Helicobacter Pylori Infection Increases the Incidence of Erosive Oral Lichen Planus and Alters the Oral Microbiome Composition


 Background Oral lichen planus (OLP), a common clinical oral disease, is associated with an increased risk for malignant transformation. The mechanism underlying the pathogenesis of OLP is unknown. Oral dysbacteriosis is reported to be one of the etiological factors for OLP. Although H. pylori infection is associated with various oral diseases, the correlation between H. pylori infection and OLP is unclear. This study aimed to investigate the effect of H. pylori infection on OLP pathogenesis and oral microbiome composition in the Chinese population, which has a high incidence of H. pylori infection.Result In this study, the saliva samples of 30 patients with OLP (OLP group) and 21 negative controls (NC group) were collected. H. pylori infection was detected using the carbon-14 urea breath test. The saliva samples were divided into the following four groups based on the H. pylori infection status: H. pylori-positive OLP (OLP+), H. pylori-positive NC (NC+), H. pylori-negative OLP (OLP−), and H. pylori-negative NC (NC−) groups. The oral microbiome composition was significantly different between the OLP and NC groups and between the OLP− and OLP+ groups. Compared with those in the OLP− group, the incidence of erosive OLP and the salivary levels of cytokines were higher in the OLP+ group. In contrast, the oral microbiome composition and cytokine levels were not significantly different between the NC− and NC+ groups.Conclusions This is the first report demonstrated that H. pylori infection is significantly correlated with the pathogenesis of erosive OLP. The alleviation of H. pylori infection may decrease the incidence of erosive OLP.

involved in the development of oral diseases [12]. The oral microbiota of patients with OLP has increased microbial diversity, decreased Streptococcus abundance, and increased Bacteroides abundance [13][14][15]. Oral dysbacteriosis is reported to be one of the etiological factors of OLP [16].
The correlation between H. pylori infection and OLP has not been elucidated. H. pylori infection has been detected in the periodontal pocket of patients with OLP. Additionally, the presence of H. pylori in the oral cavity is associated with leukoplakia and OLP oral lesions [6]. One study reported the possible connection between H. pylori and erosive OLP [17]. However, recent studies have reported that H. pylori infection was not detected in the mucosal biopsies of patients with OLP, which indicated the lack of correlation between OLP and H. pylori infection [18].
Hence, the studies evaluating the correlation between OLP and H. pylori have reported contradictory ndings. Additionally, most of these studies involved the Western population. In the Chinese population, the average H. pylori infection rate is high (more than 50%) [19]. However, the correlation between H. pylori and OLP has not been examined in the Chinese population.
In this study, the saliva samples collected from normal control volunteers and patients with OLP were grouped based on the H. pylori infection status. The clinical subtypes of OLP in H. pylori-positive and H. pylori-negative patients were determined. The ndings of this study indicated that H. pylori infection increases the incidence of erosive OLP. However, the underlying mechanism was not elucidated. So, the oral microbiome composition of OLP patients with and without H. pylori infection was examined to evaluate the effect of H. pylori infection on the oral microbiome composition.

Results
Patients with OLP exhibit enhanced prevalence of H. pylori infection The saliva samples of 30 patients with OLP (OLP group) and 21 normal control volunteers (NC group) were collected to analyze the correlation between H. pylori infection status and OLP clinical subtypes. Age and gender were not signi cantly different between the OLP and NC groups (Table 1).
H. pylori infection in the OLP and NC groups was detected using the urease breath test. The prevalence of H. pylori infection in the OLP group (70%) was markedly higher than that in the NC group (50%)( Table 1).
Erosive OLP was characterized by a red erosive mucosal surface ( Figure S1 in Additional le). The lesion severity and the risk of malignant transformation in erosive OLP are higher than those in reticular OLP [9]. In the OLP group, the incidence of erosive OLP in H. pylori-positive patients (61.9%) was signi cantly (Chisquare test) higher than that in H. pylori-negative patients (11.1%) (p=0.0041). Additionally, H. pylori infection was correlated with the OLP subtypes (Table 2).

Patients with OLP exhibit altered oral microbiome composition
Previous studies have demonstrated that the oral microbiome composition in patients with OLP was different from that in normal control individuals [20][21][22]. This study demonstrated that the α diversity of oral microbiota in the OLP group was signi cantly higher than that in the NC group ( Fig.1A and B).
Principal coordinate analysis (PCoA) revealed that the β diversity of oral microbiota in the OLP group was signi cantly different from that in the NC group (Fig. 1C).
The oral microbiome composition was signi cantly different between the OLP and NC groups at the phylum (Fig. 1D) and genus levels (Fig. 1E). The dominant phyla in the oral microbiota were Firmicutes, Proteobacteria, and Bacteroidetes (Fig. 1D). Compared with the NC group, the OLP group exhibited a signi cantly decreased abundance of Firmicutes and a signi cantly increased abundance of Bacteroidetes ( Fig. 1D). At the genus level, compared with the NC group, the OLP group exhibited a decreased abundance of Streptococcus and increased abundances of Neisseria, Prevotella, and Prevotella7 (Fig. 1E).
The bacterial genera with an average relative abundance higher than 1% in the oral microbiome of the OLP and NC groups are listed in Fig. 2A. The volcano plot was used to represent the differences in the bacterial composition at the genus level between the OLP and NC groups (Fig. 2B). Bacteria with relative abundance greater than 1% and signi cant differences in relative abundance were screened out (Fig. 2B).
Compared with the NC group, the OLP group exhibited decreased relative abundances of Streptococcus and Rothia and increased relative abundances of Alloprevotella, Prevotella, Fusobacterium, and Porphyromonas ( Fig. 2B and C).
H. pylori infection alters the salivary microbiome composition in patients with OLP.
To analyze the effect of H. pylori infection on the salivary microbiome composition of the OLP and NC groups, the saliva samples were divided into the following four groups based on the H. pylori infection status: OLP+ (n=21); OLP− (n=9); NC+ (n=10); NC− (n=11).
The α diversity of salivary microbiota in the OLP+ group was signi cantly higher than that in the OLP− group (Fig. 3A, B, C, D and E). In contrast, α diversity was not signi cantly different between the NC+ and NC− groups (Fig. 3A, B, C, D, and E). PCoA revealed that the β diversity of salivary microbiota was signi cantly different between the OLP+ and OLP− groups. However, the β diversity of salivary microbiota was not signi cantly different between the NC+ and NC− groups ( Fig. 3F and G). Additionally, the bacterial composition at the genus and phylum levels was not signi cantly different between the NC+ and the NC− groups ( Figure S2 in Additional le).
The salivary microbiome composition was signi cantly different between the OLP+ and OLP− groups at the phylum and genus levels ( Fig. 4A and B). The predominant bacterial phyla were Proteobacteria, Firmicutes, and Bacteroidetes (Fig. 4A), the relative abundance of Bacteroidetes was signi cantly high in the OLP+ group. At the genus level, the relative abundance of Alloprevotella in the OLP+ group was signi cantly higher than that in the OLP− group (Fig. 4B).
The bacteria in the salivary microbiota of the OLP+ and OLP− groups with an average relative abundance higher than 1% are listed in Fig. 4C. The volcano plot was constructed to determine the differences in the bacterial composition between the OLP+ and the OLP− groups at the genus level (Fig. 4D). The bacteria with relative abundance higher than 1% and signi cant differences in the relative abundance were screened out (Fig. 4D). Compared with the OLP− group, the OLP+ group exhibited signi cantly increased relative abundances of Alloprevotella and Haemophilus and a signi cantly decreased relative abundance of Actinomyces ( Fig. 4D and E).
Comparative analysis of salivary in ammatory factors H. pylori infection can induce the gastric mucosa to secrete in ammatory factors, such as IL-6, IL-8, and IL-17 [23]. Previous studies have reported the dysregulated expression of various in ammatory factors, such as IL-6, IL-8, IL-17, and TNF-α in patients with OLP [24]. In this study, the salivary levels of IL-6, IL-8, and IL-17 in the OLP and NC groups were analyzed using ELISA. The salivary levels of IL-6, IL-8, and IL-17 in the OLP group were signi cantly higher than those in the NC group (Fig. 5A).
Next, the effect of H. pylori infection on the salivary levels of in ammatory factors in the OLP and NC groups was evaluated. Additionally, the salivary levels of IL-6, IL-8, and IL-17 were comparatively analyzed between the following groups: OLP+ and OLP− groups; NC+ and NC− groups. Compared with those in the OLP− group, the salivary levels of IL-6, IL-8, and IL-17 were signi cantly upregulated in the OLP+ group (Fig. 5B). However, the salivary levels of IL-6, IL-8, and IL-17 were not signi cantly different between the NC+ and NC− groups (Fig. 5C).
Next, the correlation between key bacterial genera and in ammatory factors (IL-6, IL-8, and IL-17) was analyzed by constructing the heat map of Spearman's rank correlation coe cients ( Fig. 6A and B). In the OLP and NC groups, the abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated with IL-6 and IL-17, while the abundances of Prevotella and Fusobacterium genera were positively correlated with IL-8. Furthermore, the abundances of Streptococcus and Rothia genera were negatively correlated with IL-7, IL-6, and IL-8 ( Fig. 6A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were signi cantly and positively correlated with IL-17, while those of Actinomyces genus were negatively correlated with IL-7, IL-6, and IL-8 ( Fig. 6B).
Correlation of salivary microbiome function with key bacterial genera PICRUSt was used to predict the metagenome functional content based on 16S rRNA gene sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 7). Compared with those in the NC group, the expression levels of genes involved in various metabolic pathways, such as histidine metabolism, phenylalanine metabolism, novobiocin biosynthesis, lipopolysaccharide (LPS) biosynthesis, LPS biosynthesis proteins, biotin metabolism, ubiquinone, and other terpenoid-quinone biosynthesis were upregulated, while those of genes involved in galactose metabolism, phosphotransferase system, and protein kinase were downregulated in the OLP group ( Fig. 7A and B).
Furthermore, the levels of LPS biosynthesis proteins and LPS biosynthesis in the OLP+ group were upregulated when compared with those in the OLP− group ( Fig. 7C and D).
LPS can induce in ammatory reaction [25]. PICRUSt analysis predicted that the microbial metabolic pathways involved in the pathogenesis of OLP are LPS biosynthesis proteins and LPS biosynthesis. The correlation between key bacterial genera and these two metabolic pathways was examined by constructing the heat map of Spearman's rank correlation coe cients ( Fig. 8A and B). In the OLP and NC groups, the relative abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated, while those of Rothia were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were positively correlated, while those of Actinomyces were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8B).

Patients with OLP exhibit enhanced prevalence of H. pylori infection
The saliva samples of 30 patients with OLP (OLP group) and 21 normal control volunteers (NC group) were collected to analyze the correlation between H. pylori infection status and OLP clinical subtypes. Age and gender were not signi cantly different between the OLP and NC groups (Table 1).
H. pylori infection in the OLP and NC groups was detected using the urease breath test. The prevalence of H. pylori infection in the OLP group (70%) was markedly higher than that in the NC group (50%)( Table 1).
Erosive OLP was characterized by a red erosive mucosal surface ( Figure S1 in Additional le). The lesion severity and the risk of malignant transformation in erosive OLP are higher than those in reticular OLP [9]. In the OLP group, the incidence of erosive OLP in H. pylori-positive patients (61.9%) was signi cantly (Chisquare test) higher than that in H. pylori-negative patients (11.1%) (p=0.0041). Additionally, H. pylori infection was correlated with the OLP subtypes (Table 2).

Patients with OLP exhibit altered oral microbiome composition
Previous studies have demonstrated that the oral microbiome composition in patients with OLP was different from that in normal control individuals [20][21][22]. This study demonstrated that the α diversity of oral microbiota in the OLP group was signi cantly higher than that in the NC group ( Fig.1A and B). Principal coordinate analysis (PCoA) revealed that the β diversity of oral microbiota in the OLP group was signi cantly different from that in the NC group (Fig. 1C).
The oral microbiome composition was signi cantly different between the OLP and NC groups at the phylum (Fig. 1D) and genus levels (Fig. 1E). The dominant phyla in the oral microbiota were Firmicutes, Proteobacteria, and Bacteroidetes (Fig. 1D). Compared with the NC group, the OLP group exhibited a signi cantly decreased abundance of Firmicutes and a signi cantly increased abundance of Bacteroidetes ( Fig. 1D). At the genus level, compared with the NC group, the OLP group exhibited a decreased abundance of Streptococcus and increased abundances of Neisseria, Prevotella, and Prevotella7 (Fig. 1E).
The bacterial genera with an average relative abundance higher than 1% in the oral microbiome of the OLP and NC groups are listed in Fig. 2A. The volcano plot was used to represent the differences in the bacterial composition at the genus level between the OLP and NC groups (Fig. 2B). Bacteria with relative abundance greater than 1% and signi cant differences in relative abundance were screened out (Fig. 2B). Compared with the NC group, the OLP group exhibited decreased relative abundances of Streptococcus and Rothia and increased relative abundances of Alloprevotella, Prevotella, Fusobacterium, and Porphyromonas ( Fig. 2B and C).
H. pylori infection alters the salivary microbiome composition in patients with OLP.
To analyze the effect of H. pylori infection on the salivary microbiome composition of the OLP and NC groups, the saliva samples were divided into the following four groups based on the H. pylori infection status: OLP+ (n=21); OLP− (n=9); NC+ (n=10); NC− (n=11).
The α diversity of salivary microbiota in the OLP+ group was signi cantly higher than that in the OLP− group (Fig. 3A, B, C, D and E). In contrast, α diversity was not signi cantly different between the NC+ and NC− groups (Fig. 3A, B, C, D, and E). PCoA revealed that the β diversity of salivary microbiota was signi cantly different between the OLP+ and OLP− groups. However, the β diversity of salivary microbiota was not signi cantly different between the NC+ and NC− groups ( Fig. 3F and G). Additionally, the bacterial composition at the genus and phylum levels was not signi cantly different between the NC+ and the NC− groups ( Figure S2 in Additional le).
The salivary microbiome composition was signi cantly different between the OLP+ and OLP− groups at the phylum and genus levels ( Fig. 4A and B). The predominant bacterial phyla were Proteobacteria, Firmicutes, and Bacteroidetes (Fig. 4A), the relative abundance of Bacteroidetes was signi cantly high in the OLP+ group. At the genus level, the relative abundance of Alloprevotella in the OLP+ group was signi cantly higher than that in the OLP− group (Fig. 4B).
The bacteria in the salivary microbiota of the OLP+ and OLP− groups with an average relative abundance higher than 1% are listed in Fig. 4C. The volcano plot was constructed to determine the differences in the bacterial composition between the OLP+ and the OLP− groups at the genus level (Fig. 4D). The bacteria with relative abundance higher than 1% and signi cant differences in the relative abundance were screened out (Fig. 4D). Compared with the OLP− group, the OLP+ group exhibited signi cantly increased relative abundances of Alloprevotella and Haemophilus and a signi cantly decreased relative abundance of Actinomyces ( Fig. 4D and E).

Comparative analysis of salivary in ammatory factors
H. pylori infection can induce the gastric mucosa to secrete in ammatory factors, such as IL-6, IL-8, and IL-17 [23]. Previous studies have reported the dysregulated expression of various in ammatory factors, such as IL-6, IL-8, IL-17, and TNF-α in patients with OLP [24]. In this study, the salivary levels of IL-6, IL-8, and IL-17 in the OLP and NC groups were analyzed using ELISA. The salivary levels of IL-6, IL-8, and IL-17 in the OLP group were signi cantly higher than those in the NC group (Fig. 5A).
Next, the effect of H. pylori infection on the salivary levels of in ammatory factors in the OLP and NC groups was evaluated. Additionally, the salivary levels of IL-6, IL-8, and IL-17 were comparatively analyzed between the following groups: OLP+ and OLP− groups; NC+ and NC− groups. Compared with those in the OLP− group, the salivary levels of IL-6, IL-8, and IL-17 were signi cantly upregulated in the OLP+ group (Fig. 5B). However, the salivary levels of IL-6, IL-8, and IL-17 were not signi cantly different between the NC+ and NC− groups (Fig. 5C).
Next, the correlation between key bacterial genera and in ammatory factors (IL-6, IL-8, and IL-17) was analyzed by constructing the heat map of Spearman's rank correlation coe cients ( Fig. 6A and B). In the OLP and NC groups, the abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated with IL-6 and IL-17, while the abundances of Prevotella and Fusobacterium genera were positively correlated with IL-8. Furthermore, the abundances of Streptococcus and Rothia genera were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were signi cantly and positively correlated with IL-17, while those of Actinomyces genus were negatively correlated with IL-7, IL-6, and IL-8 (Fig. 6B).

Correlation of salivary microbiome function with key bacterial genera
PICRUSt was used to predict the metagenome functional content based on 16S rRNA gene sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 7). Compared with those in the NC group, the expression levels of genes involved in various metabolic pathways, such as histidine metabolism, phenylalanine metabolism, novobiocin biosynthesis, lipopolysaccharide (LPS) biosynthesis, LPS biosynthesis proteins, biotin metabolism, ubiquinone, and other terpenoid-quinone biosynthesis were upregulated, while those of genes involved in galactose metabolism, phosphotransferase system, and protein kinase were downregulated in the OLP group ( Fig. 7A and B).
Furthermore, the levels of LPS biosynthesis proteins and LPS biosynthesis in the OLP+ group were upregulated when compared with those in the OLP− group (Fig. 7C and D).
LPS can induce in ammatory reaction [25]. PICRUSt analysis predicted that the microbial metabolic pathways involved in the pathogenesis of OLP are LPS biosynthesis proteins and LPS biosynthesis. The correlation between key bacterial genera and these two metabolic pathways was examined by constructing the heat map of Spearman's rank correlation coe cients ( Fig. 8A and B). In the OLP and NC groups, the relative abundances of Alloprevotella, Porphyromonas, Fusobacterium, and Prevotella genera were positively correlated, while those of Rothia were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8A). In the OLP+ and OLP− groups, the abundances of Alloprevotella and Haemophilus genera were positively correlated, while those of Actinomyces were negatively correlated with the levels of LPS biosynthesis proteins and LPS biosynthesis (Fig. 8B).

Discussion
The pathogenesis of OLP, a common clinical oral mucosal disease, is unknown [7,8]. This study aimed to analyze the effect of H. pylori infection on the pathogenesis of OLP. The analysis of clinical data indicated that H. pylori infection increased the incidence of erosive OLP. Patients with OLP exhibited distinct salivary microbiome composition and enhanced salivary levels of in ammatory factors. H. pylori infection altered the composition and function of salivary microbiota and increased the salivary levels of in ammatory factors in patients with OLP.
Recent studies have demonstrated that the oral microbiome composition in patients with OLP is different from that in normal control individuals [20][21][22]. Consistent with the ndings of previous studies, this study demonstrated that the salivary microbiome composition in patients with OLP was signi cantly different from that in normal control individuals. Compared with that in normal controls, the diversity of salivary microbiota was signi cantly higher in patients with OLP, which was consistent with the results of Kun et al [21].
The salivary microbiome composition at the genus level was comparatively evaluated between the OLP and NC groups. The relative abundance of several bacteria in the salivary microbiota was signi cantly different in patients with OLP. Streptococcus spp., which is one of the rst microbes to colonize the oral cavity, determines the composition of oral micro ora [26]. In the oral cavity, Streptococcus produces molecules that inhibit the growth of pathogens [27]. In this study, patients with OLP exhibited a signi cantly decreased relative abundance of Streptococcus in the salivary microbiota, which can result in the impairment of the growth-inhibitory effects of Streptococcus against oral pathogens. Porphyromonas spp. and Fusobacterium spp., which are the causative agents of periodontitis, modulate the T-cell-mediated immune responses [12,28]. Recent studies have suggested that Porphyromonas spp. and Fusobacterium spp. infections can cause oral cancer [29,30]. The relative abundances of Porphyromonas spp. and Fusobacterium spp. in the salivary microbiota of patients with OLP were signi cantly high, which suggested that these microbes are involved in the pathogenesis of OLP. Prevotella spp., a member of the oral microbiota, is abundant in patients with periodontal disease and periodontal abscesses [31]. Wang et al [21] and Du et al [32] reported that the relative abundance of Prevotella increased in the salivary microbiota of patients with OLP, which was consistent with the ndings of this study. This indicated that Prevotella is an important pathogenic microorganism involved in the development of OLP. Additionally, the relative abundance of Alloprevotella increased in the salivary microbiota of patients with OLP. Alloprevotella increases the levels of H 2 S in oral microenvironment [33]. H 2 S is an in ammatory mediator that can promote the expression of pro-in ammatory factors, including IL-6, in the immune cells of humans and mice [34].
In OLP, various exogenous factors are reported to trigger the T lymphocyte-mediated in ammatory immune responses [8]. Previous studies have indicated that the secretion of in ammatory cytokines may play an important role in the OLP-associated immune dysregulation [24]. IL-6 promotes the proliferation of keratinocytes, which promotes epithelial hyperplasia in OLP [35,36]. The enhanced production of IL-8 may impair the tissue repair mechanism of the keratinocytes [37]. Recent studies have demonstrated that the expression of IL-17 was signi cantly upregulated in the mucosal tissue and peripheral blood of patients with OLP [38][39][40], which suggested that IL-17 mediates tissue damage in OLP lesions. IL-17 secreted by the keratinocytes at the lesion site of OLP may play a role in recruiting in ammatory cells to the lesion site [40].
In this study, the relative abundances of gram-negative bacteria, such as Porphyromonas, Fusobacterium, Prevotella, and Alloprevotella, were positively correlated with in ammatory cytokines, such as IL-6, IL-8, and IL-17. This indicated that these four bacterial genera mediate the pathogenesis of OLP by promoting in ammation.
The metabolic pathways of salivary microorganisms associated with LPS synthesis were signi cantly upregulated in patients with OLP and were positively correlated with the relative abundances of Porphyromonas, Fusobacterium, Prevotella, and Alloprevotella. LPS, which exerts immune-stimulatory and pro-in ammatory effects, can promote local in ammatory responses in tissues and upregulate the expression of in ammatory factors [25]. In the epithelial cells, LPS binds to TLR4 to promote the secretion of pro-in ammatory cytokines and chemokines, such as IL-6 and IL-8 through the JNK signaling pathway [41]. Additionally, LPS can stimulate the secretion of Th17-related cytokines, such as IL-17 in the immune cells [42]. Recent studies have demonstrated that LPS inhibits vitamin D receptors in the oral keratinocytes, which impairs the mucosal homeostasis and leads to epithelial barrier damage. Consequently, this may promote the occurrence and development of OLP [43].
This study, for the rst time, demonstrated that H. pylori infection is signi cantly correlated with the pathogenesis of erosive OLP. Recent studies have reported that H. pylori infection is closely associated with oral diseases, such as periodontitis, aphthous stomatitis, and oral cancer [44][45][46]. Th17 cell-related pro-in ammatory cytokines, such as IL-6 and IL-17 are reported to play an important role in H. pylori infection-induced in ammation [47][48][49][50]. In the gastric mucosa, H. pylori infection can promote the secretion of IL-17 [47]. IL-17 induces the secretion of IL-8, recruits neutrophils, and aggravates in ammation through the ERK 1/2 MAP kinase pathway [23]. Furthermore, the virulence factor of H. pylori can directly promote the secretion of IL-8 in the epithelial cells [51]. H. pylori infection-induced in ammatory cytokines in the stomach can enter the bloodstream and reach the oral cavity to modulate the immune microenvironment in the oral cavity and aggravate the in ammatory response [52].
The dysregulation of T lymphocyte balance may play an important role in the pathogenesis of OLP [8,24].
The proportion of Th17 cells in the peripheral blood of patients with erosive OLP is signi cantly higher than that in patients with reticular OLP [38,53]. The analysis of tissues derived from OLP lesions revealed that the erosive OLP lesions contained signi cantly high proportions of Th17 cells [39]. These results indicate that Th17-related cytokines, such as IL-6 and IL-17 are involved in the pathogenesis of erosive OLP [39,53].
We speculated that H. pylori-induced IL-17 may spread to the oral cavity through the bloodstream and increase the proportion of Th17 cells, which may contribute to the development of erosive OLP.
In this study, the salivary levels of IL-6, IL-8, and IL-17 in the OLP + group were higher than those in the OLP − group. This indicated that H. pylori infection alters the oral immune microenvironment in patients with OLP, which can affect the microbiome composition and promote the development of diseases [54]. Additionally, H. pylori infection enhanced the synthesis of LPS in the salivary microbiota of patients with OLP, which may be one of the reasons for the increased incidence rate of erosive OLP.
This study demonstrated that the OLP + group exhibited a decreased relative abundance of Actinomyces in the salivary microbiota. Actinomyces spp., which are the core microorganisms of oral cavity [28], are essential for maintaining the balance of bacterial ora. The decreased abundance of dominant bacteria will lead to the dysregulation of microbiome composition and promote the colonization of other bacteria [55]. In this study, the relative abundances of Alloprevotella and Haemophilus were signi cantly high in the salivary microbiota of the OLP + group. Haemophilus spp., a gram-negative bacillus, is associated with various opportunistic infections [56][57][58]. Additionally, Haemophilus spp. can activate the macrophages and promote the secretion of pro-in ammatory cytokines, such as IL-6 and IL-8 [59]. Furthermore, Haemophilus spp. infection can induce Th17 cell differentiation and IL-17 secretion, and accelerate the recruitment of neutrophils [60].
In the NC group, H. pylori infection did not enhance the secretion of salivary in ammatory cytokines, as well as the microbial community structure. Previous studies have also reported that gastric H. pylori infection does not affect the oral microbiome composition [52]. There are con icting reports on the colonization of H. pylori in the oral cavity. The number of H. pylori is low in the oral cavity [61]. A metaanalysis revealed that the number of H. pylori in the saliva was less than that in the dental plaque [62]. Chua et al. did not detect H. pylori sequences in the oral swab samples [52]. In this study, it was di cult to detect H. pylori in the saliva sample of subjects infected with H. pylori through 16S rRNA gene sequencing. This indicated that H. pylori infection was not enough to alter the oral immune microenvironment but might promote the development of OLP.
In patients with OLP, H. pylori infection may exacerbate the pathological condition of the oral immune microenvironment and disrupt the homeostasis of the salivary microbiota through the interaction with different members of the microbial community [63]. This may decrease the abundance of bene cial bacteria and increase the abundance of pathogenic bacteria in the oral cavity, which contribute to the development of severe clinical subtype of OLP.
The reason for increased incidence rates of erosive OLP in patients with H. pylori infection cannot be fully determined based on the ndings of this study. This study elucidated the correlation of H. pylori infection with the oral microbiome composition and clinical phenotype of OLP. Hence, we hypothesize that the eradication of H. pylori may relieve the clinical symptoms of OLP and reduce the incidence of erosive OLP.

Conclusions
This study elucidated the correlation of H. pylori infection with the oral microbiome composition and clinical phenotype of OLP. We rstly demonstrated that H. pylori infection is signi cantly correlated with the pathogenesis of erosive OLP Hence, we hypothesize that the eradication of H. pylori may relieve the clinical symptoms of OLP and reduce the incidence of erosive OLP. However, further clinical studies are needed to verify this hypothesis.

Sample collection
Patients with OLP(OLP group, n=30) who underwent comprehensive clinical and histopathological examinations at the Stomatology Hospital of Shandong University were randomly recruited in this study. According to the clinical diagnosis and clinical classi cation of OLP by WHO (2005) [64], OLP is divided into the following two subtypes: reticular OLP, characterized by a white papular lesion, white reticular lesion, or white plaque; erosive OLP, characterized by erythema and erosive lesions. According to the follow-up clinical examination, age-matched and gender-matched normal control volunteers were recruited (NC group, n=21). All study subjects signed the informed consent form. The study subjects did not have a history of periodontitis, dental caries, systemic diseases, smoking, and alcohol consumption.
Additionally, the subjects had not undergone antibiotic therapy or received OLP treatment before the sample was collected. This study was approved by the Medical Ethics Committee of School of Stomatology of Shandong University (Protocol Number: 20161001).
The saliva samples were collected following the guidelines of the Manual of Procedures for Human Micro-biome Project (http://hmpdacc.org/resources/tools_protocols.php). Brie y, the unstimulated saliva samples were collected from each patient between 8:00 am and 11:00 am in a sterile DNase/RNase-free conical tube. The study subjected did not consume alcohol or food for at least 2 h before sampling. The samples were transported to the laboratory and stored at −80 °C until further use.

Enzyme linked immunosorbent assay (ELISA)
The saliva samples (5 mL) were centrifuged at 4 °C and 3500 g for 20 min. The supernatant was stored in a DNase/RNase-free EP tube at −80 ° C until further use. The concentrations of IL-6, IL-8, and IL-17 were examined using the ELISA kit (Neobioscience Technology Co., Ltd. Shenzhen, China), following the manufacturer's instructions.
DNA extraction and 16S rRNA gene ampli cation The genomic DNA was isolated from the saliva samples using the QIAamp DNA micro kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. The V3 and V4 regions (336F-806R) of the 16S rRNA gene were ampli ed using polymerase chain reaction.

Sequencing and data analysis
High-throughput sequencing was performed at CloudSeq Biotech, Inc. (Shanghai, China). Brie y, the raw sequence data were obtained from samples sequenced using the Illumina MiSeq sequencer were subjected to base calling and quality ltering. The samples were separated based on the barcode. The adaptors were trimmed and the low-quality reads were removed using Trimmomatic. The paired ends were merged using FLASH. The optimized reads were used for operational taxonomic unit (OTU) clustering and the OTU matrices were generated. The most abundant sequence in each OTU was selected as the representative sequence. The sequences of representative OTU were compared with those listed in the Greengenes database and those of samples. The taxonomic abundance matrices were generated. The determination of alpha and beta diversities, as well as statistical analysis and mapping, were performed using Mother and R environment. The microbial gene function was predicted using PICRUSt.

Statistical analysis
All data are represented as mean ± standard deviation. The differences in the cytokine levels between two groups were examined using the t-test. The Kruskal-Wallis test was used to analyze the differences in microbiome composition between two groups. The correlation between variables was examined using Spearman's correlation test. All statistical analyses were performed using GraphPad Prism version 7.01 (GraphPad Software, Inc. CA, USA).  [1] Male/female 7/3 11/0 8/1 16/5 0.5 [2] Smoking habit;  [1] ANOVA; [2] Fisher's test.  [1] Erosive OLP (n=14) 1 (11.1) 13 (61.9) [1] Chi-square test. Figure 2 Comparison of the salivary microbiome composition at the genus level between the NC and OLP groups.  Comparative analysis of the relative abundance of PICRUSt-generated functional pro les of salivary microbiota. Volcano plot analysis of altered Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the following groups: NC (n=21) and OLP (n=30) groups (A); OLP− (n=9) and OLP+ (n=21) groups (C). KEGG pathways with an average relative abundance greater than 2% in the two groups were included. The p-value was calculated using the two-tailed paired t-test (B) and (D) based on volcano plot analysis ( Figure A and Figure C). The distinct gene categories were selected according to signi cant differences in gene categories at level 3 (t-test, p < 0.05). The bar plots on the left show the mean proportion of each KEGG pathway. The dot plots on the right show the differences in mean proportions between the two indicated groups using p-values. Figure 8