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2.  IL-10 control of dendritic cells in the skin 
Oncoimmunology  2013;2(3):e23186.
Interleukin-10 (IL-10) is a potent immunomodulatory cytokine, whose cellular targets have not yet been precisely identified. Dendritic cell (DC)-specific IL-10 receptor knockout mice exhibit exaggerated T-cell reactivation in the skin, highlighting a key role of DCs in the maintenance of local immune homeostasis, beyond their classical function as regulators of T-cell priming in lymph nodes.
PMCID: PMC3661155  PMID: 23802070
conditional gene targeting; contact hypersensitivity; dendritic cells; immune regulation; interleukin-10; IL-10 receptor signaling; Langerhans cells; skin
3.  Langerhans cells are negative regulators of the anti-Leishmania response 
Langerhans cells suppress the immune response to low-dose Leishmania major infection in part by inducing regulatory T cells.
Migratory skin dendritic cells (DCs) are thought to play an important role in priming T cell immune responses against Leishmania major, but DC subtypes responsible for the induction of protective immunity against this pathogen are still controversial. In this study, we analyzed the role of Langerin+ skin-derived DCs in the Leishmania model using inducible in vivo cell ablation. After physiologically relevant low-dose infection with L. major (1,000 parasites), mice depleted of all Langerin+ DCs developed significantly smaller ear lesions with decreased parasite loads and a reduced number of CD4+ Foxp3+ regulatory T cells (T reg cells) as compared with controls. This was accompanied by increased interferon γ production in lymph nodes in the absence of Langerin+ DCs. Moreover, selective depletion of Langerhans cells (LCs) demonstrated that the absence of LCs, and not Langerin+ dermal DC, was responsible for the reduced T reg cell immigration and the enhanced Th1 response, resulting in attenuated disease. Our data reveal a unique and novel suppressive role for epidermal LCs in L. major infection by driving the expansion of T reg cells. A better understanding of the various roles of different DC subsets in cutaneous leishmaniasis will improve the development of a potent therapeutic/prophylactic vaccine.
PMCID: PMC3092359  PMID: 21536741
4.  Functional Redundancy of Langerhans Cells and Langerin+ Dermal Dendritic Cells in Contact Hypersensitivity 
The relative roles of Langerhans cells (LC), dermal dendritic cells (DC), and, in particular, the recently discovered Langerin+ dermal DC subset in the induction and control of contact hypersensitivity (CHS) responses remain controversial. Using an inducible mouse model, in which LC and other Langerin+ DC can be depleted by injection of diphtheria toxin, we previously reported impaired transport of topically applied antigen to draining lymph nodes and reduced CHS in the absence of all Langerin+ skin DC. In this study, we demonstrate that mice with a selective depletion of LC exhibit attenuated CHS only upon sensitization with a low hapten dose but not with a high hapten dose. In contrast, when painting a higher concentration of hapten onto the skin, which leads to increased antigen dissemination into the dermis, CHS is still diminished in mice lacking all Langerin+ skin DC. Taken together, these data suggest that the magnitude of a CHS reaction depends on the number of skin DC, which have access to the hapten, rather than on the presence or absence of a particular skin DC population. LC and (Langerin+) dermal DC thus seem to have a redundant function in regulating CHS.
PMCID: PMC2980552  PMID: 20703247
5.  Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity 
The Journal of Cell Biology  2005;169(4):569-576.
Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.
PMCID: PMC2171694  PMID: 15897263
6.  The Late Endosomal Adaptor Molecule p14 (LAMTOR2) Regulates TGFβ1-Mediated Homeostasis of Langerhans Cells 
Langerhans cells (LCs), a sub-population of dendritic cells (DCs) in the skin, participate in the regulation of immunity and peripheral tolerance. The adaptor molecule p14 is part of the late endosomal/lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin (mTOR) activator/regulator (LAMTOR) complex, which mediates the activation of lysosome-associated extracellular signaling-regulated kinase (ERK) and the mTOR cascade. In previous work, we demonstrated that CD11c-specific deficiency of p14 disrupts LC homeostasis by affecting the LAMTOR-mediated ERK and mTOR signaling. In this study, we extended our analysis on p14 deficiency specifically in LCs. Langerin-specific ablation of p14 caused a complete loss of LCs, accompanied by an increased maturational phenotype of LCs. The absence of LCs in p14-deficient mice reduced contact hypersensitivity (CHS) responses to the contact sensitizer trinitrochlorobenzene. Analysis using bone marrow-derived DCs (BMDCs) revealed that p14 deficiency in DCs/LCs interfered with the LC-relevant transforming growth factor β1 (TGFβ1) pathway, by lowering TGFβ receptor II expression on BMDCs and LCs, as well as surface binding of TGFβ1 on BMDCs. We conclude that p14 deficiency affects TGFβ1 sensitivity of LCs, which is mandatory for their homeostasis and subsequently for their immunological function during CHS.
PMCID: PMC4285575  PMID: 25078666
7.  HIF-1α Is Essential for Myeloid Cell-Mediated Inflammation 
Cell  2003;112(5):645-657.
Granulocytes and monocytes/macrophages of the myeloid lineage are the chief cellular agents of innate immunity. Here, we have examined the inflammatory response in mice with conditional knockouts of the hypoxia responsive transcription factor HIF-1α, its negative regulator VHL, and a known downstream target, VEGF. We find that activation of HIF-1α is essential for myeloid cell infiltration and activation in vivo through a mechanism independent of VEGF. Loss of VHL leads to a large increase in acute inflammatory responses. Our results show that HIF-1α is essential for the regulation of glycolytic capacity in myeloid cells: when HIF-1α is absent, the cellular ATP pool is drastically reduced. The metabolic defect results in profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing. This role for HIF-1α demonstrates its direct regulation of survival and function in the inflammatory microenvironment.
PMCID: PMC4480774  PMID: 12628185
8.  Classical Flt3L-dependent dendritic cells control immunity to protein vaccine 
The Journal of Experimental Medicine  2014;211(9):1875-1891.
Protective immunity to protein vaccines is controlled by Flt3L-dependent classical LN-resident dendritic cells, and dampened by migratory dendritic cells.
DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin+ DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs.
PMCID: PMC4144735  PMID: 25135299
9.  ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8+ T Cells 
Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α+ DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α+ DCs in VDLNs, whereas Langerin+ DCs and CD8α− DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α+ DCs in vivo for efficient induction of CTL responses.
PMCID: PMC4285562  PMID: 21613613
10.  BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups 
BRAF-V600E expression is identified in hematopoietic progenitor and precursor myeloid dendritic cells in patients with high-risk LCH, and enforced expression of BRAF-V600E in CD11c+ cells recapitulates a high-risk LCH-like phenotype in mice.
Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207+ dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207+ DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c+ and CD14+ fractions and in bone marrow (BM) CD34+ hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207+ DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.
PMCID: PMC3978272  PMID: 24638167
11.  Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin 
Nature immunology  2012;13(8):744-752.
Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1hi monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.
PMCID: PMC4115277  PMID: 22729248
12.  Multicolor fate mapping of Langerhans cell homeostasis 
The Journal of Experimental Medicine  2013;210(9):1657-1664.
The adult epidermal Langerhans cell network is formed by adjacent proliferative units composed of dividing cells and their terminally differentiated daughter cells.
Langerhans cells (LCs) constitute a network of immune sentinels in the skin epidermis that is seeded during embryogenesis. Whereas the development of LCs has been extensively studied, much less is known about the homeostatic renewal of adult LCs in “nonmanipulated” animals. Here, we present a new multicolor fluorescent fate mapping system and quantification approach to investigate adult LC homeostasis. This novel approach enables us to propose and provide evidence for a model in which the adult epidermal LC network is not formed by mature coequal LCs endowed with proliferative capabilities, but rather constituted by adjacent proliferative units composed of “dividing” LCs and their terminally differentiated daughter cells. Altogether, our results demonstrate the general utility of our novel fate-mapping system to follow cell population dynamics in vivo and to establish an alternative model for LC homeostasis.
PMCID: PMC3754858  PMID: 23940255
13.  Alveolar epithelial cells orchestrate DC function in murine viral pneumonia 
The Journal of Clinical Investigation  2012;122(10):3652-3664.
Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSF–dependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia.
PMCID: PMC3461909  PMID: 22996662
14.  IL-10 Suppression of NK/DC Crosstalk Leads to Poor Priming of MCMV-Specific CD4 T Cells and Prolonged MCMV Persistence 
PLoS Pathogens  2012;8(8):e1002846.
IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10−/− mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.
Author Summary
Cytomegalovirus (CMV) infections are very widespread in mammalian hosts. Despite the fact that CMVs are usually well controlled by the immune system, they cause persistent life-long infection and have evolved a number of strategies to effectively modulate or hide from host immunity. Since the establishment of an immunosuppressive environment favors virus persistence, IL-10 is one of the host targets that CMVs actively use to tune the virus-host balance toward their own benefit, resulting in prolonged virus persistence and hence increased chance for horizontal transmission. Here, we delineate the mechanisms of how IL-10 exerts its powerful immune-suppressing function in the context of murine cytomegalovirus (MCMV) infection. We found that IL-10 specifically restrains the priming of MCMV-specific CD4 T cell responses by suppressing dendritic cell (DC) - natural killer cell (NK) crosstalk during acute MCMV infection. Target molecules mediating this bi-directional crosstalk between DCs and NK cells were the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 and all of them were markedly suppressed by IL-10. A consequence resulting from this impeded DC-NK cross-talk by IL-10, leading to poor priming of MCMV-specific CD4 T cell responses was increased lytic CMV persistence and reduced development of host tissue damage. Our study indicates that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive crosstalk, thereby imparting on the ensuing virus-host balance for the benefit of both the virus and the host.
PMCID: PMC3410900  PMID: 22876184
15.  Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome 
The Journal of Experimental Medicine  2011;208(13):2607-2613.
Epidermal LCs but not dermal DCs take up skin surface protein through intact tight junctions and mediate IgG1 antibody responses to bacterial antigen, conferring protective immunization.
Epidermal Langerhans cells (LCs) extend dendrites through tight junctions (TJs) to survey the skin surface, but their immunological contribution in vivo remains elusive. We show that LCs were essential for inducing IgG1 responses to patch-immunized ovalbumin in mice that lacked skin dendritic cell subsets. The significance of LC-induced humoral responses was demonstrated in a mouse model of staphylococcal scalded skin syndrome (SSSS), a severe blistering disease in which the desmosomal protein Dsg1 (desmoglein1) is cleaved by Staphylococcus aureus–derived exfoliative toxin (ET). Importantly, ET did not penetrate TJs, and patch immunization did not alter epidermal integrity. Nevertheless, neutralizing anti-ET IgG1 was induced after patch immunization and abolished upon LC depletion, indicating that antigen capture through TJs by LCs induced humoral immunity. Strikingly, the ET-patched mice were protected from developing SSSS after intraperitoneal ET challenge, whereas LC-depleted mice were susceptible to SSSS, demonstrating a vital role for LC-induced IgG1 in systemic defense against circulating toxin in vivo. Therefore, LCs elicit humoral immunity to antigens that have not yet violated the epidermal barrier, providing preemptive immunity against potentially pathogenic skin microbes. Targeting this immunological process confers protection with minimal invasiveness and should have a marked impact on future strategies for development of percutaneous vaccines.
PMCID: PMC3244045  PMID: 22143886
16.  Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate immunization requires aggregation, type I IFN, and multiple DC subsets 
The Journal of Clinical Investigation  2011;121(5):1782-1796.
The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8–DEC205+CD103–CD326– langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell–mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines.
PMCID: PMC3083762  PMID: 21540549
17.  Keratinocytes function as accessory cells for presentation of endogenous antigen expressed in the epidermis 
The precise contribution(s) of skin dendritic cells (DCs) to immune responses in the skin has not been well delineated. We developed an intradermal (i.d.) injection model in which CD8+ T (OT-I) cells that express ovalbumin (OVA)-peptide-specific T-cell receptors (Vα2/Vβ5) are delivered directly to the dermis of transgenic (Tg) mice expressing OVA in the epidermis. Following i.d. injection, these mice reliably develop skin GvHD by day 7. To determine the relative contribution of LCs to the ensuing graft-versus-host (GvHD) - like reaction, we generated K14-OVA × Langerin-Diphtheria-Toxin-Receptor (Langerin-DTR) Tg mice to allow conditional ablation of LCs in the epidermis. To delineate the role of dermal dendritic cells (dDCs) in the reaction we also generated K14-OVA Tg chimeras using beta-2-microglobulin-deficient (b2m) congenic donor bone marrow cells. Dermal DCs in these mice cannot present OVA to OT-I cells while the LC are antigen presentation competent. Unexpectedly, OT-I cell injection into diphtheria toxin (DT)-treated b2m→K14−OVA × Langerin-DTR Tg mice resulted in skin GvHD. Thus, in vivo, both LC and dDC appear to be dispensable for induction of keratinocyte-directed, CD8-mediated effector immune responses. Furthermore and surprisingly, OVA-expressing epidermal cells depleted of LCs that could not initiate allogeneic epidermal lymphocyte reactions, activated naïve OT-I cells in vitro. These results indicate that keratinocytes may function as accessory cells-competent to prime naïve skin-reactive T cells.
PMCID: PMC2784095  PMID: 19554018
Langerhans cells; Dendritic cells; GvHD; Langerin KO mice
18.  Langerhans cells and more: langerin-expressing dendritic cell subsets in the skin 
Immunological reviews  2010;234(1):120-141.
Langerhans cells (LCs) are antigen-presenting dendritic cells (DCs) that reside in epithelia. The best studied example is the LC of the epidermis. By electron microscopy, their identifying feature is the unique rod- or tennis racket-shaped Birbeck granule. The phenotypic hallmark is their expression of the C-type lectin receptor langerin/CD207. Langerin, however, is also expressed on a recently discovered population of DC in the dermis and other tissues of the body. These ‘dermal langerin+ dendritic cells’ are unrelated to LCs. The complex field of langerin-negative dermal DCs is not dealt with here. In this article, we briefly review the history, ontogeny, and homeostasis of LCs. More emphasis is laid on the discussion of functional properties in vivo. Novel models using genetically engineered mice are contributing tremendously to our understanding of the role of LCs in eliciting adaptive immune responses against pathogens or tumors and in inducing and maintaining tolerance against self antigens and innocuous substances in vivo. Also, innate effector functions are increasingly being recognized. Current activities in this area are reviewed, and possibilities for future exploitation of LC in medicine, e.g. for the improvement of vaccines, are contemplated.
PMCID: PMC2907488  PMID: 20193016
antigen presentation; cell ablation; dendritic cells; immunogenicity; Langerhans cells; langerin/CD207
19.  Dual therapeutic efficacy of vinblastine as a unique chemotherapeutic agent capable of inducing dendritic cell maturation 
Cancer research  2009;69(17):6987-6994.
Our recent unbiased functional screen of 54 chemotherapeutic drugs unveiled striking heterogeneity in their effects on dendritic cells (DCs). Most notably, vinblastine (VBL) was found to induce phenotypic and functional maturation of DCs in vitro. Here we sought to determine whether VBL exhibits “dual” therapeutic efficacy in living animals by directly killing tumor cells and by boosting host immunity via DC maturation. Local injection of VBL in a low dose into the skin of C57BL/6 mice induced in situ maturation of epidermal Langerhans cells. When co-injected with a model antigen, ovalbumin (OVA), VBL enhanced OVA-specific cellular and humoral immune responses. When injected directly into the OVA cDNA-transduced E.G7 tumors, VBL augmented clonal expansion of OVA-reactive CD8 T cells and CTL activities. In B16 melanoma model, intra-tumor VBL injection induced apoptosis of melanoma cells, phenotypic maturation of tumor-infiltrating DCs, and significant CTL activities. Although complete clearance was never achieved, growth kinetic of B16 melanoma was markedly reduced in C57BL/6 mice by intra-tumor VBL injection. Importantly, the same treatment was far less efficacious in immuno-compromised SCID mice, indicating the requirement of intact host immunity. Our results introduce a new concept that VBL may be used to design “immuno-stimulatory” chemotherapy regimens.
PMCID: PMC2737094  PMID: 19706755
chemotherapy; vinblastine; host immunity; dendritic cell
20.  Nicotinic acid– and monomethyl fumarate–induced flushing involves GPR109A expressed by keratinocytes and COX-2–dependent prostanoid formation in mice 
The Journal of Clinical Investigation  2010;120(8):2910-2919.
The antidyslipidemic drug nicotinic acid and the antipsoriatic drug monomethyl fumarate induce cutaneous flushing through activation of G protein–coupled receptor 109A (GPR109A). Flushing is a troublesome side effect of nicotinic acid, but may be a direct reflection of the wanted effects of monomethyl fumarate. Here we analyzed the mechanisms underlying GPR109A-mediated flushing and show that both Langerhans cells and keratinocytes express GPR109A in mice. Using cell ablation approaches and transgenic cell type–specific GPR109A expression in Gpr109a–/– mice, we have provided evidence that the early phase of flushing depends on GPR109A expressed on Langerhans cells, whereas the late phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the first phase of flushing was blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase was sensitive to a selective COX-2 inhibitor. Both monomethyl fumarate and nicotinic acid induced PGE2 formation in isolated keratinocytes through activation of GPR109A and COX-2. Thus, the early and late phases of the GPR109A-mediated cutaneous flushing reaction involve different epidermal cell types and prostanoid-forming enzymes. These data will help to guide new efficient approaches to mitigate nicotinic acid–induced flushing and may help to exploit the potential antipsoriatic effects of GPR109A agonists in the skin.
PMCID: PMC2912194  PMID: 20664170
21.  The lung vascular filter as a site of immune induction for T cell responses to large embolic antigen 
The Journal of Experimental Medicine  2009;206(12):2823-2835.
The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 µm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11chi cells or Ly6C/G+ cells but restored upon adoptive transfer of Ly6Chi monocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.
PMCID: PMC2806611  PMID: 19858325
22.  Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells 
The Journal of Experimental Medicine  2008;205(7):1621-1634.
Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.
PMCID: PMC2442640  PMID: 18591406
23.  Macrophages and neutrophils are the targets for immune suppression by glucocorticoids in contact allergy 
Journal of Clinical Investigation  2007;117(5):1381-1390.
Glucocorticoids (GCs) are widely used in the treatment of allergic skin conditions despite having numerous side effects. Here we use Cre/loxP-engineered tissue- and cell-specific and function-selective GC receptor (GR) mutant mice to identify responsive cell types and molecular mechanisms underlying the antiinflammatory activity of GCs in contact hypersensitivity (CHS). CHS was repressed by GCs only at the challenge phase, i.e., during reexposure to the hapten. Inactivation of the GR gene in keratinocytes or T cells of mutant mice did not attenuate the effects of GCs, but its ablation in macrophages and neutrophils abolished downregulation of the inflammatory response. Moreover, mice expressing a DNA binding–defective GR were also resistant to GC treatment. The persistent infiltration of macrophages and neutrophils in these mice is explained by an impaired repression of inflammatory cytokines and chemokines such as IL-1β, monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and IFN-γ–inducible protein 10. In contrast TNF-α repression remained intact. Consequently, injection of recombinant proteins of these cytokines and chemokines partially reversed suppression of CHS by GCs. These studies provide evidence that in contact allergy, therapeutic action of corticosteroids is in macrophages and neutrophils and that dimerization GR is required.
PMCID: PMC1849982  PMID: 17446934
24.  Inhibition of NF-κB activation in macrophages increases atherosclerosis in LDL receptor–deficient mice 
Journal of Clinical Investigation  2003;112(8):1176-1185.
Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-κB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-κB activation in macrophages during atherogenesis, we used LDL receptor–deficient mice with a macrophage-restricted deletion of IκB kinase 2 (IKK2), which is essential for NF-κB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-κB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-κB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.
PMCID: PMC213488  PMID: 14561702

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