Innate Immune Responses in Pediatric Patients with Gastritis—A Trademark of Infection or Chronic Inflammation?

The aim of this study was to define the relationship between several environmental, laboratory, and genetic factors, i.e., TLR2 and NLRP3 polymorphisms, and Helicobacter pylori (H. pylori) infection in children, by comparing three different groups of pediatric subjects: H. pylori-induced gastritis, non-H. pylori gastritis, and healthy controls. Our final study sample included 269 children, which were divided into three groups according to the histopathological exam: group 1 with 51 children with H. pylori-induced gastritis, group 2 with 103 children with H. pylori-negative gastritis, and group 3 (control group) with 115 children without any histopathological changes. All children underwent a thorough anamnesis, clinical exam, laboratory tests, and upper digestive endoscopy with gastric biopsy for rapid urease test, histopathological exam, and genetic analysis of TLR2 rs3804099, TLR2 rs3804100, and NLRP3 rs10754558 gene polymorphisms. We noticed a significant association between living conditions and the type of gastritis (p < 0.0001). Both rapid urease and serological tests were significantly associated with the presence of H. pylori (p < 0.0001). The CT variant genotype of TLR2 rs380499 was significantly associated with neutrophil count (p = 0.0325). We noticed a significant association between the CC variant genotype of NLRP3 rs10754558 and leucocytes, neutrophils, eosinophils, as well as ALT (p = 0.0185, p = 0.0379, p = 0.0483, p = 0.0356). Based on these findings, we state that poor living conditions and rural areas represent risk factors for H. pylori infection. The rapid urease test is a reliable diagnostic tool for this infection. CT and TT carriers of TLR2 rs3804099, as well as CC carriers of NLRP3 rs10754558, might display a more severe degree of systemic inflammation.


Introduction
Chronic inflammation is the hallmark of carcinogenesis, playing a crucial role in the development of a wide diversity of solid tumors. According to the World Health Organization, Helicobacter pylori (H. pylori) is a class 1 carcinogen, infecting more than 50% of the world's population. It is well-documented that this infection might lead to chronic gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue

The Demographic Analysis of the Sample
Our final sample consisted of 269 children divided into three groups according to the histopathological exam. In group 1 were 51 children with H. pylori-induced gastritis; in group 2, 103 children with H. pylori-negative gastritis, and in group 3, the control group, were 115 children without any histopathological changes. We found a similar mean age between the three groups (p = 0.4892), with a slight predominance of female gender in group 3 (p = 0.5764). Regarding the originating area, we observed that children with gastritis, irrespective of the presence of H. pylori, were mostly living in rural areas (p = 0.0100). Regarding the living conditions, we noticed a significant association between living conditions and the type of gastritis (p < 0.0001), indicating that poor living conditions predominated in the group with H. pylori-induced gastritis (35.29%), followed by the group with other types of gastritis (13.59%) versus only 6.96% in the control group. Our findings also revealed that both rapid urease and serological tests were significantly associated with the presence of H. pylori in the histopathological exam (p < 0.0001). We found no significant differences between the three groups in terms of TLR2 rs3804099, TLR2 rs3804100, and NLRP3 rs10754558 gene polymorphisms (p = 0.1339, p = 0.5971, and p = 0.5570), family history (p = 0.7700), gastroesophageal reflux (p = 0.2944), and biliary reflux (p = 0.4151). All assessed parameters are described in Table 1 and Figure 1.

The Laboratory Parameters of the Three Groups
Regarding the assessed laboratory parameters, no significant differences were observed between the three groups (Table 2), except for CRP, which varied significantly among the three groups (p = 0.0452), being significantly higher in H. pylori-negative gastritis group versus the control group, p = 0.0232 (Tables 3 and 4). We must mention that we excluded from each group the children who benefited from a qualitative assessment of CRP.

The Laboratory Parameters of the Three Groups
Regarding the assessed laboratory parameters, no significant differences were observed between the three groups (Table 2), except for CRP, which varied significantly among the three groups (p = 0.0452), being significantly higher in H. pylori-negative gastritis group versus the control group, p = 0.0232 (Tables 3 and 4). We must mention that we excluded from each group the children who benefited from a qualitative assessment of CRP.

TLR2 rs380499 Gene Polymorphism and Laboratory Parameters
In terms of TLR2 rs380499 gene polymorphism, in the CT variant genotype group, we found a significant difference between median values of neutrophils (p = 0.0325), suggesting that CT carriers of this polymorphism had an increased circulating level of neutrophils in the setting of chronic gastric inflammation. Additionally, we noted that the neutrophils in CT genotype groups are significantly higher in H. pylori-negative gastritis group versus the control group, p = 0.0190. All assessed parameters for each TLR2 rs380499 gene polymorphism are detailed in Tables 5 and 6, Figure 2.

Control Group (n = 43) Mean ± SD (Median) p-Value
Hemoglobin (g/dL) 13    We also assessed the differences in neutrophil count among each genotype of both polymorphisms TLR2 rs3804099 and NLRP3 rs10754558 for children with H. pylori-induced gastritis, but we found no significant differences (Table 7). We also assessed the differences in neutrophil count among each genotype of both polymorphisms TLR2 rs3804099 and NLRP3 rs10754558 for children with H. pylori-induced gastritis, but we found no significant differences (Table 7). Regarding the TLR2 rs380499 gene polymorphism, we found significant differences for CRP values among the three groups in children carrying the TT variant genotype (p = 0.0171). Thus, we noticed significantly higher values of CRP values in TT carriers with H. pylori-negative gastritis when compared to those with H. pylori-induced gastritis (p = 0.0264) or healthy controls carrying the same genotype (p = 0.0137) (Tables 8 and 9).

The NLRP3 rs10754558 Gene Polymorphism and Laboratory Parameters
Concerning the assessment of NLRP3 rs10754558 gene polymorphisms, we identified significant differences only for CC variant genotype of NLRP3 rs10754558 and leucocytes, neutrophils, eosinophils, as well as ALT (p = 0.0185, p = 0.0379, p = 0.0483, p = 0.0356) (Tables 10 and 11, Figure 3). Therefore, we noticed a significantly higher number of leukocytes in children carrying this genotype diagnosed with H. pylori-induced gastritis versus the control group (p = 0.0022), while neutrophils in the carriers of the same genotype were significantly higher in H. pylori-negative gastritis children (p = 0.0151) (Table 11). In addition, we found a significantly higher value for eosinophils in children carrying the CC variant genotype of NLRP3 rs10754558 from H. pylori-induced gastritis compared with H. pylori-negative gastritis (p = 0.0365) ( Table 11). The value of ALT was significantly higher in children carrying the previously mentioned genotype from the control group when compared with those included in H. pylori-negative gastritis group (p = 0.0103) ( Table 11). Table 10. The assessment of the laboratory parameters between the three groups among NLRP3 rs10754558 gene polymorphism types.
In terms of NLRP3 rs10754558 gene polymorphism, we found no significant differences for CRP values among the three groups included in the study (Table 12). In terms of NLRP3 rs10754558 gene polymorphism, we found no significant differences for CRP values among the three groups included in the study (Table 12).

Discussion
H. pylori might be defined as 'the bacterium of childhood' since it is usually acquired during early childhood, and its prevalence increases with age. Taking into account its close relationship with gastric cancer in adults, detecting factors that prolong its survival within the gastric mucosa from childhood into adulthood promoting the transformation of acute gastritis into chronic inflammation and further into gastric cancer seems to be the missing puzzle piece in terms of strategies meant to decrease the risk of carcinogenesis. Thus, our study aimed to assess children with H. pylori-induced gastritis, non-H. pylori gastritis, and healthy controls in order to define the role of several environmental, laboratory, and genetic factors that were previously associated either with the development of H. pylori infection or with the presence of gastric cancer. In terms of environmental factors, previous studies reported that improper sanitary conditions within the household, bedsharing between children and adults, along with poor socioeconomic status, increase the risk for H. pylori infection [22,23]. Similarly, our findings also pointed out that poor living conditions were significantly associated with H. pylori-induced gastritis in children. Moreover, according to a recent study performed on children, originating areas, i.e., rural areas, might also be considered a significant risk factor for developing chronic gastritis, irrespective of the presence of H. pylori [24]. Our findings also support the previously noted observation of impact and the correlation between rural areas and the development of gastric inflammation.
TLR2 was strongly related to gastric carcinogenesis. Several studies pointed out that TLR2 polymorphisms are associated with an increased risk for gastric cancer, but this association also depends on ethnicity and geographic area [3,25,26]. Nevertheless, studies on children proved that H. pylori has the ability to promote the in vivo overexpression of different TLRs such as TLR2, 4, 5, and 9, early during infection, initiating a chronic and balanced inflammation [27]. This process will continue for decades, defining the pathway towards developing H. pylori-related gastropathies during adulthood [20]. Moreover, H. pylori infection in children was also associated with an increase in pro and anti-inflammatory cytokines (IL-8, TNF-α, and IL-10) [27]. Pimentel-Nunes et al. noticed an overexpression of TLR2 and TLR4 in intestinal metaplasia and dysplasia/cancer sequence, regardless of the presence of H. pylori, but also an upregulation of these TLRs microRNA (mRNA) in individuals with H. pylori infection and normal gastric mucosa [28]. These findings suggested that H. pylori might not be the only factor that triggers the overexpression of TLRs and the secretion of pro/anti-inflammatory cytokines. These indications are further supported by Targa-Cadamuro et al., who reported an increase in TLR2 and 4 mRNA and protein expression in patients with H. pylori-induced chronic gastritis, which persisted even after the eradication therapy [20]. Contrariwise, other TLRs such as TLR10 were proven to ameliorate immune responses in the setting of this infection since their activation resulted in a suppression of proinflammatory cytokines [29], an aspect that requires further studies, especially in pediatric patients. Identifying the activation of TLRs either by H. pylori or other PAMPs and damage-associated molecular patterns (DAMPs) fills an important gap in our, still limited, understanding of the pathway towards carcinogenesis and the mechanism of tumor progression [28].
Multiple studies assessed the role of TLR2 rs380499 and TLR2 rs384100 gene polymorphisms in patients with H. pylori infection, and the results proved to be contradictory. Certain studies performed on patients with gastric cancer revealed a strong association between the previously mentioned SNPs of TLR2 and the risk for gastric carcinogenesis, as well as the prognosis of gastric cancer [30,31]. In contrast, other studies which assessed Japanese, Thai, and Saudi patients with H. pylori infection and H. pylori-associated gastropathies found no association between either of these two SNPs and the aforementioned conditions [32][33][34]. Similarly, we noticed no association between either TLR2 rs380499 or TLR2 rs384100 gene polymorphisms and the presence of chronic gastritis in children independently of the presence of H. pylori. Nevertheless, our study underlined a significant association between the CT variant genotype of TLR2 rs3804099 gene polymorphism and circulating neutrophils in children with non-H. pylori gastritis, suggesting that the carriers of this genotype might develop a more severe degree of systemic inflammation, a well-known long-term trigger for carcinogenesis.
NLRP3 is a critical factor for gastric carcinogenesis since it is the well-documented observation that its dependent pathway is crucial for the production of IL-1β in dendritic cells due to H. pylori infection [35,36]. Furthermore, IL-1β gene polymorphisms were associated with an increased risk of gastric cancer, while the overexpression of this interleukin resulted in both gastric inflammation and cancer in mice [37,38]. Perez-Figueroa et al. proved that H. pylori could increase the expression of NLRP3 inflammasome components, while the inhibitors of this inflammasome and caspase-1 induce a reduction in IL-1β production [6]. In addition, a strong partnership was noted between NLRP3 and TLR2 regarding the production of this interleukin since, according to Jang et al., this TLR is essential for the production of H. pylori-induced IL-1β in neutrophils [14]. The same authors noticed a reduction in the expression of both NLRP3 and IL-1β genes in TLR2-deficient neutrophils.
Nevertheless, NLRP3 inflammasome remains the most important host factor in neutrophils that promotes the production of IL-1β in response to H. pylori infection since both the secretion and cleavage of this interleukin were abolished in NLRP3-and caspase-1/11deficient neutrophils [14]. Neutrophils were defined as crucial innate immune cells in H. pylori-mediated gastric inflammation [37]. Moreover, they were also related to the development of gastric cancer since an increase in neutrophils recruitment in gastric cancer tissue was reported in comparison with the tissues surrounding the malignant lesion [39,40]. Aside from this local increase in neutrophils related to gastric inflammation, multiple recent studies proved that individuals with H. pylori-induced gastritis also present a significant increase in circulating neutrophils, as well as leukocytes, lymphocytes, and acute phase reactants, underlining the ability of this bacterium to trigger a low-grade systemic inflammation [41][42][43][44]. Aside from its suggested role in carcinogenesis, this subclinical inflammation was proved to be associated with several life-threatening chronic conditions such as stroke, cardiovascular diseases, diabetes, thyroid disease, glaucoma, or idiopathic thrombocytopenic purpura [45]. Similar to the findings in TLR2 rs380499 gene polymorphism, we also noticed a significant association between CC variant genotype of NLRP3 rs10754558 gene polymorphism and an increased number of circulating neutrophils in children with H. pylori-negative gastritis. Additionally, we found a significant association between this genotype and leukocytes in children diagnosed with H. pylori-induced gastritis compared with controls, eosinophils in those with H. pylori-induced gastritis compared with those with other types of gastritis, and ALT in the control group versus H. pylori-negative gastritis group.
Based on our findings, we might define a trialogue between TLR2, NLRP3, and neutrophils in developing subclinical inflammation related to H. pylori infection in children, which might represent an early leading cause for gastric carcinogenesis. Moreover, both bacterial and host factors were proven to have a synergistic role in the production of IL-1β in neutrophils [14].
The main limitation of this study consists in the relatively small number of children diagnosed with H. pylori-induced chronic gastritis that might have led to the lack of correlation between the assessed gene polymorphisms and the presence of H. pylori. The fact that we included children originating from a single area of Romania could represent another limitation since we noticed that both ethnicity and geographic area play a crucial role in the development of H. pylori-related gastropathies. It is also worth mentioning that we did not use validated tools for defining living conditions, which may be seen as another possible study limitation. Furthermore, it would have been extremely useful to assess our sample after the eradication therapy for this infection, but unfortunately, once the symptoms disappeared, the patients did not return for the follow-up. Nevertheless, several valuable strengths of this study should be emphasized: in our study, a significant number of pediatric patients were accurately diagnosed based on gastric biopsy specimens, unlike most studies reported in the literature where pediatric age implied only noninvasive methods. We assessed three gene polymorphisms previously reported to be crucial for gastric carcinogenesis, and we also included both patients with H. pylori-positive and H. pylori-negative gastritis in comparison with those with the normal gastric mucosa. To the best of our knowledge, ours is among the few studies, if not the first reported in the literature to assess three gene polymorphisms, i.e., TLR2 rs3804099, TLR2 rs3804100, and NLRP3 rs10754558, in children with H. pylori-induced gastritis, H. pylori-negative gastritis, and H. pylori-negative normal gastric mucosa in order to underline the early possible triggers of gastric carcinogenesis.

Conclusions
Our findings indicated that poor living conditions and rural areas might be considered important risk factors for developing pediatric chronic gastritis, whether induced by H. pylori infection or not. Rapid urease test proved to be reliable for the early detection of H. pylori infection. Our study revealed that both CT and TT carriers of TLR2 rs3804099 gene polymorphism might display a more severe degree of systemic inflammation considering their increased level of circulating neutrophils and CRP in the setting of gastric inflammation. Similarly, the CC genotype carriers of NLRP3 rs10754558 polymorphism detected with H. pylori-negative gastritis were also found with significantly increased levels of circulating neutrophils. Moreover, these carriers associated with significantly higher circulating levels of leukocytes and eosinophils in the setting of H. pylori-induced gastritis. Therefore, children with gastritis carrying NLRP3 rs10754558 polymorphism might have an increased risk for developing subclinical systemic inflammation, irrespective of the presence of H. pylori. The significant association between CC genotype of NLRP3 rs10754558 polymorphism and ALT in children with H. pylori-negative gastritis suggests a possible susceptibility of these carriers to associate liver impairment in the context of gastric inflammation. Taking into account the association between both TLR2 rs380499 and NLRP3 rs10754558 gene polymorphisms and increased level of circulating neutrophils, also known as the key innate immune cells, involved in both local gastric and systemic inflammation, it is conceivable that host genetic factors are crucial for the development of H. pylori and non-H. pylori-induced gastropathies. These findings represent a solid basis for further studies to determine the precise role of these polymorphisms in children with H. pyloriand non-H. pylori-gastritis. Informed Consent Statement: Moreover, we obtained the signed informed consent of all the parents/caregivers and the assent of all children prior to their inclusion in the study.
Data Availability Statement: Not applicable.