Is There a Similarity in Serum Cytokine Profile between Patients with Periodontitis or 2019-Novel Coronavirus Infection?—A Scoping Review

Simple Summary This scoping review highlights the immune-related similarities between the COVID-19 infection and periodontal disease, especially focusing on serum cytokine levels, such as IL-1β, IL-6, and TNF-α levels. Overall, higher IL-1β, IL-6, and TNF- α levels were reported in COVID-19-infected patients compared to patients with periodontitis. However, most of the included studies indicated elevated serum pro-inflammatory cytokine (IL-1β, IL-6 and TNF-α) levels in diseases (COVID-19/periodontitis) compared to healthy controls included in the same study. This shows the strong immunopathogenic role of these proinflammatory cytokines in the rationale and progressive destruction of these two conditions, and this review aims to highlight the role of a robust immune response in inflammation progression and to educate the readers about the importance of oral hygiene during the pandemic era. Abstract On 11 March 2020, the WHO declared a global emergency as a result of the ‘novel coronavirus infection’, which emerged from Wuhan, China, and rapidly spread across international borders. There is vast evidence that supports a direct link between oral cavities and this systemic circulation, but it is still unclear if oral conditions like periodontitis influenced the COVID-19 disease outcome. This scoping review highlights the fact that both periodontitis and COVID-19 independently increase serum pro-inflammatory cytokine levels, however there is a lack of documentation on if this biochemical profile synergizes with COVID-19 and/or periodontal severity in the same individuals. The purpose of this scoping review is to accumulate existing data on the serums IL-1β, IL-6, and TNF-α in COVID-19 and periodontitis patients and check if periodontitis negatively impacts the COVID-19 outcome, educating the population about the implications of COVID-19-related complications on their oral health, and vice versa, and motivating patients towards oral hygiene maintenance.


Introduction
Periodontitis is an inflammatory condition that damages the alveolar bone and soft tissues around the teeth, and compromises tooth stability [1]. It is one of the most common causes of partial to complete edentulousness; it remains a major contributing factor towards oro-functional abnormality and psychological challenges [2]. Periodontitis is established by the formation of a thin biofilm around the tooth necks, which induces inflammation caused by host-bacterial interactions and the production of pro-inflammatory cytokines. Following this, a strong immune response is initiated by the further secretion of inflammatory cytokines to combat pathogenic gram-negative bacteria, along with several adverse effects that manifest clinically (as tissue dissolution), immunologically (as increased immune cell infiltrate), and bio-chemically (as a pro-inflammatory cytokine milieu). Periodontal destruction is a sequelae of inflammation, vascular endothelial dilatation, leukocytic trans-inflammatory cytokine milieu). Periodontal destruction is a sequelae of inflammation, vascular endothelial dilatation, leukocytic trans-endothelial migration, cytokine release, and chemotaxis, that cumulatively generates a continuous positive feedback loop ( Figure  1). Increased vascular leakiness following periodontal microvascular dilatation causes cytokines to enter the systemic circulation, leading to systemic implications for periodontitis related to the heart, lungs, and brain apart from other complications such as pre-term birth, acute respiratory distress syndrome (ARDS), and "cytokine storm" as in the case of covid-19 infection. [3,4]. Periodontal inflammation is directed by an imbalance in the Th1/Th2/Th17 milieu [5]. A systemic link between periodontitis and bacterial pneumonia is established due to the accidental aspiration of periodontopathic bacteria into the respiratory tract, and some reports suggest that adequate oral hygiene maintenance could prevent the bacterial spread to the lungs [6,7]. Respiratory distress in COVID-19 leads to clinical hypoxia, which in turn increases reactive oxygen species that damage periodontal and lung tissues via apoptotic cell death [8]. Hypoxia also increases leucocytic infiltration and mast cell degranulation, which activates further chemokine secretion [9].

Research Questions
If IL-1β, IL-6, AND TNF-α are elevated in the serum of patients with periodontal disease OR COVID-19 infection.
(ii) If elevated serum cytokines influence the severity of periodontitis or the COVID-19 infection. Increased vascular leakiness following periodontal microvascular dilatation causes cytokines to enter the systemic circulation, leading to systemic implications for periodontitis related to the heart, lungs, and brain apart from other complications such as pre-term birth, acute respiratory distress syndrome (ARDS), and "cytokine storm" as in the case of covid-19 infection. [3,4]. Periodontal inflammation is directed by an imbalance in the Th1/Th2/Th17 milieu [5]. A systemic link between periodontitis and bacterial pneumonia is established due to the accidental aspiration of periodontopathic bacteria into the respiratory tract, and some reports suggest that adequate oral hygiene maintenance could prevent the bacterial spread to the lungs [6,7]. Respiratory distress in COVID-19 leads to clinical hypoxia, which in turn increases reactive oxygen species that damage periodontal and lung tissues via apoptotic cell death [8]. Hypoxia also increases leucocytic infiltration and mast cell degranulation, which activates further chemokine secretion [9].

Research Questions
(i) If IL-1β, IL-6, AND TNF-α are elevated in the serum of patients with periodontal disease OR COVID-19 infection.
(ii) If elevated serum cytokines influence the severity of periodontitis or the COVID-19 infection.

Methodology
A detailed keyword search (Supplementary Table S1) in the PubMed, LitCovid, Cochrane, and Google Scholar databases was performed, and relevant articles were selected based on their titles and abstracts. Articles published on COVID-19 between December 2019 and March 2022 were included, whereas no time period was followed for the periodontitis articles. The full texts of the included studies were further scrutinized based on the inclusion criteria. Finally, the references of the selected articles were hand-searched to locate additional studies.
Inclusion criteria. Studies using patient samples for in-vitro analysis to estimate the release of IL-1β, IL-6 and TNF-α.

3.
Samples where materials other than serum/blood/plasma/placental blood were used.

4.
General reviews, systematic reviews, case reports, case series, and animal studies.

6.
Articles published in languages other than English. 7.
Studies which do not compare cytokine levels with control groups.

Results
The search strategy for COVID-19 and periodontal disease yielded 19,338 and 781 articles, respectively, and were narrowed down to 351 studies and 381 studies on COVID-19 and periodontitis, respectively, based on their titles and abstracts ( Figure 2). Systemic inflammatory conditions not only hasten disease severity but alter cytokine levels in periodontal and lung tissues; studies including patients with systemic conditions were excluded, and these excluded studies are shown in supplementary Table S2. Further full-text analysis based on the inclusion and exclusion criteria lead to the inclusion of 15 individual articles on COVID-19 and periodontitis. The included studies were critically screened to extract data, such as author name, country, journal, number of participants, study groups, study type, sample used for cytokine evaluation, and results of the serum/plasma cytokine (IL-1β, TNF-α and IL-6) levels (if reported). Since both Aggressive periodontitis (AgP) and Chronic periodontitis (CP) cases were included, studies with COVID-19 infection cases and controls of all of the age groups were also included, wherein three studies included participants aged < 18 y old [18][19][20].

Methodology
A detailed keyword search (Supplementary Table S1) in the PubMed, LitCovid, Cochrane, and Google Scholar databases was performed, and relevant articles were selected based on their titles and abstracts. Articles published on COVID-19 between December 2019 and March 2022 were included, whereas no time period was followed for the periodontitis articles. The full texts of the included studies were further scrutinized based on the inclusion criteria. Finally, the references of the selected articles were handsearched to locate additional studies.
Inclusion criteria. Samples where materials other than serum/blood/plasma/placental blood were used. 4. General reviews, systematic reviews, case reports, case series, and animal studies. 5. Analysis of the serums IL-1β, (or) IL-6, (or) TNF-α in patients with periimplantitis. 6. Articles published in languages other than English. 7. Studies which do not compare cytokine levels with control groups.

Results
The search strategy for COVID-19 and periodontal disease yielded 19,338 and 781 articles, respectively, and were narrowed down to 351 studies and 381 studies on COVID-19 and periodontitis, respectively, based on their titles and abstracts ( Figure 2). Systemic inflammatory conditions not only hasten disease severity but alter cytokine levels in periodontal and lung tissues; studies including patients with systemic conditions were excluded, and these excluded studies are shown in supplementary Table S2. Further fulltext analysis based on the inclusion and exclusion criteria lead to the inclusion of 15 individual articles on COVID-19 and periodontitis. The included studies were critically screened to extract data, such as author name, country, journal, number of participants, study groups, study type, sample used for cytokine evaluation, and results of the serum/plasma cytokine (IL-1β, TNF-α and IL-6) levels (if reported). Since both Aggressive periodontitis (AgP) and Chronic periodontitis (CP) cases were included, studies with COVID-19 infection cases and controls of all of the age groups were also included, wherein three studies included participants aged < 18 y old [18][19][20].  In the periodontitis group, nine studies showed all of the cytokines were elevated in periodontitis compared to the controls; six studies showed no difference between the controls and periodontitis, and one study only reported elevated IL-6 in severe periodontitis compared to the mild periodontitis group (Figure 3a) [21][22][23][24][25][26][27][28][29][30][31][32][33]. Data reporting methods were highly heterogeneous between the included studies; hence, further statistical analysis of these data was not possible. (Table 1) In the periodontitis group, nine studies showed all of the cytokines were elevated in periodontitis compared to the controls; six studies showed no difference between the controls and periodontitis, and one study only reported elevated IL-6 in severe periodontitis compared to the mild periodontitis group (Figure 3a) [21][22][23][24][25][26][27][28][29][30][31][32][33]. Data reporting methods were highly heterogeneous between the included studies; hence, further statistical analysis of these data was not possible. (Table 1)      Regarding COVID-19, eight studies showed increased IL-1β, IL-6, and TNF-α in these cases compared to the controls [18,19,[34][35][36][37][38][39], five studies showed elevated IL-6 alone, and one study indicated higher TNF-α alone (Figure 3b) [40][41][42][43][44][45]. In addition, one study reported no significant difference between the cases and controls in all three of the cytokine levels, and one study reported no difference in all three IL-6, IL-1β, and TNF-α between cases and controls [41], and interestingly two studies showed decreased IL-1β and TNF-α in the cases compared to the controls [20,40,41,[43][44][45]. A single study reported that in COVID-19 cases, high serum IL-1β and IL-6 in the disease were correlated with critical in-hospital deaths (p = 0.01) [36] (Table 2).   IL-1β and TNF-α were higher in controls than cases, and IL-6 was higher in cases than controls NR in cases versus controls NR in cases versus controls After the data analysis, serum cytokine concentration was extracted from 11 periodontitis studies and the highest reported concentration for IL-1 β level was 114 pg/mL, whilst IL-6's was 125.4 pg/mL, and TNF-α's was 202.71 pg/mL. Among COVID-19 patients, the highest reported serum concentration of IL-1 β level was 140 pg/mL, whilst IL-6's was 249.0 pg/mL, and TNF-α's was 151.59 pg/mL. Overall, higher serum IL-1 β, IL-6, and TNF-α levels were reported in COVID-19 compared to periodontitis, (Figure 3c). Another group of researchers suggested that patients showing IL-6 levels above the 50th percentile (IL-6 cut-off value above 163.4 pg/mL) had a 91.7% probability of dying (p = 0.0018), and TNF-α levels above the 50th percentile (cut-off level > 33.91 pg/mL) had a 75% probability of dying (p = 0.0648) [38]. On the other hand, a 1mm increase in PD is associated with a 25.06 pg/mL increase in IL-1β, a 1.72 pg/mL increase in IL-6, and a 1.70 pg/mL increase in TNF-α in saliva, supporting their role in tissue destruction [46,47]. Apart from these findings, it should be noted that IL-6 was the single cytokine that consistently increased in COVID-19 and was also used as a marker to distinguish levels of COVID-19 severity [41].

Discussion
Several periodontal studies indicated an increase in all three of the cytokines; however, a few studies found a decrease in the overall cytokine levels. Studies showing decreased cytokines were greater than the number of studies reporting increased IL-6 alone. Regarding COVID-19, most studies showed increased cytokines, whereas only one study reported a decrease in all cytokines. The number of periodontitis studies showing decreased cytokines was just over half of the number of COVID-19 studies showing decreased cytokines. Nevertheless, overall data supported the hypothesis that high IL-1β, IL-6, and TNF-α in the same patient play a role in COVID-19 and periodontitis pathogenicity independently. Interestingly, when studies reported only one cytokine increase, IL-6 increased more frequently than IL-1 and TNF-α. In fact, a few studies reported a decrease in IL-1β and/or TNF-α but not IL-6, which explains why IL-6 could be the sole determinant of severity in COVID-19 [20].
Apart from periodontitis and COVID-19, local (salivary) and systemic (serum) TNFα/IL-6/IL-1β levels also increased in other inflammatory conditions, such as chronic oral erosive lesions and ulcers, and these cytokine levels were inversely proportional to the IL-10 that regulated epithelial healing [48,49]. Interplay between IL-1β, IL-6, and THF-α allows for seamless disease progression. (Figure 4). For instance, TNF-α upregulates PMN infiltration, disrupts the epithelial integrity, and positively influences IL-1β and IL-6 to downregulate epithelial growth [50][51][52]. Following an epithelial breach, IL-1β and TNF-α increase gingival fibroblastic MMP and activate the protease pathway for tissue dissolution [53]. IL-6 and TNF-α interplay by raising IL-1 levels for advancing bone demineralization [54]. IL-1 β is largely associated with bone destruction, osteoclastic maturation, inhibition of ALP activity, and collagen synthesis [55,56]. In lung epithelium, bacterial LPS stimulate TNF-α to prime cells to produce more TNF-α and IL-1β to continue the destructive pathway [57]. Lastly, bacterio-viral interactions diminish respiratory ciliary activity and enhance bacterial adhesion, which facilitate pathogenic aspiration from oral cavities [58]. Table 3 shows that the occurrence of periodontitis is associated with COVID-19 and COVID-19 worsens the severity of periodontal disease and COVID-19-related hospitalization. Therefore, periodontal disease management and a decreased pathogenic load may decrease the accidental aspiration of periodontal pathogens into the lungs and decrease hospitalization or COVID-19-related complications [59].  Lastly, bacterio-viral interactions diminish respiratory ciliary activity and enhance bacterial adhesion, which facilitate pathogenic aspiration from oral cavities [58]. Table 3 shows that the occurrence of periodontitis is associated with COVID-19 and COVID-19 worsens the severity of periodontal disease and COVID-19-related hospitalization. Therefore, periodontal disease management and a decreased pathogenic load may decrease the accidental aspiration of periodontal pathogens into the lungs and decrease hospitalization or COVID-19-related complications [59].

Further Scope for Scrutiny
Currently, data reporting methods on the cytokine levels in periodontitis/COVID-19 are highly ambiguous, with limited available literature. The lack of homogeneity in the methodology, ethnicity, and sample size prevents generalizability and conclusions of a definitive outcome. Future studies with age-matched groups and control over possible confounding factors will lead to homogenous studies assessing the data with a more detailed meta-analysis. Table 4 enlists research areas that need focus to develop a further understanding of an association between periodontitis and COVID-19.
Studies without control groups were not included in the methodology; hence, a large number of studies were excluded and the bias of the included studies has not been analysed, which are major limitations of this scoping review. In addition, the authors of the selected studies were not contacted to procure the serum cytokine values in the diseased patients when this was not mentioned in the manuscript, which might contribute to a skewed data analysis and could be an added limiting factor. Table 4. Scope for further research and unanswered questions.

Scope for Further Research and Unanswered Questions
Chemical profile -Report the cytokine range in population of specific age groups -Familial H/o inflammatory comorbidities that influence cytokine level in the same patients with COVID-19 infection/periodontitis -If the COVID-19 infection altered the GCF/gingival cytokine concentration -Cytokine serum level threshold beyond which the patient dies.
Disease outcome -Respiratory symptoms corresponding to the serum cytokine level -PD/CAL/defect size corresponding to the serum cytokine level -Patterns in bone loss corresponding to the serum cytokine levels -Influence of serum cytokine levels on the disease prognosis Disease management -Occurrence of coronavirus in intrabony defects/gingival tissues -Occurrence of periodontopathic bacteria in the lung lesions of COVID-19-infected subjects, and if it is increased with periodontitis severity -If COVID-19/periodontitis increases the susceptibility to other inflammatory conditions -If COVID-19 infection alters the periodontal treatment outcome/tissue healing/dental implant stability -Serum cytokine comparison among COVID-19/periodontitis/periodontitis + COVID-19 to find an association.
-If systemic anti-cytokine therapy could decrease disease severity

Conclusions
Most studies showed greater increases in IL-6 in cases than controls, which suggests that IL-6 is a strong potential diagnostic and prognostic biomarker. However, this pattern has been more often reported in cases of COVID-19, and warrants additional research to conclude its role in periodontal pathology. Overall, serum pro-inflammatory cytokines are elevated in the presence of periodontopathic bacteria, so it could be derived that periodontal disease negatively influences the serum cytokine profile, which could further affect COVID-19 disease outcomes. Moreover, reports suggest that recent periodontal therapy/wellcontrolled periodontitis result in lesser COVID-19 complications. Furthermore, a two-way link is established between periodontitis and lung conditions due to the aspiration of oral bacteria into the respiratory tract and the dislodgement of coronavirus into the periodontal pockets. This may worsen the disease outcome of periodontitis and COVID-19 thereby underlining the importance of the maintenance of proper periodontal hygiene and prevent complications related to each other.