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Editorial Note (16 ^th June 2023): Since publication, it has been brought to the attention of the Editorial Team that the article was missing key information regarding adverse events, anaesthetic and euthanasia procedures, and ethical approval. The Editorial Team requested further detail and an explanation from the authors in March 2023. The authors provided an adequate response and were requested by the Editorial Team to create a new version of the article to include the additional details. Peer review activity has been suspended until the authors publish a new version of this article.

The oral cavity is the habitat of numerous bacteria, including Porphyromonas gingivalis (Pg). Pg is a gram negative, facultative anaerobic pathogen, which is responsible in causing gingivitis or periodontitis. ¹ In low-income countries, gingivitis and periodontitis can affect up to 90% of the adult population. ² Rather than alveolar bone and ligament destruction, Pg is believed to be involved with the development of atopic responses in a susceptible host. ³ Following Pg infection, hosts’ adaptive immune response (both cell-mediated and humoral-mediated) could induce a systemic inflammatory reaction, not only just local destruction of tooth-supporting tissues. ^{4– 5} Although periodontal pathogens, such as Pg, play a major role in the initiation of local and systemic inflammatory reaction, ⁶ the host aberrant immune responses require further study. Since humoral immune responses are stimulated following Pg infection, there might be a link to the occurrence of atopy.

Despite long-standing research about hygiene hypothesis for several decades, there is an unequivocally accepted fact that the prevalence of atopy increases more among children who have periodontal pathogen colonization or infection. ⁷ While endorsing these hygiene hypothesis approaches, there is an alternative hypothesis in which exposure to some periodontal pathogens will exclusively trigger an “immunoglobulin-E skew” rather than reducing it. ⁸ Within the context of the hygiene hypothesis, the most essential microbial exposures needed to be studied is the biomolecular relationship between host antibody and regulatory T-cell with lipopolysaccharide (LPS), an endotoxin released by Pg to affect host immune reaction.

Hygiene hypothesis principles might not be able to answer all phenomenon of increasing incidence of atopy among children with poor oral hygiene. ⁹ Some studies report a positive association between the colonization/infection of Pg with the development of allergic diseases, ^{10– 15} whereas some studies report no association. ^{16– 21} Due to lack of conclusive evidence about the association between Pg and allergic diseases, ²² we try to measure the level of atopic biomarkers following Pg infection.

To the best of our knowledge, measuring IgG ₄ and IgE antibody may have a closer association to atopic profiles, since IgG ₄ and IgE are released after activation of mature B cells following the modulation of IL-4 and IL-5 released by Th-2 cells during type I hypersensitivity. ²³ By looking at the alteration of IgG ₄ and IgE antibodies level after exposure to these selected components of Pg in a rat model, we hope to understand more deeply the biological mechanism of B-cell production antibodies pattern and humoral immune responses before the clinical manifestation of atopy. We chose a rat model since they are inbred so they are almost identical genetically and their genetic, biological and behavior characteristics closely resemble those of humans.

Several mechanisms have been suggested to alter atopic inflammatory responses following LPS Pg infection. One of the mechanisms proven in this study is an elevation of IgE antibody and reduction of IgG ₄/IgE ratio. ²⁴ As far as we have known, Th-1 and Th-2 cells are not two different CD4+ T-cell subsets, but it represents polarized forms of the highly heterogenous CD4+ Th cell–mediated immune response. Host genetic and microenvironmental factors could have contributed with series of modulatory factors including: ¹ the ligation of T-cell receptor (TCR); ² the activation of costimulatory molecules and its particular components; ³ the predominance of an inflammatory cytokine in the local environment; and ⁴ the number of postactivation cell divisions following exposure to antigens. Down-regulation of the Th-1 cell is associated with depression of cell-mediated immune response and stimulation of humoral immune response, thus pathogens are able to evade immune clearance. ²⁵

Porphyromonas gingivalis possess very sophisticated defense mechanisms against host immune responses. These pathogens produce capsules containing long chain LPS which is designed effectively to counter membrane attack complex. These long chain LPS can also downgrade cell-mediated immunity by shifting Th-1 into Th-2 which less dangerous to pathogens. ²⁶ LPS may have an essential role in switching cell-mediated to humoral-mediated immune responses. ²⁷ LPS Pg antigen is processed and presented on its surface with MHC-II molecule. Recent studies suggest an activation of alternative complement pathway, disruption of classical complement pathway, modulation of antigen presenting cells, and downregulation of anti-inflammatory cytokines are responsible for the Th2-skewed immune response following exposure to LPS Pg. Predominance shifting from Th-1 into Th-2 occurs in several extra-lymphoid tissues; the ideal site for Porphyromonas gingivalis is the oral cavity. ²⁸

Interleukin-4 (IL-4), which is produced by naive T cells, acts as autocrine manner known to be responsible for the differentiation and activation of Th-2 phenotype. ²⁹ Guo et al (2014) shows upon the occurrence and development of allergic diseases, there is a complex pathobiology which results in an imbalance of Th-1/Th-2. ³⁰ In an atopic disease such as bronchial asthma or urticaria, naive T cell can differentiate into Th-2 under IL-4–induced STAT6 and GATA-3 transcription factors. ³⁰ Th-2 predominant immune response will automatically stimulate plasma cell to release IgE and IgG ₄. ³¹ Upon re-exposure of antigen or allergen, binding of the allergen to IgE orchestrates the adaptive immune system to initiate rapid sensitization. Frequent sensitization is a major risk factor for the development of allergic diseases such as urticaria, bronchial asthma, hay fever or atopic dermatitis/eczema. ³²

Our previous study used whole-cell body of Porphyromonas gingivalis to study different molecular responses in Wistar rats. Our first project studied the association between periodontal pathogen and host innate immunity. Exposure to Porphyromonas gingivalis had been shown to stimulate level of TLR2 and depress level of TLR4. ³³ Our findings might indicate that several bacterial properties can turn-off host innate immunity and host inflammatory response. Our second project studied the association between periodontal pathogen and host adaptive immunity. We summarized that high dose CFU of Pg stimulates fold increase of Th-2 cytokines (IL-4, IL-5 and IL-13) and decrease of Th-1 cytokines (IFN-γ and IL-17). ³⁴ These were the cornerstone to continue our project in studying LPS as the most important component of these bacteria.

At this moment, both total IgE or specific IgE antibodies have little diagnostic value in the occurrence of allergic manifestation. Even total or specific IgE is increasing, yet the manifestation of allergy doesn’t usually develop, since IgG ₄ level also increases as a counter-regulator. ³⁵ It means that even human or rat become susceptible to atopic allergy due to the increasing level of IgE, body mechanism is able to provide protection, with increased IgG ₄ as a counter response to prevent manifestation of allergic diseases and immediate hypersensitivity. Thus, exposure of LPS Pg will develop chance of atopic and hypersensitivity markers, but manifestation of allergic reaction is a complex pattern. ³⁶ IgG ₄/IgE ratio has closer accuracy to detect any alteration of atopic inflammatory pathway. Increase level of IgE, which isn’t accompanied by IgG ₄, can be seen in patients with urticaria or atopic dermatitis. ³⁷ IgE-switched B cells are much more likely to differentiate into plasma cells, whereas IgG ₄-switched B cells are less likely to differentiate. ³⁸ This reason would explain why IgE antibody is the most dominant antibody in the development of atopic inflammatory pathway, whereas IgG ₄ antibodies become prominent later during chronic non-atopic stimulation. ³⁹ According to this reason, IgG ₄/IgE ratio may predict atopic responses more accurately than total or specific IgE level.

Several experiments in rats indicate that exposure to LPS Pg may have a tendency to increase levels of IgE and IgG ₄. On the contrary, declining IgG ₄/IgE ratio following exposure to LPS Pg suggests the potential role of LPS Pg for isotype switching from IgG ₄ to IgE. The results of the present study favor indirect isotype switching route for most IgE as secondary responses from LPS Pg infection that leads to systemic atopic inflammatory pathway.