Biomedical subjects

Meredith O'Keeffe

Publications and source records attributed to Meredith O'Keeffe.

At least 19 recordsLinked to original sources

PubMed2006

Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells.

The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-gamma (IFN-gamma) or IFN-alpha was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN-gamma production. However, an early developmental form of the CD4-8+ cDC subtype, and the Ly6C- Ly49Q- pDC subtype, both were able to produce moderate amounts of IFN-gamma, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C- Ly49Q- pDC subtypes were effective producers of IFN-alpha. However, IKDCs, which efficiently produced IFN-gamma and showed immediate cytotoxicity on NK target cells, did not produce IFN-alpha under these conditions.

Animals↗

PubMed2006

Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes.

Immediate precursors of the many subtypes of dendritic cells (DCs) remain obscure. Here we purified a splenic precursor population that produced all splenic CD8+ and CD8- conventional DCs (cDCs) but not plasmacytoid DCs or other lineages. This 'pre-cDC' population included cells 'precommitted' to form either CD8+ or CD8- cDCs. The pre-cDCs, which comprised 0.05% of splenocytes, expressed a CD11c(int) CD45RA(lo) CD43(int) SIRP-alpha(int) CD4- CD8- major histocompatibility complex class II-negative surface phenotype. The pre-cDCs were not monocytes. Monocytes generated few cDCs in steady-state recipient mice. However, when transferred into mice with an inflammatory milieu dependent on granulocyte-macrophage colony-stimulating factor, monocytes produced a distinct type of splenic DC. Thus, the inflammatory status of the host influences the developmental origin and type of DC present in lymphoid tissues.

Animals↗

PubMed2006

The long-term but not the short-term antiviral effect of IFN-alpha depends on Flt3 ligand and pDC.

The cooperation between IFN-alpha/beta and FL, the ligand of Fms-like tyrosine kinase 3 (Flt3), plays an important role in the defense against herpes simplex virus type 1 (HSV-1) in neonates. Treatment of neonatal mice with recombinant IFN-alpha has a short-term, FL-independent and a long-term, FL-dependent protective effect against HSV-1. In mice lacking FL, neonatal resistance against HSV-1 is very low and DC numbers in the spleen are reduced. The treatment of these mice with rIFN-alpha at day 6 resulted in an increased resistance against infection with HSV-1 at day 7. In C57BL/6 mice, treatment with rIFN-alpha at birth induced both FL and plasmacytoid DC (pDC), resulting in enhanced resistance against HSV-1 at day 7. In contrast, in mice lacking FL, IFN-alpha treatment at birth did not influence the splenic cell composition and had no effect on viral protection. The transfer of pDC to mice lacking FL enhanced viral resistance. Therefore, the induction and function of pDC, normally controlled by IFN-alpha/beta and FL, are decisive for viral resistance in neonatal mice.

Animals↗

PubMed2006

Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production.

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.

Adenoviridae↗

PubMed2005

Distinct roles for the NF-kappaB1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals.

Reticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-kappaB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type-specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-kappaB1 and C-Rel, Rel/NF-kappaB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1(-/-)C-Rel(-/-) cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1(-/-)C-Rel(-/-) pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9-mediated death of Nfkb1(-/-)C-Rel(-/-) pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-x(L)) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-kappaB transcription factors independently regulate TLR-9-mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-kappaB1 and c-Rel, while largely dispensable for TLR-9-induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.

Animals↗

PubMed2005

Cutting edge: generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures.

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) cDC subsets were present, and these subsets showed equivalent properties to splenic CD8(+) and CD8(-) cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-alpha; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-alpha, MIP-1alpha, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24(high) subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.

Animals↗

PubMed2005

Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines.

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 mug of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.

Animals↗

PubMed2005

Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development.

A dendritic cell (DC) imbalance with a marked deficiency in CD4- 8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4- 8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4- 8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tyrosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.

Animals↗

PubMed2005

Activation of plasmacytoid dendritic cells.

Four years after the discovery of mouse plasmacytoid dendritic cells (pDC), pDC are still very much an 'enigmatic' cell type. It is clear that pDC are potent producers of type I IFN in response to viral, bacterial and even mammalian nucleotides. The role that they play in vivo before and after activation is still under scrutiny. This review concentrates on the pathways to activation of pDC, examining the activating ligands, receptors and signalling molecules that are known to be involved, and the relevance of these activation pathways to human disease.

Animals↗

PubMed2004

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways.

Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-alpha, and whether the production of IFN-alpha in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-alpha in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-alpha was produced by other cell types. Moreover, IFN-alpha production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-alpha in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-alpha production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-alpha in response to HSV-1 via both TLR9-independent and -dependent pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Development of the dendritic cell system during mouse ontogeny.

Based on the view that the efficacy of the immune system is associated with the maturation state of the immune cells, including dendritic cells (DC), we investigated the development and functional potential of conventional DC and plasmacytoid pre-DC (p-preDC) in spleen, thymus, and lymph nodes during mouse development. Both CD11c+ DC and CD45RA+ p-preDC were detected in small numbers in the thymus as early as embryonic day 17. The ratio of DC to thymocytes reached adult levels by 1 wk, although the normal CD8alpha+ phenotype was not acquired until later. Significant, but low, numbers of DC and p-preDC were present in the spleen of day 1 newborn mice. The full complement of DC and p-preDC was not acquired until 5 wk of age. The composition of DC populations in the spleen of young mice differed significantly from that found in adult mice, with a much higher percentage (50-60% compared with 20-25%) of the CD4-CD8alpha+ DC population and a much lower percentage (10-20% compared with 50-60%) of the CD4+CD8alpha- DC population. Although the p-preDC of young mice showed a capacity to produce IFN-alpha comparable with that of adult mice, the conventional DC of young mice were less efficient than those of their adult counterparts in IL-12p70 and IFN-gamma production and in Ag presentation. These results suggest that the neonatal DC system is not fully developed, and innate immunity is the dominant form of response. The complete DC system required for adaptive immunity in the mouse is not fully developed until 5 wk of age.

Aging↗

PubMed2004

Treatment of neonatal mice with Flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced IL-12 and IFN-alpha production.

Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8alpha and MHC class II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-alpha. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines.

Animals↗

PubMed2004

Differential production of inflammatory chemokines by murine dendritic cell subsets.

Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate naïve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4(-)CD8(-) (DN), CD4(+)CD8(-) (CD4+) or CD4(-)CD8(+) (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1alpha and Mip-1beta in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes.

Animals↗

PubMed2004

Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes.

Using a metacyclic promastigote ear infection model of cutaneous leishmaniasis, we examined the phenotype, parasite load, and cytokine production of dendritic cells in the skin and draining lymph nodes of resistant C57BL/6J and susceptible BALB/c mice. Five dendritic cell populations were isolated from the skin and lymph nodes, and the main difference between the groups of mice was an increased number of plasmacytoid dendritic cells in the lymph nodes of the susceptible mice. Although similar cell types were present in the skin emigrants of both strains, there was a 10-fold larger number of cells in BALB/c mouse skin early in infection than in C57BL/6J mouse skin. None of the dendritic cells in the lymph nodes harbored parasites until 3 weeks after infection, with the Langerhans cells having the largest load and the plasmacytoid dendritic cells having the smallest load but the longest lasting infection. Although parasites could be detected in the lymph nodes a few hours after infection, none of the skin emigrants harbored parasites, indicating that they are not the vehicle that ferries the parasites from the skin to the lymph nodes. The presence of larger numbers of plasmacytoid cells in infected BALB/c mice, the more protracted infection of these cells, and their production of alpha interferon point to a complex and important role for the plasmacytoid cells in leishmaniasis.

Animals↗

PubMed2003

The lymphoid past of mouse plasmacytoid cells and thymic dendritic cells.

There has been controversy over the possible lymphoid origin of certain dendritic cell (DC) subtypes. To resolve this issue, DC and plasmacytoid pre-DC isolated from normal mouse tissues were analyzed for transient (mRNA) and permanent (DNA rearrangement) markers of early stages of lymphoid development. About 27% of the DNA of CD8(+) DC from thymus, and 22-35% of the DNA of plasmacytoid pre-DC from spleen and thymus, was found to contain IgH gene D-J rearrangements, compared with 40% for T cells. However, the DC DNA did not contain IgH gene V-D-J rearrangements nor T cell Ag receptor beta gene D-J rearrangements. The same DC lineage populations containing IgH D-J rearrangements expressed mRNA for CD3 chains, and for pre-T alpha. In contrast, little of the DNA of the conventional DC derived from spleen, lymph nodes, or skin, whether CD8(+) or CD8(-), contained IgH D-J rearrangements and splenic conventional DC expressed very little CD3 epsilon or pre-T alpha mRNA. Therefore, many plasmacytoid pre-DC and thymic CD8(+) DC have shared early steps of development with the lymphoid lineages, and differ in origin from conventional peripheral DC.

Animals↗

PubMed2003

CD8alpha+ mouse spleen dendritic cells do not originate from the CD8alpha- dendritic cell subset.

Although previous studies had indicated that the CD8alpha- and CD8alpha+ subtypes of murine dendritic cells (DCs) differ in immediate origin, a recent study found that intravenous transfer of CD8alpha- DCs led to CD8alpha+ DCs in the spleen several days later, suggesting a direct precursor-product relationship. We have repeated these experiments with a balance sheet approach. We find that though a few CD8alpha+ DCs can be generated in such experiments, this is a rare event and could be the result of a contaminant precursor. Most of the immediate precursors of CD8alpha+ DCs are cells that lack the phenotype of a recognizable DC. CD8alpha- DCs and CD8alpha+ DCs are not precursor-product related, though these sublineages may be connected further upstream.

Animals↗

PubMed2003

Flt3 ligand-treated neonatal mice have increased innate immunity against intracellular pathogens and efficiently control virus infections.

Flt-3 ligand (FL), a hematopoetic growth factor, increases the number of dendritic cells (DCs), B cells, and natural killer cells in adult mice but the effect in neonates was unknown. We show that FL treatment of newborn mice induced a >100-fold increase in the innate resistance against infection with herpes simplex virus type 1 and Listeria monocytogenes. This resistance required interferon (IFN)-alpha/beta for viral and interleukin (IL)-12 for bacterial infections. Long-term survival after viral but not bacterial infection was increased approximately 100-fold by FL treatment. After treatment, CD11c(+)/major histocompatibility complex type II(+) and CD11c(+)/B220(+) DC lineage cells were the only cell populations increased in the spleen, liver, peritoneum, and skin. DC induction was independent of IFNs, IL-2, -4, -7, -9, -15, and mature T and B cells. The data suggest that FL increases the number of DCs in neonates and possibly in other immune-compromised individuals, which in turn improves IFN-alpha/beta- and IL-12-associated immune responses.

Animals↗

PubMed2002

Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8(+) dendritic cells only after microbial stimulus.

The CD45RA(hi)CD11c(int) plasmacytoid predendritic cells (p-preDCs) of mouse lymphoid organs, when stimulated in culture with CpG or influenza virus, produce large amounts of type I interferons and transform without division into CD8(+)CD205(-) DCs. P-preDCs express CIRE, the murine equivalent of DC-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN). P-preDCs are divisible by CD4 expression into two subgroups differing in turnover rate and in response to Staphylococcus aureus. The kinetics of bromodeoxyuridine labeling and the results of transfer to normal recipient mice indicate that CD4(-) p-preDCs are the immediate precursors of CD4(+) p-preDCs. Similar experiments indicate that p-preDCs are normally long lived and are not the precursors of the short-lived steady-state conventional DCs. However, in line with the culture studies on transfer to influenza virus-stimulated mice the p-preDCs transform into CD8(+)CD205(-) DCs, distinct from conventional CD8(+)CD205(+) DCs. Hence as well as activating preexistant DCs, microbial infection induces a wave of production of a new DC subtype. The functional implications of this shift in the DC network remain to be determined.

Animals↗