Stella E. Autenrieth

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

Staphylococcus aureus Phenol-Soluble Modulin Peptides Modulate Dendritic Cell Functions and Increase In Vitro Priming of Regulatory T Cells

The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4+ T cells, characterized by reduced Th1 but increased frequency of FOXP3+ regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-β. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2−/−) and the cognate G protein (p110γ−/−). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.

Yersinia enterocolitica YopP inhibits MAP kinase‐mediated antigen uptake in dendritic cells

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild‐type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)‐deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP‐C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin‐mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA‐specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits thispathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

Significance Invasive pulmonary aspergillosis (IPA) is a frequently fatal lung disease of immunocompromised patients, and is being increasingly reported in individuals with underlying respiratory diseases. Proven diagnosis of IPA currently relies on lung biopsy and detection of diagnostic biomarkers in serum, or in bronchoalveolar lavage fluids. This study supports the use of immunoPET/MR imaging for the diagnosis of IPA, which is so far not used for diagnosis. The antibody-guided imaging technique allows accurate, noninvasive and rapid detection of fungal lung infection and discrimination of IPA from bacterial lung infections and general inflammatory responses. This work demonstrates the applicability of molecular imaging for IPA detection and its potential for aiding clinical diagnosis and management of the disease in the neutropenic host. Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of κB kinase (IKK)-β resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential ΔΨm and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.

Immune evasion by Yersinia enterocolitica: Differential targeting of dendritic cell subpopulations in vivo

CD4+ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4+ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4+ T cells was markedly reduced when cultured with splenic CD8α+ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4+ or CD4−CD8α− DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α+ DCs, but not in CD4+ and CD4−CD8α− DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α+ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α+ DCs. Three days post infection with Ye the number of splenic CD8α+ and CD4+ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4+ and CD8α+ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.

Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.

Insights how monocytes and dendritic cells contribute and regulate immune defense against microbial pathogens.

The immune system protects from infections primarily by detecting and eliminating invading pathogens. Beside neutrophils, monocytes and dendritic cells (DCs) have been recently identified as important sentinels and effectors in combating microbial pathogens. In the steady state mononuclear phagocytes like monocytes and DCs patrol the blood and the tissues. Mammalian monocytes contribute to antimicrobial defense by supplying tissues with macrophage and DC precursors. DCs recognize pathogens and are essential in presenting antigens to initiate antigen-specific adaptive immune responses, thereby bridging the innate and adaptive immune systems. Both, monocytes and DCs play distinct roles in the shaping of immune response. In this review we will focus on the contributions of monocytes and lymphoid organ DCs to immune defense against microbial pathogens in the mouse and their dynamic regulation from steady state to infection.

Variable antigen uptake due to different expression of the macrophage mannose receptor by dendritic cells in various inbred mouse strains

Antigen uptake by dendritic cells is essential for the induction of antigen‐specific T‐cell responses. Here, we investigate the ability of dendritic cells from different mouse strains to endocytose antigens. The uptake of different fluorescently labelled soluble antigens by bone marrow‐derived dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice was analysed by flow cytometry. Using transferrin as a specific marker for clathrin‐mediated endocytosis, we observed no significant differences of transferrin uptake by dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice. Similar results were obtained by analysing macropinocytosis with lucifer yellow. In contrast, analysing the uptake of ovalbumin, which is predominantly mediated by clathrin‐mediated endocytosis via the macrophage mannose receptor, we found that dendritic cells from C3H/HeN mice take up three‐ to fivefold more ovalbumin than dendritic cells from BALB/c or C57BL/6 mice. Blocking the uptake of ovalbumin via the macrophage mannose receptor by using mannan led to a comparable uptake of ovalbumin by dendritic cells from all three mouse strains. Consistently, dendritic cells from C3H/HeN mice displayed significantly increased expression of the macrophage mannose receptor compared to dendritic cells from BALB/c or C57BL/6 mice. In conclusion, receptors involved in antigen uptake such as the macrophage mannose receptor may be differentially expressed and may explain variations of T‐cell responses after vaccination in different individuals.

Y. enterocolitica inhibits antigen degradation in dendritic cells.

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.