Salivary Th17 cytokine, human β-defensin 1–3, and salivary scavenger and agglutinin levels in Crohn’s disease

Crohn’s disease is a type of inflammatory bowel disease characterized by the chronic transmural intestinal inflammation. It can affect any part of the gastrointestinal track from the oral cavity to the anus [1]. It has a relapsing–remitting character, and its prevalence varies between 3 and 20 in 100,000 individuals, being highest in the industrialized countries [2]. Major risk factors of the Crohn’s disease are environmental exposures (diet, life style, smoking, and medications), dyscolonization of microbiota, disrupted immune response, and genetic susceptibility. Genetic risk ratio of Crohn’s disease is generally higher than that of other complex diseases, including type 2 diabetes mellitus, schizophrenia, or celiac disease [3]. Single nucleotide polymorphism (SNP)-based genome wide association studies pointed out over 140 loci that are related to the Crohn’s disease [4]. Among the Crohn’s disease-associated genes, a strong and replicated association was observed in IL23R gene, which takes part in Th17 lymphocyte differentiation [5]. In turn, high levels of serum interleukin (IL)-6, IL-17A, IL-23, and interferon-γ are commonly observed Crohn’s disease patients, especially when they are at the active state of the disease [6]. Earlier studies also indicated that human beta-defensins (hBDs), which are chemotactic to T cells and induce the mRNA expression of IL-1β, IL-6, and IL-22, may have copy number variations in Crohn’s disease [7]. When tissue and circulating hBD protein levels were analyzed, elevated hBD-3 levels were detected in terminal ileum, and elevated hBD-2 levels were detected in serum samples of Crohn’s disease patients [8]. IL-22 overexpression exacerbates mucosal inflammation and upregulates the expression of the salivary scavenger and agglutinin (SALSA) protein, which is also known as salivary agglutinin SAG, gp340, or DMBT1 [9]. SALSA is an innate immune protein found on mucosal surfaces and in secretions. It interacts with variety of microbial and host ligands, and appears to support immunological homeostasis [9‐11]. Indeed, a deletion variant of DMBT1 with a reduced number of scavenger receptor cysteine-rich domain coding exons is associated with increased risk of Crohn’s disease [12], and diminished SALSA function has been associated with Crohn’s disease [9].

Periodontal diseases and caries are infection-induced diseases of the oral cavity. While dysbiotic biofilms are the prerequisite for the onset of these diseases, systemic and environmental conditions that dysregulate host immune responses contribute to the disease pathogenesis as well [13]. Crohn’s disease patients usually suffer from oral infectious diseases, including apthous-ulcerative lesions, caries, and periodontal diseases [14]. In fact, recent evidence suggests that Crohn’s disease is associated with severe periodontitis and early teeth-loss [15, 16]. On one hand, extension of gut mucosal inflammation was proposed as an explanatory factor for the high prevalence of periodontitis observed in Crohn’s disease [16]. On the other hand, both periodontitis and Crohn’s disease are multifactorial diseases and share similarities in their pathogenesis, including poor oral hygiene, smoking, diet, psychosocial stress, disturbed cytokine network or disrupted antimicrobial response [15, 17]. Indeed, previous studies suggested associations between periodontal inflammation and disrupted intraoral SALSA and hBD levels [18, 19]. Nevertheless, the observed changes on the oral immune response can arouse from the simultaneous impact of Crohn’s disease and oral inflammatory diseases, which is not yet fully elucidated.

Saliva contains large variety of molecules that originate from oral cavity as well as serum-derived markers. Non-invasively collected saliva contains biological markers that are useful to diagnose periodontal diseases and caries, detect oral infection and inflammation in a non-dental setting, and present information about the overall health of the patient [20]. Two common infectious diseases of the oral cavity, periodontitis and caries, can regulate the levels of various salivary biomarkers either by stimulating immune cell responses, or by affecting the oral environment (pH, oxidative stress) or by modulating the salivary microbial composition [21, 22]. Our group recently demonstrated the independent associations between Crohn’s disease and salivary immunoglobulin (Ig) antibody responses against two periodontitis-associated bacteria, Porphyromonas gingivalis and Prevotella intermedia [23]. In the present study, we hypothesized that the genetic background of Crohn’s disease causes defects in oral immune and antimicrobial protein responses. Thus, we analyzed the salivary Th17 cytokine, epithelial hBD 1–3, and SALSA levels in relation to Crohn’s disease. As the salivary antimicrobial response protein levels can be affected by periodontitis [21] and caries [22], regression models were applied to demonstrate independent associations between Crohn’s disease and salivary Th17 cytokine, epithelial hBD 1–3, and SALSA levels.

Nineteen participants were diagnosed with gingivitis, 11 with initial periodontitis, and 12 with periodontal health in the Crohn’s disease group, and 15 participants with gingivitis, 4 with initial periodontitis, and 15 with periodontal health in the control group.

No significant difference was observed between the Crohn’s disease and control groups in terms of their age, gender, number of teeth, VPI%, BOP%, number of teeth with PPD ≥ 4 mm, and saliva flow rates. DMFT index scores, however, were significantly higher in Crohn’s disease group compared to the controls (p = 0.001, Table 1).

Elevated salivary IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-23, IL-25, IL-35, IFN-γ, and sCD40 levels were observed in Crohn’s disease group, in comparison to controls (Table 2). No difference in salivary levels of IL-1β, IL-31, hBD-1, hBD-2, hBD-3, or SALSA was observed between Crohn’s disease and control groups (Table 3).

In western blots, SALSA appears as slightly diffuse bands, due to its glycosylation. Yet, SALSA proteins with different sizes (270–317 kDa) of one or double bands, were evident in individual saliva samples (examples shown in Fig. 1), but no difference in size patterns was seen between groups.

Linear regression models revealed significant associations between salivary IL-6 and IL-10 with Crohn’s disease; however, those associations were diminished after adjusting the model for age, DMFT, BOP%, and medication use (Table 4). Associations of BOP% and DMFT with salivary Th17 cytokine, hBD 1–3, and SALSA levels are presented in Table 5.

To our knowledge, this study is the first to analyze salivary hBD-1, hBD-2, hBD-3, and SALSA levels in Crohn’s disease patients. According to our results, salivary hBD-1, hBD-2, hBD-3, and SALSA levels of Crohn’s disease patients are similar to those of systemically healthy controls, while salivary Th17 cytokine levels were found higher in Crohn’s disease patients. Nevertheless, according to the regression analysis, there is no independent association between elevated salivary Th17 cytokine levels and Crohn’s disease.

Comprehensive analysis of salivary Th17 cytokine, hBDs, and SALSA levels were the main strength of this study. Previous studies indicated that there are reciprocal interactions between the expressions of Th17 cytokine, hBD 1–3, and SALSA levels [7‐9]. One limitation of this study was the relatively low participant numbers. Participants with Crohn’s disease were recruited from the Crohn and Colitis patient organization (IBD Association of Finland) between January 2017 and May 2018. All members of the association received the invitation and all who fit to the inclusion criteria were recruited. Therefore, no sample size calculation was performed. Secondly, the localization of the lesions and disease behavior according to the Montreal classification were not available; therefore, this information was not implemented into the current study. Thirdly, even though the periodontal disease diagnosis of participants was determined according to the 2018 Periodontal Disease Classification [25], use of WHO probe and the lack of radiographs restrained us to define the grades of periodontitis. As the maximum probing pocket depth measured from each periodontitis patient was 4 mm, these participants were diagnosed as initial periodontitis. Finally, majority of the Crohn’s disease patients were receiving anti-inflammatory or immunosuppressive drugs. While this was a standard procedure for this group of patients, it may still affect the cytokine and antimicrobial peptide expression profiles.

Th17 cytokine levels were previously evaluated in saliva, gingival crevicular fluid, dental biofilm, and gingival tissue samples of Crohn’s disease patients. Elevated gingival IL-10 RNA expression was documented in Crohn’s disease patients in relation to healthy controls, while no difference was observed for IL-2 and IL-6 [26]. When the gingival crevicular fluid Th17 cytokine protein expressions were analyzed in Crohn’s patients, similar expression profiles of IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IFN-γ, and tumor necrosis factor (TNF)-α were observed in Crohn’s disease patients and in systemically healthy controls [27]. According to our findings, salivary Th17 cytokine levels were elevated in Crohn’s disease patients; however, there are no independent associations between Crohn’s disease and elevated cytokine levels. Although a previous study showed higher salivary IL-1β and TNF-α levels in Crohn’s patients in comparison to systemically healthy controls, no information on the oral health status of the participants was given [28]. Indeed, similar to our findings, when the salivary samples were collected from individuals with healthy periodontium, no significant difference was observed in IL-1β, IL-10, IL-17A, and TNF-α levels between Crohn’s patients and healthy controls [29]. In inflammatory bowel disease patients, gingival and intestinal tissue Th17 cytokine expression profiles differ from each other [30], which indicates that the systemic condition is not the only determinant of oral cytokine expression, but the local environment, i.e., oral microbiome, also plays a significant role.

As mentioned above, to our knowledge, there is no available information on the oral hBD or SALSA levels of Crohn’s disease patients. hBDs are antimicrobial peptides with wide-range of functions, including the regulation of T cell chemotaxis, epithelial cell proliferation, and wound healing. In the oral cavity, hBDs are mainly secreted by epithelial cells [31]. Early studies associated Crohn’s disease with low copy number defects at the hBD-2 gene (DEFB4) region [7, 32]. Interestingly, studies by Fellermann et al., [7] and Bentley et al., [32] found the associations in opposite directions. However, neither genome wide association studies nor replication studies found evidence on the association between DEFB4 gene copy number variation and Crohn’s disease, indicating the false-positive results were possibly an outcome of limited statistical power and technological limitations [32, 33]. Similarly, an earlier study proposed association between Crohn’s disease and copy number defect at the gene DMBT1 [12] but this was not confirmed in a later study with other cohort [34]. In our study, we could see size variations of the salivary SALSA molecules between individuals, but no difference was observed between Crohn’s disease patients and control group. The noted size variation of SALSA may reflect to differences in protein core, but also the heavy glycosylation of SALSA affects the protein size and thus hampers the interpretation. Increased SALSA levels are reported in the intestine of Crohn’s disease patients [12], but based on our findings, salivary hBD-1, hBD-2, hBD-3, or SALSA levels did not differ between Crohn’s disease patients and systemically healthy controls. This is in line with the recent evidence that defects in the hBD or SALSA expressions do not necessarily exists in Crohn’s disease, as there is no genetic susceptibility. Yet, confounding factors, such as disease-specific quality of life, may significantly contribute to the dual interactions between Crohn’s disease and periodontal immune responses [35].

Our findings suggest that there is a tendency of increased salivary Th17 cytokine levels in Crohn’s disease patients; however, an independent association between Crohn’s disease and Th17 cytokine profile is lacking. The shifts in the oral cytokine levels of Crohn’s disease patients can be either an outcome of genetic background, or explained by the existence of periodontal diseases and mucosal lesions that are related to Crohn’s disease, or both.