The function of dendritic cells in modulating the host response

SUMMARY

Dendritic cells (DCs) are antigen-presenting cells that capture, process and present antigens to lymphocytes to initiate and regulate the adaptive immune response. DCs detect bacteria in skin and mucosa and migrate into regional lymph nodes, where they stimulate antigen-specific T and B lymphocyte activation and proliferation. DCs direct CD4 T cells to differentiate to T cell subsets such as Th1, Th2, Th17 and Treg cells. The periodontium is chronically exposed to oral bacteria that stimulate an inflammatory response to induce gingivitis or periodontitis. DCs play both a protective and destructive role through activation of the acquired immune response and are also reported to be a source of osteoclast precursors that promote bone resorption. FOXO1, a member of the forkhead box O family of transcription factors, plays a significant role in the activation of DCs. The function of DCs in periodontal inflammation has been investigated in a mouse model by lineage specific deletion of FOXO1 in these cells. Deletion of FOXO1 reduces DC protective function and enhances susceptibility to periodontitis. The kinase Akt, phosphorylates FOXO1 to inhibit FOXO activity. Thus the Akt-FOXO1 axis may play a key role in regulating DCs to have a significant impact on periodontal disease.

INTRODUCTION

Dendritic cells (DCs) are antigen-presenting cells (APCs), which capture, process and present antigens to lymphocytes to initiate and regulate the adaptive immune response ¹. Based on function and phenotype, DCs can be divided into plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs). Langerhans cells are another member of the DC family found in oral mucosal epithelium and the skin epidermis ². DCs play a key role in immune defense; when immune homeostasis mediated by DCs is disrupted, inflammatory destruction may occur ³. There is ample evidence that DCs are activated in experimental periodontitis in animals and in humans with periodontitis. Interestingly, dendritic cells have the potential to play protective or destructive roles or both. Due to the limited number of studies examining cause and effect relationships this issue is not entirely clear. However there are reports in which deletion of Langerhans cells or reduced DC function through lineage specific FOXO1 deletion have been shown to increase susceptibility to periodontitis ^4–6, suggesting that in animal models DCs have an overall effect in the periodontium which is protective.

DCs and subsets

DCs are derived from CD34⁺ hematopoietic stem cells (HSCs) in the bone marrow ⁷. These progenitor cells initially differentiate to immature DCs, which have high endocytic activity and low T cell activation potential ^8,9. Immature DCs express various pattern recognition receptors (PRRs) that constantly sample the surrounding environment for pathogens such as viruses and bacteria ¹⁰. Maturation of dendritic cells is induced by captured microbes or their components^11,12, inflammatory cytokines and ligation of select cell surface receptors. ^11–13 Once DCs take-up antigen they become activated into mature dendritic cells and present pathogen fragments at their cell surface using MHC molecules. DCs initiate the acquired immune response by interacting with lymphocytes after migrating to lymph nodes ¹⁴.

Dendritic cells can be subdivided into plasmacytoid (pDC) and conventional (cDC). pDCs are derived from lymphoid progenitors and have characteristics that resemble a cross between B cells and cDCs ¹⁵. pDCs predominantly recognize viral antigens ¹⁶, specializing in the production and secretion of type I interferons, IL-12, IL-6, TNF-α and pro-inflammatory chemokines ¹⁷. pDCs enter the lymphoid nodes directly via the bloodstream ¹⁸. Murine pDCs express B220, SiglecH, Ly6c and low amounts of CD11c. Human pDCs express CD4, CD123, HLA-DR, and blood derived cell antigen-2 (BDCA-2), as well as toll-like receptor (TLR)-7 and -9 but do not express the cDC-marker CD11c ^17,19. pDCs can act as antigen presenting cells, but are much less efficient than cDCs.

cDCs also called classical dendritic cells or myeloid dendritic cells, express high levels of CD11c and display a characteristic morphology of long dendrite extensions ²⁰. There are two primary cDCs subpopulations in mice: CD11b⁻ and CD11b⁺. CD103⁺CD11b⁻ cDCs populate most connective tissues and share their origin and function with lymphoid tissue CD8⁺ cDCs ²¹. The CD11b⁺ cDC subset most often lacks the integrin CD103 ²². In human, cDCs express CD11b, CD11c, CD1a, CD1c, CD14, CD45, CD141, CD172a, CD206, Clec9a/DNGR1, XCR1, HLA-DR, FceRI. cDCs present antigens and produce the stimulatory mediators TNF, NO and IL-12 ²³. cDCs recognize both bacterial and viral antigens ^24,25. Immature cDCs patrol various tissues to sample foreign antigens. During maturation cDCs migrate to draining lymph nodes to present pathogen-derived peptides to T cells ²⁶.

Langerhans cells are a special member of the DC family that is usually situated in the oral mucosal epithelium and the skin epidermis ² and can be distinguished by expression of langerin/CD207 ²⁷. Langerhans cells play an important role in immune surveillance by promoting immunity or tolerance through activation of antigen specific T cells ²⁷.

DCs modulate specific lymphocyte responses

The interaction between DCs and pathogens contributes to different types of adaptive immunity. The specific cytokines expressed by DCs are dependent on stimulation by pathogen-associated molecular patterns (PAMPS). As a result under certain circumstances DCs can stimulate an immune response along a particular direction such as Th1, Th2, Th17, or Treg. DCs promote Th1 polarization by expressing IL-12 ²⁸. IL-12 secretion from DCs is induced by CD40 engagement ²⁹. DCs primed with antigen stimulate naïve T cells to differentiate to Th1 cells ³⁰. Interferon regulatory factor 4 plays an important role in DC initiation of Th2 cell responses ³¹. High-mobility group nucleosome-binding protein 1 (HMGN1) modulates DCs to induce Th2 responses in a TLR4-dependent manner ³². DCs primed with antigens of Streptococcus mitis increase differentiation of Th2 cells ³⁰. DCs promote B cells through release of B-cell activating factor (BAFF) ³³ and a proliferation-inducing ligand (APRIL) ³⁴. They also promote B cell differentiation to plasma cells both via the secretion of IFN-α and IL-6 ³⁵ and via direct cell-cell contact ³⁶. DCs promote Th17 cell differentiation through secretion of the cytokines TGF-β, IL-23 and IL-1β ³⁷. DCs can induce the differentiation of Treg cells via TGF-β and IL-10 ³⁸. For example, DCs incubated with Propionibacterium acnes antigens induce differentiation of lymphocytes to Tregs ³⁰. DCs also regulate the recruitment and activation of NK cells through secretion of IL-27 ³⁹.

Evidence of DCs in periodontal disease

DC stimulated by oral pathogens can contribute to different types of adaptive immunity and can lead to reduced proteolytic or osteolytic activity through Th2 or Treg responses or an increase through induction of Th1 or Th17 lymphocytes (Fig. 1). Although it is well recognized that dendritic cells play a key role in initiating an adaptive immune response there are relatively few studies which provide a causal link between dendritic cells and periodontal breakdown, particularly in animal models where specific hypotheses can be tested ⁴⁰.

Figure 1.

DCs activate T cells and innate immune response in periodontal disease. Immature DCs capture oral bacteria, which induces migration to lymph nodes and maturation. Mature DCs can attract neutrophils and macrophage to sites of inflammation through IL-8 or TNF-α and present bacterial antigen to lymphocytes. DCs in turn may stimulate naïve T cells to differentiate along several pathways including Th1, Th17, Th2 and Treg cells. Cytokines produced by Th1 and Th17 cells may up-regulate TNF-α and IL-1β and cause greater MMP and RANKL expression, more osteoclast formation and more bone resorption. Th2 and Tregs have the opposite effect and may restrain inflammatory cytokine production to limit bone loss.

DCs may promote periodontal disease through induction of Th1- or Th17-lymphocytes

DCs can potentially enhance periodontal bone loss through up regulation of Th1 or Th17 response. Th1 activity is correlated with the number of mature DCs in gingiva in periodontitis ⁴¹. Porphyromonas gingivalis stimulates mature cDCs derived from individuals with chronic periodontitis to secrete IL-12 and IFN-γ ⁴². Both IL-12 and IFN-γ can promote Th1 responses and sustain inflammation ⁴³. IFN-γ is the signature cytokine of Th1-type responses ⁴⁴ associated with activating phagocytosis and the production of inflammatory cytokines and chemokines ⁴⁵. Th1 responses have also been linked to increased RANKL expression ⁴⁶, promotion of osteoclast formation and alveolar bone loss in vivo ⁴⁷. Moreover, IFN-γ is present at high levels in periodontal lesions and is associated with progressive lesions or more severe periodontal disease⁴⁸.

Oral infection with P. gingivalis in mice increases the number of cDCs, which is positively correlated with the generation of a Th17 response ^47,49. Oral infection with P. gingivalis and Fusobacterium nucleatum stimulate migration of cDCs to the lymph nodes and gingiva and is associated with increased IL-17 levels, as well as other pro-inflammatory factors such as TNF, IL-6 and IL-1β, which contribute to alveolar bone loss ^4,50. In humans IL-17 levels are correlated with more mature cDCs and increased periodontal bone loss ⁴¹. IL-17 induces pro-inflammatory and osteoclastogenic mediators such as RANKL or TNF ^51,52. Thus, cDCs are upregulated in response to periodontal infection and are associated with increased inflammation and bone loss. However, a cause and effect relationship has not been established.

Although Th17 and Th1 responses that are typically thought to promote periodontal bone loss, there is some indication that these responses may be protective. For example IFN-γ produced during a Th1 response can inhibit RANKL induced osteoclastogenesis ⁵³. Th1 leukocytes also have the ability of enhance recruitment and the killing activity of neutrophils and macrophages, which promote bacterial clearance ^54,55.

DCs may induce a protective response through induction of Th2-lymphocytes

DCs triggered by P. gingivalis LPS induce a Th2 response through the TLR pathway ^56,57. IL-4, the major Th2 type cytokine ^58–60 inhibits the production of MMPs and RANKL and up-regulates the MMP inhibitor TIMP and the RANKL inhibitor OPG ^61,62. Inhibition of MMPs and RANKL leads to reduced severity of experimental periodontitis ⁶³. IL-4 levels are reduced in patients with chronic periodontitis compared to healthy persons ⁶⁴. IL-4 levels increase after non-surgical periodontal therapy, suggesting a protective role of Th2 cells ⁶⁵. Although Th2 responses are generally thought of as being protective, there is also evidence that Th2 lymphocytes can play destructive role in periodontal disease. Mice infected with Tannerella forsythia causes a pronounced Th2 bias and induce alveolar bone loss ⁶⁶.

DCs have the ability to stimulate naïve B cells ^67,68. In a murine model, oral infection stimulated cDCs to migrate to lymph nodes, inducing formation of plasma cells and the production of bacteria-specific antibodies. When DC function is reduced there is less DC migration to the lymph nodes, reduced numbers of plasma cells and less bacteria-specific IgG1 production, leading to more alveolar bone loss compared to control mice ⁴. This supports the contention that DCs activate a protective antibody response that leads to less bone loss.

DCs protective role in periodontal disease through Treg response

Treg cells limit excessive inflammation and bone loss ⁵⁴. In active periodontal lesions, Treg-related cytokine levels are inversely related to RANKL levels ^69,70. Inhibition of Treg function leads to higher levels of IFN-γ, TNF-α and RANKL and more alveolar bone loss ⁷¹. Oral DCs have the capacity to induce a Treg response through the secretion of IL-10 and TGF-β ⁷². The importance of IL-10 is illustrated by the finding that in IL-10 knockout mice, susceptibility to P. gingivalis-induced alveolar bone loss is greatly increased ⁷³. Moreover, after periodontal treatment, inflammation is reduced and IL-10 expression is increased ⁷⁴.

DCs may affect periodontal disease through enhancement or attenuation of the innate immune response

cDCs produce a number of mediators that can modulate the innate immune response. P. gingivalis and LPS stimulate DCs to produce TNF-α ⁷⁵ and IL-8 ¹². P gingivalis binds to TLR-2 to activate complement receptor-3 (CR3), and subsequent binding to CR3 by P gingivalis fimbriae contributes to the induction of inflammatory cytokines such as TNF ⁷⁶. In addition, P gingivalis may affect dendritic cells by affecting autophagy and intracellular killing^77,78. Chemokines produced by DC can attract neutrophils and monocytes to sites of inflammation ⁷⁹ enhancing inflammation and the expression of factors that stimulate osteoclastogenesis ⁸⁰ (Fig. 1). Meanwhile IL-10 and TGF-β from DCs can potentially have the opposite effect, reducing inflammation ⁸¹.

The role of Langerhans cells in periodontal disease

Langerhans cells, a subset of DCs have been linked to periodontal disease. There is a positive correlation between the number of Langerhans cells in the gingival epithelium of chronic periodontitis patients versus healthy persons ^82,83 and increased numbers in individuals with chronic gingivitis compared to non-inflamed gingiva ⁸⁴. There is increased migration of Langerhans cells to the gingival epithelium in response to the accumulation of bacterial plaque ⁸⁵ and a decrease following periodontal treatment ⁸⁶. In one of the few cause and effect studies, ablation of Langerhans cells in experimental periodontitis in mice results in reduced numbers of Tregs, elevated production of RANKL and enhanced alveolar bone loss ⁶. Taken together these results suggest that Langerhans cells are increased by periodontal inflammation and have a protective effect through the generation of Treg cells. The results are also consistent with findings that reduction of cDC activity through deletion of FOXO1 increases periodontal disease susceptibility in both young and aged mice ^4,5, suggesting that DCs have a protective function against increased periodontal inflammation.

Evidence of DC to osteoclast differentiation in periodontal disease

DCs as a source of osteoclast precursors

DCs are the professional antigen-presenting cells that also share several features with osteoclasts. Both DCs and osteoclasts are derived from a common hematopoietic precursor and exhibit phagocytic activities ⁸⁷. In vitro, these precursors can be stimulated to differentiate to DCs by granulocyte monocyte colony stimulating factor (GM-CSF) and IL-4 or osteoclast precursors by M-CSF ⁸⁸. There is increasing evidence that committed immature cDCs in some inflammatory conditions such as rheumatoid arthritis or periodontitis can transdifferentiate toward osteoclasts in the presence of RANKL ⁸⁷. The co-culture of CD11c⁺ DCs with CD4⁺ T cells and Aggregatibacter actonimycetemcomitans produces osteoclasts that can induce bone resorption in vitro and in vivo ⁸⁹. Moreover, the differentiation of DCs to osteoclasts could potentially be enhanced by bacteria induced inflammation. Thus, immature cDCs may be directly involved in osteoclastogenesis and contribute to bone loss in rheumatoid arthritis, histiocytosis, or periodontitis ⁹⁰ (Fig. 2).

Figure 2.

The pathway of DCs transdifferentiating to osteoclast.

DCs and monocytes are derived from a common myeloid progenitor under the stimulation of GM-CSF and IL-4 (for DCs) or M-CSF (for monocytes). Immature cDCs can transdifferentiate to osteoclasts under the influence of M-CSF and RANKL. Thus, cDCs can transdifferentiate into osteoclasts and cause bone resorption in some inflammatory environments such as periodontitis and rheumatoid arthritis.

Immature cDCs either from bone marrow or from spleen are able to develop into osteoclasts, but mature cDCs do not have this ability. cDC-derived osteoclast formation is positively regulated by pro-inflammatory cytokines such as IL-1β, TNF-α and negatively regulated by IFN-α, IFN-γ, IL-2 and IL-4 ⁹¹. In vivo, immature CD11c⁺ cDCs can form osteoclasts when injected into osteopetrotic mice that normally lacked osteoclasts ⁹². mRNA profiling compared DC-derived and monocyte-derived osteoclasts and showed that both formed molecularly indistinguishable mature osteoclasts. Interestingly, fewer genes need to be up- or down- regulated when osteoclasts are formed from cDCs compared to monocytic precursors ⁹³.

In humans, cDCs are contributors to inflammatory bone destruction. Synovial fluid from arthritic patients increases the differentiation of osteoclasts from immature but committed cDCs⁹⁰. Moreover, the presence of Th17 cells is associated with the emergence of cDC-derived osteoclasts ⁹². In myeloproliferative diseases, the participation of DC to osteoclast formation has also been suggested. Langerhans cell histiocytosis (LCH) is a rare disease caused by the clonal accumulation of dendritic Langerhans cells. LCH is frequently associated with osteolytic lesion and multinucleated giant cells expressing osteoclast markers (TRAP, cathepsin K, MMP9). Bone loss in this disease can be accounted for the transdifferentiation of cDCs to functional osteoclasts both in vivo and in vitro ⁹⁴.

PI3K/Akt-FOXO1 signaling pathway in DCs

FOXO1 promotes DC activity and Akt enhances DC survival and proliferation

FOXO1, a member of forkhead transcription factor family, plays an important role in the regulation of many cellular and biological processes that include protection against oxidative stress, apoptosis and progression through the cell cycle⁹⁵. Surprisingly FOXO1 can have opposite effects depending upon the microenvironment and the specific cell type ⁹⁶. FOXO1 affects immune responses by controlling cytokine production ⁹⁷, protecting hematopoietic stems cells from oxidative stress ⁹⁵ and by promoting lymphocyte homeostasis through development of T regulatory lymphocytes and maintenance of naïve T cells ⁹⁸. Lineage specific deletion of FOXO1 in cDCs reduces their capacity for bacterial phagocytosis, migration and binding to lymphocytes ⁹⁹. FOXO1 is needed for efficient DC homing to lymph nodes and regulates CCR7, a key receptor needed for the DC homing ⁹⁹. FOXO1 participates in cDC activation of both T and B lymphocytes and regulates expression of ICAM-1, which is needed to form an immune synapse. FOXO1 regulates these key molecules by binding to their promoters to transactivate transcription ⁹⁹. In DCs, FOXO1 mediates LPS induced IL-6 and IL-12 expression, but reduces IL-10 production ¹⁰⁰. In keratinocytes, P. gingivalis induces loss of barrier function and apoptosis by reducing expression of genes associated with epithelial barriers (integrin beta-1, -3 and -6) and increasing those that stimulate apotposis ¹⁰¹. In addition P. gingivalis-induces reactive oxygen species (ROS) that activate FOXO transcription factors through Jun-N-terminal kinase (JNK) signaling, which may modulate oxidative stress responses in gingival keratinocytes ¹⁰².

Forkhead box O (FOXO) transcription factors are downstream targets of the serine/threonine protein kinase B (PKB)/Akt ¹⁰³. Akt is a kinase, which regulates processes of cellular proliferation and survival. The PI3K-Akt pathway has been reported to regulate cDC proliferation and survival ¹⁰⁴. Phosphorylation of FOXOs by Akt inhibits the transcriptional function of FOXOs and contributes to cell survival, growth and proliferation ¹⁰³. FOXO signaling is regulated by interactions with other intracellular proteins as well as post-translational modifications such as phosphorylation ¹⁰³. In addition to FOXOs, Akt phosphorylates and inhibits glycogen synthase kinase-3β (GSK-3β)¹⁰⁵. GSK3 is a serine protein kinase involved in regulation of DCs ¹⁰⁶. PI3K reduces GSK3 activity via Akt phosphorylation^107,108,109. In plasmacytoid dendritic cells, PI3K-Akt pathway is an important regulator of type I interferon production via activation of the interferon-regulatory factor 7 ^110,111.

PI3K/Akt-FOXO1 signaling pathway in DCs in periodontal disease

It has been shown that FOXO1 activation promotes the expression of inflammatory cytokines in a number of cell types including dendritic cells ⁴, macrophages ¹¹² and epithelial cells ¹¹³. This result suggests that deletion of FOXO1 in DC could reduce periodontal bone loss through reduced inflammatory cytokine expression. However experimental evidence is the opposite; deletion of FOXO1 in DC increases susceptibility to periodontitis.

P. gingivalis increases FOXO1 gene expression in cDCs through TLR signaling ^99,114. Oral infection stimulates recruitment of cDCs to oral mucosal epithelium and connective tissue ⁴. Deletion of FOXO1 specifically in cDCs blocks enhanced recruitment of cDCs and increases susceptibility to periodontitis ⁴. Oral infection stimulates migration of cDCs to the cervical lymph nodes, the formation of plasma cells and bacteria-specific antibody production ⁴. Each of these is significantly blocked with lineage specific FOXO1 deletion in cDCs by CD11c driven Cre recombinase ⁴. When FOXO1-deleted cDCs exhibited a compensatory increase in IL-1β and IL-17 in response to oral infection in vivo, which likely occurred from a reduced protective antibody response ⁴. This hypothesis is supported by a failure to upregulate APRIL and BAFF mRNA when FOXO1 in DC is ablated whereas both mediators are increased by wild type cDCs in response to bacterial stimulation ⁹⁹. This is significant since cDCs regulate B cells through production of BAFF and APRIL. Moreover, deletion of FOXO1 in DC leads to reduced antibody production ⁴ and reduced lymphocyte activity. Thus, the principal effect of FOXO1 may be due to a reduced ability of DC to stimulate a protective acquired immune response.

There have been few studies that have examined the Akt-FOXO1 pathway in dendritic cells. Those studies that have been reported have not used a lineage-specific approach, limiting interpretation of the data. For example, it has been shown that a global deletion of transforming growth factor beta-activated kinase 1 (TAK1) disrupts Akt-FOXO in DC and may disrupt generation of Tregs¹¹⁵. However, the direct linkage to the Akt FOXO1 axis in DC was not demonstrated due to the global nature of the gene deletion and since TAK1 has other substrates such as NF-kB that may also be responsible for the effect. In another study the compound piperlongumine was shown to inhibit DC maturation and reduce the symptoms of rheumatoid arthritis. Although these changes were associated with an inhibition of Akt pathway in DC a cause and effect relationship was not established because piperlongumine affects several other pathways including NF-kB¹¹⁶.

CONCLUSION

DCs are antigen-presenting cells, which capture, process and present antigens to lymphocytes to initiate and regulate the adaptive immune response ¹. DCs play a critical role in the adaptive immune response and can modulate lymphocyte polarization that is potentially destructive (Th1, Th17) or protective (Th2, Treg). DCs can transdifferentiate into osteoclasts and cause bone resorption during periodontal inflammation. However it is not clear whether immature DCs represent a significant source of osteoclast precursors in periodontal inflammation. Recent evidence makes it clear that the FOXO1 signaling plays an important role in DCs (Fig. 3). Our results are consistent with the hypothesis that FOXO1 deletion leads to loss of protection against bacterial challenge in the periodontium. We hypothesize that this may be due to a reduced capacity to upregulate the acquired immune response when FOXO1 is deleted. Alternatively, if DC are an important source of osteoclast precursors, deletion of FOXO1 could potentially increase DC to osteoclast differentiation to enhance bone loss. However, the precise mechanisms through which FOXO1 modulates periodontal disease susceptibility through altering DC function remains to be established. It is possible that FOXO1 enhances formation of Tregs to limit periodontal bone loss or increases formation of plasma cells to generate a protective antibody response. Moreover, the potential role of Akt in modulating FOXO1 may also be an important component of DC function that remains to be investigated in the context of periodontal disease.

Figure 3.

The Akt-FOXO1 signaling pathway maintains DC’s protective or destructive function to control periodontal inflammation. FOXO1 is essential for DCs to maintain their protective function and control periodontitis. Phosphorylation of FOXO1 by Akt inhibits FOXO1 activity and reduces the protective function of DCs enhancing susceptibilities to periodontitis.