Ravi Jotwani

Mature Dendritic Cells Infiltrate the T Cell-Rich Region of Oral Mucosa in Chronic Periodontitis: In Situ, In Vivo, and In Vitro Studies

Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined. We show that in human CP tissues, CD1a+ immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83+ mature dendritic cells (DCs) specifically infiltrate the CD4+ lymphoid-rich lamina propria. In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response. Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1β, PGE2) and Th (IL-10, IL-12) cytokines. Interestingly, the IL-10:IL-12 ratio elicited from P. gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs. This may account for the significantly (p < 0.05) lower proliferation of autologous CD4+ T cells and reduced release of IFN-γ elicited by P. gingivalis-pulsed DCs. Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P. gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.

Dendritic Cells at the Oral Mucosal Interface

The mucosal lining of the respiratory and digestive systems contains the largest and most complex immune system in the body, but surprisingly little is known of the immune system that serves the oral mucosa. This review focuses on dendritic cells, particularly powerful arbiters of immunity, in response to antigens of microbial or tumor origin, but also of tolerance to self-antigens and commensal microbes. Although first discovered in 1868, the epidermal dendritic Langerhans cells remained enigmatic for over a century, until they were identified as the most peripheral outpost of the immune system. Investigators’ ability to isolate, enrich, and culture dendritic cells has led to an explosion in the field. Presented herein is a review of dendritic cell history, ontogeny, function, and phenotype, and the role of different dendritic cell subsets in the oral mucosa and its diseases. Particular emphasis is placed on the mechanisms of recognition and capture of microbes by dendritic cells. Also emphasized is how dendritic cells may regulate immunity/tolerance in response to oral microbes.

Oral Mucosal Endotoxin Tolerance Induction in Chronic Periodontitis

ABSTRACT The oral mucosa is exposed to a high density and diversity of gram-positive and gram-negative bacteria, but very little is known about how immune homeostasis is maintained in this environment, particularly in the inflammatory disease chronic periodontitis (CP). The cells of the innate immune response recognize bacterial structures via the Toll-like receptors (TLR). This activates intracellular signaling and transcription of proteins essential for the induction of an adaptive immune response; however, if unregulated, it can lead to destructive inflammatory responses. Using single-immunoenzyme labeling, we show that the human oral mucosa (gingiva) is infiltrated by large numbers of TLR2+ and TLR4+ cells and that their numbers increase significantly in CP, relative to health (P < 0.05, Students t test). We also show that the numbers of TLR2+ but not TLR4+ cells increase linearly with inflammation (r2 = 0.33, P < 0.05). Double-immunofluorescence analysis confirms that TLR2 is coexpressed by monocytes (MC)/macrophages (mφ) in situ. Further analysis of gingival tissues by quantitative real-time PCR, however, indicates that despite a threefold increase in the expression of interleukin-1β (IL-1β) mRNA during CP, there is significant (30-fold) downregulation of TLR2 mRNA (P < 0.05, Students t test). Also showing similar trends are the levels of TLR4 (ninefold reduction), TLR5 (twofold reduction), and MD-2 (sevenfold reduction) mRNA in CP patients compared to healthy persons, while the level of CD14 was unchanged. In vitro studies with human MC indicate that MC respond to an initial stimulus of lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) or Escherichia coli (EcLPS) by upregulation of TLR2 and TLR4 mRNA and protein; moreover, IL-1β mRNA is induced and tumor necrosis factor alpha (TNF-α), IL-10, IL-6, and IL-8 proteins are secreted. However, restimulation of MC with either PgLPS or EcLPS downregulates TLR2 and TLR4 mRNA and protein and IL-1β mRNA and induces a ca. 10-fold reduction in TNF-α secretion, suggesting the induction of endotoxin tolerance by either LPS. Less susceptible to tolerance than TNF-α were IL-6, IL-10, and IL-8. These studies suggest that certain components of the innate oral mucosal immune response, most notably TLRs and inflammatory cytokines, may become tolerized during sustained exposure to bacterial structures such as LPS and that this may be one mechanism used in the oral mucosa to attempt to regulate local immune responses.

Human dendritic cells respond to Porphyromonas gingivalis LPS by promoting a Th2 effector response in vitro

Understanding how mucosal pathogens modulate the immune response may facilitate the development of vaccines for disparate human diseases. In the present study, human monocyte‐derived DC (MDDC)were pulsed with LPS of the oral pathogen Porphyromonas gingivalis and Escherichia coli 25922 and analyzed for: (i) production of Th‐biasing/inflammatory cytokines; (ii) maturation/costimulatory molecules; and (iii) induction of allogeneic CD4+ and naive CD45RA+ T cell proliferation and release of Th1 or Th2 cytokines. We show that E. coli LPS‐pulsed MDDC released Th1‐biasing cytokines — consisting of high levels of IL‐12 p70, IFN‐γ‐inducible protein 10 (IP‐10) – but also TNF‐α, IL‐10, IL‐6 and IL‐1β. In contrast, no IL‐12 p70 or IP‐10, and lower levels of TNF‐α and IL‐10 were induced by P. gingivalis LPS. These differences were sustained at LPS doses that yielded nearly equivalent maturation of MDDC; moreover the T cell response was consistent: E. coli LPS‐pulsed MDDC induced higher T cell proliferation, and T cells released more IFN‐γ and IL‐2, but less IL‐5 than T cells co‐cultured with P. gingivalis LPS pulsed‐MDDC. IL‐13 was secreted by naive CD45RA+CD45RO–CD4+ T cells in response to P. gingivalisLPS‐pulsed MDDC. These results suggest that human MDDC can be polarized by LPS from the mucosal pathogen P. gingivalis to induce a Th2 effector response in vitro.

Fimbriated Porphyromonas gingivalis Is More Efficient than Fimbria-Deficient P. gingivalis in Entering Human Dendritic Cells In Vitro and Induces an Inflammatory Th1 Effector Response

ABSTRACT Porphyromonas gingivalis is a fimbriated mucosal pathogen implicated in chronic periodontitis (CP). The fimbriae are required for invasion of the gingival mucosa and for induction of CP in animal models of periodontitis. CP is associated with infection of immature dendritic cells (DCs) by P. gingivalis in situ and with increased numbers of dermal DCs (DDCs) and mature DCs in the lamina propria. The role of fimbriae in gaining entry into human DCs and how this modulates the inflammatory and effector immune responses, however, have not been explored. To address this, we generated monocyte-derived DCs (MDDCs) in vitro which phenotypically and functionally resemble DDCs. We show here that virulent fimbriated P. gingivalis 381, in contrast to its fimbria-deficient mutant, P. gingivalis DPG3, efficiently gains entry to MDDCs in a manner dependent on active cell metabolism and cytoskeletal rearrangement. In addition, uptake of 381, unlike DPG3, induces DCs to undergo maturation, upregulate costimulatory molecules, and secrete inflammation cytokines interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, IL-10, and IL-12. Moreover, MDDCs pulsed with 381 also stimulated a higher autologous mixed lymphocyte reaction and induced a Th1-type response, with gamma interferon (IFN-γ) being the main cytokine. Monocytes used as controls demonstrated fimbria-dependent uptake of 381 as well but produced low levels of inflammatory cytokines compared to MDDCs. When MDDCs were pulsed with recombinant fimbrillin of P. gingivalis (10 μg/ml), maturation of MDDCs was also induced; moreover, matured MDDCs induced proliferation of autologous CD4+ T cells and release of IFN-γ. Thus, these results establish the significance of P. gingivalis fimbriae in the uptake of P. gingivalis by MDDCs and in induction of immunostimulatory Th1 responses.

Targeting of DC-SIGN on Human Dendritic Cells by Minor Fimbriated Porphyromonas gingivalis Strains Elicits a Distinct Effector T Cell Response

The oral mucosal pathogen Porphyromonas gingivalis expresses at least two adhesins: the 67-kDa mfa-1 (minor) fimbriae and the 41-kDa fimA (major) fimbriae. In periodontal disease, P. gingivalis associates in situ with dermal dendritic cells (DCs), many of which express DC-SIGN (DC-specific ICAM-3 grabbing nonintegrin; CD209). The cellular receptors present on DCs that are involved in the uptake of minor/major fimbriated P. gingivalis, along with the effector immune response induced, are presently unclear. In this study, stably transfected human DC-SIGN+/− Raji cell lines and monocyte-derived DCs (MoDCs) were pulsed with whole, live, wild-type Pg381 or isogenic major (DPG-3)-, minor (MFI)-, or double fimbriae (MFB)-deficient mutant P. gingivalis strains. The influence of blocking Abs, carbohydrates, full-length glycosylated HIV-1 gp120 envelope protein, and cytochalasin D on the uptake of strains and on the immune responses was determined in vitro. We show that the binding of minor fimbriated P. gingivalis strains to Raji cells and MoDCs is dependent on DC-SIGN, whereas the double fimbriae mutant strain does not bind. Binding to DC-SIGN on MoDCs is followed by the internalization of P. gingivalis into DC-SIGN-rich intracellular compartments, and MoDCs secrete low levels of inflammatory cytokines and remain relatively immature. Blocking DC-SIGN with HIV-1 gp120 prevents the uptake of minor fimbriated strains and deregulates the expression of inflammatory cytokines. Moreover, MoDCs promote a Th2 or Th1 effector response, depending on whether they are pulsed with minor or major fimbriated P. gingivalis strains, respectively, suggesting distinct immunomodulatory roles for the two adhesins of P. gingivalis.

Increase in HIV Receptors/Co-receptors/α-defensins in Inflamed Human Gingiva

Transmission of HIV-1 through the oral cavity is considered to be a rare event. To identify factors in resistance/susceptibility to oral HIV-1 infection, we analyzed expression in human gingiva of HIV-1 receptors Langerin, DC-SIGN, MR, and GalCer, HIV-1 co-receptors CCCR5, CXCR4, and anti-microbial protein α-defensin-1. Our results show that healthy gingiva is infiltrated with cells expressing all HIV-1 receptors tested; however, there are very few CCR5+ cells and a complete absence of CXCR4+ cells in the lamina propria. In chronic periodontitis (CP), DC-SIGN, MR, CXCR4, and CCR5 increase, but this was accompanied by a ten-fold increase in α-defensin-1 mRNA. The CCR5+ cells were revealed to be T-cells, macrophages, and dermal dendritic cells. Moreover, epithelial expression of GalCer and CXCR4 together was not apical and showed no trend with underlying inflammation. Thus, low expression of HIV-1 co-receptors in health and high expression of α-defensin during CP may comprise endogenous factors that provide protection from oral HIV-1 infection.

Dendritic Cells: Immune Saviors or Achilles' Heel?

Dendritic cells (DCs) consist of a family of antigen-presenting cells (APC) that patrol all tissues of the body with the possible exceptions of the brain and testes. DCs function to capture bacteria and other pathogens for processing and presentation to T cells in the secondary lymphoid organs ([2][

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Nuclear factor-κB p50 subunits in chronic periodontitis and Porphyromonas gingivalis lipopolysaccharide-pulsed dendritic cells

The pathogenesis of a number of chronic inflammatory diseases has been linked to dysregulated functioning of nuclear transcription factor (NF)‐κB. In the present study, we examine NF‐κB activation in human oral mucosal tissues of chronic periodontitis (CP) patients. Electrophoretic mobility shift assay and DNA‐binding enzyme‐linked immunosorbent assays indicate elevated levels of transcriptionally repressive p50 subunits and an increased p50/p65 ratio in CP tissues compared to healthy controls. Because Porphyromonas gingivalis has been recognized to be a causal factor in CP, we used P. gingivalis lipopolysaccharide (LPS) for in vitro studies with monocyte‐derived dendritic cells (MoDCs). Porphyromonas gingivalis LPS, unlike Escherichia coli LPS, induced an increased p50/p65 ratio in MoDCs. Using blocking antibodies, we demonstrated that while both Toll‐like receptor 2 (TLR2) and TLR4 are required for MoDC maturation by P. gingivalis LPS, only TLR4 signaling is sufficient to induce cytokine secretion. Our results suggest that increased levels of transcriptionally repressive p50 may be characteristic of CP and might be a result of suboptimal NF‐κB activation and dendritic cell maturation by P. gingivalis, a bacterium implicated in CP.