Separation of Plasmacytoid Dendritic Cells from B-Cell-Biased Lymphoid Progenitor (BLP) and Pre-Pro B Cells Using PDCA-1

B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1⁺Siglec H⁺ plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1⁺ pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP^low/neg pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1⁺ pDCs. Thus, removal of PDCA-1⁺ pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220⁺CD19⁻ fraction demonstrated that AA4.1⁺Ly6D⁺PDCA-1⁻ Pre-pro B cells gave rise to CD19⁺ B cells at high frequency, while PDCA-1⁺ pDCs in this fraction did not. Interestingly, the presence of PDCA-1⁺ pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.     

Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand

Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are critical for initiating and controlling the immune response. However, study of DC, particularly pDC, function is hampered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation preferentially favor the production of cDCs over pDCs. Here, we demonstrated that pDCs could be efficiently generated in vitro from common lymphoid progenitors (CLPs) using Flt3 ligand (FL) in three different culture systems, namely feeder-free, BM-feeder and AC-6-feeder. This was in stark contrast to common DC progenitors (CDPs), in which cDCs were prominently generated under the same conditions. Moreover, the efficiency and function of pDCs generated from these three systems varied. While AC-6 system showed the greatest ability to support pDC development from CLPs, BM-feeder system was able to develop pDCs with better functionality. pDCs could also be expanded in vivo using hydrodynamic gene transfer of FL, which was further enhanced by the combined treatment of FL and IFN-α. Interestingly, IFN-α selectively promoted the proliferation of CLPs and not CDPs, which might contribute to enhanced pDC development. Together, we have defined conditions for in vitro and in vivo generation of pDCs, which may be useful for investigating the biology of pDCs.     

The balance between plasmacytoid DC versus conventional DC determines pulmonary immunity to virus infections

Background

Respiratory syncytial virus (RSV) infects nearly all infants by age 2 and is a leading cause of bronchiolitis. RSV may employ several mechanisms to induce immune dysregulation, including dendritic cell (DC) modulation during the immune response to RSV.

Methods and Findings

Expansion of cDC and pDC by Flt3L treatment promoted an anti-viral response with reduced pathophysiology characterized by decreased airway hyperreactivity, reduced Th2 cytokines, increased Th1 cytokines, and a reduction in airway inflammation and mucus overexpression. These protective aspects of DC expansion could be completely reversed by depleting pDCs during the RSV infection. Expansion of DCs by Flt3L treatment enhanced in CD8+ T cell responses, which was reversed by depletion of pDC.

Conclusions

These results indicate that a balance between cDC and pDC in the lung and its lymph nodes is crucial for the outcome of a pulmonary infection. Increased pDC numbers induced by Flt3L treatment have a protective impact on the nature of the overall immune environment.     

Oral Administration of Lactococcus lactis Subsp. lactis JCM5805 Enhances Lung Immune Response Resulting in Protection from Murine Parainfluenza Virus Infection

When activated by viral infection, plasmacytoid dendritic cells (pDCs) play a primary role in the immune response through secretion of IFN-α. Lactococcus lactis subsp. lactis JCM5805 (JCM5805) is a strain of lactic acid bacteria (LAB) that activates murine and human pDCs to express type I and type III interferons (IFNs). JCM5805 has also been shown to activate pDCs via a Toll-like receptor 9 (TLR9) dependent pathway. In this study, we investigated the anti-viral effects of oral administration of JCM5805 using a mouse model of murine parainfluenza virus (mPIV1) infection. JCM5805-fed mice showed a drastic improvement in survival rate, prevention of weight loss, and reduction in lung histopathology scores compared to control mice. We further examined the mechanism of anti-viral effects elicited by JCM5805 administration using naive mice. Microscopic observations showed that JCM5805 was incorporated into CD11c⁺ immune cells in Peyer’s patches (PP) and PP pDCs were significantly activated and the expression levels of IFNs were significantly increased. Interestingly, nevertheless resident pDCs at lung were not activated and expressions levels of IFNs at whole lung tissue were not influenced, the expressions of anti-viral factors induced by IFNs, such as Isg15, Oasl2, and Viperin, at lung were up-regulated in JCM5805-fed mice compared to control mice. Therefore expressed IFNs from intestine might be delivered to lung and IFN stimulated genes might be induced. Furthermore, elevated expressions of type I IFNs from lung lymphocytes were observed in response to mPIV1 ex vivo stimulation in JCM5805-fed mice compared to control. This might be due to increased ratio of pDCs located in lung were significantly increased in JCM5805 group. Taken together, a specific LAB strain might be able to affect anti-viral immunological profile in lung via activation of intestinal pDC leading to enhanced anti-viral phenotype in vivo.     

Orally administered <Emphasis Type="Italic">Bifidobacterium</Emphasis> triggers immune responses following capture by CD11c<Superscript>+</Superscript> cells in Peyer’s patches and cecal patches

We have investigated the immunomodulatory mechanisms of Bifidobacterium pseudocatenulatum JCM7041 (Bp) as model of probiotics following oral administration to mice. This study was conducted with the aim of clarifying the mechanism of immunomodulation induced by oral administration of probiotic bacteria through elucidation of the detailed mechanism of transfer of orally administered bacterial cells within the body and the interaction between bacterial cells and cells of the immune tissues. We observed the localization of Bp in mice following oral administration, showing that Bp was surrounded by CD11c⁺ cells in Peyer’s patches (PP) and cecal patches (CP). These results indicated that Bp might induce CD11c⁺ cell-mediated immune responses directly. Furthermore, IL-10 and IL-12p40 production by Thy1.2⁻ cells, including CD11c⁺ cells, increased significantly. Production of IL-10 and IL-12p40 by bone marrow-derived dendritic cells (BMDC) was significantly increased by Bp stimulation. These results suggest that oral administration of Bp induces immune responses directly following capture by CD11c⁺ dendritic cells (DCs). Subsequently, we observed oral administration of Bp for 1 week induced IgA and IgA-associated cytokine production by CP and PP cells, suggesting that Bp induced DC-mediated immune responses on CP as well as PP.     

Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model

Splenic plasmacytoid dendritic cells (pDC) possess the capability to harbor live replicative Plasmodium parasite. Isolated splenic pDC from infected mice causes malaria when transferred to naïve mice. Incomplete autophagic degradation might cause poor antigen processing and poor immune response. Induction of autophagic flux by rapamycin treatment led to better prognosis by boosting pDC centered immune response against the pathogen. Splenic pDC from rapamycin-treated infected mice, caused less parasitemia in naïve mice. The downregulation of adhesion with unaltered phagocytic potential of the cells post autophagic induction restricted excessive parasite burden within them. Rapamycin-treated pDC played a better role in antigen presentation. They showed higher expression of co-stimulatory molecules CD80, CD86, DEC205, MHCI. Rapamycin-treated pDC induced CD28 expression on CD8⁺ T cells and suppressed FasL level. This cells also influenced differentiation of effector, memory T cell population. The increase in IL10: TNFα ratio, Treg: Th17 ratio and lowering of myeloid DC: plasmacytoid DC ratio was observed. It shifted the overaggressive inflammation mediated Th1 pathway that is reported to incur host damage, to a better well-balanced cytokine profile exhibiting Th2 pathway. Autophagic flux induction within pDC proved to be beneficial in combating malarial pathogenicity.     

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.     

EBI2 Is a Negative Regulator of Type I Interferons in Plasmacytoid and Myeloid Dendritic Cells

Epstein-Barr virus induced receptor 2 (EBI2), a Gα_i-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b⁺ myeloid cells. Activation of EBI2^−/− pDCs and CD11b⁺ cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, in vivo challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2^−/− mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b⁺ cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.     

Plasmacytoid Dendritic Cells Mediate the Regulation of Inflammatory Type T Cell Response for Optimal Immunity against Respiratory Chlamydia Pneumoniae Infection

Chlamydia pneumoniae (Cpn) infection is a leading cause for a variety of respiratory diseases and has been implicated in the pathogenesis of chronic inflammatory diseases. The regulatory mechanisms in host defense against Cpn infection are less understood. In this study, we investigated the role of plasmacytoid dendritic cells (pDCs) in immune regulation in Cpn respiratory tract infection. We found that in vivo depletion of pDCs increased the severity of infection and lung pathology. Mice depleted of pDC had greater body weight loss, higher lung bacterial burden and excessive tissue inflammation compared to the control mice. Analysis of specific T cell cytokine production pattern in the lung following Cpn infection revealed that pDC depleted mice produced significantly higher amounts of inflammatory cytokines, especially TNF-α, but lower IL-10 compared to the controls. In particular, pDC depleted mice showed pathogenic T cell responses characterized by inflammatory type-1 (CD8 and CD4) and inflammatory Th2 cell responses. Moreover, pDC depletion dramatically reduced CD4 regulatory T cells (Tregs) in the lungs and draining lymph nodes. Furthermore, pDC-T cell co-culture experiments showed that pDCs isolated from Cpn infected mice were potent in inducing IL-10 producing CD4 Tregs. Together, these findings provide in vivo evidence for a critical role of pDCs in homeostatic regulation of immunity during Cpn infection. Our findings highlight the importance of a ‘balanced’ immune response for host protective immunity and preventing detrimental immunopathology during microbial infections.     

Relationships between IL-17+ Subsets,Tregs and pDCs That Distinguish among SIV Infected Elite Controllers,Low, Medium and High Viral Load Rhesus Macaques

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4⁺ Tregs, CD8⁺ Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4⁺ Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8⁺ Tregs was also noted with a moderate increase in ECs but further increases of CD8⁺ Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4⁺ Tregs and increases in the levels of CD8⁺ Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17⁺ subsets in GIT as CD3^−/CD8⁺/NKG2a⁻ and CD3⁺/CD8⁺/NKG2a⁺ subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design.     

RAG-1 and Ly6D Independently Reflect Progression in the B Lymphoid Lineage

Common lymphoid progenitors (CLPs) are thought to represent major intermediates in the transition of hematopoietic stem cells (HSCs) to B lineage lymphocytes. However, it has been obvious for some time that CLPs are heterogeneous, and there has been controversy concerning their differentiation potential. We have now resolved four Flt3⁺ CLP subsets that are relatively homogenous and capable of forming B cells. Differentiation potential and gene expression patterns suggest Flt3⁺ CLPs lacking both Ly6D and RAG-1 are the least differentiated. In addition to B cells, they generate natural killer (NK) and dendritic cells (DCs). At the other extreme is a subset of the recently described Flt3⁺ Ly6D⁺ CLPs that have a history of RAG-1 expression and are B lineage restricted. These relatively abundant and potent CLPs were depleted within 48 hours of acute in vivo estrogen elevation, suggesting they descend from hormone regulated progenitors. This contrasts with the hormone insensitivity of other CLP subsets that include NK lineage progenitors. This progenitor heterogeneity and differentiation complexity may add flexibility in response to environmental changes. Expression of RAG-1 and display of Ly6D are both milestone events, but they are neither synchronized nor dependent on each other.     

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.     

Modulation of respiratory dendritic cells during Klebsiella pneumonia infection

Background

Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity.

Method

By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection.

Results

Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103⁺ DC, CD11b⁺ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103⁺ DC and IL-19 and IL-12p35 in CD11b⁺ DC subsets in comparison to CD11c⁺ MHC-class II^low cells indicating distinct functional roles. Antigen-specific naive CD4⁺ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4⁺ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103⁺ DC and CD11b⁺ DC subsets represented the most potent naive CD4⁺ T helper cell activators.

Conclusion

These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration.     

Similar functional activity of dendritic cells recruited to the mesenteric lymph nodes of newborn and adult mice after the rectal delivery of Mycobacterium bovis BCG

BCG rectal administration to newborn and adult mice induced protective immune responses against tuberculosis. BCG reaches the sub-epithelial site and the draining mesenteric lymph nodes (MLNs), and dendritic cells (DC) could be recruited to these sites. Using polarized Caco-2 epithelial cells, we showed that BCG translocates epithelial cells to basolateral compartment. Delayed in newborn BALB/c mice, an important recruitment of CD11c⁺ DCs, was documented in the rectal lamina propria and the MLNs during the first two weeks after rectal BCG delivery. In MLNs, two major DC subtypes were observed: conventional DCs (cDCs) (B220⁻) and plasmacytoid DCs (pDCs) (B220⁺). CIRE, mouse DC-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is predominantly expressed on pDCs and at a higher level on pDCs from the adult compared to newborn MLNs. cDCs with a higher capacity to induce the proliferation of naïve CD4⁺ T cells than pDCs, triggered CD4⁺ T cells to produce interferon-γ whereas pDCs triggered them to release interleukin-10. Both DC subtypes equilibrates T cells as a source of microbicidal/microbiostatic signals and those acting as source of counter-inflammatory signals, preventing tissue damage and/or accelerating tissue repair. Thus, rectal delivery of BCG could be a safe and efficient route of vaccination against tuberculosis.