[show abstract][hide abstract] ABSTRACT: The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C(-) macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C(+) macrophages. Such Ly6C(+) macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C(-) macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.
[show abstract][hide abstract] ABSTRACT: Knauf et al. demonstrate that prolonged activation of the intrarenal inflammasome is responsible for the loss of kidney function in oxalate crystal nephropathy. These findings suggest new therapeutic opportunities for patients suffering from severe hereditary kidney diseases such as primary hyperoxaluria, and reveal a previously unappreciated general mechanism of kidney disease progression that may also contribute to conditions other than crystal nephropathy.
Kidney International 11/2013; 84(5):859-61. · 7.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: The DC-derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α(-) CD11b(+) LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α-galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α(-) and CD8α(+) splenic DC subsets and enhances cross-presentation of exogenous antigens. Based on genome-wide expression profiling, we now show that splenic CD11b(+) DCs are susceptible to IFN-γ-mediated suppression of CCL17, whereas LN CD11b(+) CCL17(+) DCs downregulate the IFN-γR and are much less responsive to IFN-γ. Under inflammatory conditions, particularly in the absence of IFN-γ signaling in IFN-γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM-CSF in concert with IL-4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression. This article is protected by copyright. All rights reserved.
European Journal of Immunology 10/2013; · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The kidneys are frequently targeted by pathogenic immune responses against renal autoantigens or by local manifestations of systemic autoimmunity. Recent studies in rodent models and humans have uncovered several underlying mechanisms that can be used to explain the previously enigmatic immunopathology of many kidney diseases. These mechanisms include kidney-specific damage-associated molecular patterns that cause sterile inflammation, the crosstalk between renal dendritic cells and T cells, the development of kidney-targeting autoantibodies and molecular mimicry with microbial pathogens. Conversely, kidney failure affects general immunity, causing intestinal barrier dysfunction, systemic inflammation and immunodeficiency that contribute to the morbidity and mortality of patients with kidney disease. In this Review, we summarize the recent findings regarding the interactions between the kidneys and the immune system.
[show abstract][hide abstract] ABSTRACT: DCs and macrophages both express the chemokine receptor CX3CR1. Here we demonstrate that its ligand, CX3CL1, is highly expressed in the murine kidney and intestine. CX3CR1 deficiency markedly reduced DC numbers in the healthy and inflamed kidney cortex, and to a lesser degree in the kidney medulla and intestine, but not in other organs. CX3CR1 also promoted influx of DC precursors in crescentic glomerulonephritis, a DC-dependent aggressive type of nephritis. Disease severity was strongly attenuated in CX3CR1-deficient mice. Primarily CX3CR1-dependent DCs in the kidney cortex processed antigen for the intrarenal stimulation of T helper cells, a function important for glomerulonephritis progression. In contrast, medullary DCs played a specialized role in inducing innate immunity against bacterial pyelonephritis by recruiting neutrophils through rapid chemokine production. CX3CR1 deficiency had little effect on the immune defense against pyelonephritis, as medullary DCs were less CX3CR1 dependent than cortical DCs and because recruited neutrophils produced chemokines to compensate for the DC paucity. These findings demonstrate that cortical and medullary DCs play specialized roles in their respective kidney compartments. We identify CX3CR1 as a potential therapeutic target in glomerulonephritis that may involve fewer adverse side effects, such as impaired anti-infectious defense or compromised DC functions in other organs.
The Journal of clinical investigation 09/2013; · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: The JAK-inhibitor Ruxolitinib decreases constitutional symptoms and spleen size of myelofibrosis (MF) patients by mechanisms distinct from its anti-clonal activity. Here we investigated whether Ruxolitinib affects dendritic cell (DC) biology. The in vitro development of monocyte-derived DCs was almost completely blocked when the compound was added throughout the differentiation period. Furthermore, when applied solely during the final LPS-induced maturation step, Ruxolitinib reduced DC activation as demonstrated by decreased IL-12 production and attenuated expression of activation markers. Ruxolitinib also impaired both in vitro and in vivo DC migration. Dysfunction of Ruxolitinib-exposed DCs was further underlined by their impaired induction of allogeneic and antigen-specific T cell responses. Ruxolitinib-treated mice immunized with OVA/CpG induced markedly reduced in vivo activation and proliferation of OVA-specific CD8(+) T cells compared to vehicle-treated controls. Finally, using an adenoviral infection model, we show that Ruxolitinib-exposed mice exhibit delayed adenoviral clearance. Our results demonstrate that Ruxolitinib significantly affects DC differentiation and function leading to impaired T cell activation. DC dysfunction may result in increased infection rates in Ruxolitinib-treated patients. However, our findings may also explain the outstanding anti-inflammatory and immunomodulating activity of JAK-inhibitors currently used in the treatment of MF and autoimmune diseases.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS: We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS: Mature peripheral blood monocytes co-cultured with HSCs down-regulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS: Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.
Journal of Hepatology 05/2013; · 9.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.
[show abstract][hide abstract] ABSTRACT: Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.
[show abstract][hide abstract] ABSTRACT: Although the spleen is a major site where immune tolerance to circulating innocuous antigens occurs, the kidney also contributes. Circulating antigens smaller than albumin are constitutively filtered and concentrated in the kidney and reach the renal lymph node by lymphatic drainage, where resident dendritic cells (DCs) capture them and induce tolerance of specific cytotoxic T cells through unknown mechanisms. Here, we found that the coinhibitory cell surface receptor programmed death 1 (PD-1) on cytotoxic T cells mediates to their tolerance. Renal lymph node DCs of the CD8(+) XCR1(+) subset, which depend on the transcription factor Batf3, expressed the PD-1 cognate ligand PD-L1. Batf3-dependent DCs in the renal lymph node presented antigen that had been concentrated in the kidney and used PD-L1 to induce apoptosis of cytotoxic T cells. In contrast, T cell tolerance in the spleen was independent of PD-1, PD-L1, and Batf3. In summary, these results clarify how the kidney/renal lymph node system tolerizes the immune system against circulating innocuous antigens.
Journal of the American Society of Nephrology 02/2013; · 8.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Processing and presentation of antigen on MHC-I class I molecules serves to present peptides derived from cytosolic proteins to CD8(+) T cells. Infection with bacteria that remain in phagosomal compartments, such as Mycobacterium tuberculosis (Mtb), provides a challenge to this immune recognition as bacterial proteins are segregated from the cytosol. Previously we identified the Mtb phagosome itself as an organelle capable of loading MHC Class I molecules with Mtb antigens. Here, we find that the TAP transporter, responsible for importing peptides into the ER for loading in Class I molecules, is both present and functional in Mtb phagosomes. Furthermore, we describe a novel peptide reagent, representing the N-terminal domain of the bovine herpes virus UL49.5 protein, which is capable of specifically inhibiting the lumenal face of TAP. Together, these results provide insight into the mechanism by which peptides from intra-phagosomal pathogens are loaded onto Class I molecules.
PLoS ONE 01/2013; 8(11):e79571. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Professional antigen-presenting cells such as dendritic cells (DCs) and macrophages internalize extracellular antigens, process them intracellularly, and present the resulting antigen-derived peptides in the context of MHC I or MHC II molecules. Since the intracellular routing of the antigen determines whether antigens are presented on MHC I or MHC II molecules, a profound analysis of the intracellular distribution of the internalized antigens is of high interest. Here, we describe an immunofluorescence protocol to monitor the intracellular routing of the model-antigen Ovalbumin in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with antibodies against different endosomal markers, such as EEA1 and LAMP1, and can be easily adopted to other antigen-presenting cells or antigens.
Methods in molecular biology (Clifton, N.J.) 01/2013; 960:371-377.
[show abstract][hide abstract] ABSTRACT: Isolation and characterization of antigen-containing endosomes remains difficult utilizing standard purification techniques. Here, we describe a method, which allows isolation of antigen-loaded endosomes, that is based on flow cytometrical analysis and sorting. We specifically isolated antigen-containing endosomes from cells that had taken up fluorochrome-labeled ovalbumin via mannose receptor-mediated endocytosis. The protocol described here allows for the isolation of pure fractions of ovalbumin-containing endosomes and the extraction of proteins from these endosomes for analysis by western blot. Importantly, this protocol can be easily extended to other fluorochrome-labeled antigens and to primary antigen-presenting cells like bone marrow-derived dendritic cells.
Methods in molecular biology (Clifton, N.J.) 01/2013; 960:379-388.
[show abstract][hide abstract] ABSTRACT: Introduction: Standardized intestinal manipulation (IM) leads to generalized postoperative ileus (POI). Previously, we demonstrated that POI is immune-mediated. Aim of this study was to investigate the role of secondary lymphoid organs (mesenteric lymph nodes/MLN, GALT) in IM-mediated POI by employing lymphatic-deficient mice (aly/aly, MLN ex) or by administration of immunosuppressants FTY720. Materials and Methods: Jejunal and colonic muscularis from wild-type (WT) mice as control, from aly/aly mice, from FTY720 treated mice (daily dose 1.0 mg/kg mouse i.p. starting 3 days before surgical procedure) and mice after removal of mesenteric lymph nodes prior to IM (MLN ex mice) were obtained after IM or sham operation. The POI was analyzed by measuring the gastrointestinal transit time, colonic transit time, infiltration of myeloperoxidase positive cells, and circular smooth muscle contractility. Furthermore, the mRNA levels of inflammatory cytokines (IL-6, TNF-α, MIP-1α) were determined by Taqman®-PCR. Results: We observed a significantly reduced upregulation of proinflammatory cytokines (IL-6, TNF-α, MIP-1α) in colonic muscularis of lymphatic-deficient mice and FTY720 treated mice. The colonic, but not the jejunal contractility was significantly improved in these mice. Additionally, the colonic inflammation and the colonic transit time were significantly improved. In summary, the lack of secondary lymphoid organs cancelled the POI. Conclusion: These data demonstrate that secondary lymphoid organs are involved in the propagation of POI. FTY720 indirectly affects POI by inhibiting migration of activated T cells from the jejunum and adjacent secondary lymphoid organs to the colon. These findings support the crucial role of the adaptive immune system in POI.
AJP Gastrointestinal and Liver Physiology 12/2012; · 3.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.
Cancer Immunology and Immunotherapy 11/2012; · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Immature renal dendritic cells (DCs) are protective early in murine crescentic GN, but the mechanisms underlying this protection are unknown. Here, depletion of DCs reduced the recruitment of invariant natural killer T (iNKT) cells, which attenuate GN, into the kidney in the early stage of experimental crescentic GN. More than 90% of renal iNKT cells expressed the chemokine receptor CXCR6, and renal DCs produced high amounts of the cognate ligand CXCL16 early after induction of nephritis, suggesting that renal DC-derived CXCL16 might attract protective CXCR6(+) iNKT cells. Consistent with this finding, CXCR6-deficient mice exhibited less iNKT cell recruitment and developed nephritis that was more severe, similar to the aggravated nephritis observed in mice depleted of immature DCs. Finally, adoptive transfer of CXCR6-competent NKT cells ameliorated nephritis. Taken together, these results suggest an immunoprotective mechanism involving immature DCs, CXCL16, CXCR6, and regulatory iNKT cells, which might stimulate the development of new therapeutic strategies for GN.
Journal of the American Society of Nephrology 11/2012; · 8.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria.
[show abstract][hide abstract] ABSTRACT: Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF.