Matthias Bartneck

Age-dependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults

BackgroundRecent experimental approaches have unraveled essential migratory and functional differences of monocyte subpopulations in mice. In order to possibly translate these findings into human physiology and pathophysiology, human monocyte subsets need to be carefully revisited in health and disease. In analogy to murine studies, we hypothesized that human monocyte subsets dynamically change during ageing, potentially influencing their functionality and contributing to immunosenescence.ResultsCirculating monocyte subsets, surface marker and chemokine receptor expression were analyzed in 181 healthy volunteers (median age 42, range 18-88). Unlike the unaffected total leukocyte or total monocyte counts, non-classical CD14+CD16+ monocytes significantly increased with age, but displayed reduced HLA-DR and CX3CR1 surface expression in the elderly. Classical CD14++CD16- monocyte counts did not vary dependent on age. Serum MCP-1 (CCL2), but not MIP1α (CCL3), MIP1β (CCL4) or fractalkine (CX3CL1) concentrations increased with age. Monocyte-derived macrophages from old or young individuals did not differ with respect to cytokine release in vitro at steady state or upon LPS stimulation.ConclusionsOur study demonstrates dynamic changes of circulating monocytes during ageing in humans. The expansion of the non-classical CD14+CD16+ subtype, alterations of surface protein and chemokine receptor expression as well as circulating monocyte-related chemokines possibly contribute to the preserved functionality of the monocyte pool throughout adulthood.

Rapid uptake of gold nanorods by primary human blood phagocytes and immunomodulatory effects of surface chemistry.

Nanoparticle-based in vivo applications should consider the omnipresence of the phagocytes in the bloodstream and tissue. We have studied the nanoparticle uptake capacities of the most important human primary leukocyte populations using a nanoparticle library encompassing both rod-shaped and spherical gold nanoparticles with diameters between 15 and 50 nm and a variety of surface chemistries. Cetyltrimethylammoniumbromide (CTAB)-stabilized nanoparticles were internalized rapidly within 15 min and in large amounts by macrophages and to a lower extent also by monocytes. Interestingly, we found that the uptake of nanorods by macrophages was more efficient than that of nanospheres. Blocking experiments and electron microscopic studies revealed macropinocytosis as the major uptake mechanism. Grafting of poly(ethylene oxide) (PEO) onto the nanorods was found to significantly delay their internalization for several hours. The long-term uptake of PEO-coated nanoparticles with positively or negatively charged end groups was almost identical. Particle surface chemistry strongly influenced the expression of inflammation-related genes within 1 day. Furthermore, the macrophage phenotype was significantly affected after 7 days of culture with nanorods depending on the surface chemistry. Thus, in vivo application of nanoparticles with certain surface functionalities may lead to inflammation upon particle accumulation. However, our data also suggest that chemical modifications of nanoparticles may be useful for immunomodulation.

Phagocytosis Independent Extracellular Nanoparticle Clearance by Human Immune Cells

It has recently been discovered that human immune cells, especially neutrophil granulocytes, form neutrophil extracellular traps (NETs) that abolish pathogens. Our study provides evidence that extracellular traps formed by neutrophils, monocytes and macrophages act as physical barriers for nanoparticles, thus presenting a new nanomaterial clearance mechanism of the human immune system. While particle shape is of minor importance, positive charges significantly enhance particle trapping.

CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

Objectives In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. Design Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. Results Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. Conclusions Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis

Non‐alcoholic fatty liver disease (NAFLD) represents the most common liver disease in Western countries and often progresses to non‐alcoholic steatohepatitis (NASH) leading ultimately to liver fibrosis and liver cancer. The occurrence of hepatocyte cell death—so far characterized as hepatocyte apoptosis—represents a fundamental step from benign steatosis toward progressive steatohepatitis. In contrast, the function of RIP3‐dependent “necroptosis” in NASH and NASH‐induced fibrosis is currently unknown. We show that RIP3 is upregulated in human NASH and in a dietary mouse model of steatohepatitis. RIP3 mediates liver injury, inflammation, induction of hepatic progenitor cells/activated cholangiocytes, and liver fibrosis through a pathway suppressed by Caspase‐8. This function of RIP3 is mediated by a positive feedback loop involving activation of Jun‐(N)‐terminal Kinase (JNK). Furthermore, RIP3‐dependent JNK activation promotes the release of pro‐inflammatory mediators like MCP‐1, thereby attracting macrophages to the injured liver and further augmenting RIP3‐dependent signaling, cell death, and liver fibrosis. Thus, RIP3‐dependent necroptosis controls NASH‐induced liver fibrosis. This pathway might represent a novel and specific target for pharmacological strategies in patients with NASH.

Pharmacological inhibition of the chemokine C‐C motif chemokine ligand 2 (monocyte chemoattractant protein 1) accelerates liver fibrosis regression by suppressing Ly‐6C+ macrophage infiltration in mice

Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified “restorative” macrophages as Ly‐6C (Ly6C, Gr1) low‐expressing, monocyte‐derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer‐based inhibitor of the chemokine, C‐C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX‐E36, in the regression phase of two murine models of toxic (CCl4) and metabolic (methionine‐choline–deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly‐6C+ infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up‐regulated expression of CCR2, and the pro‐inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly‐6C+ iMΦ by the CCL2 inhibitor, mNOX‐E36, the intrahepatic macrophage equilibration shifted toward the “restorative” Ly‐6C‐ subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX‐E36 administration in both models. Blocking transient recruitment of infiltrating Ly‐6C+ monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines. Conclusion: Transient CCL2‐dependent recruitment of infiltrating Ly‐6C+ monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly‐6C+ monocyte recruitment using the CCL2‐inhibitor, mNOX‐E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models. (Hepatology 2014;59:1060–1072)

Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C‐C motif chemokine receptor 8 in mice

Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte‐derived dendritic cells (DCs) and T‐helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8−/− and wild‐type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl4) administration and surgical bile duct ligation (BDL). CCR8 was strongly up‐regulated in the injured liver. Ccr8−/− mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4+ T cells increased. The main CCR8‐expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1‐directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8−/− animals was altered with increased expression of DC markers and enhanced expression of T‐cell‐attracting chemokine macrophage inflammatory protein 1‐alpha (MIP‐1α/CCL3). Correspondingly, hepatic CD4+ T cells showed increased Th1 polarization and reduced Th2 cells in CCR8‐deficient animals. Liver fibrosis progression, but also subsequent T‐cell alterations, could be restored by adoptively transferring CCR8‐expressing monocytes/macrophages into ccr8−/− mice during experimental injury. Conclusions: CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression. (Hepatology 2012)

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Inducing healing-like human primary macrophage phenotypes by 3D hydrogel coated nanofibres

Immune cells are present in the blood and in resident tissues, and the nature of their reaction towards biomaterials is decisive for materials success or failure. Macrophages may for example be classically activated to trigger inflammation (M1), or alternatively activated which supports healing and vascularisation (M2). Here, we have generated 3D nanofibrous meshes in different porosities and precisely controlled surface chemistries comprising PLGA, hydrogel-coated protein repellant and protein repellant endowed with the bioactive peptide sequences GRGDS or GLF. We also prepared 2D substrates with corresponding surface chemistry for a systematic evaluation of primary human macrophage adhesion, migration, transcriptome expression, cytokine release and surface marker expression. Our data show that material morphology is a powerful means in biomaterial design to influence immune cell response. Flat substrates lead to an increased number of M2 classified CD163(+) macrophages. However, these M2 cells released large amounts of pro-inflammatory cytokines. In contrast, 3D nanofibres with corresponding surface chemistry yielded M1 classified 27E10(+) macrophages with a significantly increased release of pro-angiogenic chemokines and angiogenesis related molecules and a strong decrease of pro-inflammatory cytokines. We thus suggest that, for macrophages in contact with biomaterials, cytokine release is taken as main criterion instead of surface-markers for macrophage classifications.

Peptide-Functionalized Gold Nanorods Increase Liver Injury in Hepatitis

Targeted nanomedicine holds enormous potential for advanced diagnostics and therapy. Although it is known that nanoparticles accumulate in liver in vivo, the impact of cell-targeting particles on the liver, especially in disease conditions, is largely obscure. We had previously demonstrated that peptide-conjugated nanoparticles differentially impact macrophage activation in vitro. We thus comprehensively studied the distribution of gold nanorods (AuNR) in mice in vivo and assessed their hepatotoxicity and impact on systemic and hepatic immune cells in healthy animals and experimental liver disease models. Gold nanorods were stabilized with either cetyltrimethylammonium bromide or poly(ethylene glycol) and additional bioactive tripeptides RGD or GLF. Gold nanorods mostly accumulated in liver upon systemic injection in mice, as evidenced by inductively coupled plasma mass spectrometry from different organs and by non-invasive microcomputerized tomography whole-body imaging. In liver, AuNR were only found in macrophages by seedless deposition and electron microscopy. In healthy animals, AuNR did not cause significant hepatotoxicity as evidenced by biochemical and histological analyses, even at high AuNR doses. However, flow cytometry and gene expression studies revealed that AuNR polarized hepatic macrophages, even at low doses, dependent on the respective peptide sequence, toward M1 or M2 activation. While peptide-modified AuNR did not influence liver scarring, termed fibrosis, in chronic hepatic injury models, AuNR-induced preactivation of hepatic macrophages significantly exacerbated liver damage and disease activity in experimental immune-mediated hepatitis in mice. Bioactively targeted gold nanoparticles are thus potentially harmful in clinically relevant settings of liver injury, as they can aggravate hepatitis severity.