Constitutive plasmacytoid dendritic cell migration to the splenic white pulp is cooperatively regulated by CCR7- and CXCR4-mediated signaling

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.     

IFN-alphabeta-mediated inflammatory responses and antiviral defense in liver is TLR9-independent but MyD88-dependent during murine cytomegalovirus infection

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.     

Plasmacytoid dendritic cells of different origins have distinct characteristics and function: studies of lymphoid progenitors versus myeloid progenitors

Plasmacytoid dendritic cells (pDCs) play a central role in host innate and adaptive immunity and are thought to be of lymphoid origin. However, in IL-7Ralpha-/- mice, which are deficient in T and B lymphocytes, pDCs are still found in lymphoid organs, which suggests that there is a lymphoid-independent pathway for the development of pDCs. Previous work has demonstrated that pDCs originate from both lymphoid and myeloid progenitors (MPs). However, it is not clear whether the function of pDCs is different relative to their origin. In an effort to compare the characteristics and functions between pDCs generated from different progenitors, we performed adoptive transfer studies using highly enriched populations of common lymphoid progenitors (CLPs) and MPs from the bone marrow of control mice and examined their potential and developmental kinetics for the generation of pDCs. Interestingly, although CLPs were polarized to generate pDCs, MPs were polarized to generate conventional dendritic cells and the kinetics of pDC generation from MPs was reached earlier than from CLPs. Furthermore, CLPs have the potential to generate more pDCs on a per cell basis. Moreover, MP-derived pDCs produce relatively higher levels of IFN-alpha than CLP-derived pDCs following CpG stimulation. These data indicate that MPs are multipotential and have the capacity to develop into not only myeloid cells, but also pDCs, which have distinct characteristics and function compared to that of lymphoid origin and, therefore, imply a more important role for MP-derived pDCs in conditions where the function of lymphoid progenitors is impaired or compromised.     

DAP12 Overexpression Induces Osteopenia and Impaired Early Hematopoiesis

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis.     

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.     

Murine plasmacytoid pre-dendritic cells generated from Flt3 ligand-supplemented bone marrow cultures are immature APCs

The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c(+)B220(+) pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c(+)B220(+) pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c(+)B220(+) pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c(+)B220(-) dendritic cells and were poor T cell stimulators. Stimulation of CD11c(+)B220(+) pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8alpha expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-alpha and IL-12 secretion, and to the acquisition of priming capacities for both CD4(+) and CD8(+) OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.     

Cutting edge: Overlapping functions of TLR7 and TLR9 for innate defense against a herpesvirus infection

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-alpha/beta in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-alpha/beta production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-alpha/beta, IL-12p40, and TNF-alpha production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-alpha/beta levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.     

Absence of Siglec-H in MCMV Infection Elevates Interferon Alpha Production but Does Not Enhance Viral Clearance

Plasmacytoid dendritic cells (pDCs) express the I-type lectin receptor Siglec-H and produce interferon α (IFNα), a critical anti-viral cytokine during the acute phase of murine cytomegalovirus (MCMV) infection. The ligands and biological functions of Siglec-H still remain incompletely defined in vivo. Thus, we generated a novel bacterial artificial chromosome (BAC)-transgenic “pDCre” mouse which expresses Cre recombinase under the control of the Siglec-H promoter. By crossing these mice with a Rosa26 reporter strain, a representative fraction of Siglec-H⁺ pDCs is terminally labeled with red fluorescent protein (RFP). Interestingly, systemic MCMV infection of these mice causes the downregulation of Siglec-H surface expression. This decline occurs in a TLR9- and MyD88-dependent manner. To elucidate the functional role of Siglec-H during MCMV infection, we utilized a novel Siglec-H deficient mouse strain. In the absence of Siglec-H, the low infection rate of pDCs with MCMV remained unchanged, and pDC activation was still intact. Strikingly, Siglec-H deficiency induced a significant increase in serum IFNα levels following systemic MCMV infection. Although Siglec-H modulates anti-viral IFNα production, the control of viral replication was unchanged in vivo. The novel mouse models will be valuable to shed further light on pDC biology in future studies.     

Increased and long-term generation of dendritic cells with reduced function from IL-6-deficient bone marrow

The importance of IL-6 in dendritic cell (DC) development and function has not been well defined. To establish the role of IL-6, we studied bone marrow-derived DC (BMDC) and freshly isolated splenic DC from IL-6(-/-)-transgenic mice. We found that although IL-6(-/-) bone marrow had a similar composition to that of wild-type (WT) mice, it generated up to 10 times more DC when cultured in GM-CSF. The difference persisted even when IL-6(-/-) and WT bone marrow were cultured together, excluding the possibility that the effects were simply due to different cytokine microenvironments. In comparison to WT BMDC, IL-6(-/-) BMDC captured at least as much Ag, had an equivalent surface phenotype, and matured similarly in response to LPS or CpG. However, IL-6(-/-) BMDC induced less T cell allostimulation and Ag-specific T cell activation, but only the former was related to their inability to generate IL-6. Although WT bone marrow cultures died within 4 wk, IL-6(-/-) cultures continued to generate BMDC for >120 days, although the BMDC became immature and less functional. In vivo, we found that IL-6(-/-) mice had similar numbers and types of splenic DC as WT mice, both normally and after treatment with either Flt-3 ligand or GM-CSF. These findings demonstrate that IL-6 has profound effects on DC development in vitro, although the number and subtype composition of DC are unaffected by the absence of IL-6 in vivo. Furthermore, secretion of IL-6 is critical to certain DC functions.     

IL-27 production and STAT3-dependent upregulation of B7-H1 mediate immune regulatory functions of liver plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are highly specialized APCs that, in addition to their well-recognized role in anti-viral immunity, also regulate immune responses. Liver-resident pDCs are considerably less immunostimulatory than those from secondary lymphoid tissues and are equipped to promote immune tolerance/regulation through various mechanisms. IL-27 is an IL-12 family cytokine that regulates the function of both APCs and T cells, although little is known about its role in pDC immunobiology. In this study, we show that mouse liver pDCs express higher levels of IL-27p28 and EBV-induced protein 3 (Ebi3) compared with those of splenic pDCs. Both populations of pDCs express the IL-27Rα/WSX-1; however, only liver pDCs significantly upregulate expression of the coregulatory molecule B7 homolog-1 (B7-H1) in response to IL-27. Inhibition of STAT3 activation completely abrogates IL-27-induced upregulation of B7-H1 expression on liver pDCs. Liver pDCs treated with IL-27 increase the percentage of CD4(+)Foxp3(+) T cells in MLR, which is dependent upon expression of B7-H1. pDCs from Ebi3-deficient mice lacking functional IL-27 show increased capacity to stimulate allogeneic T cell proliferation and IFN-γ production in MLR. Liver but not spleen pDCs suppress delayed-type hypersensitivity responses to OVA, an effect that is lost with Ebi3(-/-) and B7-H1(-/-) liver pDCs compared with wild-type liver pDCs. These data suggest that IL-27 signaling in pDCs promotes their immunoregulatory function and that IL-27 produced by pDCs contributes to their capacity to regulate immune responses in vitro and in vivo.     

Effective TRAIL-based immunotherapy requires both plasmacytoid and CD8α dendritic cells

It is now appreciated that there are distinct subsets of dendritic cells (DC) with specialized functions. Plasmacytoid DC (pDC) and CD8α DC can contribute to the priming, activation and function of antitumor CD8 T cells; however, their specific roles and necessity in stimulating antitumor immunity are not clearly understood. We examined the importance of pDC and CD8α DC during immunotherapy of an orthotopic model of metastatic renal cell carcinoma. Immunotherapy that utilizes a recombinant adenovirus encoding tumor necrosis factor-related apoptosis-inducing ligand (Ad5-TRAIL) in combination with an immunostimulatory CpG-containing oligodeoxynucleotide (CpG) resulted in the clearance of primary and metastatic tumors in wild-type (WT) replete BALB/c mice and prolonged survival. In comparison, mice deficient in either pDC (accomplished using a depleting mAb specific for PDCA1) or CD8α DC (through utilization of CD8α DC-deficient Batf3 ^?/? BALB/c mice) had uncontrolled tumor growth and high mortality after Ad5-TRAIL/CpG administration. The ineffectiveness of Ad5-TRAIL/CpG therapy in the anti-PDCA1-treated and Batf3 ^?/? BALB/c mice was marked by an altered activation phenotype of the DC, as well as significantly reduced expression of type I IFN-stimulated genes and IL-15/IL-15R complex production. In addition, pDC-depleted and Batf3 ^?/? BALB/c mice had significantly decreased effector CD8 T cell infiltration in the primary tumor site compared with WT mice after therapy. These data collectively suggest that pDC and CD8α DC carry out independent, but complementary, roles that are necessary to initiate an efficacious antitumor immune response after Ad5-TRAIL/CpG therapy.     

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.     

A role for IFNgamma in differential superantigen stimulation of conventional versus plasmacytoid DCs

Superantigens (SAgs) are known to play a role in food poisoning, toxic shock syndrome and have been identified as a potential mediator of autoimmunity. Although much is known about the effects of SAgs on T cells, by comparison few studies have investigated how SAgs influence innate immune cells. In particular no study has examined how SAgs affect murine plasmacytoid dendritic cells (pDC). We report that in vivo administration of staphylococcal enterotoxin A (SEA) increased the number of pDCs in secondary lymphoid organs, and induced CD86 and CD40 expression. Similar to SEA activation of conventional DCs (cDCs), pDCs relied on T cells, but not on CD40. Nonetheless, pDCs strictly required IFNgamma for upregulation of CD86 and CD40, but cDCs did not depend upon IFNgamma for activation. Further, even though IFNgamma deficient pDCs were not activated by SEA, they were still capable of producing wild-type levels of IFNalpha in response to CpG oligodeoxynucleotide (ODN). The source of IFNgamma for pDC activation was not T cells, nor did pDCs themselves have to synthesize or bind IFNgamma, but the presence of IFNgamma was essential. After SEA stimulation, IFNgamma deficient mice fail to induce expression of the pDC dependent chemokines CXCL9, and demonstrated a defect in recruitment of pDCs to marginal zones of lymphoid organs. Thus, SEA exerts its combined effect on pDC activation, recruitment and chemokine induction through the action of IFNgamma. This fundamental dichotomy of the effects of SAgs on pDCs versus cDCs show how a non-PAMP from bacteria, can selectively and indirectly stimulate innate cell subpopulations much in the same way that differential TLR expression influences cells of the innate immune system.     

Rat plasmacytoid dendritic cells are an abundant subset of MHC class II+ CD4+CD11b-OX62- and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG

We have identified in the rat a new subset of MHC class II(+) CD4(+)CD3(-)CD11b(-) leukocytes that produce high amounts of type I IFN upon viral stimulation and that appeared homologous to plasmacytoid DC (pDC) previously described in humans and mice. These cells exhibited the following phenotype: CD5(+),CD90(+),CD45R(+),CD45RC(+),CD11c(-),CD161a(+),CD200(+),CD172a(+),CD32(+),CD86(+). Rat pDC did not express the DC-specific marker OX62 and were more abundant in the spleen than the classical CD4(+) and CD4(-) subsets of OX62(+)CD11b(+) DC we previously described that produced very little, if any, type I IFN. Spleen pDC exhibited an undifferentiated morphology and rapidly died in vitro, but showed extensive dendrite formation, survival, maturation, and moderate type I IFN production upon stimulation by oligonucleotides containing type B CpG motifs (CpG ODN). Type A CpG ODN and CD40 ligand induced pDC to produce large amounts of type I IFN, but did not promote maturation. CpG ODN and CD40 ligand, but not influenza virus, induced IL-12p40 and IL-6 secretion. Spleen pDC did not produce IL-12p70, TNF-alpha, IL-1beta, or IL-10 using these stimulation conditions. Correlating with their strong responsiveness to virus and CpG ODN, rat pDC specifically expressed Toll-like receptor 7 and 9 mRNA. Fresh spleen pDC were poor stimulators of allogenic CD4(+) and CD8(+) T cells, but became potent inducers of allogenic T cell proliferation as well as Th1 differentiation after stimulation by type B CpG. Therefore, rat pDC appear very similar to human pDC, indicating that the specific phenotype and functions of pDC have been highly conserved between species.     

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN‐I) during viral infections, in response to triggering of endosomal Toll‐like receptors (TLRs) 7 or 9 by viral single‐stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN‐I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN‐I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN‐I downstream of the TLR9‐to‐MyD88‐to‐IRF7 signaling pathway without requiring IFN‐I positive feedback, high IRF7 expression, or AP3‐driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN‐I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.     

Aberrant DAP12 signaling in the 129 strain of mice: implications for the analysis of gene-targeted mice

NK cells are implicated in antiviral responses, bone marrow transplantation and tumor immunosurveillance. Their function is controlled, in part, through the Ly49 family of class I binding receptors. Inhibitory Ly49s suppress signaling, while activating Ly49s (i.e., Ly49D) activate NK cells via the DAP12 signaling chain. Activating Ly49 signaling has been studied primarily in C57BL/6 mice, however, 129 substrains are commonly used in gene-targeting experiments. In this study, we show that in contrast to C57BL/6 NK cells, cross-linking of DAP12-coupled receptors in 129/J mice induces phosphorylation of DAP12 but not calcium mobilization or cytokine production. Consistent with poor-activating Ly49 function, 129/J mice reject bone marrow less efficiently than C57BL/6 mice. Sequence analysis of receptors and DAP12 suggests no structural basis for inactivity, and both the 129/J and C57BL/6 receptors demonstrate normal function in a reconstituted receptor system. Most importantly, reconstitution of Ly49D in 129/J NK cells demonstrated that the signaling deficit is within the NK cells themselves. These unexpected findings bring into question any NK analysis of 129/J, 129Sv, or gene-targeted mice derived from these strains before complete backcrossing, and provide a possible explanation for the differences observed in the immune response of 129 mice in a variety of models.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.