Helicobacter pylori (HP) is a spiral gram-negative flagellated bacterium colonizing the human gastric mucosa about half of world’s population.¹ This infection occurs during childhood but silently persists for decades. It can progress from the normal gastric mucosa infected with HP to chronic active gastritis, chronic atrophic gastritis (AG), intestinal metaplasia (IM), dysplasia, and adenocarcinoma. Long-lasting infection and inflammation related to HP result in peptic ulcers in approximately 15% to 20% of infected individuals, gastric mucosa-associated lymphoid tissue lymphoma in less than 0.01%, and gastric cancer (GC) in 1% to 3%.¹

HP-associated inflammation undergoes complex interaction between the host immune responses and bacterial-specific virulence factors. Specific virulence factors of HP such as cytotoxin-associated gene A (cagA) and vacuolating cytotoxin gene (vacA) s1/m1 are particularly virulent and have been revealed to be related to severe gastric epithelial injury.^2,3 The host immune responses such as both innate and adaptive immunity, play important roles in the activation of the immune cells and subsequent synthesis and secretion of a variety of inflammatory cytokines.⁴ Among them, an individual’s susceptibility to the development of gastroduodenal disease depends on genetic predisposition, which includes genetic aberrations, such as single-nucleotide polymorphisms (SNPs) in various cytokines, growth factors, their receptors and enzymes.⁵ Epidemiological and genome-wide studies revealed an association between various SNPs and representative HP-related diseases, including GC.⁶ The most frequently studied inflammation-related genes of GC include gene-related interleukin (IL)-1β, IL-8, IL-10, tumor necrosis factor (TNF)-α, and Toll-like receptor 4, however, the results are inconsistent and conflicting.^7,8 Previous studies comprehensively evaluated possible GC-related SNPs associated with the risk of AG.^9,10 A recent meta-analysis of 33 case-control studies reported that TLR1, TLR4, IL-10-819, IL-8-251, and prostate stem cell antigen are associated with AG risk.¹⁰ Although most researches on the associations between SNPs and various gastroduodenal diseases focused on the risk of gastroduodenal disease development, there are still limited studies on the association between inflammation severity and SNPs.

It is generally understood that the age-adjusted incidence rates of GC are 2- to 3-fold higher in males than in females in most populations¹¹ of patients aged >40 years.¹² And there have been reports that AG and IM tend to be more severe in males than females.^13,14 In a Japanese case-control study, AG and IM scores of the HP-infected corpus appeared more severe in males than in females, especially among older patients, although no difference in IL-8 messenger RNA levels was detected between sexes.¹⁴ In a Korean nationwide multicenter prospective study, the prevalence of endoscopic AG and IM in males diagnosed were significantly higher than that in females, and the most important risk factor for AG was age, followed by sex.¹³ Our research team previously reported that the greatest risk factors for both AG and IM diagnosed by histological examination were HP infection and age (61 years or older), and bacterial virulence factors including cagA and vacA m1, were risk factors for only AG.¹⁵ However, we did not analyze these factors according to sex, mainly because we lacked the concept of sex-specific medicine, which is based on the recognition of the differences between males and females and is actively utilized to achieve more accurate diagnosis and treatment.¹⁶

Although most studies on the effects of cytokine genetic polymorphisms in gastroduodenal diseases focused on the risk of associated disease development, there are still limited studies on the association between inflammation severity and SNPs. Considering our previous research, we hypothesized that genetic polymorphism responsible for cytokine production could be sex-specific and it might influence the degree of gastric inflammation. Based on this background, an association study for SNPs, including IL-1B-511 C/T, IL-1RN variable number of tandem repeats (VNTR), IL-6-572 G/C, IL-8-251 A/T, IL-8-781 C/T, IL-10-1082 G/A, IL-10-592 C/A, TNF-A-308 G/A, and transforming growth factor (TGF)-B-509 C/T, and HP-associated gastric inflammation was conducted according to sex in Koreans without significant gastroduodenal diseases.

1. Study participants

We consecutively enrolled 636 individuals registered at Seoul National University Bundang Hospital between 2013 and 2022. All of them were Koreans and had undergone esophagogastroduodenoscopy (EGD) because of symptoms such as epigastric soreness or indigestion or a screening program for GC and/or gastric precancerous lesions. Participants were enrolled as controls if EGD revealed no significant gastroduodenal diseases, such as gastric ulcer (GU), duodenal ulcer (DU), GC, gastric adenoma, gastric mucosa-associated lymphoid tissue lymphoma, or esophageal cancer despite current HP infection. GU, DU, gastric adenoma, and GC had been diagnosed by EGD, and gastric adenoma and GC had been confirmed histologically. All participants who were older than 18 years provided informed consent to participate, and the ethical approval was given by the Ethics Committee of Seoul National University Bundang Hospital (number: B-0602-030-001).

2. HP testing, histology, and serum pepsinogen testing

In all participants, 10 gastric biopsy specimens were obtained during EGD for histological examination,¹⁷ and a Campylobacter-like organism test (CLOtest; Delta West, Bentley, Australia) and culturing were performed to confirm the current HP infection. This method was described previously.¹⁸ Two gastric biopsy specimens each were obtained from both the antrum and the corpus, then they were fixed in formalin to check whether HP exists using modified Giemsa staining. In addition, to access the degree of inflammatory cells (neutrophil and lymphocyte) infiltration, AG, and IM hematoxylin and eosin staining was performed. The updated Sydney system was used for the grade of these histological features of the gastric specimen (0, none; 1, mild; 2, moderate; and 3, severe).^17,19 No clinical information was provided to one pathologist who examined all biopsy specimens. Another biopsy specimen taken from the lesser curvatures of the antrum and corpus was subjected to rapid urease testing (CLOtest), while two from the antrum and two from the corpus were subjected to culturing. The remaining biopsy specimens which were obtained from non-cancerous and cancer tissues were immediately frozen at –70°C. This study included only individuals with a current HP infection, which was defined when at least one of the results of HP test such as modified Giemsa stain, culture, CLOtest, and ¹³C-urea breath (UBiTkit; Otsuka Pharmaceutical, Tokyo, Japan) was positive.

3. Genetic polymorphisms and HP genotype

The extraction of genomic DNA was performed from tissue of gastric antrum.²⁰ The homogenization of the specimens was done using proteinase K solution with sterile micropestle and incubation was done for 3 hours at 52°C.²⁰ The isolation of DNA was performed from the homogenates after phenol/chloroform extraction and ethanol precipitation.

To determine the polymorphisms of IL-1B-511, IL-1B RN, IL-6-572, IL-8-251, IL-8-781, IL-10-592, IL-10-1082, TNF-A-308, and TGF-B-509, polymerase chain reaction (PCR)–restriction fragment length polymorphism analysis was performed using a GeneAmp 9600 PCR system (PerkinElmer Co., Norwalk, CT, USA). All restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA, USA) and the primers used for PCR are listed in Supplementary Table 1. Regarding the IL-1RN penta-allelic VNTR, the alleles were coded as follows: allele 1 (4 repeats, 410 bp); allele 2 (2 repeats, 240 bp); allele 3 (5 repeats, 500 bp); allele 4 (t3 repeats, 325 bp); and allele 5 (6 repeats, 595 bp). They were classified into the short (allele *2=2) and long alleles (alleles *1, *3, *4, and *5 = L).

Analyses of cagA, vacA, and oipA were done after PCR amplifications as described previously.^21,22 The primers used for PCR to detect these virulence factors are listed in Supplementary Table 1.

4. Statistical analysis

Statistical analyses were conducted using the chi-square test or Fisher exact test, and Student t-test as appropriate. A goodness-of-fit chi-square test was used for Hardy-Weinberg equilibrium with one degree of freedom. The association between the SNPs and the presence of moderate/severe gastric inflammation was evaluated using a logistic regression analysis adjusted for confounders. A multivariate logistic regression analysis was done to evaluate factors associated with moderate/severe gastric inflammation. Age and all significant variables in the univariate analysis were included in the full multivariate analysis. All statistical analyses were performed using SPSS for Windows (version 23.0; SPSS Inc., Armonk, NY, USA). p-values of less than 0.05 were regarded as statistically significant.

1. Baseline characteristics of study population and risk of development of gastroduodenal diseases according to SNPs

Table 1 shows the general characteristics of study population. The study population includes 636 controls, 210 patients with GU, 202 patients with DU, 246 patients with gastric adenoma, and 795 patients with GC. The risk of development of GU, DU, gastric adenoma, and GC was analyzed adjusted by age and sex (Table 2). IL-1B-511 CT and CC genotypes were associated with an increased risk of GC (74.9% vs 68.3%: adjusted hazard ratio [HR], 1.391; 95% confidence interval [CI], 1.086 to 1.783; p=0.009). Additionally, TNF-A-308 GA and AA genotype and TGF-B-509 CT and TT genotype were associated with an increased risk of gastric adenoma (19.8% vs 13.7%: adjusted HR, 1.591; 95% CI, 1.026 to 2.467; p=0.038 and 77.0% vs. 68.8%: adjusted HR, 1.719; 95% CI, 1.173 to 2.518; p=0.005). There was a positive association between IL-8-251 AT and AA genotype and an increased risk of DU (64.4% vs 54.5%: adjusted HR, 1.471; 95% CI, 1.042 to 2.078; p=0.028). However, IL-6-572 GG genotype was associated with decreased risk of GU (5.1% vs 10.8%: adjusted HR, 0.440; 95% CI, 0.211 to 0.917; p=0.028), DU (2.6% vs 10.8%: adjusted HR, 0.225; 95% CI, 0.088 to 0.574; p=0.002), gastric adenoma (4.7% vs 10.8%: adjusted HR, 0.456; 95% CI, 0.220 to 0.944; p=0.034), and GC (5.6% vs 10.8%: adjusted HR, 0.538; 95% CI, 0.354 to 0.818; p=0.004).

Table 1 . Clinical Characteristics of Helicobacter pylori-Positive Control, Patients with Gastric Ulcer, Duodenal Ulcer, Gastric Adenoma, and Gastric Cancer.

Diagnosis	No.	Age, yr	Male sex
Control	636	53.97±11.55	232 (36.5)
Gastric ulcer	210	58.72±13.57	158 (75.2)
Duodenal ulcer	202	51.59±14.85	131 (64.9)
Gastric adenoma	246	62.82±9.63	166 (67.5)
Gastric cancer	795	60.09±26.60	523 (65.8)

Data are presented as mean±SD or number (%)..

Table 2 . Genotype Frequencies and Risk of Development of Gastric Cancer in Helicobacter pylori-Positive Subjects.

Genetic polymorphism	Genotypes	Control		Gastric ulcer				Duodenal ulcer				Gastric adenoma				Gastric cancer
		No. (%)		No. (%)	OR (95% CI)*	p-value		No. (%)	OR (95% CI)*	p-value		No. (%)	OR (95% CI)*	p- value		No. (%)	OR (95% CI)*	p- value
IL-1B-511	TT CT+CC	199 (31.7) 427 (68.3)		71 (34.0) 138 (66.0)	0.868 (0.605–1.246)	0.443		47 (23.3) 155 (76.7)	1.595 (1.091–2.332)	0.016		71 (29.1) 173 (70.9)	1.111 (0.774–1.594)	0.568		198 (25.1) 592 (74.9)	1.391 (1.086–1.783)	0.009
IL-1RN VNTR	L/L L/2+2/2	550 (86.6) 85 (13.4)		176 (83.8) 34 (16.2)	1.131 (0.707–1.811)	0.607		167 (83.1) 34 (16.9)	1.307 (0.834–2.050)	0.243		217 (88.9) 27 (11.1)	0.746 (0.450–1.238)	0.257		700 (88.3) 93 (11.7)	0.851 (0.608–1.191)	0.346
IL-8-251	TT AT+AA	280 (45.5) 335 (54.5)		76 (37.8) 125 (62.2)	1.263 (0.886–1.801)	0.197		69 (35.6) 125 (64.4)	1.471 (1.042–2.078)	0.028		92 (39.1) 143 (60.9)	1.186 (0.843–1.669)	0.328		328 (41.6) 461 (58.4)	1.197 (0.954–1.502)	0.121
IL-8-781	CC+CT TT	544 (87.9) 75 (12.1)		175 (87.5) 25 (12.5)	0.964 (0.568–1.635)	0.891		169 (87.6) 24 (12.4)	1.056 (0.634–1.759)	0.833		208 (89.7) 24 (10.3)	0.744 (0.434–1.275)	0.282		683 (87.3) 99 (12.7)	1.009 (0.718–1.417)	0.959
IL-6-572	GG+GC GG	551 (89.2) 67 (10.8)		198 (94.9) 10 (5.1)	0.440 (0.211–0.917)	0.028		189 (97.4) 5 (2.6)	0.225 (0.088–0.574)	0.002		222 (95.3) 11 (4.7)	0.456 (0.220–0.944)	0.034		737 (94.4) 44 (5.6)	0.538 (0.354–0.818)	0.004
IL-10-1082	GA+AA GG	596 (99.0) 6 (1.0)		197 (99.0) 2 (1.0)	1.043 (0.167–6.538)	0.964		187 (98.4) 3 (1.6)	1.782 (0.415–7.645)	0.437		233 (99.6) 1 (0.4)	0.511 (0.053–4.978)	0.564		769 (99.2) 6 (0.8)	0.976 (0.289–3.296)	0.968
IL-10-592	AA+CA CC	532 (85.8) 88 (14.2)		185 (92.0) 16 (8.0)	1.038 (1.023–1.053)	0.055		173 (89.2) 21 (10.8)	0.829 (0.492–1.398)	0.482		210 (89.7) 24 (10.3)	0.611 (0.359–1.041)	0.070		690 (88.1) 93 (11.9)	0.809 (0.579–1.130)	0.214
TNF-A-308	GG GA+AA	543 (86.3) 86 (13.7)		170 (82.1) 37 (17.9)	1.543 (0.972–2.448)	0.066		169 (83.7) 33 (16.3)	1.153 (0.732–1.816)	0.540		195 (80.2) 48 (19.8)	1.591 (1.026–2.467)	0.038		661 (84.1) 125 (15.9)	1.266 (0.923–1.738)	0.143
TGF-B-509	CC CT+TT	190 (31.3) 418 (68.8)		48 (24.5) 148 (75.5)	1.326 (0.893–1.969)	0.162		53 (27.5) 140 (72.5)	1.169 (0.807–1.695)	0.409		56 (23.0) 188 (77.0)	1.719 (1.173–2.518)	0.005		199 (25.6) 578 (74.4)	1.258 (0.981–1.614)	0.071

OR, adjusted odds ratio; CI, confidence interval..

*Adjusted for age and sex..

2. Clinicopathologic characteristics of controls

To determine whether the presence of GC-associated SNPs influenced the susceptibility to severe gastric inflammation in the adult Korean population, we examined its association with histological inflammation in a large cohort of healthy controls. In total, 636 controls with current HP infection were genotyped for GC-associated SNPs. The clinical details are shown in Table 3. All SNPs included in this study were in Hardy-Weinberg equilibrium. The mean ages of the male and female patients were 53.57±12.15 and 54.20±11.21 years, respectively. Ex/current smoking and alcohol consumption statuses were more frequent among male than among female patients. A significant difference in the degree of monocyte infiltration of the corpus was noted between the male and female patients; however, the severity of gastric inflammation, such as monocyte infiltration of the antrum, neutrophil infiltration of the antrum and corpus, and AG and IM of the antrum and corpus, did not differ significantly between them, nor did the frequency distributions of the evaluated SNPs.

Table 3 . Comparison of Baseline Characteristics between Helicobacter pylori-Positive Males and Females.

Characteristic	Male (n=232)	Female (n=404)	p-value
Age, yr	53.57±12.15	54.20±11.21	0.510
<40 yr	31 (13.4)	36 (8.9)	0.105
40–59 yr	117 (50.4)	233 (57.7)
≥60 yr	84 (36.2)	135 (33.4)
Smoking*
Never	73 (32.3)	374 (95.9)	<0.001
Ex/current	153 (67.7)	16 (4.1)
Alcohol consumption*
No	45 (19.9)	212 (54.6)	<0.001
Ex/current	181 (80.1)	176 (45.4)
Body mass index, kg/m2	24.12±2.73	22.79±3.08	<0.001
Degree of gastric inflammation*
Neutrophil infiltration
Antrum
No/mild	82 (35.5)	143 (35.9)	0.913
Moderate/severe	149 (64.5)	255 (64.1)
Body
No/mild	76 (32.9)	117 (29.1)	0.318
Moderate/severe	155 (67.1)	285 (70.9)
Monocyte infiltration
Antrum
No/mild	52 (22.5)	65 (16.3)	0.053
Moderate/severe	179 (77.5)	334 (83.7)
Body
No/mild	61 (26.4)	76 (18.9)	0.027
Moderate/severe	170 (73.6)	326 (81.1)
Atrophic gastritis
Antrum
No/mild	132 (83.0)	234 (88.0)	0.153
Moderate/severe	27 (17.0)	32 (12.0)
Body
No/mild	143 (87.2)	272 (91.3)	0.165
Moderate/severe	21 (12.8)	26 (8.7)
Intestinal metaplasia
Antrum
No/mild	191 (83.0)	334 (83.5)	0.882
Moderate/severe	39 (17.0)	66 (16.5)
Body
No/mild	209 (90.5)	377 (93.8)	0.127
Moderate/severe	22 (9.5)	25 (6.2)
Genetic polymorphism*
IL-1B-511			0.538
CC	40 (17.5)	84 (21.1)
CT	115 (50.4)	188 (47.2)
TT	73 (32.0)	126 (31.7)
IL-1RN VNTR			0.330
L/L†	196 (84.5)	354 (87.8)
L/2	36 (15.5)	48 (11.9)
2/2	0	1 (0.2)
IL-8-251			0.345
TT	97 (43.5)	183 (46.7)
AT	102 (45.7)	157 (40.1)
AA	24 (10.8)	52 (13.3)
IL-8-781			0.905
CC	109 (48.4)	193 (49)
CT	87 (38.7)	155 (39.3)
TT	29 (12.9)	46 (11.7)
IL-10-1082			0.094
AA	200 (91.3)	327 (85.4)
GA	18 (8.2)	51 (13.3)
GG	1 (0.5)	5 (1.3)
IL-10-592			0.052
AA	118 (51.8)	165 (42.1)
CA	84 (36.8)	165 (42.1)
CC	26 (11.4)	62 (5.8)
IL-6-572			0.438
CC	118 (52.4)	187 (47.6)
GC	86 (38.2)	160 (40.7)
GG	21 (9.3)	46 (11.7)
TNF-A-308			0.987
G/G	198 (86.5)	345 (86.3)
G/A	29 (12.7)	51 (12.8)
A/A	2 (0.9)	4 (1.0)
TGF-B-509			0.473
CC	67 (29.8)	123 (32.1)
CT	113 (50.2)	173 (45.2)
TT	45 (20.0)	87 (22.7)
Bacterial virulence factor*
vacA s1			0.721
Positive	80 (37.0)	141 (38.5)
Negative	136 (63.0)	225 (61.5)
vacA m1			0.756
Positive	91 (47.4)	155 (46.0)
Negative	101 (52.6)	182 (54.0)
vacA m2			0.970
Positive	175 (91.6)	310 (91.7)
Negative	16 (8.4)	28 (8.3)
cagA			0.982
Positive	106 (46.3)	182 (46.2)
Negative	123 (53.7)	212 (53.8)
oipA			0.886
Positive	101 (44.1)	177 (44.7)
Negative	128 (55.9)	219 (55.3)

Data are presented as mean±SD or number (%)..

*Some data were missing; ^†L indicates a long variable number tandem repeat consisting of more than two 86-bp repeats. Allele 1 of this polymorphism consists of four repeats and is the commonest allele. Alleles 3 (five repeats), 4 (three repeats), and 5 (six repeats) are very rare. Alleles 3, 4, and 5 grouped with allele 1 are referred to as “L” (long alleles) for analysis purposes..

3. Effect of SNPs on AG in the antrum and corpus

The degree of gastric inflammation was divided into no/mild and moderate/severe. The genotypes of polymorphism were compared, and an association analysis was performed separately for each sex (Table 4). In males, age, IL-1B-511 CC genotype, non-drinker status, and oipA–negative HP status were significantly associated with moderate/severe AG in the corpus (p=0.022). Of the IL-1B-511 CC patients, 26.9% had moderate or severe corporal AG compared with 10% of CT and TT patients. IL-1B-511 CC genotype was associated with an HR of 4.377 (95% CI, 1.387 to 13.814; p=0.012) for the development of moderate or severe AG of the corpus, independent of age, alcohol consumption, and oipA–negative HP infection (Table 5). In females, moderate/severe AG of the corpus was significantly higher in the IL-8-251 AA genotype than in the AT and TT genotypes (21.4% vs 6.0%, p=0.001) and in the IL-8-781 TT genotype than in the IL-8-781 CT and CC genotypes (19.4% vs 7.0%, p=0.018). IL-8-251 AA genotype remained a significant independent predictor of moderate/severe AG after adjusting for other confounders (adjusted HR, 3.799; 95% CI, 1.515 to 9.523; p=0.004) (Table 5). No significant differences were noted between SNPs and moderate/severe AG in the antrum.

Table 4 . The Association of Clinical, Bacterial and Genetic Factors with Severity of Atrophic Gastritis of the Corpus.

Variable	Male				Female
	No/mild	Moderate/severe	p-value		No/mild	Moderate/severe	p-value
Age, yr	51.94±12.55	59.67±8.87	0.001		53.36±11.15	60.19±7.98	<0.001
Smoking
Never	42 (84.0)	8 (16.0)	0.481		253 (91.3)	24 (8.7)	0.361
Ex/current	96 (88.1)	13 (11.9)			12 (85.7)	2 (14.3)
Drinking
No	21 (72.4)	8 (27.6)	0.028		132 (89.2)	16 (10.8)	0.261
Yes	117 (90.0)	13 (10.0)			132 (93.0)	10 (7.0)
Body mass index, kg/m2	24.27±2.85	23.67±2.82	0.373		28.12±87.40	24.34±4.16	0.829
IL-1B 511
CT+TT	121 (89.6)	14 (10.4)	0.022		210 (90.9)	21 (9.1)	0.489
CC	19 (73.1)	7 (26.9)			59 (93.7)	4 (6.3)
IL-1RN VNTR
2/2	0	0			1 (100)	0	1.000
L/2+ L/La	143 (84.2)	21 (12.8)			270 (91.2)	26 (8.8)
IL-8-251
AT+TT	126 (88.1)	17 (11.9)	0.943		234 (94.0)	15 (6.0)	0.001
AA	14 (87.5)	2 (12.5)			33 (78.6)	9 (21.4)
IL-8-781
CC+CT	128 (88.9)	16 (11.1)	0.370		240 (93.0)	18 (7.0)	0.018
TT	13 (81.3)	3 (18.8)			25 (80.6)	6 (19.4)
IL-10-1082
GA+AA	137 (88.4)	18 (11.6)	1.000		258 (91.8)	23 (8.2)	0.357
GG	1 (100)	0			4 (80.0)	1 (20.0)
IL-10-592
AA+CA	128 (87.1)	19 (12.9)	0.483		225 (91.1)	22 (8.9)	0.368
CC	14 (93.3)	1 (6.7)			40 (95.2)	2 (4.8)
IL-6-572
CC+GC	128 (87.1)	19 (12.9)	0.531		238 (91.9)	21 (8.1)	0.722
GG	13 (92.9)	1 (7.1)			27 (90.0)	3 (10.0)
TNF-A-308
GG+GA	139 (86.9)	21 (13.1)	1.000		267 (91.8)	24 (8.2)	0.235
AA	1 (100)	0			2 (66.7)	1 (33.3)
TGF-B-509
CC+CT	107 (84.9)	19 (15.1)	0.063		201 (92.2)	17 (7.8)	0.711
TT	32 (97.0)	1 (3.0)			59 (90.8)	6 (9.2)
vacA s1
Negative	51 (82.3)	11 (17.7)	0.150		92 (88.5)	12 (11.5)	0.155
Positive	83 (90.2)	9 (9.8)			156 (93.4)	11 (6.6)
vacA m1
Negative	64 (97.7)	9 (12.3)	0.893		111 (93.3)	8 (6.7)	0.276
Positive	61 (88.4)	8 (11.6)			127 (89.4)	15 (10.6)
vacA m2
Negative	115 (87.8)	16 (12.2)	1.000		218 (90.8)	22 (9.2)	0.704
Positive	10 (90.9)	1 (9.1)			21 (95.5)	1 (4.5)
cagA
Negative	64 (84.2)	12 (15.8)	0.314		124 (91.2)	12 (8.8)	0.895
Positive	77 (89.5)	9 (10.5)			142 (91.6)	13 (8.4)
oipA
Negative	64 (81.0)	15 (19.0)	0.026		117 (90.7)	12 (9.3)	0.844
Positive	77 (92.8)	6 (7.2)			148 (91.4)	14 (8.6)

Data are presented as mean±SD or number (%)..

Table 5 . Independent Predictors of Moderate to Severe Atrophic Gastritis and Intestinal Metaplasia of the Corpus at Logistic Regression Analysis.

Variable	No. of subjects (%) or mean±SD	HR	95% CI	p-value
Atrophic gastritis
Male
Age, yr	59.67±8.87	1.038	0.990–1.088	0.126
Drinking (no vs yes)	8 (27.6) vs 13 (10.0)	0.221	0.073–0.666	0.007
IL-1B-511 (CT+TT vs CC)	14 (10.4) vs 7 (26.9)	4.377	1.387–13.814	0.012
oipA (negative vs positive)	15 (19.0) vs 6 (7.2)	0.349	0.123–0.991	0.048
Female
Age, yr	60.19±7.98	1.061	1.014–1.110	0.010
IL-8-251 (AT+TT vs AA)	15 (6.0) vs 9 (21.4)	3.799	1.515–9.523	0.004
IL-8-781 (CC+CT vs TT)	18 (7.0) vs 6 (19.4)	1.182	0.316–4.426	0.804
Intestinal metaplasia
Male
Age, yr	60.73±10.77	1.064	1.017–1.113	0.007
IL-8-251 (AT+AA vs TT)	7 (5.6) vs 13 (13.4)	3.128	1.149–8.512	0.026
IL-10-592 (AA vs CA+CC)	6 (5.1) vs 14 (12.7)	2.334	0.837–6.503	0.105
Female
Age, yr	53.36±10.23	1.048	1.008–1.090	0.019

HR, hazard ratio; CI, confidence interval..

4. Effect of SNPs on IM and monocyte and neutrophil infiltration in the antrum and corpus

The logistic regression analysis considering the variables associated with moderate/severe IM and monocyte and neutrophil infiltration in the univariate analysis as independent variables is shown in Tables 5-7. In males, the IL-8-251 TT genotype was a significant and independent predictor of an increased risk of the development of moderate/severe IM of the corpus compared with the IL-8-251 AT and AA genotypes (13.4% vs 5.6%: adjusted HR, 3.128; 95% CI, 1.149 to 8.512; p=0.026) (Table 5). Moderate/severe monocyte infiltration of the antrum was associated with IL-10-592 CA and CC genotypes independent of the HP virulence factor in males (83.6% vs 71.8%: adjusted HR, 2.227; 95% CI, 1.125 to 4.407; p=0.022) (Table 6). In females, the IL-8-251 AT and AA genotypes were significantly associated with moderate/severe monocyte infiltration of the antrum; however, after adjusting for significant variables, this association disappeared (87.4% vs 79.6%: adjusted HR, 1.592; 95% CI, 0.841 to 3.015; p=0.153). IL-8-781 CC and CT genotypes were independent and significant factors for moderate/severe neutrophil infiltration of the antrum in females (adjusted HR, 2.981; 95% CI, 1.297 to 6.852; p=0.010) and the corpus in males (adjusted HR, 3.516; 95% CI, 1.388 to 8.905; p=0.008) (Table 7). No significant associations were observed between the frequencies of the genotypes of IL-10-1082 G/A, TNF-α-308 G/A, TGF-β-509 C/T, and IL-1RN VNTR with the severity of gastric inflammation in the antrum and corpus.

Table 6 . Independent Predictors of Moderate to Severe Monocyte Infiltration of the Antrum.

Variable	No. of subjects (%)	HR	95% CI	p-value
Male
IL-10-592 (AA vs CA+CC)	84 (71.8) vs 92 (83.6)	2.227	1.125–4.407	0.022
vacA s1 (negative vs positive)	51 (63.8) vs 114 (84.4)	3.233	1.664–6.283	0.001
cagA (negative vs positive)	74 (68.8) vs 102 (83.6)	1.270	0.607–2.657	0.013
oipA (negative vs positive)	71 (70.3) vs 105 (82.7)	1.634	0.819–3.260	0.163
Female
IL-8-251 (TT vs AT+AA)	144 (79.6) vs 181 (87.4)	1.592	0.841–3.015	0.153
vacA m2 (negative vs positive)	125 (81.2) vs 162 (90.0)	1.762	0.893–3.487	0.021
cagA (negative vs positive)	75 (80.6) vs 128 (95.5)	2.097	1.054–4.197	0.037

HR, hazard ratio; CI, confidence interval..

Table 7 . Independent Predictors of Moderate to Severe Neutrophil Infiltration of the Antrum and Corpus.

Variable	No. of subjects (%) or mean±SD	HR	95% CI	p-value
Antrum
Male
vacA s1 (negative vs positive)	37 (46.3) vs 100 (74.1)	1.918	0.957–3.824	0.066
vacA m1 (negative vs positive)	50 (54.9) vs 73 (72.3)	0.886	0.423–1.854	0.747
cagA (negative vs positive)	54 (50.9) vs 94 (77.0)	2.209	1.105–4.420	0.025
oipA (negative vs positive)	49 (48.5) vs 98 (77.2)	2.719	1.407–5.253	0.003
Female
Age, yr	53.15±11.38	0.968	0.945–0.991	0.007
IL-8-781 (TT vs CC+CT)	21 (46.7) vs 231 (67.3)	2.981	1.297–6.852	0.010
vacA s1 (negative vs positive)	68 (29.1) vs 166 (70.9)	2.212	1.212–4.036	0.010
vacA m1 (negative vs positive)	88 (40.6) vs 129 (59.4)	1.701	0.928–3.118	0.086
vacA m2 (negative vs positive)	194 (89.0) vs 24 (11.0)	10.420	2.230–48.701	0.003
cagA (negative vs positive)	96 (37.8) vs 158 (62.2)	1.306	0.701–2.436	0.401
oipA (negative vs positive)	99 (39.1) vs 154 (60.9)	1.353	0.771–2.373	0.292
Corpus
Male
Age, yr	54.70±11.93	1.025	0.998–1.052	0.070
Smoking (negative vs positive)	56 (76.7) vs 95 (62.5)	0.381	0.178–0.813	0.013
IL-8-781 (TT vs CC+CT)	13 (46.4) vs 137 (69.9)	3.516	1.388–8.905	0.008
vacA s1 (negative vs positive)	43 (53.8) vs 98 (72.6)	2.138	1.080–4.232	0.029
cagA (negative vs positive)	60 (56.6) vs 94 (77.0)	1.866	0.956–3.643	0.067
Female
vacA s1 (negative vs positive)	86 (61.0) vs 173 (77.6)	1.915	1.160–3.163	0.011
vacA m1 (negative vs positive)	101 (65.2) vs 139 (76.8)	1.375	0.768–2.460	0.283

HR, hazard ratio; CI, confidence interval..

Cytokine-mediated inflammatory responses to HP infection play key roles in the pathogenesis of GC.²³ Genetic polymorphisms related to inflammatory cytokines are often associated with susceptibility to gastric disease owing to differences in the expression levels that interfere with chemotaxis and amplify the immune response. In addition to their risk of developing gastric disease, genetic polymorphisms in cytokines influence the extent of gastric inflammation. Previous studies have showed the significant associations between some SNPs such as IL-1B-511,^24,25 IL-1B-31,^26,27 IL-1-RN*2,²⁶ and IL-8-251^28,29 and gastric inflammation severity. However, these studies included a limited number of patients and did not consider age and the virulence factors of HP.

Herein, we reported that the IL-8-251 AA genotype is associated with moderate/severe AG in the female corpus. IL-8 is a potent chemotactic factor, it activates neutrophils and lymphocytes in the HP-infected gastric mucosa, contributing to an inflammatory response.³⁰ The A allele of the IL8-251 polymorphism was known to be associated with increased production and expression of IL-8 and consequently with an amplified inflammatory response with recruiting neutrophils and monocytes.³¹ This results in severe and advanced gastritis, and these lesions ultimately predisposes to the GC development.^31-34 IL-8-251 A allele has been reported to be associated with the progression of gastric atrophy in HP-infected patients and with an increased risk of GC in South Korea and Japan.^28,29,31 However, several Western studies did not report any significant associations of IL-8-251 A/T polymorphism with risk of GC.^35,36

Another important polymorphism is IL-8-781 C allele, for which the role of the IL-8-781 polymorphism for gastric disease is still unclear. Kamangar et al.³⁷ suggested that IL-8-781 polymorphism was not significantly associated with the incidence of GC and Song et al.³⁸ studied the association between the IL-8-781 T allele and the susceptibility to cachexia in GC. To the best of our knowledge, the association between the IL-8-781 polymorphism and gastric inflammation has not been reported thus far, and our study is the first to show that the IL-8-781 C allele is significantly associated with severe neutrophil infiltration in males and females. However, this association requires confirmation by large-scale studies.

Contrary to the results for females, the IL-8-251 TT genotype was associated with moderate/severe IM in males. Although the reasons for these discrepancies are not well understood, some possibilities can be considered. The relevance of sex to polymorphic alleles was found in only one sex or to be a risk or protection factor differently according to sex. Barboza et al.³⁹ indicated that sex might play a pivotal role in influencing the severity of gastric inflammations, demonstrating that the IL-6-174 C and IL-8-251 T alleles showed a protective effect against active chronic gastritis development in females, while the IL-8-251 polymorphism was associated with an increased risk of gastritis in males. They suggested that the IL-8-251 polymorphism plays a dual role in the development of active chronic gastritis according to sex, which might be caused by hormonal and/or lifestyle differences.⁴⁰ Otherwise, more potent and important factors may determine the severity of gastric inflammation in males. Thus, further studies, including more individuals, are required to determine the relevance of sex to genetic polymorphisms.

The present study reported that male carriers of the IL-1B-511 CC genotype had significantly higher degrees of atrophy in the corpus than those of the IL-1B-511 CT and TT genotypes, which contradicts the previous results.^24,25 IL-1β, the most potent pro-inflammatory cytokine that inhibits gastric acid secretion and bolsters inflammation, it is increased in the gastric mucosa of HP-infected patients. Hwang et al.²⁵ revealed that carriers of the IL-1B-511T, IL-1B-31C, and ILRN*2 alleles had significantly higher IL-1β levels than individuals with the other genotypes. On the contrary, Rech et al.²⁷ reported the association between IL-1B-31 T allele and an increased risk of moderate/severe gastric antral inflammation in Brazilian patients with functional dyspepsia, which was consistent with our results because IL-1B-511 CT and IL-1B-31 CT are known to be completely in linkage disequilibrium.⁴¹ The exact reason why the IL-1β-511 CC genotype increases moderate/severe AG in the corpus was not revealed. These polymorphisms may be involved in the early stage rather than in the late stage of the histological inflammatory response, as they are related to cytokine expression levels, grades of inflammation, and grades of atrophy.²⁵ In addition, the genotypes of inflammation-related cytokines are suggested to be associated with the development of AG or IM, but not with the severity of AG or IM. Severe AG and IM are the late stages of gastric inflammation, and the effects of SNPs may be diminished in advanced gastric diseases.

As a complex multifunctional anti-inflammatory cytokine, IL-10 downregulates cytotoxic inflammatory responses and cell-medicated immune responses.⁴² IL-10-1082 G/-819 T/-592 C have been reported to be associated with increased production of IL-10.⁴³ A low level of IL-10 is associated with enhanced gastric inflammation and an increased GC risk in HP-infected patients but there are ethnic differences. In Asian populations, low IL-10 production (IL-10-1082 A/-819 C/-592 A) was associated with GC; however, in Western populations, no association was found.^44-47 In our study, the presence of the IL-10-592 C allele was associated with moderate/severe monocyte infiltration of the corpus in males.

Studies of the effects of cytokine polymorphisms on HP-induced inflammation and diseases have led to inconsistent results in different cohorts. Small sample sizes, variations in genotyping methods, and variations in minor allelic frequencies across ethnicities lead to a lack of consistency in the results. Possible contributions of these genetic variants to the risk of disease profile would be relatively limited in this population. Therefore, the sample size was extremely small to determine a risk estimate, and possible associations between the SNPs and gastric inflammation or AG might have been missed.

Susceptibility to advanced gastric inflammation is likely determined by the epigenetic interaction of various genes, environmental factors, and bacterial factors, similar to in many other complex diseases.⁴⁸ In addition, the activation of innate and adaptive immune systems can lead to the recruitment of a variety of inflammatory cells (including dendritic cells, macrophages, neutrophils, mast cells, T cells, and B cells) into the stomach of HP-infected individuals in which T cells are the main coordinator of immunity.⁴⁹ T helper cells are important type of T cells, which are thought to be differentiated into CD4+ cell types with different functions under the stimulation of different cytokines. However, it is difficult or might be impossible to control for all subtle inter-patient genetic or environmental variability that interact to determine the development or severity of the disease in clinical studies.⁴⁸ Nevertheless, the strength of our study is that an extensive sampling of Korean healthy controls infected with HP was conducted, and separate analyses were performed according to sex with adjusting for age, environmental factors such as smoking and alcohol consumption, and HP virulence factors. To the best of our knowledge, this is the first study reporting the association between and SNPs considering HP virulence factor. Our sample size is large enough to detect SNPs with low frequency. In addition, the degree of inflammation was evaluated from several aspects such as infiltration of neutrophil and monocyte, AG and IM.

This study also has some limitations. First, the expression levels of cytokine in gastric mucosa were not evaluated. The expression levels of gastric mucosal cytokine might be helpful to determine the actual differences in cytokine production. Second, the effects of the combined SNPs on gastric inflammation were not evaluated. Third, we tried to adjust for various confounding factors in the statistical analysis. However there may have been residual confounders. Fourth, we have not investigated the function of T helper cells. Finally, the crosstalk between function of T helper cells, virulence factors, and cytokine production could not be explained according to sex difference.

In conclusion, the current study highlighted that the IL-1B-511 C/T, IL-8-251 A/T, IL-8-781 C/T, and IL-10-592 G/C polymorphisms are significantly associated with moderate/severe gastric inflammation in HP-positive controls, and that these SNPs influence the severity of gastric inflammation with sex differences. The association between SNPs and inflammation severity may provide new insights into the clinical value of predictive markers. Further studies with larger sample sizes are required to confirm the effects of these SNPs on gastric inflammation and elucidate the exact putative biological functions of these SNPs in gastric inflammation.

Research data can be shared if it is specifically requested to the corresponding author.

This work was supported by grant no 02-2020-041 from the Seoul National University Bundang Hospital Research fund. This research was supported by the Korea Center for Gendered Innovations for Science and Technology Research (GISTeR), through the Center for Women in Science, Engineering and Technology (WISET) funded by the Ministry of Science and ICT (No. WISET-202203GI01).

No potential conflict of interest relevant to this article was reported.

Study concept and design: N.K. Data acquisition: N.K. Data analysis and interpretation: H.J.K. Drafting of the manuscript: H.J.K. Critical revision of the manuscript for important intellectual content: N.K., Statistical analysis: H.J.K. Obtained funding: N.K. Administrative, technical, or material support; study supervision: J.Y.J., S.K., J.L., H.J.O. Approval of final manuscript: all authors.

Supplementary materials can be accessed at https://doi.org/10.5009/gnl230359.