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Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow–derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1− CD127− CX3CR1− Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

A retrospective case-control study found significantly increased levels of interleukin-5 and interleukin-8, critical cytokines of the innate immune system, in the aqueous humor of patients undergoing repeat DMEK for graft failure, compared to primary DMEK.

Background Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. Methods Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. Results Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. Conclusions The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

The important role of macrophages in host defense against a variety of pathogens has long been recognized and has been documented and reviewed in numerous publications. Recently, it has become clear that tissue macrophages are not entirely derived from monocytes, as has been assumed for a long time, but rather show an ontogenetic dichotomy in most tissues: while part of the tissue macrophages are derived from monocytes, a major subset is prenatally seeded from the yolk sac. The latter subset shows a remarkable longevity and is maintained by self-renewal in the adult animal. This paradigm shift poses interesting questions: are these two macrophage subsets functionally equivalent cells that are recruited into the tissue at different development stages, or are both macrophage subsets discrete cell types with distinct functions, which have to exist side by side? Is the functional specialization that can be observed in most macrophages due to their lineage or due to their anatomical niche? This review will give an overview about what we know of macrophage ontogeny and will discuss the influence of the macrophage lineage and location on their functional specialization.

Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown.Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector.Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality.The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therapy.

The important role of macrophages in host defense against a variety of pathogens has long been recognized and has been documented and reviewed in numerous publications. Recently, it has become clear that tissue macrophages are not entirely derived from monocytes, as has been assumed for a long time, but rather show an ontogenetic dichotomy in most tissues: while part of the tissue macrophages are derived from monocytes, a major subset is prenatally seeded from the yolk sac. The latter subset shows a remarkable longevity and is maintained by self-renewal in the adult animal. This paradigm shift poses interesting questions: are these two macrophage subsets functionally equivalent cells that are recruited into the tissue at different development stages, or are both macrophage subsets discrete cell types with distinct functions, which have to exist side by side? Is the functional specialization that can be observed in most macrophages due to their lineage or due to their anatomical niche? This review will give an overview about what we know of macrophage ontogeny and will discuss the influence of the macrophage lineage and location on their functional specialization.

A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.

Neutrophils are potent immune effectors against bacterial infections. Macrophages are important in infections as effectors and regulators, but their exact roles, phenotypic characterization and their relation to neutrophils is incompletely understood. Here we report in a model of bacterial urinary tract infection, one of the most prevalent bacterial infections that tissue-resident Ly6C− macrophages recruited circulating neutrophils and inflammatory Ly6C+ macrophages through chemokines. Neutrophils were primarily recruited through ligands of the chemokine receptor CXCR2, in particular by CXCL1 and less by macrophage migration inhibitory factor (MIF), but not through CXCL5 and CXCL2. Neutrophils, but not Ly6C+ macrophages, cleared the bacteria by phagocytosis. Ly6C+ macrophages instead performed a regulatory function: in response to the infection, they produced the cytokine tumor necrosis factor (TNF), which in turn caused the resident macrophages to secrete CXCL2. This chemokine induced the secretion of matrix metalloproteinase-9 (MMP-9) in neutrophils and allowed these cells to degrade the uroepithelial basement membrane, in order to enter the uroepithelium, the mucosal interface from where the bacteria invade the bladder. Thus, the phagocyte response against bacteria is a highly coordinated event, in which Ly6C− macrophages act as sentinels and Ly6C+ macrophages as innate helper cells. In analogy with T helper cells (Th), we propose to name these helper macrophages (Ph) as they provide a second signal on whether to unleash the principal effector phagocytes, the neutrophils. This cellular triage may prevent ‘false-positive’ immune responses. The role of TNF as innate ‘licensing’ factor contributes to its central role in antibacterial immunity.

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.

Antigen-presenting cells (APCs), especially macrophages and dendritic cells (DCs), are important for the induction of an adaptive immune response through their phagocytic capacity. APCs internalize extracellular antigens and, dependent on their intracellular localization, antigen-derived peptides are presented on MHC I or MHC II molecules. In context of antigen presentation and T cell activation tracking of internalized antigens is of high interest. In this article, we provide an immunofluorescence protocol and illustrate the analysis of intracellular routing of internalized antigens using the example of the model-antigen ovalbumin (OVA) in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with an antibody against EEA-1, a marker for early endosomes, which can be easily adapted to other endosome markers, antigen-presenting cells, or antigens.

Dendritic cells (DCs) are the main inducers and regulators of cytotoxic T lymphocyte (CTL) responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and “licensed” by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (“classical licensing”) or by natural killer T cells (“alternative licensing”). Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.