Metabolic Interaction of Helicobacter pylori Infection and Gut Microbiota

As a barrier, gut commensal microbiota can protect against potential pathogenic microbes in the gastrointestinal tract. Crosstalk between gut microbes and immune cells promotes human intestinal homeostasis. Dysbiosis of gut microbiota has been implicated in the development of many human metabolic disorders like obesity, hepatic steatohepatitis, and insulin resistance in type 2 diabetes (T2D). Certain microbes, such as butyrate-producing bacteria, are lower in T2D patients. The transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome, but the exact pathogenesis remains unclear. H. pylori in the human stomach cause chronic gastritis, peptic ulcers, and gastric cancers. H. pylori infection also induces insulin resistance and has been defined as a predisposing factor to T2D development. Gastric and fecal microbiota may have been changed in H. pylori-infected persons and mice to promote gastric inflammation and specific diseases. However, the interaction of H. pylori and gut microbiota in regulating host metabolism also remains unknown. Further studies aim to identify the H. pylori-microbiota-host metabolism axis and to test if H. pylori eradication or modification of gut microbiota can improve the control of human metabolic disorders.


Introduction
The human gut, including the stomach and intestine, is inhabited by a vast number of microorganisms that host microbiota [1]. Gut microbiota starts after birth through contact with the mother's vaginal, skin, and fecal microorganisms [2,3] and its ecology is influenced by the delivery type, maternal diet, gestational age, and antibiotic exposure [4][5][6]. Using new metagenomics techniques, researchers have demonstrated that gut microbiota regulate the host immune homeostasis and are related to many human metabolic disorders [7]. In an animal study, the gut microbiome increased the capacity of obese mice to harvest energy from their diet [8]. Moreover, the transmission of microbiota from obese mice to germ-free mice led to increased total body fat [8]. Thus, the integrity and balance of gut microbiota play a major role in the metabolic interaction between the host and the microbial community.
H. pylori can persistently colonize the gastric epithelium by interacting with bacterial adhesion molecules and gastric receptors [9,10]. The metabolic consequences of H. pylori infection have been reported to change the microbial-origin fatty acid and lipid profiles in host blood [11]. In an animal study that used NMR-based metabolic analysis, H. pylori infection was shown to disturb the carbohydrate and amino acid metabolism of the host [12]. In addition, metabolic changes are associated with the diversity of gut microbiota. Taken together, these studies indicate that the homeostasis and systemic metabolism between gut microbiota and the host may be altered by H. pylori. However, the exact mechanism remains unclear. This review article focused on the metabolic interaction between gut microbiota and H. pylori, as well as the related consequences of such interaction on the host's health.

Crosstalk between Microbiota and Intestines
Gut microbial communities are known to be inherited from the mother [2,3]. Several studies have shown that human health is susceptible to interactions between gut microbiota diversity or composition and intestinal cells [1,3]. Mucosal and immune homeostasis can be established by a balanced interaction between microbial signals and host immune cells [2,13]. In mice studies, Ivanov et al. demonstrated that a single segmented filamentous bacterium can induce effector T cell differentiation in the lamina propria [14]. Other reports show that certain commensal bacteria modulate intestinal inflammatory responses by inducing regulatory T (Treg) cells and the downstream production of transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) [15,16]. Mortha et al. reported that intestinal macrophages could sense microbial signals to induce the RORγt + innate lymphoid cells (ILCs) to produce colony-stimulating factor 2 (CSF2), thereby promoting intestinal homeostasis [13]. All of these suggest that autoimmune diseases caused by the innate-adaptive immune activation are likely to be affected by the microbial environment [17].

Metabolic Roles of Gut Microbiota
Crosstalk and signaling between the host and microbiota occur at both the cellular and metabolic levels. Ktsoyan et al. showed that significant concentrations of microbial long chain fatty acids (LCFAs) were present in human blood and corresponded to specific microbial compounds [11]. Furthermore, the profile of these LCFAs is distinguished between healthy and pathologic states. Commensal microbiota can break down indigestible polysaccharides in the diet, thereby serving as 70% of the energy source. Using genomic analysis, studies revealed that human colonic microbes like Bacteroides and Bifidobacterium possessed abundant polysaccharides and starch breakdown genes [18][19][20].
The other important energy source of bacterial colonizers of the epithelium, especially butyrate fermenting bacteria, is short chain fatty acids (SCFA) [21]. Microbe-producing butyrate may serve as nutrients for cell growth but also as augmentation of the barrier function to prevent carcinogenesis of the colonic epithelium [22,23]. Using the 16S rRNA sequencing method, Pryde et al. demonstrated that the most common butyrate-producing bacteria were Clostridium spp., particular in clusters XIVa and IV in the human feces [21]. Decreased production of SCFAs can also be correlated to colonic inflammation and clinical diseases. Kelly et al. revealed that microbe-derived SCFAs, particularly butyrate, stimulated epithelial metabolism and decreased intracellular O 2 , resulting in the stabilization of the transcription factor hypoxia-inducible factor-1 (HIF-1) and epithelial barrier function [22]. In a rat model of colon cancer, Mclntyre et al. reported that rats fed a high butyrate-producing fiber diet (wheat bran) had significantly fewer tumors and less tumor mass than those given low butyrate-production fiber diet [23]. These results imply that gut microbiota influences local and systemic metabolites, and closely determinates immunity and other protective mechanisms in humans ( Figure 1).

Gut Dysbiosis and Human Metabolic Disorders
An altered balance between gut microbiota and the host contributes to a spectrum of immune, inflammatory, and metabolic disorders. A metagenome-wide association study using deep shotgun sequencing of the gut microbial DNA demonstrated that type 2 diabetes (T2D) in Chinese patients had moderate degrees of gut microbiota dysbiosis, particularly decreased butyrate-producing bacteria [24]. Based on the close association between microbiota and diabetes, Vrieze et al. transferred intestinal microbiota from lean donors to recipient males with metabolic syndrome. Six weeks later, the insulin sensitivity of the recipients increased along with levels of butyrate-producing intestinal microbiota [25].
Wen et al. used a type 1 diabetes (T1D) non-obese diabetic (NOD) mice model to suggest that signaling through the MyD88 adaptor was critical for T1D development. This effect depended on commensal microbes because germ-free MyD88-negative NOD mice developed robust diabetes [26]. Moreover, the transplantation of microbiota from specific pathogen-free MyD88-negative NOD donors to germ-free NOD recipients attenuated the T1D. Taken together, alterations in intestinal microbiota are associated with insulin resistance and diabetes. The restoration of "healthy microbiota" (microbiota in healthy condition) may be a promising therapeutic strategy for controlling metabolic syndrome.

H. pylori Infection and Metabolic Diseases
Identified by Marshall and Warren in 1984, H. pylori can cause chronic gastritis and peptic ulcer disease [27,28]. The World Health Organization (WHO) has categorized H. pylori as a group I carcinogen, emphasizing its association with gastric cancer [29]. H. pylori have also been associated with several extra-gastric diseases like iron deficiency anemia, idiopathic thrombocytopenic purpura, and childhood growth [30][31][32]. Although the exact relationship between H. pylori and diseases is still being debated, bacterial eradication results in long-term benefits [33,34]. Recently, studies demonstrated that H. pylori infection was also related to lipid and glucose metabolism [35,36]. A large-scale cross-sectional study revealed that males who were H. pylori-seropositive exhibited significantly higher low-density lipoprotein (LDL) cholesterol levels and significantly lower high-density lipoprotein (HDL) cholesterol levels than H. pylori-seronegative subjects [37]. Jia et al. suggested that lower HDL cholesterol was associated with H. pylori infection but did not correlate with the severity of coronary atherosclerosis [38].
A randomized clinical trial is needed to prove that the eradication of H. pylori can prevent or improve hyperlipidemia and atherosclerosis.
The influence of H. pylori on the host's glucose metabolism remains controversial. In a hospital-based study, dyspeptic patients with chronic H. pylori infection had significantly higher HbA1c level and type 2 diabetes in participants older than 65 years of age, but had decreased insulin secretion and sensitivity in participants younger than 45 years old [39]. A large-scale cross-sectional National Health and Nutrition Examination Survey (NHANES) study revealed that H. pylori infection was associated with higher HbA1c, but not self-reported diabetes [40]. However, this correlation was not present in healthy Lebanese adults [41].
The underlying mechanisms of H. pylori-induced insulin resistance and diabetes development were studied in a mice model. Zhou et al. reported that H. pylori infection caused hepatic insulin resistance by mediating the c-Jun/miR-203/SOCS3 signaling pathway [42]. However, there is a paucity of clinical trials that identify the efficacy of H. pylori eradication on glycemic control. Vafaeimanesh et al. treated 93 consecutive patients with type 2 diabetes mellitus (T2DM) and found that H. pylori eradication had no role in glycemic control [43]. In contrast, Bégué et al. suggested that the eradication of H. pylori in T1DM patients could lead to a short-term decrease in HbA1c level [44]. However, despite these suggestions, testing and treating H. pylori in patients with insulin resistance and diabetes are still not routinely done in clinical practice. Further large-scale investigations are needed to clarify the association of H. pylori and diabetes.

H. pylori Infection and Gut Microbiota
H. pylori and gut microbiota both influence the host's metabolism. Stearns et al. studied bacterial bio-geography from different GI sites in four healthy volunteers. Their results revealed larger Shannon and phylogenetic diversities in mouth samples than in samples from the stomach, duodenum, and colon [45]. They also identified that stomach microbes were the least diverse compared than those from the colon and stool. The impact of H. pylori infection on changes in stomach or colon microbiota has been studied in animals and humans [46][47][48][49][50]. Martin et al. investigated stomach microbes by deep sequencing of 16S-rRNA in rhesus macaque pre-and post-inoculation of H. pylori. They found no significant difference in the average relative abundance of non-Helicobacter taxa in the antrum or corpus [46]. A culture method applied by Yin et al. demonstrated an increment in Enterococcus spp. and Staphylococcus aureus in the stomach and duodenum, and a downshift of Lactobacillus spp. in the stomach after H. pylori inoculation in Mongolian gerbils [47]. In the same model, Heimesaat et al. demonstrated that mice infected with immuno-pathologic H. pylori B8 could increase the luminal load of E. coli and enterococci in the cecum and of Bacteroides/Prevotella spp. in the colon [48].
Using deeper sequencing methods, human studies have tried to determine the differences in gastric microbiota between H. pylori-infected and non-infected persons [49,50]. Bik et al. found that the gastric bacterial composition is similar between H. pylori-infected and non-infected subjects at the phylotype level [49]. In contrast, Maldonado-Contreras et al. reveal marked differences in the structure of gastric bacterial community in H. pylori-infected stomach, with an increase in Proteobacteria and a decrease in Actinobacteria, Bacteroidetes, and Firmicutes [50]. Since the association between H. pylori infection and changes in gastric and colonic microbiota is still being debated, more studies on the influence of interactions between commensal colonization and the microbiota-host-environment are critical for understanding how H. pylori influences the gut microbiota and human health [51,52].

Inflammatory Bowel Diseases and Intestinal Microbiota
Crohn's disease (CD) and ulcerative colitis (UC) are both immune-related chronic inflammatory bowel diseases (IBD). Clinical trials have shown that a dysbiosis of fecal microbiota, including changes in microbial diversity and an abundance of certain genus are closely related to the development and maintenance of IBD in adults and children [53][54][55][56]. The diversity of microbiota includes a significant decrease in the genus Faecalibacterium and an increase in the genus Bacteroides in adults with CD and active disease [53]. Unlike findings in adults, there is less abundance and diversity of the bifidobacterial population in children with IBD [55], implying a close relationship between intestinal microbiota and human IBD. Thus, re-establishing gut homeostasis in patients with IBD is desired in order to improve the disease severity and prevent relapse.
Recurrent and serious Clostridium difficile infections have good responses in colonic and frozencapsulized fecal microbiota transplantation (FMT) [57,58]. Moayyedi et al. reported that FMT enema once weekly for six weeks significantly improved disease remission by week 7 in patients with UC (24% vs. 5%) [59]. Moreover, fecal donor and disease duration can affect outcomes. Although this trial is an original study, some limits, such as the duration, route, and dosage of FMT administration, as well as donor selection, still warrant further clarification.

Inflammatory Bowel Diseases and Helicobacter Infection
If H. pylori infection and IBD are both related to changes in gut microbiota, is there a link between H. pylori infection and IBD through alterations in gut microbiota? Luther et al. conducted a meta-analysis and concluded a significantly lower rate of H. pylori infection in IBD patients than in controls (27.1% vs. 40.9%), with a relative risk of 0.64 (95% confidence interval [CI]: 0.54-0.75) [60]. The potential mechanisms of H. pylori protection in IBD were fully discussed by Papamichael et al. [61]. Like genetic and environmental factors, H. pylori can induce a Treg response and down-regulate the pro-inflammatory Th1/Th17 pathway on the gastric mucosa and in peripheral blood [61][62][63].
Theoretically, such immune changes may be beneficial in protecting against the development of CD, a characteristic disease that up-regulates type 1 helper-T immune responses [64]. In animal studies, administration of H. pylori DNA or live bacteria to experimental mice can ameliorate colonic inflammation induced by dextran sulfate sodium (DSS) and Salmonella, respectively [65,66]. Protection against DSS-induced colitis in mice is dependent on NLRP3 inflammasome and interleukin-18 signaling [67]. However, to answer the causative and protective roles of H. pylori infection against IBD, evidence on the increased development of IBD in patients after the eradication of H. pylori is needed.
Recent studies focused on the association between non-H. pylori Helicobacter species (NHPH) and human gastrointestinal diseases, including Crohn's disease, hepatobiliary disease, and gastritis [68][69][70]. Entero-hepatic Helicobacteraceae were detected by PCR sequencing in 59% of fecal samples from children with Crohn's disease, compared to 9% in healthy controls [69]. Nonetheless, among 160 Chinese patients with inflammatory bowel disease (10%) and 80 controls (6.3%), there was no difference in the detection rate of Helicobacter spp. in intestinal specimens [70]. The role of gut microbiota in humans, with focus on the interaction between H. pylori and IBD, should be investigated in the future. Nagalingam et al. reported that H. hepaticus triggered colonic inflammation only in conventional raised IL-10´{´C57BL/6 mice, but not in germ-free mice [71], implying that conventional microbiota are essential for H. hepaticus-induced colonic inflammation. Moreover, H. hepaticus infection altered the structure of intestinal microbiota even with prior antibiotic treatment.
Interestingly, Yang et al. demonstrated that H. hepaticus-induced colonic inflammation was different in the IL-10´{´C57BL/6 mice obtained from two facilities. They further revealed different susceptibilities of H. hepaticus-induced colonic inflammation that originated from distinct microbiota among the groups. Further studies are therefore warranted in order to compare the discrepancy in microbiota structure between pre-and post-H. pylori eradication, and to investigate the structure susceptible to IBD development after H. pylori eradication.

Metabolic Interaction of H. pylori and Gut Microbiota
Although numerous studies suggest that gastric and colonic microbiota communities are changed after H. pylori infection [12,47,50], only one animal study has shown that H. pylori alters gut microbiota, with simultaneous changes in carbohydrate and amino acid metabolisms [12]. Recently, Khosravi et al. demonstrated that H. pylori-infected specific pathogen-free (SPF) mice had elevated levels of leptin, insulin, and peptide YY metabolites. Nonetheless, their growth curves remained inconclusive.
Interestingly, germ-free mice with H. pylori infection also have increased levels of ghrelin and peptide YY, but with concomitant weight loss and malnutrition [72]. Experience has shown that H. pylori infection alters the composition of fecal Bifidobacterium/E. coli in children, which, in turn, is improved by ingesting probiotics-containing yogurt [73]. Lastly, the eradication of H. pylori increases the growth in weight and height among children, together with increased serum acylated ghrelin level [32]. These findings suggest that H. pylori infection alters host metabolism and that commensal microbiota can attenuate the growth delay caused by H. pylori. The detailed mechanisms of metabolic interaction between H. pylori and gut microbiota in hosts warrant more studies.

Conclusions
Both H. pylori and gut microbiota regulate the host metabolism. Some clinical observations and animal studies support the relationship of H. pylori infection and dysbiosis of gut microbiota to metabolic disorders like insulin resistance and diabetes [24][25][26]. However, how H. pylori and gut microbiota interact with each other to regulate the host metabolism remains unknown. Further studies are warranted to identify the H. pylori-microbiota-host metabolism axis and to test if the eradication of H. pylori or the modification of gut microbiota can control or treat human metabolic disorders.
Author Contributions: Both Yao-Jong Yang and Bor-Shyang Sheu contributed to the study design and data interpretation. Yao-Jong Yang wrote draft of the manuscript. Bor-Shyang Sheu contributed to the revision and improvement of the English writing.

Conflicts of Interest:
The authors declare no conflict of interest.