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1.
Nat Commun ; 14(1): 5628, 2023 09 12.
Article in English | MEDLINE | ID: mdl-37699897

ABSTRACT

The postnatal interaction between microbiota and the immune system establishes lifelong homeostasis at mucosal epithelial barriers, however, the barrier-specific physiological activities that drive the equilibrium are hardly known. During weaning, the oral epithelium, which is monitored by Langerhans cells (LC), is challenged by the development of a microbial plaque and the initiation of masticatory forces capable of damaging the epithelium. Here we show that microbial colonization following birth facilitates the differentiation of oral LCs, setting the stage for the weaning period, in which adaptive immunity develops. Despite the presence of the challenging microbial plaque, LCs mainly respond to masticatory mechanical forces, inducing adaptive immunity, to maintain epithelial integrity that is also associated with naturally occurring alveolar bone loss. Mechanistically, masticatory forces induce the migration of LCs to the lymph nodes, and in return, LCs support the development of immunity to maintain epithelial integrity in a microbiota-independent manner. Unlike in adult life, this bone loss is IL-17-independent, suggesting that the establishment of oral mucosal homeostasis after birth and its maintenance in adult life involve distinct mechanisms.


Subject(s)
Langerhans Cells , Microbiota , Adult , Humans , Interleukin-17 , Homeostasis , Adaptive Immunity , Plaque, Amyloid
2.
PLoS Pathog ; 19(4): e1011284, 2023 04.
Article in English | MEDLINE | ID: mdl-37023213

ABSTRACT

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.


Subject(s)
Porphyromonas gingivalis , Toll-Like Receptor 2 , Porphyromonas gingivalis/genetics , Toll-Like Receptor 2/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Immune Evasion , Vinculin/metabolism , Base Composition , Phylogeny , RNA, Ribosomal, 16S , Sequence Analysis, DNA
3.
Cell Rep ; 42(1): 111981, 2023 01 31.
Article in English | MEDLINE | ID: mdl-36640306

ABSTRACT

While saliva regulates the interplay between the microbiota and the oral immune system, the mechanisms establishing postnatal salivary immunity are ill-defined. Here, we show that high levels of neutrophils and neonatal Fc receptor (FcRn)-transferred maternal IgG are temporarily present in the neonatal murine salivary glands in a microbiota-independent manner. During weaning, neutrophils, FcRn, and IgG decrease in the salivary glands, while the polymeric immunoglobulin receptor (pIgR) is upregulated in a growth arrest-specific 6 (GAS6)-dependent manner independent of the microbiota. Production of salivary IgA begins following weaning and relies on CD4-help, IL-17, and the microbiota. The weaning phase is characterized by a transient accumulation of dendritic cells capable of migrating from the oral mucosa to the salivary glands upon exposure to microbial challenges and activating T cells. This study reveals the postnatal mechanisms developed in the salivary glands to induce immunity and proposes the salivary glands as an immune inductive site.


Subject(s)
Microbiota , Receptors, Polymeric Immunoglobulin , Mice , Animals , Saliva , Salivary Glands , Immunoglobulin G
4.
Proc Natl Acad Sci U S A ; 119(3)2022 01 18.
Article in English | MEDLINE | ID: mdl-35012988

ABSTRACT

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.


Subject(s)
Antineoplastic Agents/metabolism , Carcinogens/toxicity , Langerhans Cells/metabolism , 4-Nitroquinoline-1-oxide/toxicity , Cell Line, Tumor , Dendritic Cells/drug effects , Dendritic Cells/pathology , Epithelial Cells/metabolism , Epithelium/drug effects , Epithelium/pathology , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/pathology , Histones/metabolism , Humans , Immunity/drug effects , Langerhans Cells/drug effects , Phagocytes/drug effects , Phagocytes/metabolism , Phagocytes/pathology , Quinolones/toxicity , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/pathology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Tongue/pathology , Transcriptome/genetics
5.
Cell Host Microbe ; 29(2): 197-209.e5, 2021 02 10.
Article in English | MEDLINE | ID: mdl-33412104

ABSTRACT

Postnatal host-microbiota interplay governs mucosal homeostasis and is considered to have life-long health consequences. The intestine monolayer epithelium is critically involved in such early-life processes; nevertheless, the role of the oral multilayer epithelium remains ill defined. We demonstrate that unlike the intestine, the neonate oral cavity is immensely colonized by the microbiota that decline to adult levels during weaning. Neutrophils are present in the oral epithelium prenatally, and exposure to the microbiota postnatally further recruits them to the preamble neonatal epithelium by γδT17 cells. These neutrophils virtually disappear during weaning as the epithelium seals. The neonate and adult epithelium display distinct turnover kinetics and transcriptomic signatures, with neonate epithelium reminiscent of the signature found in germ-free mice. Microbial reduction during weaning is mediated by the upregulation of saliva production and induction of salivary antimicrobial components by the microbiota. Collectively, unique postnatal interactions between the multilayer epithelium and microbiota shape oral homeostasis.


Subject(s)
Bacterial Load , Mouth Mucosa/immunology , Mouth Mucosa/microbiology , Neutrophils/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Saliva/microbiology , Animals , Animals, Newborn/growth & development , Animals, Newborn/microbiology , Interleukin-17/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mouth Mucosa/cytology , Mouth Mucosa/growth & development , Th17 Cells/immunology
6.
Cancers (Basel) ; 12(8)2020 Aug 18.
Article in English | MEDLINE | ID: mdl-32824786

ABSTRACT

Porphyromonas gingivalis is a member of the dysbiotic oral microbiome associated with oral inflammation and periodontal disease. Intriguingly, epidemiological studies link P. gingivalis to an increased risk of pancreatic cancer. Given that oral bacteria are detected in human pancreatic cancer, and both mouse and human pancreata harbor microbiota, we explored the involvement of P. gingivalis in pancreatic tumorigenesis using cell lines and a xenograft model. Live P. gingivalis induced proliferation of pancreatic cancer cells; however, surprisingly, this effect was independent of Toll-like receptor 2, the innate immune receptor that is engaged in response to P. gingivalis on other cancer and immune cells, and is required for P. gingivalis to induce alveolar bone resorption. Instead, we found that P. gingivalis survives inside pancreatic cancer cells, a trait that can be enhanced in vitro and is increased by hypoxia, a central characteristic of pancreatic cancer. Increased tumor cell proliferation was related to the degree of intracellular persistence, and infection of tumor cells with P. gingivalis led to enhanced growth in vivo. To the best of our knowledge, this study is the first to demonstrate the direct effect of exposure to P. gingivalis on the tumorigenic behavior of pancreatic cancer cell lines. Our findings shed light on potential mechanisms underlying the pancreatic cancer-periodontitis link.

7.
Mucosal Immunol ; 13(5): 767-776, 2020 09.
Article in English | MEDLINE | ID: mdl-32457449

ABSTRACT

Unlike epidermal Langerhans cells (LCs) that originate from embryonic precursors and are self-renewed locally, mucosal LCs arise and are replaced by circulating bone marrow (BM) precursors throughout life. While the unique lifecycle of epidermal LCs is associated with an age-dependent decrease in their numbers, whether and how aging has an impact on mucosal LCs remains unclear. Focusing on gingival LCs we found that mucosal LCs are reduced with age but exhibit altered morphology with that observed in aged epidermal LCs. The reduction of gingival but not epidermal LCs in aged mice was microbiota-dependent; nevertheless, the impact of the microbiota on gingival LCs was indirect. We next compared the ability of young and aged BM precursors to differentiate to mucosal LCs. Mixed BM chimeras, as well as differentiation cultures, demonstrated that aged BM has intact if not superior capacity to differentiate into LCs than young BM. This was in line with the higher percentages of mucosal LC precursors, pre-DCs, and monocytes, detected in aged BM. These findings suggest that while aging is associated with reduced LC numbers, the niche rather than the origin controls this process in mucosal barriers.


Subject(s)
Cell Differentiation , Cellular Microenvironment/immunology , Langerhans Cells/immunology , Langerhans Cells/metabolism , Mucous Membrane/immunology , Mucous Membrane/metabolism , Age Factors , Aging/physiology , Animals , Biomarkers , Bone Morphogenetic Protein 7/genetics , Bone Morphogenetic Protein 7/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Cellular Microenvironment/genetics , Cellular Senescence/genetics , Cellular Senescence/immunology , Epidermal Cells/immunology , Epidermal Cells/metabolism , Epidermis/immunology , Epidermis/metabolism , Epidermis/microbiology , Gene Expression , Gingiva/immunology , Gingiva/metabolism , Gingiva/microbiology , Immunophenotyping , Langerhans Cells/cytology , Mice , Microbiota , Mucous Membrane/microbiology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism
8.
Pain ; 160(6): 1281-1296, 2019 06.
Article in English | MEDLINE | ID: mdl-30933959

ABSTRACT

Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.


Subject(s)
Inflammation/drug therapy , Nociceptors/physiology , Platelet-Derived Growth Factor/metabolism , Sensory Receptor Cells/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Animals , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiopathology , Hyperalgesia/metabolism , Hyperalgesia/physiopathology , Male , Nociceptors/drug effects , Pain/metabolism , Pain/physiopathology , Pain Measurement/drug effects , Pain Measurement/methods , Platelet-Derived Growth Factor/pharmacology , Rats, Sprague-Dawley , Sensory Receptor Cells/drug effects
9.
J Periodontal Res ; 54(4): 396-404, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30793777

ABSTRACT

AIM: To explore the role of keratinocyte myeloid differentiation primary response 88 (MyD88) expression in the adhesion of Porphyromonas gingivalis to the cells and its subsequent invasion and intracellular survival. MATERIALS AND METHODS: Primary mouse keratinocytes from wild-type (WT) or Myd88-/- mice were infected with P gingivalis alone or co-infected with Fusobacterium nucleatum. Bacterial adhesion and invasion were measured using fluorescent microscopy and flow cytometry, and intracellular survival in keratinocytes was quantified by an antibiotic protection assay. Keratinocyte expression of antimicrobial peptides was measured by real-time PCR. RESULTS: In the absence of MyD88, P gingivalis adherence, invasion, and intracellular survival were enhanced compared with WT keratinocytes. The presence of F nucleatum during infection increased the adhesion of P gingivalis to WT keratinocytes but reduced the adhesion to Myd88-/- keratinocytes. Fusobacterium nucleatum improved mildly the invasion and survival of P gingivalis in both cell types. Baseline expression of beta-defensin 2, 3, 4 and RegIII-γ was elevated in Myd88-/- keratinocytes compared to WT cells; however, following infection beta-defensin expression was strongly induced in WT cells but decreased dramatically in the MyD88 deficient cells. CONCLUSION: In the absence of MyD88 expression, P gingivalis adhesion to keratinocytes is improved, and invasion and intracellular survival are increased. Furthermore, keratinocyte infection by P gingivalis induces antimicrobial peptide expression in a MyD88-dependent manner. Thus, MyD88 plays a key role in the interaction between P gingivalis and keratinocytes.


Subject(s)
Bacteroidaceae Infections/immunology , Keratinocytes/microbiology , Myeloid Differentiation Factor 88/immunology , Porphyromonas gingivalis , Animals , Antimicrobial Cationic Peptides/immunology , Bacterial Adhesion , Fusobacterium nucleatum , Keratinocytes/immunology , Mice , Mice, Knockout
10.
Proc Natl Acad Sci U S A ; 116(7): 2652-2661, 2019 02 12.
Article in English | MEDLINE | ID: mdl-30692259

ABSTRACT

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6+ cells, and to a lesser extent Vγ1+ and Vγ4+ cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6+ cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6+ and Vγ4+ cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.


Subject(s)
Homeostasis , Interleukin-17/biosynthesis , Microbiota , Mouth Mucosa/microbiology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocytes/metabolism , Animals , Biofilms , Gingiva/immunology , Gingiva/microbiology , Inflammation/immunology , Mice
11.
Front Immunol ; 9: 1374, 2018.
Article in English | MEDLINE | ID: mdl-29967614

ABSTRACT

Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6-/- mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.

12.
Proc Natl Acad Sci U S A ; 115(25): E5736-E5745, 2018 06 19.
Article in English | MEDLINE | ID: mdl-29871951

ABSTRACT

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.


Subject(s)
Carrier Proteins/metabolism , Epidermis/metabolism , Langerhans Cells/metabolism , Protein S/metabolism , Animals , Bone Marrow/metabolism , Calcium-Binding Proteins , Cell Differentiation/physiology , Homeostasis/physiology , Keratinocytes/metabolism , Mice , Mice, Inbred C57BL , Monocytes/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , c-Mer Tyrosine Kinase/metabolism
13.
J Exp Med ; 215(2): 481-500, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29343501

ABSTRACT

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However, the mechanisms involved in their in situ development remain unclear. Here, we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria, signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium, TGF-ß1 finalizes LC differentiation, and ALK5 is crucial to this process. Moreover, the local microbiota has a major impact on the development of mucosal LCs, whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-ß1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota.


Subject(s)
Bone Morphogenetic Protein 7/immunology , Langerhans Cells/immunology , Microbiota/immunology , Transforming Growth Factor beta1/immunology , Animals , Antigens, Surface/genetics , Antigens, Surface/metabolism , Bone Morphogenetic Protein 7/genetics , Bone Morphogenetic Protein Receptors, Type I/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Humans , Immunity, Mucosal , Langerhans Cells/cytology , Langerhans Cells/metabolism , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Male , Mannose-Binding Lectins/deficiency , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mouth Mucosa/cytology , Mouth Mucosa/immunology , Receptor, Transforming Growth Factor-beta Type I/metabolism , Signal Transduction/immunology , Stem Cells/cytology , Stem Cells/immunology , Transcriptome , Transforming Growth Factor beta1/genetics , Up-Regulation
14.
Cell Rep ; 18(2): 419-431, 2017 01 10.
Article in English | MEDLINE | ID: mdl-28076786

ABSTRACT

Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ T cells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.


Subject(s)
Interferon Type I/biosynthesis , Myeloid Differentiation Factor 88/metabolism , Porphyromonas gingivalis/physiology , Proteolysis , Receptors, Cell Surface/metabolism , Signal Transduction , Alveolar Bone Loss/complications , Alveolar Bone Loss/immunology , Alveolar Bone Loss/pathology , Animals , Bacteroidaceae Infections/complications , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/microbiology , Bone Resorption/complications , Bone Resorption/immunology , Bone Resorption/pathology , Dendritic Cells/immunology , Gingiva/microbiology , Gingiva/pathology , Humans , Interferon Type I/metabolism , Leukocytes/pathology , Lymph Nodes/pathology , Mice , Mouth Mucosa/microbiology , Mouth Mucosa/pathology , Periodontitis/immunology , Periodontitis/microbiology , Periodontitis/pathology
15.
J Infect Dis ; 213(9): 1505-15, 2016 May 01.
Article in English | MEDLINE | ID: mdl-26704610

ABSTRACT

Porphyromonas gingivalis,an anaerobic bacterium strongly linked to infection-driven inflammatory bone erosion, thrives within a highly inflamed milieu and disseminates to distant sites, such as atherosclerotic plaque. We examined the role of monocyte/macrophages in determining the outcome of infection with P. gingivalis. Surprisingly, transient monocyte/macrophage depletion led to greatly improved clearance of P. gingivalis. The chemokine receptors CCR2 and CX3CR1 play a major role in monocyte recruitment and differentiation to Ly6C(hi) vs CX3CR1(hi) subsets, respectively. To determine the contribution of particular monocyte/macrophage subsets to bacterial survival, we challenged chemokine receptor knockout mice and found that P. gingivalis clearance is significantly improved in the absence of CX3CR1. CX3CR1(hi) monocyte/macrophages promote P. gingivalis survival by downregulating neutrophil phagocytosis. Furthermore, CX3CR1 knockout mice resist bone resorption in the oral cavity following challenge with P. gingivalis Our findings provide an explanation for bacterial coexistence alongside an activate neutrophil infiltrate.


Subject(s)
Alveolar Bone Loss/immunology , Alveolar Bone Loss/microbiology , Macrophages/immunology , Monocytes/immunology , Porphyromonas gingivalis , Receptors, CCR2/metabolism , Receptors, Chemokine/metabolism , Animals , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/microbiology , CX3C Chemokine Receptor 1 , Host-Pathogen Interactions/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Periodontitis/immunology , Periodontitis/microbiology , Porphyromonas gingivalis/immunology , Porphyromonas gingivalis/pathogenicity , Receptors, CCR2/genetics , Receptors, Chemokine/genetics
16.
J Immunol ; 192(4): 1395-403, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24420922

ABSTRACT

In vivo studies questioned the ability of Langerhans cells (LCs) to mediate CD8(+) T cell priming. To address this issue, we used intradermal immunization with plasmid DNA, a system in which activation of CD8(+) T cells depends on delayed kinetics of Ag presentation. We found that dendritic cells (DCs) located in the skin at the time of immunization have limited ability to activate CD8(+) T cells. This activity was mediated by a second generation of DCs that differentiated in the skin several days after immunization, as well as by lymph node-resident DCs. Intriguingly, CD8(+) T cell responses were not affected following treatment with clodronate liposomes, immunization of CCR2(-/-) mice, or local neutralization of CCL20. This suggests that local, rather than blood-derived, DC precursors mediate CD8(+) T cell priming. Analysis of DC differentiation in the immunized skin revealed a gradual increase in the number of CD11c(+) cells, which reached their maximum 2 wk after immunization. A similar differentiation kinetics was observed for LCs, with the majority of differentiating LCs proliferating in situ from epidermal precursors. By using B6/Langerin-diphtheria toxin receptor chimeric mice and LC ablation, we demonstrated that epidermal LCs were crucial for the elicitation of CD8(+) T cell responses in vivo. Furthermore, LCs isolated from lymph nodes 2 wk after immunization contained the immunization plasmid and directly activated Ag-specific CD8(+) T cells ex vivo. Thus, these results indicate that second-generation Ag-expressing LCs differentiating from epidermal precursors directly prime CD8(+) T cells and are essential for optimal cellular immune responses following immunization with plasmid DNA.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Giant Cells, Langhans/immunology , Lymphocyte Activation/immunology , Animals , CD11c Antigen/metabolism , Cell Differentiation/immunology , Chemokine CCL20/immunology , Clodronic Acid , Dendritic Cells/metabolism , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasmids/genetics , Receptors, CCR2/deficiency , Receptors, CCR2/genetics , Receptors, CCR2/immunology , Skin/cytology , Skin/immunology
17.
PLoS One ; 8(6): e66488, 2013.
Article in English | MEDLINE | ID: mdl-23824049

ABSTRACT

Memory CD8(+) T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+) T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+) T-cell expansion.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Genetic Vectors , Immunologic Memory , Lentivirus/genetics , Animals , Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Proliferation , Kinetics , Lentivirus/immunology , Mice , Mice, Inbred C57BL
18.
Proc Natl Acad Sci U S A ; 109(18): 7043-8, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22509018

ABSTRACT

Excessive bone resorption is frequently associated with chronic infections and inflammatory diseases. Whereas T cells were demonstrated to facilitate osteoclastogenesis in such diseases, the role of dendritic cells, the most potent activators of naive T cells, remains unclear. Using a model involving inflammation-driven alveolar bone loss attributable to infection, we showed that in vivo ablation of Langerhans cells (LCs) resulted in enhanced bone loss. An increased infiltration of B and T lymphocytes into the tissue surrounding the bone was observed in LC-ablated mice, including receptor activator of NF-κB ligand (RANKL)-expressing CD4(+) T cells with known capabilities of altering bone homeostasis. In addition, the absence of LCs significantly reduced the numbers of CD4(+)Foxp3(+) T-regulatory cells in the tissue. Further investigation revealed that LCs were not directly involved in presenting antigens to T cells. Nevertheless, despite their low numbers in the tissue, the absence of LCs resulted in an elevated activation of CD4(+) but not CD8(+) T cells. This activation involved elevated production of IFN-γ but not IL-17 or IL-10 cytokines. Our data, thus, reveal a protective immunoregulatory role for LCs in inflammation-induced alveolar bone resorption, by inhibiting IFN-γ secretion and excessive activation of RANKL(+)CD4(+) T cells with a capability of promoting osteoclastogenesis.


Subject(s)
Alveolar Bone Loss/immunology , Alveolar Bone Loss/prevention & control , Langerhans Cells/immunology , Alveolar Bone Loss/etiology , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Bacteroidaceae Infections/complications , Bacteroidaceae Infections/immunology , Base Sequence , CD4-Positive T-Lymphocytes/immunology , DNA Primers/genetics , Disease Models, Animal , Down-Regulation/immunology , Heparin-binding EGF-like Growth Factor , Humans , Inflammation/complications , Inflammation/immunology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/immunology , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-17/metabolism , Langerhans Cells/classification , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Lymphocyte Activation , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Porphyromonas gingivalis/immunology , RANK Ligand/metabolism , T-Lymphocytes, Regulatory/immunology
19.
Physiol Genomics ; 34(1): 78-87, 2008 Jun 12.
Article in English | MEDLINE | ID: mdl-18430807

ABSTRACT

Sporadic findings in humans suggest that reinduction of heat acclimation (AC) after its loss occurs markedly faster than that during the initial AC session. Animal studies substantiated that the underlying acclimatory processes are molecular. Here we test the hypothesis that faster reinduction of AC (ReAC) implicates "molecular memory." In vivo measurements of colonic temperature profiles during heat stress and ex vivo assessment of cross-tolerance to ischemia-reperfusion or anoxia insults in the heart demonstrated that ReAC only needs 2 days vs. the 30 days required for the initial development of AC. Stress gene profiling in the experimental groups highlighted clusters of transcriptionally activated genes (37%), which included heat shock protein (HSP) genes, antiapoptotic genes, and chromatin remodeling genes. Despite a return of the physiological phenotype to its preacclimation state, after a 1 mo deacclimation (DeAC) period, the gene transcripts did not resume their preacclimation levels, suggesting a dichotomy between genotype and phenotype in this system. Individual detection of hsp70 and hsf1 transcripts agreed with these findings. HSP72, HSF1/P-HSF1, and Bcl-xL protein profiles followed the observed dichotomized genomic response. In contrast, HSP90, an essential cytoprotective component mismatched transcriptional activation upon DeAC. The uniform activation of the similarly responding gene clusters upon De-/ReAC implies that reacclimatory phenotypic plasticity is associated with upstream denominators. During AC, DeAC, and ReAC, the maintenance of elevated/phosphorylated HSF1 protein levels and transcriptionally active chromatin remodeling genes implies that chromatin remodeling plays a pivotal role in the transcriptome profile and in preconditioning to rapid cytoprotective acclimatory memory.


Subject(s)
Acclimatization/physiology , Heart/physiopathology , Heat-Shock Response , Hot Temperature , Myocardial Ischemia/physiopathology , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genotype , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Heat Shock Transcription Factors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Male , Memory , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocytes, Cardiac/pathology , Phenotype , Phosphorylation , Rats , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolism
20.
J Appl Physiol (1985) ; 100(6): 1992-2003, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16469936

ABSTRACT

The induction of the heat-acclimated phenotype involves reprogramming the expression of genes encoding both constitutive and inducible proteins. In this investigation, we studied the global genomic response in the hypothalamus during heat acclimation, with and without combined hypohydration stress. Rats were acclimated for 2 days (STHA) or for 30 days (LTHA) at 34 degrees C. Hypohydration (10% decrease in body weight) was attained by water deprivation. 32P-labeled RNA samples from the hypothalamus were hybridized onto cDNA Atlas array (Clontech no. 1.2) membranes. Clustering and functional analyses of the expression profile of a battery of genes representing various central regulatory functions of body homeostasis demonstrated a biphasic acclimation profile with a transient upregulation of genes encoding ion channels, transporters, and transmitter signaling upon STHA. After LTHA, most genes returned to their preacclimation expression levels. In both STHA and LTHA, genes encoding hormones and neuropeptides, linked with metabolic rate and food intake, were downregulated. This genomic profile, demonstrating an enhanced transcription of genes linked with neuronal excitability during STHA and enhanced metabolic efficiency upon LTHA, is consistent with our previously established integrative acclimation model. The response to hypohydration was characterized by an upregulation of a large number of genes primarily associated with the regulation of ion channels, cell volume, and neuronal excitability. During STHA, the response was transiently desensitized, recovering upon LTHA. We conclude that hypohydration overrides the heat acclimatory status. It is notable that STHA and hypohydration gene profiles are analogous with the physiological profile described in the response to various types of brain injury.


Subject(s)
Acclimatization/genetics , Acclimatization/physiology , Dehydration/physiopathology , Gene Expression Regulation/physiology , Hot Temperature , Hypothalamus/physiopathology , Animals , Appetite Regulation/genetics , Appetite Regulation/physiology , Carrier Proteins/genetics , Carrier Proteins/physiology , Dehydration/genetics , Energy Metabolism/genetics , Energy Metabolism/physiology , Heat Stress Disorders/genetics , Heat Stress Disorders/physiopathology , Hypothalamus/chemistry , Ion Channels/genetics , Ion Channels/physiology , Male , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Rats, Inbred Strains , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/physiology , Synaptic Transmission/genetics , Synaptic Transmission/physiology
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