Dysbiosis From a Microbial and Host Perspective Relative to Oral Health and Disease.

The significance of microbiology and immunology with regard to caries and periodontal disease gained substantial clinical or research consideration in the mid 1960's. This enhanced emphasis related to several simple but elegant experiments illustrating the relevance of bacteria to oral infections. Since that point, the understanding of oral diseases has become increasingly sophisticated and many of the original hypotheses related to disease causality have either been abandoned or amplified. The COVID pandemic has reminded us of the importance of history relative to infectious diseases and in the words of Churchill "those who fail to learn from history are condemned to repeat it." This review is designed to present an overview of broad general directions of research over the last 60 years in oral microbiology and immunology, reviewing significant contributions, indicating emerging foci of interest, and proposing future directions based on technical advances and new understandings. Our goal is to review this rich history (standard microbiology and immunology) and point to potential directions in the future (omics) that can lead to a better understanding of disease. Over the years, research scientists have moved from a position of downplaying the role of bacteria in oral disease to one implicating bacteria as true pathogens that cause disease. More recently it has been proposed that bacteria form the ecological first line of defense against "foreign" invaders and also serve to train the immune system as an acquired host defensive stimulus. While early immunological research was focused on immunological exposure as a modulator of disease, the "hygiene hypothesis," and now the "old friends hypothesis" suggest that the immune response could be trained by bacteria for long-term health. Advanced "omics" technologies are currently being used to address changes that occur in the host and the microbiome in oral disease. The "omics" methodologies have shaped the detection of quantifiable biomarkers to define human physiology and pathologies. In summary, this review will emphasize the role that commensals and pathobionts play in their interaction with the immune status of the host, with a prediction that current "omic" technologies will allow researchers to better understand disease in the future.

A comparison of Aggregatibacter actinomycetemcomitans (Aa) virulence traits in a rat model for periodontal disease.

Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4(+) T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease.

Regulatory T cells as central regulators of both autoimmunity and B cell malignancy in New Zealand Black mice.

Regulatory T cells (Tregs) play an important role in protection against autoimmune disease and are also known to be potent inhibitors of anti-tumor immune responses. The New Zealand Black (NZB) mouse is a murine model for both autoimmune diseases, since high levels of autoantibodies are present, and human CLL, due to the expansion of malignant B-1 cells. In this study, we examined the functional role of CD4(+)CD25(+) Foxp3(+) Tregs in these different manifestations. Flow cytometric analysis showed increased levels of Tregs in NZB mice compared to healthy C57Bl/6 controls. Aged NZB mice that have developed a B-1 cell malignancy identified as IgM(+)CD5(+), have the most pronounced increase in Tregs. Ex vivo treatment of splenocytes from NZB mice with IFN-alpha resulted in a decrease in the frequency of Tregs and malignant B-1 cells. In vivo treatment of both NZB and C57Bl/6 mice with poly (I:C), a potent inducer of IFN-alpha, also led to a decrease in the levels of Tregs and malignant B-1 cells (NZB only) while amplifying autoimmune manifestations. These results indicate that while high levels of Tregs found in NZB mice might suppress a more severe autoimmune disease, they may also contribute to the development of the B cell malignancy.

Latent TGF-beta1-transduced CD4+ T cells suppress the progression of allergic encephalomyelitis.

Systemic injection of small amounts of transforming growth factor-beta (TGF-beta), a cytokine produced by lymphoid and other cells, has a profound effect in protecting mice from the inflammatory demyelinating lesions of experimental allergic encephalomyelitis (EAE; an animal model for multiple sclerosis). However, TGF-beta has side-effects, which might be avoided if the cells producing TGF-beta can be delivered to the affected site in the nervous system to insure its local release in small amounts. Myelin basic protein (MBP)-specific, cloned CD4+ T cells were engineered by retroviral transduction to produce latent TGF-beta. Studies about the spontaneous form of EAE in T cell receptor (TCR)-transgenic recombination-activating gene (RAG)-1(-/-) mice showed that essentially all of the MBP-specific, TCR-transgenic RAG-1(-/-) (BALB/cxB10.PL)F1 mice develop spontaneous EAE by the age of 11 weeks. By 12 weeks, 25-50% of the mice have died from disease. A single injection of TGF-beta1-transduced T helper cell type 1 (Th1) cells significantly protected the mice from EAE, and untransduced Th1 cells did not protect. MBP-specific BALB/c Th2 clones, transduced with TGF-beta1-internal ribosome entry site-green fluorescent protein (GFP) significantly reduced EAE induction by untransduced Th1 cells in RAG-1(-/-) B10.PL mice. Furthermore, the GFP+ TGF-beta1-producing Th2 cells were detectable in the spinal cords of the injected mice.

Identification of oligoclonal CD4 T cells in diffuse large B cell lymphomas.

Human B cell lymphomas often contain CD4 T cells. Here we show that, in diffuse large B cell lymphomas (DLCL), such T cells are oligoclonal. The CDR3 lengths and nucleotide sequences of oligoclonal TCRBV of CD4 T cells in an original and relapsed lymphoma from one patient were compared. Three BV23 sequences were identical (12/17 and 16/16 clones in primary and relapsed lymphomas, respectively), but were absent in CD4 T cells from another patient's DLCL. Two of the repetitive BV23 sequences were found in peripheral blood CD4 T cells (5/17 clones); gamma-irradiated DLCL from this patient stimulated syngeneic BV23 response in CD4 cells (92% of BV23 had the same CDR3 length). Skew in TCRBV representation was observed in CD4 T cells from all the DLCL. One DLCL, with overrepresentation of BV13S1 in CD4 cells, stimulated the same TCR in CD4 cells from three unrelated individuals. These findings support the conclusion that there is clonal selection of CD4 T cells in DLCL.

Regulation of mouse mammary tumor virus env transcriptional activator initiated mammary tumor virus superantigen transcripts in lymphomas of SJL/J mice: role of Ikaros, demethylation, and chromatin structural change in the transcriptional activation of mammary tumor virus superantigen.

Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+V16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29- lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+V16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.