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BackgroundThe linkage between periodontal disease and rheumatoid arthritis is well established. Commonalities among the two are that both are chronic inflammatory diseases characterized by bone loss, an association with the shared epitope susceptibility allele, and anti-citrullinated protein antibodies.MethodsTo explore immune mechanisms that may connect the two seemingly disparate disorders, we measured host immune responses including T-cell phenotype and anti-citrullinated protein antibody production in human leukocyte antigen (HLA)-DR1 humanized C57BL/6 mice following exposure to the Gram-negative anaerobic periodontal disease pathogen Porphyromonas gingivalis. We measured autoimmune arthritis disease expression in mice exposed to P. gingivalis, and also in arthritis-resistant mice by flow cytometry and multiplex cytokine-linked and enzyme-linked immunosorbent assays. We also measured femoral bone density by microcomputed tomography and systemic cytokine production.ResultsExposure of the gingiva of DR1 mice to P. gingivalis results in a transient increase in the percentage of Th17 cells, both in peripheral blood and cervical lymph nodes, a burst of systemic cytokine activity, a loss in femoral bone density, and the generation of anti-citrullinated protein antibodies. Importantly, these antibodies are not produced in response to P. gingivalis treatment of wild-type C57BL/6 mice, and P. gingivalis exposure triggered expression of arthritis in arthritis-resistant mice.ConclusionsExposure of gingival tissues to P. gingivalis has systemic effects that can result in disease pathology in tissues that are spatially removed from the initial site of infection, providing evidence for systemic effects of this periodontal pathogen. The elicitation of anti-citrullinated protein antibodies in an HLA-DR1-restricted fashion by mice exposed to P. gingivalis provides support for the role of the shared epitope in both periodontal disease and rheumatoid arthritis. The ability of P. gingivalis to induce disease expression in arthritis-resistant mice provides support for the idea that periodontal infection may be able to trigger autoimmunity if other disease-eliciting factors are already present.

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Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P T7 ) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P T7 -driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene α-tubulin locus. A plasmid construct with P T7 and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the noncoding strand of an 18SrRNA gene, resulted in tetracyclineinducible expression of the reporter as much as 16 and 150 fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P T7 augmented the reporter's expression 2 fold more in comparison to P T7 -less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.

Our previous studies have shown that inoculation of the oral cavity of “humanized” B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity.

Regulatory T cells (Tregs) are key players in tolerance and immune homeostasis. The identification of this unique cell subset was originally established through identification of CD4+ T cells expressing high levels of the IL-2 receptor α-chain CD25. CD4+CD25+ cells from naïve mice are predominantly Tregs. CD4+CD25- cells are predominantly conventional T cells. With the identification of the nuclear transcription factor Foxp3 as a protein that plays a key role in the biology of Tregs and with GFP as a means of selection, pure populations of Foxp3 expressing cells can be obtained. Among murine CD4+ T cells that express Foxp3, there is a continuum of CD25 expression from negative (or very dim) to very bright. Our analyses of peripheral Foxp3+CD4+CD25dim/- cells suggest that while their transcription of many cytokine, chemokine and receptor genes are similar to Foxp3+CD4+CD25bright T cells, they do not have identical phenotypic and functional properties, with the latter capable of a greater degree of suppression and an enhanced transcription of Interleukin-7. Parenteral administration of a heterologous type II collagen to induce tolerance in a TcR tg mouse model of Collagen-Induced Arthritis increases the percentage of Foxp3+CD4+CD25bright T cells and protects the mice from disease. Adoptive transfer of a Foxp3+cells from CII-tolerized mice confers a greater degree of protection when compared with transfer of equivalent numbers of Tregs from naïve mice.

Periodontal disease (PD) is a chronic pro-inflammatory response to oral pathogens such as Porphyromonas gingivalis which culminates in alveolar bone loss and loss of dental structure and function. Like rheumatoid arthritis (RA), periodontal disease is influenced strongly by lifestyle factors such as tobacco use, but also greatly influenced by genetic components, such as the “shared epitope,” which refers to a conserved linear sequence of amino acids in the DRβ1 chain of the HLA-DRα/β heterodimer between amino acids 67 and 74. Aggressive PD (the most destructive form of PD) is also now thought to be associated with specific HLA haplotypes including DRβ1. Using IL-17Frfp and Foxp3gfpreporters engineered into I-A° C57BL/6 mice expressing chimeric mouse/human HLA-DRβ1 (B6.DR1 mice), we find that gingival brushing with P gingivalis results in a rapid Th17 response in the peripheral blood and cervical lymph nodes, which decreases shortly after brushing. Evidence of a chronic infection can be detected in these nodes three months after oral inoculation. In addition to Th17 responses, we also find a concomitant increase in Th17-related cytokine production that parallels the Th17 activity. Furthermore, we measured a brisk production of antibodies directed against P. gingivalis and also against cyclic citrullinated peptides (these ACPAs are widely used as the principle early diagnostic for RA) as well as a loss in trabecular bone that can be detected in peri-articular bone structures, e.g. distal to the alveolar bone structures normally measured in PD models. Our findings suggest that periodontal disease can have far-reaching effects in bone morphology which may be driven by Th17/cytokine/ACPA activity even in the absence of other pathologies.