S100-alarmin-induced innate immune programming protects newborn infants from sepsis.

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

How to measure the immunosuppressive activity of MDSC: assays, problems and potential solutions.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of mononuclear and polymorphonuclear myeloid cells, which are present at very low numbers in healthy subjects, but can expand substantially under disease conditions. Depending on disease type and stage, MDSC comprise varying amounts of immature and mature differentiation stages of myeloid cells. Validated unique phenotypic markers for MDSC are still lacking. Therefore, the functional analysis of these cells is of central importance for their identification and characterization. Various disease-promoting and immunosuppressive functions of MDSC are reported in the literature. Among those, the capacity to modulate the activity of T cells is by far the most often used and best-established read-out system. In this review, we critically evaluate the assays available for the functional analysis of human and murine MDSC under in vitro and in vivo conditions. We also discuss critical issues and controls associated with those assays. We aim at providing suggestions and recommendations useful for the contemporary biological characterization of MDSC.

Activation-dependent cell death of human monocytes is a novel mechanism of fine-tuning inflammation and autoimmunity.

In patients with juvenile idiopathic arthritis (JIA), increased release of IFN-γ and GM-CSF in cells infiltrating synovial tissue can be a potent driver of monocyte activation. Given the fundamental role of monocyte activation in remodeling the early phases of inflammatory responses, here we analyze the GM-CSF/IFN-γ induced activity of human monocytes in such a situation in vitro and in vivo. Monocytes from healthy donors were isolated and stimulated with GM-CSF ± IFN-γ. Monocyte activation and death were analyzed by flow cytometry, immunofluorescence microscopy, ELISA, and qPCR. T-cell GM-CSF/IFN-γ expression and monocyte function were determined in synovial fluid and peripheral blood from 15 patients with active JIA and 21 healthy controls. Simultaneous treatment with GM-CSF and IFN-γ induces cell death of monocytes. This cell death is partly cathepsin B-associated and has morphological characteristics of necrosis. Monocytes responding to costimulation with strong proinflammatory activities are consequently eliminated. Monocytes surviving this form of hyperactivation retain normal cytokine production. Cathepsin B activity is increased in monocytes isolated from synovial fluid from patients with active arthritis. Our data suggest GM-CSF/IFN-γ induced cell death of monocytes as a novel mechanism to eliminate overactivated monocytes, thereby potentially balancing inflammation and autoimmunity in JIA.

Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1ß, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

Bacterial Lipoproteins Shift Cellular Metabolism to Glycolysis in Macrophages Causing Bone Erosion.

Belonging to a group of membrane proteins, bacterial lipoproteins (LPPs) are defined by a unique lipid structure at their N-terminus providing the anchor in the bacterial cell membrane. In Gram-positive bacteria, LPPs play a key role in host immune activation triggered through a Toll-like receptor 2 (TLR2)-mediated action resulting in macrophage stimulation and subsequent tissue damage demonstrated in in vivo experimental models. Yet the physiologic links between LPP activation, cytokine release, and any underlying switches in cellular metabolism remain unclear. In this study, we demonstrate that Staphylococcus aureus Lpl1 not only triggers cytokine production but also confers a shift toward fermentative metabolism in bone marrow-derived macrophages (BMDMs). Lpl1 consists of di- and tri-acylated LPP variants; hence, the synthetic P2C and P3C, mimicking di-and tri-acylated LPPs, were employed to reveal their effect on BMDMs. Compared to P3C, P2C was found to shift the metabolism of BMDMs and the human mature monocytic MonoMac 6 (MM6) cells more profoundly toward the fermentative pathway, as indicated by lactate accumulation, glucose consumption, pH reduction, and oxygen consumption. In vivo, P2C caused more severe joint inflammation, bone erosion, and lactate and malate accumulation than P3C. These observed P2C effects were completely abrogated in monocyte/macrophage-depleted mice. Taken together, these findings now solidly confirm the hypothesized link between LPP exposure, a macrophage metabolic shift toward fermentation, and ensuing bone destruction. IMPORTANCE Osteomyelitis caused by S. aureus is a severe infection of the bone, typically associated with severe bone function impairment, therapeutic failure, high morbidity, invalidity, and occasionally even death. The hallmark of staphylococcal osteomyelitis is the destruction of the cortical bone structures, yet the mechanisms contributing to this pathology are hitherto poorly understood. One bacterial membrane constituent found in all bacteria is bacterial lipoproteins (LPPs). Previously, we have shown that injection of purified S. aureus LPPs into wild-type mouse knee joints caused a TLR2-dependent chronic destructive arthritis but failed to elicit such effect in monocyte/macrophage-depleted mice. This observation stirred our interest in investigating the interaction of LPPs and macrophages and analyzing the underlying physiological mechanisms. This ascertainment of LPP-induced changes in the physiology of macrophages provides an important clue in the understanding of the mechanisms of bone disintegration, opening novel avenues to manage the course of S. aureus disease.

UVA1 radiation attenuates pro-inflammatory functions in human monocytes.

UVA1 therapy is effective in the treatment of inflammatory and autoimmune skin diseases. The mode of action of UVA1 therapy is not completely understood and especially data on cells of the innate immune system like monocytes, which are critically involved in many inflammatory processes, are sparse. We wanted to answer the question whether UVA1 irradiation alters functional properties of human monocytes. We treated human peripheral blood monocytes in vitro with 2 J/cm2 UVA1 light, incubated the cells for 48 h and examined both functional properties and alterations in the gene and protein expression profile. While UVA1 did not alter cell viability or susceptibility to apoptosis inducing agents, it decreased the capacity of monocytes for phagocytosis and to eliminate infectious agents like Leishmania major. Moreover, we measured a significantly reduced production of interleukin (IL)-1ß mRNA in lipopolysaccharide activated monocytes after UVA1 treatment. Importantly, UVA1-treated monocytes not only produce less IL-1ß, but also upregulate expression of the anti-inflammatory IL-1ß decoy receptor. Our data provide evidence that UVA1 radiation not only interferes with fundamental monocyte properties like phagocytosis, pathogen killing and activation, but could also specifically attenuate pro-inflammatory IL-1 effects. This might constitute a hitherto unknown anti-inflammatory mechanism of UVA1 in human monocytes.

The Good and the Bad: Monocytes' and Macrophages' Diverse Functions in Inflammation.

Monocytes and macrophages are central players of the innate immune response and play a pivotal role in the regulation of inflammation. Thereby, they actively participate in all phases of the immune response, from initiating inflammation and triggering the adaptive immune response, through to the clearance of cell debris and resolution of inflammation. In this review, we described the mechanisms of monocyte and macrophage adaptation to rapidly changing microenvironmental conditions and discussed different forms of macrophage polarization depending on the environmental cues or pathophysiological condition. Therefore, special focus was placed on the tight regulation of the pro- and anti-inflammatory immune response, and the diverse functions of S100A8/S100A9 proteins and the scavenger receptor CD163 were highlighted, respectively. We paid special attention to the function of pro- and anti-inflammatory macrophages under pathological conditions.

Tracking of Tumor Cell-Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis.

PURPOSE: Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis. PROCEDURES: Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell-derived versus extracellular vesicles from healthy serum. RESULTS: In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV. CONCLUSIONS: In conclusion, distribution of tumor cell-derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.

Target-Specific Imaging of Cathepsin and S100A8/A9 Reflects Specific Features of Malignancy and Enables Estimation of Tumor Malignancy.

PURPOSE: Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential. PROCEDURES: Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry. RESULTS: At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results. CONCLUSIONS: Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors' local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.

More Than Suppression: Glucocorticoid Action on Monocytes and Macrophages.

Uncontrolled inflammation is a leading cause of many clinically relevant diseases. Current therapeutic strategies focus mainly on immunosuppression rather than on the mechanisms of inflammatory resolution. Glucocorticoids (GCs) are still the most widely used anti-inflammatory drugs. GCs affect most immune cells but there is growing evidence for cell type specific mechanisms. Different subtypes of monocytes and macrophages play a pivotal role both in generation as well as resolution of inflammation. Activation of these cells by microbial products or endogenous danger signals results in production of pro-inflammatory mediators and initiation of an inflammatory response. GCs efficiently inhibit these processes by down-regulating pro-inflammatory mediators from macrophages and monocytes. On the other hand, GCs act on "naïve" monocytes and macrophages and induce anti-inflammatory mediators and differentiation of anti-inflammatory phenotypes. GC-induced anti-inflammatory monocytes have an increased ability to migrate toward inflammatory stimuli. They remove endo- and exogenous danger signals by an increased phagocytic capacity, produce anti-inflammatory mediators and limit T-cell activation. Thus, GCs limit amplification of inflammation by repressing pro-inflammatory macrophage activation and additionally induce anti-inflammatory monocyte and macrophage populations actively promoting resolution of inflammation. Further investigation of these mechanisms should lead to the development of novel therapeutic strategies to modulate undesirable inflammation with fewer side effects via induction of inflammatory resolution rather than non-specific immunosuppression.

Peroxisome Proliferator-Activated Receptor-γ Modulates the Response of Macrophages to Lipopolysaccharide and Glucocorticoids.

Although glucocorticoids (GC) represent the most frequently used immunosuppressive drugs, their effects are still not well understood. In our previous studies, we have shown that treatment of monocytes with GC does not cause a global suppression of monocytic effector functions, but rather induces differentiation of a specific anti-inflammatory phenotype. The anti-inflammatory role of peroxisome proliferator-activated receptor (PPAR)-γ has been extensively studied during recent years. However, a relationship between GC treatment and PPAR-γ expression in macrophages has not been investigated so far. Studies using PPAR-γ-deficient mice have frequently provided controversial results. A potential reason is the use of primary cells, which commonly represent inhomogeneous populations burdened with side effects and influenced by bystander cells. To overcome this constraint, we established ER-Hoxb8-immortalized bone marrow-derived macrophages from Ppargfl/fl and LysM-Cre Ppargfl/fl mice in this study. In contrast to primary macrophages, the ER-Hoxb8 system allows the generation of a homogeneous and well-defined population of resting macrophages. We could show that the loss of PPAR-γ resulted in delayed kinetic of differentiation of monocytes into macrophages as assessed by reduced F4/80, but increased Ly6C expression in early phases of differentiation. As expected, PPAR-γ-deficient macrophages displayed an increased pro-inflammatory phenotype upon long-term LPS stimulation characterized by an elevated production of pro-inflammatory cytokines TNF-α, IL1-ß, IL-6, IL-12 and a reduced production of anti-inflammatory cytokine IL-10 compared to PPAR-γ WT cells. Moreover, PPAR-γ-deficient macrophages showed impaired phagocytosis. GC treatment of macrophages led to the upregulation of PPAR-γ expression. However, there were no differences in GC-induced suppression of cytokines between both cell types, implicating a PPAR-γ-independent mechanism. Intriguingly, GC treatment resulted in an increased in vitro migration only in PPAR-γ-deficient macrophages. Performing a newly developed in vivo cell-tracking experiment, we could confirm that GC induces an increased recruitment of PPAR-γ KO, but not PPAR-γ WT macrophages to the site of inflammation. Our findings suggest a specific effect of PPAR-γ on GC-induced migration in macrophages. In conclusion, we could demonstrate that PPAR-γ exerts anti-inflammatory activities and shapes macrophage functions. Moreover, we identified a molecular link between GC and PPAR-γ and could show for the first time that PPAR-γ modulates GC-induced migration in macrophages.

Alarmins MRP8 and MRP14 induce stress tolerance in phagocytes under sterile inflammatory conditions.

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation.