A potential therapeutic strategy for prostatic disease by targeting the oral microbiome

Abstract Nowadays, human microbiome research is rapidly growing and emerging evidence has witnessed the critical role that oral microbiome plays in the process of human health and disease. Oral microbial dysbiosis has been confirmed as a contributory cause for diseases in multiple body systems, ranging from the oral cavity to the gastrointestinal, endocrine, immune, cardiovascular, and even nervous system. As research progressing, oral microbiome‐based diagnosis and therapy are proposed and applied, which may represent potential drug targets in systemic diseases. Recent studies have uncovered the possible association between periodontal disease and prostatic disease, suggesting new prevention and therapeutic treatment for the disease by targeting periodontal pathogens. Thus, we performed this review to first explore the association between the oral microbiome and prostatic disease, according to current knowledge based on published articles, and then mainly focus on the underlying molecular and cellular mechanisms and the potential prevention and treatment derived from these mechanistic studies.

based on published articles, and then mainly focus on the underlying molecular and cellular mechanisms and the potential prevention and treatment derived from these mechanistic studies.

| INTRODUCTION
Benign diseases of the prostate, including prostatitis and benign prostatic hyperplasia (BPH), as well as prostate cancer, account for a large proportion of male urologic disease. Prostatitis has been the most frequent urologic condition in young-and middle-aged men, which is characterized by inflammation of the prostate gland. 1 The prevalence of prostatitis diagnosed medically is approximated as 9%, and the overall lifetime prevalence in contemporary research is reported to be 14%. 2 BPH, another common urinary disease, is mainly prevalent in the elder male population, with an increased amount of epithelial and stromal cells in the periurethral area of the prostate. 3 It is known that the prevalence and diagnostic rate of BPH in males increase with age 4 and a recent meta-analysis proposed a prevalence of BPH of 26.2% in a lifetime despite ethnic background. 5 Prostate cancer, the second most frequently diagnosed cancer among men worldwide, accounting for 7.1% of total cancer cases. 6 Inflammation has been noticed for years in the process of prostatic diseases and considered to be a possible causal factor in benign disease and prostate cancer. 7,8 Histological evidence of inflammatory infiltrates has almost been commonly detected in patients with BPH, who nearly all having symptoms of prostatitis. 9 However, the pathophysiologic mechanisms of the benign prostatic diseases remain incompletely characterized and the relationship between the two disease conditions is still unclear. Numerous epidemiological studies suggest that men with a history of chronic inflammation or prostatitis have increased risks of having prostate cancer. 10,11 However, the epidemiological links between prostatitis and prostate cancer development remain arguable. Current therapeutic treatments for prostatic disease include pharmacological and surgical therapies. Unfortunately, multidrug-resistance and post-surgery adverse effects remain big challenges for these treatments. [12][13][14] Therefore, determining the key contributors in the involvement of prostate biology is crucial in establishing preventive treatments and therapeutic strategies.
On the basis of findings of the National Institute of Health-Human Microbiome Project (NIH-HMP), the association between the human microbiome and health is likely to be a fast-moving domain in the research area. 15 To date, the majority of studies 7,16,17 have tried to investigate the link between genitourinary microbiota and urologic disease, focusing on their roles in pathophysiology and management of these common prostatic dysfunctions. However, indirect internal interactions between the microbiome and prostatic disease, for example, the impacts of the oral or gastrointestinal microbiome on prostatic disease are poorly understood. Herein, we have summarized a constructive table showing the prostatic disease-associated microbes presented in the prostate gland or prostatic secretion samples from all nine studies [18][19][20][21][22][23][24][25][26] (Table 1). Except for the microbiota of the genitourinary tract, the oral microbiota deserves more attention according to the strong evidence of epidemiology association between periodontal disease and prostatic disease. Recently, most studies including our findings [27][28][29][30] have shown that periodontal disease also increases the risk of prostatic disease. The microbial dysbiosis, defined as the change of microbial composition or alteration of bacterial pathogenicity, is important in determining the activity of the periodontal disease. 31 To control supragingival plaque and prevent disease progression, periodontal treatment adopted a combination of therapeutic interventions and lifelong maintenance of periodontal health, including individually oral hygiene and dietary instructions, removing subgingival plaque and calculus, local and FANG ET AL. Prostatic secretion and dental plaque samples PCR (1) A total of 8 of 14 patients with CP had no less than one pathogen in their prostatic secretion samples [18] (2) Porphyromona sgingivalis, Treponema denticola, and Escherichia coli were identified in both prostatic secretion samples and dental plaques within the sample individual Benign prostatic hyperplasia (BPH) Estemalik 2017 USA Case series

BPH
Prostatic secretion and dental plaque samples PCR (1) A total of 9 of 10 patients with BPH had no less than one pathogen in their prostatic secretion samples [18] (2) Porphyromonas gingivalis, Treponema denticola and Escherichia coli were identified in both prostatic secretion samples and dental plaques within the sample individual Jain 2020  [20] T A B L E 1 (Continued) Author ( [22] (2) Three prostate cancer-specific microbiome signatures were identified and related to the stages, grades and scores of PCa Pseudomonas were the most frequent genera [23] (2) The core tumor tissues were enriched for and Pseudomonas were abundant and the core prostate microbiome [24] (2) The microbiome biodiversity could not be differentiated between the tumor and adjacent benign tissues.
(3) Ten Pseudomonas genes were strongly associated with host small RNA genes; three of which may negatively correlate with cancer metastasis.

Ma 2019
China Case control 32 PCa and 27
systemic pharmacotherapy, and various types of surgery. 32 Studies have demonstrated that periodontal treatment can reduce systemic inflammation and markers of endothelial dysfunction within 2-6 months, 33 which may act as novel therapeutic targets for inflammation-linked diseases, such as prostatic disease.
However, there is no review, according to our knowledge, to summarize the association between the oral microbiome and prostatic diseases and the applications of the oral microbiome in the management of prostatic disease. Here, we present this review to summarize the findings related to periodontal disease and prostatic diseases, and then mainly focus on underlying the molecular mechanism roles of the oral microbiome in the diseases, so as to provide potential therapeutic targets. We, therefore, believe this review will be timely and necessary that could both provoke new ideas and promote relevant research and practice on this topic.

| Oral microbiome and prostatic inflammation
The microflora of the oral cavity is composed of a unique group of bacteria, fungi, viruses, archaea, and protozoa, generally known as the microbiome. 34,35 The term microbiome encompassing the summary of all microbiotic genetic information, thus, is becoming more popular. 36 The primary habitats of oral surfaces are predominantly facultative anaerobes such as Streptococci and Actinomyces species. 37 Nowadays, more than 1000 different taxonomic bacteria have been proved in the human oral cavity and research employing next-generation sequencing (NGS) methods demonstrate that the coverage of diversity of oral bacteria maybe even much larger. 38 Bacteria, the major microorganisms that inhabit the oral cavity, are, therefore, the focus of this review.
Periodontal disease is an inflammatory disorder that refers to the impairment of the supportive tissues around the teeth, which is triggered by the accumulation of bacteria in a microbial biofilm on the teeth and gingiva. 31,39 Culture-and molecular-based research revealed that the periodontal bacterial pathogens are significantly linked to clinical parameters of periodontal diseases, such as gingival redness, bleeding on probing (BOP), and periodontal destruction. To simplify the description of the microbiota from multiple individual species to complexes of bacteria, the microbial composition of supragingival plaque has been classified into six major complexes: the "red complex" and "orange complex" are predominantly correlated with periodontal disease, the other four complexes (green complex, yellow complex, purple complex, and Actinomyces complex) are auxiliary pathogens. 31,[40][41][42] The species of "red complex" comprise Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola; the "orange complex" was mainly composed of Fusobacterium nucleatum, Prevotella nigrescens, Prevotella intermedia, Campylobacter rectus, and Peptostreptococcus micros. There is amount of microbiota in the oral cavity, we believe many as-yet uncultivated phylotypes to be associated with periodontal disease apart from the above six complexes; in fact, emerging oral pathogens are continuous to be identified. 43,44 Hence, the current "6 major complexes" classification would be modified in the future.
There is compelling evidence that the oral microbiome can extend beyond the oral cavity, and systemic conditions such as Alzheimer's disease, coronary artery disease, arthritis, and colitis are linked to the oral microbiome. [45][46][47][48] Chronic prostatitis (CP) and BPH are complex inflammatory conditions, however, the relevant etiology of the inflammation is far from being clearly understood. The clinical observation that bacteriuria and urinary tract infections are common sequelae of BPH and the frequent occurrence of CP and BPH together in the aging male may indicate that the chronic inflammation in the prostate is likely caused by chronic transmissible infections. 49 This inflammatory condition may present a potential novel diagnostic and therapeutic target for these diseases.
Periodontal disease and prostatic diseases are distinct types and seemly unrelated diseases. However, several common risk factors, such as age, metabolic disorders, psychological factors, are all related to diseases. [50][51][52] Both diseases affect the middle-aged and elderly people, and long disease course and unsatisfactory treatment results have brought a huge burden to the healthcare system. 5,53,54 Furthermore, chronic inflammation has been shown to contribute to or associate with the development of both periodontal and prostatic diseases. 7,55,56 Table 2 summarizes previous epidemiological studies 18,27-30,57-69 exploring the associations between periodontal disease and prostatic disease. Thus, it is of great clinical significance to explore the interaction mechanism between these diseases and whether it affects the development and prognosis of the diseases.

| Periodontal disease, porphyromonas gingivalis, and prostatitis
In terms of the NIH classification, prostatitis is grouped into four categories as follows: category I, acute bacterial prostatitis; Category II, chronic bacterial prostatitis; Category III, CP/chronic pelvic pain syndrome; and Category IV, asymptomatic inflammatory prostatitis. 70 Men suffered from CP always presenting the symptoms of perineal or pelvic pain, and some patients also experience dysfunctional voiding symptoms. 71,72 The prostate is susceptible to infections by pathogenic microorganisms in the male reproductive system. Gram-negative bacteria, such as Escherichia coli and Klebsiella spp, were involved in the etiology of prostate inflammation. 27,73 As noted above, Gram-negative bacteria (i.e., Porphyromonas gingivalis) also act as etiologic agents for periodontal disease.
A recent study 68 involving 100 patients diagnosed with CP and periodontal diseases found that the mean clinical attachment level (CAL) was obviously higher in males suffered from moderate-to-severe prostatitis than mild prostatitis patients, which provides a possible pathological correlation between the two diseases. Furthermore, Estemalik et al. 18 identified oral pathogens from the patients' prostatic secretions and dental plaque, who suffered periodontal disease and CP or BPH simultaneously using polymerase chain reaction (PCR). They found that 17 (

| Periodontal disease, porphyromonas gingivalis, and BPH
BPH, the most frequent urologic condition afflicting elder males, is a popular noncommunicable disease (chronic noncommunicable diseases [CNCDs]) in humans. 3,7,74 It is substantially a histological diagnosis, which is usually clinically defined on the basis of symptoms (lower urinary tract symptoms, LUTS), due to mechanical pressure on the bladder and urethra. 75 A previous study conducted in the USA demonstrated that individuals suffering from periodontal disease could enhance the risk of BPH by 1.50 times. 28 Our recent study 29 on 2171 participants also showed an increased risk of BPH (odds ratio = 1.68, 95% confidence interval = 1.26-2.24) in men with periodontal disease, and patients suffering from periodontitis displayed a higher risk. A significant association was also observed when adjusted by age, body mass index, or blood pressure status, which would strongly suggest a relationship between the two diseases.
Furthermore, periodontal disease obviously increased greater risk in the patients whose prostate volume greater than 60 g. Moreover, Matsumoto et al. 63 investigated the association between chronic periodontal disease and LUTS, and confirmed that urgency and weak stream score of international prostate symptom score (IPSS) was obviously associated with the severity of chronic periodontal disease in both genders. Also, a significant association between the severity of periodontal disease and the existence of overactive bladder syndrome was observed in male patients. Estemalik et al. 18 also investigated the oral pathogen in patients with BPH, and found that 9 of 10 patients with BPH presented no less than one oral pathogen in their prostatic secretion samples, such as Porphyromonas gingivalis and Treponema denticola. There was a bit of pity that the study only focused on the T A B L E 2 Epidemiological studies explore the associations between periodontal disease and prostatic disease Author ( [62] (2) Men with >4 ng/ml levels of PSA at baseline, displayed obvious reduction in PSA after periodontal treatment.  [63] (2) Significant association between the CPD severity and and 9 of 10 patients with BPH and had no less than one oral pathogen in their prostatic secretion samples [18] (2) Porphyromonas gingivalis, Treponema denticola, and Escherichia coli were found in both prostatic secretion samples and dental plaques showed an association with tPSA levels [66] (2) PSA testing before and after CP treatment revealed no significant differences in tPSA, fPSA, or  [30] T A B L E 2 (Continued) Author ( [69] (2) Participants with higher severity of periodontal disease were more able to posses PSA > 4.0 ng/ml compared with those free from periodontal disease, although the adjusted OR was insignificant  [29] (2) Stratified analysis showed that periodontal disease significantly increased higher risk in the group with a prostate volume > 60 g | 1823 detection of three well-recognized periodontal bacteria. In addition, a detailed and comprehensive analysis of the microbial diversity (e.g., abundance) has not been reported in this study, because the authors only used PCR.
Therefore, we suggest performing a study using both sequencing analysis and PCR to obtain proportion and microbial diversity, which can confirm the link between the two diseases better.

| Periodontal disease and prostate-specific antigen
Prostate-specific antigen (PSA), synthesized and secreted by the epithelial cells of the prostate acini, is characterized as an inflammatory marker. 76 It is most frequently employed for prostate cancer screening despite other interventions or exposure can also lead to increased levels of PSA, such as digital rectal examination, prostate biopsy, prostatitis, and BPH. 77,78 Numerous studies 79,80 have revealed that prostatic inflammation contributes greatly to elevated serum levels of PSA.
Previously, Joshi et al. 27 had studied the correlation between PSA and periodontal disease in CP populations and found that patients with CAL ≥ 2.7 mm and concurrent moderate/severe prostatitis displayed enhanced PSA levels than those with one of these conditions. Consistent with the above results, the recent study 68 involving subjects with CP and periodontal diseases observed a significant and positive correlation between PSA and periodontal parameters, including CAL scores, probe depth (PD) scores, gingival index (GI), and plaque index values. Whereas, in a cross-sectional study of 1236 men without prostate cancer diagnosis or inflammation or infection of the prostate, the PSA levels did not increase in periodontal disease patients when taken age and other factors into account.
Interestingly, patients with higher severity of periodontal disease were more probably to possess PSA greater than 4.0 ng/ml compared with those free from periodontal disease, although the multivariable-adjusted ORs revealed insignificant. 69 Moreover, Alwithanani et al. 62 reported that periodontal treatment reduced the PSA value in men afflicted with abnormal prostate on digital rectal examination. A statistical correlation between the alteration of PSA levels and periodontal parameters, such as CAL, GI, BOP, and gingival recession, was also observed after periodontal treatment. However, in a prospective trial of 47 chronic periodontitis patients who did not receive any prostate diagnostics or treatment, the periodontal treatment showed no impacts on PSA reduction. 66 Taken together, the findings above suggest that PSA levels may be correlated with the severity degree of periodontal disease in participants with symptomatic prostatic disease. Whereas, such a potential link seems to be excludable in asymptomatic men. Of course, prospective research with a large sample size should be designed to further explore the relationship.

| Possible mechanisms
Increasing evidence supports that bacterial infections, inflammatory stimuli, and environmental factors are considered key contributors to the development of prostatic diseases. 21 46 Similarly, Témoin et al. 47 detected the oral bacteria DNA from synovial fluid of subjects with arthritis. The authors suggested that oral pathogens had moved to the extraoral site through the bloodstream. As mentioned above, Estemalik et al. 18 isolated Porphyromonas gingivalis and Treponema denticola from patients' dental plaque and prostatic fluid simultaneously. Thus, we purposed that the oral pathogens presented within the same individual may spread from their regular residence and arrived at the prostate gland via hematogenous spread. These microbes might play the same roles as in the oral cavity once they reach the prostate, resulting in a histological alteration in the organ.

| Inflammation
There is substantial evidence to establish that periodontal disease leads to amplified levels of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, and interferon-γ. 56 The effect of these proinflammatory cytokines in the ontogenesis of the prostatic disease has also been recognized, therefore making it possible for periodontal disease to indirectly relate to prostate inflammation through the diffusion of cytokines.
As is reported, Porphyromonas gingivalis and other periodontitis-associated bacteria were highly presented in atherosclerotic plaques and might enhance the persistence of inflammation. 84 Thus, it is highly likely that oral pathogens may initiate or sustain a prostatic inflammatory response, which eventually results in the progression of

| Metabolism
Epidemiology study has revealed the link between periodontal disease and metabolic disorders. 91 A recent study has demonstrated that patients suffering from metabolic syndrome are at an increased risk of BPH. 92 Moreover, metabolic syndrome is correlated with larger prostate size, thus supporting the role of metabolic derangements in the pathogenesis and development of a benign prostatic enlargement. 93 Available evidence further showed that bacterial infection, which could be autosustained or exacerbated by a metabolic disorder, might induce prostatic inflammation. The mechanical obstruction, accompanied by the abnormal metabolic and hormonal milieu on the neck of the bladder, contributing to BPH/LUTS. 51 It is interesting to note that chronic inflammation might exhibit a common relationship between metabolic syndrome and BPH development. 51 Metabolites, such as short-chain fatty acids are commonly produced by gut microbiota during fermentation of dietary fiber, which have similar chemical FANG ET AL.
| 1825 structures as hormones. 94 Since hormones are essential for the growth and survival of prostate cells, metabolites may be correlated with prostate growth. Moreover, SFCAs were reported to appear to enhance regulatory T-cell frequency in the gut, which may influence immune surveillance and involved in carcinogenesis. 95 Thus, we proposed that the oral pathogens may translocate to the prostate via hematogenous spread and influence the proliferation and apoptosis of the prostate based on inflammatory and metabolism mechanisms (Figure 1).

| Immune dysregulation
If the microenvironment of the oral cavity is dysbiotic, immune dysfunction may be induced and the person could suffer periodontal disease, the oral microbiome plays an important role in this immune response. 96 Of these microorganisms, the Porphyromonas gingivalis was reported to trigger oral inflammation and lead to immune The potential mechanism role of oral microbiota in prostatic disease. The oral microbiota may disseminate from the oral cavity and reached the prostate gland via the hematogenous spread, causing direct tissue injury. Meanwhile, oral pathogens may indirectly induce the prostatic inflammatory process through the dissemination of proinflammatory cytokines, such as IL-1β, IL-6, IL-8, TNF-α, and IFN-γ. Then, prostate inflammation could be auto-sustained or exacerbated by a metabolic disorder, thus contributing to prostatic disease. IFN-γ, interferon-γ; IL, interleukin; TNF-α, tumor necrosis factor-α [Color figure can be viewed at wileyonlinelibrary.com] dysregulation in mice 97 ; alterations of the immune response in the mediation of periodontal disease and cancer were also revealed in other published studies. [98][99][100] Accordingly, oral dysbiosis may be overall immunosuppression instead of causing prostatic diseases. The evidence between oral microbiome and efficacy of cancer therapy lacked, but the gut microbiome was found to impact the therapeutic efficacy of certain cancer immunotherapy, including cyclophosphamide, 101 anti-PD-L1, 102 and CTLA-4 blockade. 103 It is speculated that the microbiome may serve as a therapy target to be modulated to enhance treatment responses. Due to the lack of relevant studies, we suggest carrying out human or animal research to investigate the interplay between the oral microbiota and immunotherapy response in prostate cancer, also its mechanisms.

| Periodontal intervention and changes in prostatic symptoms
As mentioned above, the study by Alwithanani et al. 62 reported a periodontal treatment reduced the PSA levels in men afflicted with abnormal prostate on digital rectal examination. The improvement of IPSS after periodontal treatment was also noticed in this study. The authors suggest that rebuilding the integrity of the glandular epithelium of the prostate may decrease the leakage of PSA to enter the blood. Such linkage warrants further investigation since periodontal treatment may eliminate certain periodontal pathogens.

| Manipulation of the oral microbiota in early adult
In light of the findings described above, the manipulation of the oral microbiota composition may be proposed as a novel approach to reduce prostatic inflammatory conditions and prevent prostatic disease. A recent systematic review 104

| Periodontal and prostatic synchronous treatment
As mentioned above, the periodontal treatment improved prostate symptom score and lowered PSA value in men afflicted with chronic periodontitis and prostatic disease. Periodontal disease can influence systematic disease and vice versa. It is known that certain microbiome has the ability to catabolize estrogens and androgens, thereby affecting hormones levels. 107,108 Although the causal relationship between periodontal disease and prostatic disease remains unclear, synchronous treatment for the patients with periodontal and prostatic diseases may be proposed as a novel strategy in certain situations and it is vital to further explore and understand the relationships that are involved.

| Prognosis and self-management of patients
The main objectives of prostatic disease treatment are to alleviate symptoms, improve life quality, and promote the recovery of related functions. As for BPH, simultaneous therapy using alpha-blockers and 5-alpha-reductase inhibitors has obviously decreased disease progression, whereas, 12.6% of patients show clinical progression after 4-year treatment, and 5% needing surgical intervention. 13 It has been indicated that periodontal disease can be successfully treated by active periodontal therapy (APT) and regular obedience during periodontal maintenance therapy (PMT) is important to maintain the remission of periodontal condition gained after APT. 109,110 In a 6-year follow-up prospective study 111 involving 212 participants in PMT, the author reported that patient compliance positively influenced the composition of subgingival microbes and the steady-state of periodontal condition. Since oral microbiota may act as potential targets not only for periodontal disease but also for prostatic disease. Thus, receiving PMT and improving patients' awareness of oral healthcare may have a positive impact on the prognosis of the two diseases.

| PERSPECTIVE AND FUTURE DIRECTIONS
Current epidemiological studies have demonstrated that periodontal disease is related to prostatic disease. The oral microbiota in the oral cavity, peripheral circulation, and urinary system (including tissue, urine, and prostatic fluid) can be detected and quantified using PCR and NGS detection methods. Furthermore, multifunctional nanomaterials, like immunomagnetic beads, can be efficiently used to separate oral microbiota from peripheral circulation or urinary system and enhance the accuracy of PCR or NGS detection. Better early-stage diagnosis or prognosis models based on oral microbiota may be established for prostatic disease, providing new alternative opportunities for disease management. In addition to utilizing the information in PCR or NGS data from the oral microbiome, enzyme-linked immunosorbent assay can also be used to detect the specific antigens or antibodies of oral microbiota in blood samples ( Figure 2).

| CONCLUSION
Although correlations between oral microbial dysbiosis and various diseases have been reported in both experimental animals and humans, the mechanistic role of oral microbiota in disease progression remains to be elucidated. In recent years, the epidemiology correlation between periodontal disease and prostatic disease has attracted increasing attention despite controversies still exist in these studies due to limited sample size, different diagnostic criteria for diseases, and various ethnic backgrounds of included populations. The causative relationship between the oral microbiome and prostatic disease, although needs to be solidly established at the molecular level, would have important consequences for disease prevention and therapy. Prospective trials to estimate the preventive impacts of periodontal therapy on prostatic disease should be designed. Ideally, we try to find the specific oral bacteria, but current evidence support that multiple species of microorganism can induce prostatic inflammation, including the known pathogens (i.e., Escherichia coli) that causing symptomatic bacterial prostatitis (Table 1), none of them is unique. Furthermore, animal studies report that prostatic inflammation persists for months or years even after clearance of infectious pathogens. Thus, it is difficult to identify a unique infectious microorganism as a causative agent of chronic inflammation in the prostates or as a driver of prostate carcinogenesis. In fact, the microbes that are linked with inflammation in the prostate gland could be further connected with BPH and cancer; the definite mechanisms that remain need to be explored. We also suggest performing studies to investigate which periodontal pathogens involved in and the underlying mechanisms of carcinogenesis in the prostate gland. Furthermore, modulation of oral microbiota has emerged as a potential intervention that could provide a novel strategy to manage periodontal diseases and their systemic effects. With further investigation into the impacts of the oral microbiome on prostatic disease, novel therapies for these diseases will be achieved.

ACKNOWLEDGMENTS
The study was supported (in part) by the Nature Science