significantly different with different histopathological findings of gastritis patients (acute gastritis, chronic gastritis, and atrophic gastritis; Table 5). In the GC group, gastric lymphoma had the lowest IL-11 expression level of 0.36 (0.33–0.39), and adenocarcinoma grade III had the highest level of 1.09 (0.61–1.68). Both values were significantly different from the IL-11 expression level in other histopathological findings in GC patients (Table 5).

Studying the association of IL-11 expression level with different upper endoscopic findings

According to endoscopic findings of study subjects, in gastritis group, the IL-11 expression level was the highest, 0.21 (0.14–0.26), for gastric ulcer, while the lowest level, 0.00 (0.00–0.31), was found for congestive gastropathy. The difference between both endoscopic findings was significant (P = 0.011; Table 6).

As noticed with LIF, gastric fold thickening was an endoscopic finding with the highest IL-11 expression level of 1.743 (1.741–1.744) among other endoscopic findings of the GC group. Statistically significant differences between this LIF expression level and its level among other GC endoscopic findings, namely, fungating gastric mass, gastric ulcer, and malignant gastric ulcer, were noticed (P = <0.0001, 0.001, and <0.0001, respectively; Table 6).

Correlation between H. pylori load, LIF, and IL-11 expression levels among gastritis and GC patients

One of the objectives of the present study was to find how the H. pylori bacterial load within the gastric mucosa can affect IL-11 and LIF mRNA expression levels. A positive significant correlation was detected between mucosal levels of LIF and H. pylori load in gastritis and GC patients (r = 0.873; P < 0.0001 and r = 0.597; P < 0.0001, respectively). Also, mucosal level of IL-11 was positively correlated with H. pylori load in gastritis and GC patients (r = 0.868; P < 0.0001 and r = 0.661; P < 0.0001, respectively; Table 7).

Table 7.

Correlation Between Helicobacter pylori Load, Leukemia Inhibitory Factor, and Interleukin-11 in Gastritis and Gastric Cancer Patients

	All patients (N = 147)		Gastritis (N = 88)		Gastric cancer (N = 59)
	H. pylori load		H. pylori load		H. pylori load
	r	P	r	P	r	P
LIF expression level	0.945	<0.0001^a	0.873	<0.0001^a	0.597	<0.0001^a
IL-11 expression level	0.939	<0.0001^a	0.868	<0.0001^a	0.661	<0.0001^a

Significant.

IL-11, interleukin 11; LIF, leukemia inhibitory factor.

Discussion

GC is the fifth most common malignancy and the third cause of cancer-induced death in the world (Ferlay and others 2015). The World Health Organization (WHO) declared in 1994 that H. pylori infection is considered a class I carcinogen (WHO 1994). About 3% of H. pylori-induced chronic gastritis will progress to GC (Tran and others 2017). The association of H. pylori with GC has received much attention and careful study. The susceptibility and severity of disease are determined by a complex interaction of genetic, bacterial, and environmental factors (Tran and others 2017).

Through the current work, H. pylori was investigated in endoscopically biopsied gastric tissues from patients with gastrointestinal complaints. H. pylori was detected in all patients with either gastritis or GC. This correlates with Correa's model of GC development, which has described a sequence of events beginning with H. pylori-associated gastritis and ending with cancer (Howlett and others 2009).

Until today there has been no consensus regarding a gold standard for the diagnosis of H. pylori infection; so we used 3 different methods for H. pylori detection (Al-Moayad and others 2014). The first was RUT, which is based on the activity of H. pylori urease enzyme. RUT was positive for all patients, which reflect a high bacterial load in the specimens, because a minimum of 10⁴ organisms is usually required for a positive RUT result. In general, commercial RUTs have very good specificity and sensitivity (Moon and others 2012). The second method was histology, which is generally considered a gold standard for the detection of H. pylori infection directly (Wang and others 2015). In addition, the immunohistochemical stain was used; this is considered the most specific and sensitive stain. The third method was qRT-PCR, which provides excellent sensitivity and specificity, more than 95% compared to other conventional tests (Wang and others 2015).

A significant difference in the mean value of H. pylori load between gastritis and GC patients (P < 0.0001) was approved. This agrees with findings of Wang et al. (2016), who reported that H. pylori load was significantly higher in GC compared to chronic gastritis patients (Wang and others 2016). However, till now it is unclear whether bacterial overgrowth is a potential factor that promotes cancer (Correa 1988), or whether it is a consequence of cancer as malignant mucosa may favor bacterial growth. Further studies are needed to clarify this debate. H. pylori interacts with host cells within the gastric mucosa, resulting in the production of pro-inflammatory cytokines and recruitment of immune cells, leading to increased epithelial turnover and, ultimately, cellular transformation (Kim and others 2011).

The present study investigated the molecular aspects of host immune responses to H. pylori gastric infection. H. pylori-dependent regulation of IL-11 and LIF genes in gastric mucosa was identified. This could highlight the possible link among IL-11, LIF, and H. pylori-associated pathological complications. Mucosal level of IL-11 was firmly correlated with H. pylori load in gastritis and GC patients (r = 0.914; P < 0.0001 and r = 0.825; P < 0.0001, respectively). This comes in accordance with many reports that described the involvement of IL-11 in H. pylori-associated inflammation during the chronic phase (Mudter and others 2002; Yamaoka and others 2002; Dossumbekova and others 2006; Sugimoto and others 2011). In addition, IL-11/IL-11R pathway plays an important role in the development and differentiation of human GC (Mudter and others 2002; Howlett and others 2005, 2009). According to our findings, a significant difference was detected between the mean values of IL-11 among gastritis and GC patients (P < 0.0001).

The current work has found a significant difference in the mean values of expression levels of LIF between the studied groups (P < 0.0001). LIF has a complex role in the progression and development of tumors. LIF expression can be induced at mRNA level under hypoxia and inflammatory stress (Kamohara and others 1997, 2007; Yue and others 2015). LIF increases cell proliferation in tumor cell lines, such as medulloblastoma (Liu and others 1999), skin (Szepietowski and others 2001), breast, kidney, and prostate (Kellokumpu-Lehtinen and others 1996). Noteworthy, the presence of leukemia inhibitory factor receptor has been linked to the growth of cancer cells (Dhingra and others 1998). Limited reports referred to the association between LIF and H. pylori in gastric tissues (Zeaiter and others 2011). However, data from these previous reports support our results. In contrast, our findings disagreed with those of Wen et al. (2004), who addressed that LIF gene expression was not associated with H. pylori infection (Wen and others 2004).

By investigating the differential expression levels of LIF and IL-11 in gastric tissues in different gastric pathological features that have been diagnosed by either histopathological or endoscopic examinations, we found that both cytokines almost have the same pattern of expression in gastric tissues with different types of gastritis and grades of GC. This may be explained by the fact that both cytokines are related to the same family (IL-6 family) that use the subunit gp130 for signal transduction, with overlapping of their actions.

To our knowledge, this is the first report that has recognized the differential expression of LIF in association with H. pylori in gastritis and GC patients. LIF has been shown to stimulate growth, inhibit differentiation, and induce metastasis of many tumors (Wu and others 2015). Moreover, it increases resistance toward cancer therapy, including chemotherapy and radiotherapy (Liu and others 2013; Yu and others 2014). LIF overexpression is often associated with poor prognosis in many human carcinomas, including colorectal cancers, breast cancers, and nasopharyngeal carcinoma (Liu and others 2013; Li and others 2014; Yu and others 2014). This attracts attention toward the potential role of LIF in GC in the presence of H. pylori infection, which has been very scarcely studied.

Conclusively, this work is the first report to investigate the increased level of expression of IL-11 and LIF in gastric mucosa of GC associated with H. pylori infection. This is considered a potential target for immunotherapeutic strategy for H. pylori infection-associated GC.

Conclusion

The current report underlines the molecular events related to the immune response against H. pylori infection and H. pylori-associated pathology. A strong correlation was detected between mucosal levels of LIF, IL-11, and H. pylori load for both groups. This finding together with the emergence of antibiotic-resistant strains has underscored the need for immunotherapy strategies against H. pylori-induced cytokines and/or its receptors and signaling pathways to prevent H. pylori-associated complications.

Footnotes

Acknowledgment

All authors acknowledge help from all paramedical personals to accomplish and finalize the clinical research work.

Author Disclosure Statement

No competing financial interests exist.

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