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Abstract
Plaque Psoriasis (PP) and periodontitis are inflammatory disorders with a bidirectional association. They both have a qualitatively similar immune-modulatory cascade, cytokine profile, and a recently described dysbiosis. Different oral bacterial species compositions in the periodontal pocket might play a role in the development of PP. To describe the subgingival microbiota of the Mexican population with PP and the periodontal conditions. Subjects were divided into two groups: periodontal health (PH) (PH-non-PP, PH-PP) and periodontitis (PD) (P-non-PP, PD-PP). Following clinical examination, the patients were classified into three groups according to the degree of psoriasis as measured by the Psoriasis Area Severity Index (PASI) and the periodontal status according to the parameters of the American Academy of Periodontology (AAP). Subgingival microbiota samples of each patient were used to determine 40 species of periodontal bacteria by checkerboard DNA-DNA hybridization. IL-2 and IL-6 were measured by ELISA. Of the forty-eight patients with PP, 21 patients had PH and 27 patients had PD. PD-PP group has a significant increase in the percentage of plaque, gingival redness, pocket probing depth, and clinical attachment loss (P<0.001) compared to PH-PP group. Microbiologically PD-PP exhibited significantly higher mean counts for A. georgiae, A. israelii, A. naeslundii from blue complex (P<0.001) than PD-non-PP. Moreover, the counts of these Actinomyces in PD-PP increased according to the severity of index PASI. The concentration of IL-2 and IL-6 were increased in saliva from PH-PP and PD-PP patients compared to PH non-PP. PP individuals harbored a particular sub-gingival microbiota profile different from non-PP. The severity of psoriasis was related to dysbiosis of microbiota —PASI > 5 related to periodontitis with the predominance of Actinomyces periodontal, irrespective of their periodontal condition. Finally, the severity of psoriasis could be unbalanced in subgingival microbiota and increase the risk to develop periodontitis.
Keywords: Dysbiosis, microbiota, periodontitis, plaque psoriasis
Background 
Plaque psoriasis (PP) is an autoimmune skin disease characterized by keratinocyte hyperproliferation and thickening of the stratum corneum, which causes an increase in lesions of variable clinical characteristics that mainly affect the entire body surface.[1] The pathology of PP is unknown but different studies show that genetic susceptibility and environmental factors contribute to the development of this disease.[2] Recently it has been suggested that dysbiosis[3] may be a key factor for activating plasmacytoid dendritic cells which triggers an imbalance in the immune-modulatory cascade and the cytokine expression profile feeding the pro-inflammatory process.[4] Currently, PP affects approximately 2.5 million adults in Mexico, with 25–30% of patients showing moderate to severe disease. Moreover, epidemiological data indicate a positive association between psoriasis and chronic diseases (diabetes, obesity, and periodontitis).[5] In this context, some articles have shown that periodontitis develops an imbalance in skin microbiota and increases the release of pro-inflammatory molecules associated with the earlier onset of psoriasis.[6]
Dysbiosis has become a key research topic on the pathophysiology of microbiota-associated chronic inflammatory diseases.[7] An imbalance in oral bacterial species in the periodontal pocket during periodontitis has been correlated to chronic disease (arthritis, diabetes, cancer, and hypertension).[8] Additionally, it has been shown that an increase of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia in a periodontal pocket is associated with both psoriatic and rheumatoid arthritis.[9],[10] At the molecular level, these bacteria present in their membrane peptidoglycans (PG), lipopolysaccharide (LPS), or lipoteichoic acid (LTA) which induce the activation of NF-κB- to produce pro-inflammatory cytokines IL-2 and IL-6 to generate positive feedback in the psoriatic lesions.[10],[11],[12]
The findings support the idea that dysbiosis of subgingival microbiota contributes to the pathogenesis of psoriasis, and that the severity of psoriasis could affect the subgingival microbiota to trigger periodontitis. The present study aimed to characterize the subgingival microbiota in PP patients with and without periodontitis regarding psoriasis severity. We hypothesized that the imbalance in different subgingival bacterial species in the periodontal pocket might play a role in the severity of PP.
Materials and Methods Study population
Patients with PP were enrolled in the study from October to December 2019. PP patients were included in the study if the following criteria were met: exclusion criteria included systemic diseases other than PP that might influence the course of periodontitis such as human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), rheumatoid arthritis, autoimmune diseases, type 2 diabetes mellitus, and other types of psoriasis (Guttate Psoriasis, Pustular Psoriasis, Inverse Psoriasis, Erythrodermic Psoriasis), no antibiotic therapy or antioxidant drugs for at least 3 months, no history of tobacco usage or alcoholism, that had not received any form of periodontal therapy in the past, having at least 20 natural teeth (excluding third molars). Pregnant or lactating women were not included in the study. General information included a medical history, age, gender, weight, height, and body mass index was defined (Kg weight/m2 Height). Patients with heart failure, any type of dementia diagnosed during the study, ictus, or stroke were immediately excluded.
Diagnosis of psoriasis
The diagnosis of PP was established by a calibrated dermatologist from the Mexican Association against psoriasis. The severity of PP was determined by the Psoriasis Area and Severity Index (PASI), examining four body regions: I) head and neck, II) hands and arms, III) chest, abdomen, and back and IV) buttocks, thighs, and legs. The area score ranged from 0 (Health) to 6 (all of the skin affected). The lesions were graded based on their redness, thickness, and scale, each ranging from 0 to 4, so there could be a maximum of 12. An area and severity score for each region was calculated by multiplying the area score by the severity score (maximum 6 × 12 = 72). The patients were classified by severity as mild if the PASI is below 4.9, moderate from 5 to 9.9, and severe if it is 10 or above.[13]
Clinical monitoring
Complete clinical oral examinations were carried out by a calibrated dentist (Kappa coefficient ≥0.85). Six clinical measurements were taken per tooth in a single visit (mesio-buccal, buccal, disto-buccal, disto-lingual, lingual, and mesio-lingual), for all the teeth present, excluding the third molars (a maximum of 168 sites per person) following the method described by Haffajee.[14]
Clinical assessment included plaque accumulation (0/1; undetected/detected), gingival redness (0/1), bleeding on probing (0/1), suppuration (0/1), pocket probing depth, and clinical attachment loss.[14] The pocket depth and clinical attachment loss measurements were taken twice by the same examiner at the same visit, and the mean of the two measurements was recorded to the nearest millimeter using a North Carolina periodontal probe (Hu-Friedy, Chicago, IL). All measurements for a given subject were performed by the same examiner at each visit.
Periodontitis classification
The periodontal status was performed according to the parameters of the American Academy of Periodontology (AAP). All patients with periodontal disease had at least 20 teeth. Periodontal Health (PH) was determined by an absence or minimal levels of clinical inflammation. Periodontitis (PD) was established when it was detected at least eight sites with a pocket depth >4 mm and/or more than 10% of sites with loss of attachment >3 mm.
Sample collection and DNA-DNA hybridization
After drying and isolation with cotton rolls of the teeth, a supragingival microbiota was removed with curettes, and subgingival microbiota samples were obtained with individual sterile Gracey curette from the mesio-buccal site, of Ranford teething (first upper right molar, upper left center, first upper left premolar, first lower right molar, lower right-center, first lower left premolar). Samples were placed in individual tubes containing 150 μl of TE buffer (pH 7.6), 100 μl NaOH at 0.5 M were added to each tube, and the samples were dispersed for a 2 min vortex mixer and the samples were frozen (−20°C) until used. The samples were individually analyzed by checkerboard DNA-DNA hybridization.[15] The list of specimen's bacterial strains employed is presented in [Table 1]. Bacteria DNA was isolated from the American Type Culture Collection lyophilized stocks.
Table 1: Stains employed for the development of DNA probes. All strains were obtained from the American Type Culture Collection (ATCC)Saliva sample collection
Saliva samples of the patients included in this study met the following criteria: no eating, drinking, or teeth brushing in the morning before sample collection and before the clinical examination. Whole saliva samples (WSS) were collected in sterile tubes by paraffin stimulation and after carefully rinsing their mouths with 10 mL of distilled water to eliminate exfoliated cells. Approximately 5 mL of WSS was immediately centrifuged (700 xg for 15 min at 4°C) to remove cells without lysis. The supernatant was collected and centrifuged at 12,000 xg for 10 min at 4°C to remove all suspended insoluble debris. Samples from each participant were collected in one session between 9 and 11 a.m. The samples were stored at − 80°C without thawing until analysis.
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of interleukin-2 (IL-2) and IL-6 in saliva were determined using ELISA (R and D Systems, Minneapolis, MN USA) in accordance with the manufacturer's instructions. Analysis results of concentrations of interleukin were calculated using the standard curves constructed from each assay of saliva volume, given as pg/mL.
Statistical analysis
Microbiological data available for each subject were the counts of each of the 40 test species from six subgingival microbiota samples per subject. The analyses compared microbial data expressed in three ways: counts (levels), % DNA probe count (proportions), and prevalence (% of sites colonized). To compare the counts of each of the bacterial species, the data were expressed as counts ×105 at each site, averaged within a subject, and then averaged across subjects. Similarly, the percentage of DNA probe count and prevalence of each species were computed at each site, averaged across sites within each subject and then across subjects, in each group. Microbial differences were sought between paired comparisons between psoriatic groups, separately for PH and PD, and between the periodontal groups (non-PP and PP), assessed by the Mann–Whitney U (MW) test. To examine the relationship between microbial species in subgingival microbiota samples and pocket depth, sampled sites were subset at the quartiles into categories of, <3, 3-5, and >5 mm pocket depth. Counts and proportions of each species were averaged for each pocket depth category within a subject and averaged across subjects in the three categories. The significance of differences between counts or proportions of bacterial species for each pocket depth category was tested using the Quade test. The significance of differences between counts of the same microbial species in the three pocket depth categories was tested using the Wilcoxon signed ranks test. Adjustments were made for multiple comparisons as described above.
Probing pocket depth and clinical attachment loss were assessed by repeated-measures analysis of variance followed by the Bonferroni test (Prism 5, GraphPad Software Inc., San Diego, USA) Values of P < 0.05 were considered statically significant. Data are presented as mean ± standard deviation of the mean (SD). Microbial differences were sought between paired comparisons between psoriatic groups, separately for PH and PD, and between the periodontal groups (non-PP and PP) assessed by Mann–Whitney U test. Finally, the IL-2 and IL-6 levels data were compared using Kruskal–Wallis test.
Results Clinical parameters
The population consisted of 96 subjects classified as Periodontal Health (PH-non-PP; n = 22, PH-PP; n = 25) and Periodontitis (PD-non-PP; n = 21, PD-PP; n = 27). The PP subjects enrolled were analyzed during the period from October to December 2019. Following the inclusion, exclusion, and elimination criteria. The final sample had 52 PP patients included, 37 females and 15 males, with a mean age of 51.35 (±8.7 SD). The average years with PP were 10 (±8.3 SD) for PH-PP and 11.6 (±6.7) for PD-PP. The use of the drugs Methotrexate was 10 (47.6%) for the PH-PP group and 11 (52.38%) PD-PP and topical corticosteroids use was 5 (50%) PH-PP and 5 (50%) for PD-PP. A significant increase in the PASI score was found in PD-PP 15 (±3.48) compared to PH-PP 9.26 (±2.6). The body mass index was 24 (±3.5) for PH and 26.8 (±3.8) for PD-PP. Furthermore, teeth loss, teeth filled, and caries were higher in PD-PP patients than in PH-PP patients. The main differences between periodontal groups showed that PP harbored a higher mean percentage of plaque (P < 0.05), gingival redness (P < 0.01), pocket probing depth (P < 0.05), and clinical attachment loss (P < 0.01) in PD patients compared to PH [Table 2].
Microbiological findings
Subgingival microbiota samples were analyzed by DNA-DNA hybridization to identify 40 bacterial species [Table 1]. All tested species were detected in each of the four clinical groups representing a considerable number of samples for microbial comparison and all these test specimens were detected in non-PP and PP samples. The bacterial species were grouped in the complexes previously described by Socransky.[15] The mean proportions (% total DNA probe count) of microbial complexes of each clinical group are presented in [Figure 1].
Figure 1: Pie Charts of the mean % DNA probe counts of microbial groups in subgingival microbiota sample from (A) Periodontal Health non-Plaque Psoriasis (PH non-PP) (B) Periodontal Health with Plaque Psoriasis (PH-PP), (C) Periodontitis non-Plaque Psoriasis (PD non-PP), (D) Periodontitis with Plaque Psoriasis (PD-PP). The significance of differences was tested using the Mann–Whitney test signed rank test. *P < 0.05 ** P < 0.001, after adjusting for multiple comparisonsThe present study found higher proportions of blue-complex species (P < 0.05) in the PD-PP individuals than in the PH-PP and PD non-PP. Moreover, the red-complex species showed a decreased proportion in the PD-PP than PD non-PP (P < 0.001). No statistically significant differences were observed between PH-PP and PD-PP in the proportions of the red-complex [Figure 1].
The profiles of the mean counts (1 × 105, ±SEM) of the main 40 taxa in subgingival microbiota samples of PP and non-PP [Figure 2]. PP patients presented a higher level in all periodontal species. The difference was statistically significant in the PP groups, which harbored higher mean individual levels than the non-PP of species Actinomyces georgiae, Actinomyces israelii, Actinomyces naeslundii, Actinomyces odontolyticus, A. viscosus (PD-PP versus PH-PP, P < 0.05). Moreover, the species from the orange-complex showed higher mean individual levels of Corynebacterium matruchotii, Eubacterium saburreum, Parvimonas micra, Prevotella intermedia, Prevotella nigrescens, Streptococcus constellatus in the PD-PP than PH-PP (P < 0.05). Furthermore, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis from the yellow-complex showed higher levels in PD-PP compared to PH-PP and non-PP groups (P < 0.05). Finally, the purple-complex showed an increase of Veillonella parvula levels in PP groups (PD and PH) versus non-PP groups (P < 0.05).
Figure 2: Profiles of the mean counts (1 × 105) of 40 taxa in subgingival microbiota samples in Periodontal Health non-Plaque Psoriasis (PH non-PP) Periodontal Health with Plaque Psoriasis (PH-PP), Periodontitis non-Plaque Psoriasis (PD non-PP), Periodontitis with Plaque Psoriasis (PD-PP). The significance of differences was tested using the Mann–Whitney test signed rank test. PH and PD non-PP versus PP. *P < 0.05, PH versus PD non-PP, and PP Φ P < 0.05, after adjusting for multiple comparisonsThe severity of PP increases periodontal dysbiosis
The subgingival microbiota samples previously analyzed by DNA-DNA hybridization were grouped based on the severity of psoriasis as mild (PASI ≤4.9), moderate (PASI 5-9.9), and severe (PASI ≥10). The results show a significant increase in three species from the blue-complex group: A. georgiae, A. israelii, and A. naeslundii in severe PP compared with mild plaque psoriasis [Figure 3]. Nevertheless, no statistically significant difference was found to periodonto-pathogens from the red-complex (P. gingivalis, T. forsythia, and T. denticola) according to the severity of PP [Figure 4].
Figure 3: Profiles of the mean counts (1 × 105) from blue-complex (a) and red-complex (b) in subgingival microbiota samples in PP according to the severity of psoriasis as mild (<4.9), moderate (5–9.9), and severe (>10) score. The significance of differences was tested using Wilcoxon signed rank test. *P < 0.05, after adjusting for multiple comparisons
Figure 4: Levels of inflammatory cytokines in saliva of PH and Periodontitis (PD) with and without PP. (a) Interleukin 2 (IL-2) (b) IL-6 were measured by ELISA. *Statistically different compared to subjects without PD within the same group. P < 0.05, Kruskal–Walli's testThe salivary concentration of inflammatory cytokines in PP patients
To investigate whether the above-mentioned dysbiosis in subgingival microbiota could be associated with a systemic altered inflammatory response, cytokines levels were measured in the saliva of PP and non-PP subjects [Figure 4]. The concentration of IL-2 and IL-6 was increased in saliva from PH-PP and PD-PP patients compared to PH non-PP (P < 0.05). Therefore, the increased levels of the cytokines IL-2 and IL-6 were positively correlated with the severity of PASI.
Discussion The current study compared and analyzed the periodontal status, and microbiota in the periodontal pocket of four groups (PD-PP, PH-PP, PD non-PP, and PH non-PP) and the severity of PP for a psoriatic group. The clinical periodontal evaluation exhibited significantly more sites with plaque, gingival redness, bleeding on probing, imbalance of blue-complex regarding the severity of PP patients, and the real periodontal damage is evaluated with periodontal clinical attachment loss as part of the periodontal diagnosis.[16] In this sense, clinical attachment loss of severe PP with periodontitis in the present evaluation exhibited significantly higher mean values than the mild PP. The rest of the paired differences showed a similar distribution between PH and periodontitis in the PP groups. Although our results add to the state of clinical, epidemiological, and microbiological knowledge, there are some limitations in our research primarily, that cross-sectional studies cannot establish causal relationships between dependent and independent variables due to their temporal ambiguity.
PP or psoriasis vulgaris is the most common form of psoriasis, and an estimated 80–90% of people with psoriasis have PP. This disease is characterized by thick red patches of skin, often with a silver or white scaly layer. Moreover, periodontitis is the most common comorbidity of psoriasis and epidemiologic studies have been showing that periodontitis increases the risk of early onset of psoriasis[17],[18] and the severity of psoriasis increase the prevalence of periodontitis.[19] In this sense, our results indicated that a higher score from PASI is related to a periodontal clinical parameter impaired.[20] Moreover, PP patients with periodontitis showed a higher score from PASI than periodontally healthy. These changes in characteristics of clinical periodontal and PASI scores are similar to those previously reported.[21] Thus, psoriasis and periodontitis could have a bidirectional relationship in the development and severity of both diseases.
The dysbiosis in the periodontal pocket is considered a main factor in a bidirectional relationship in chronic disease[11],[22],[23],[24] promoting the production of interleukins,[25] prostaglandins,[26] leukotrienes,[27] activation of osteoclast,[28] bone destruction, and creation of a gateway to different oral bacteria to the body.[29] Moreover, oral dysbiosis has been associated with the imbalance of the microbiota in other areas of the body.[30] In this sense, oral and gut microbiota are closely linked to the etiology of inflammatory,[31] immune-mediated disease and trigger trouble in nutrient absorption.[22],[32] Some studies have demonstrated a change in periodonto-pathogen counts from red-complex (P. gingivalis, T. forsythia, and T. denticola) from oral microbiota are related to the development of diabetes mellitus,[33] endocarditis,[34] rheumatoid arthritis, and psoriatic arthritis.[35] However, the periodontal microbiota in PP has not yet been evaluated.
The severity of psoriasis has been determined by the immunologic and microbiologic status of the skin system by controlling the keratinocyte hyperproliferation and thickening of the stratum corneum.[36] In this sense, microbiota dysbiosis in the gut and oral cavity has been related to the severity of psoriasis.[37] Our results indicated that while some differences could be detected consistently between PH and periodontitis in PP subjects, significant differences in subgingival microbial profiles were observed between the severity of psoriasis.[38] In the PP subjects, it was found the highest proportion of the orange-complex, blue-complex, and a lesser proportion of the red-complex in both periodontal statuses. These main results indicated a dysbiosis of the subgingival microbiota of PP individuals, which is certainly related to their systemic condition. Nevertheless, contradictory recent reports using molecular techniques have suggested a high prevalence of specific microorganisms, including the “non-periodontal pathogenic” species S. sanguinis, S. oralis, S. intermedius, and Actinomyces spp in subgingival microbiota of chronic diseases.[39] Our results are following these studies, showing higher levels of the purple and green complexes were predominant in PH patients, whereas PD patients showed increase in the blue-complex particularly A. georgiae, A. israeli, and A. naeslundi. Moreover, the proportion of species from red-complex has no significant difference between PD and PH, these results suggest that periodontitis is not directly related to increasing the counts from periodonto-pathogen (P. gingivalis, V. parvulla, and F. nucleatum) in PP with periodontitis.
The severity of PP is determined by different immunologic, microbiologic, and genetic factors, and could modify the response in gingival tissues. The subgingival microbiota in periodontitis has been characterized by higher proportions of red-complex and low proportions of Actinomyces spp compared to PH in individuals with non-metabolic risk factors. Contradictorily, other studies showed a lower prevalence of red-complex in periodontitis.[40] Our results indicated that the differences between the severity of PP were without signification in the proportion of the pathogenic species P. gingivalis, T. forsythia, and T. denticola. However, the blue-complex (A. georgiae, A. israelii, A. naeslundii, A. odontolyticus and A. viscosus) showed increased counts in severe PP compared to mild PP. These results could suggest a decrease in the immune tolerance to Actinomyces to increase the inflammatory condition in PP patients to develop periodontitis.
PP and periodontitis present common cytokines that have been reported to play an important role in the severity of both diseases and shared inflammatory pathways; factors that may contribute to this relationship.[41] Moreover, recent studies showed that mild psoriasis is less affected by comorbid diseases than those with severe psoriasis.[42] Higher levels of cytokines (IL-2, IL-6, IL-12, interferon-gamma, TNF-α) are also considered important risk factors of psoriasis.[43] Moreover, IL-6 concentrations are higher in patients with periodontitis than in healthy individuals.[44],[45]
The present study shows a higher concentration of salivary IL-2 and IL-6 regardless of the periodontal status in PP patients compared to those without PP. These results suggested that psoriasis is defined as an inflammatory systemic disorder, and these could induce the early onset of periodontal disease regarding the severity of psoriasis.
Therefore, research on the tolerance of these microorganisms in patients with PP is necessary to evaluate. Our results indicated that microbial dysbiosis of PPs was related to clinical parameters. However, we could infer from the present study that subjects with severe psoriasis influenced microbial changes of psoriasis, with a higher proportion of Actinomyces spp, under the subgingival microbiota of previous reports.[39],[46] Therefore, the need for a longitudinal study to evaluate the transition of subgingival microbiota in the severity of psoriasis is evident. It could be expected that PP might share certain immunological traits that could lead to comparable changes in the subgingival microbial composition.
Conclusion In conclusion, the PP uncontrolled showed distinct clinical alterations in periodontal tissue, including an increase in the gingival redness, clinical attachment loss, imbalance of Actinomyces counts, and increase the salivary IL-2 and IL-6. Accelerated periodontitis development in PP likely results from the altered response of host defenses and dysregulated immune-inflammatory by the presence of Actinomyces, these changes could enhance the severity of psoriasis. Therefore, the need for a longitudinal study to evaluate the transition of subgingival microbiota from the oral cavity to the gut to skin and the dysregulated immune-inflammatory in PP patients is evident.
Acknowledgement
The authors thank and acknowledge that this study was largely by Dra. Gladys Leon Dorantes who rests in peace.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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