Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis.

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2(-/-) mice were exposed to short term application of CCl4, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl4 intoxication showed increased liver damage in LCN2(-/-) mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1ß, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2(-/-) mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4weeks bile duct ligation or 8weeks CCl4 application), LCN2(-/-) mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

OBJECTIVE: Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C-C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of steatohepatitis and fibrosis. It was recently shown that monocyte/macrophage infiltration into the liver upon injury is critically regulated by the CCL2/CCR2 axis and is functionally important for perpetuating hepatic inflammation and fibrogenesis. The structured L-enantiomeric RNA oligonucleotide mNOX-E36 (a so-called Spiegelmer) potently binds and inhibits murine MCP-1. Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated in two murine models of chronic liver diseases. METHODS: Pharmacological inhibition of MCP-1 by thrice-weekly mNOX-E36 subcutaneously was tested in murine models of acute or chronic carbon tetrachloride (CCl(4))- and methionine-choline-deficient (MCD) diet-induced chronic hepatic injury in vivo. RESULTS: Antagonising MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro as well as migration of Gr1(+) (Ly6C(+)) blood monocytes into the liver upon acute toxic injury in vivo. In murine models of CCl(4)- and MCD diet-induced hepatic injury, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1-treated mice as found by fluorescence-activated cell sorting (FACS) analysis and immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (tumour necrosis factor α, interferon γ and interleukin 6) were significantly reduced in liver tissue. Overall fibrosis progression over 6 (CCl(4)) or 8 weeks (MCD diet) was not significantly altered by anti-MCP-1 treatment. However, upon MCD diet challenge a lower level of fatty liver degeneration (histology score, Oil red O staining, hepatic triglyceride content, lipogenesis genes) was detected in mNOX-E36-treated animals. mNOX-E36 also ameliorated hepatic steatosis upon therapeutic administration. CONCLUSIONS: These results demonstrate the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration by blocking MCP-1 during chronic liver damage in two in vivo models. The associated ameliorated steatosis development suggests that inhibition of MCP-1 is an interesting novel approach for pharmacological treatment in liver inflammation and steatohepatitis.

Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury.

BACKGROUND & AIMS: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.

The fractalkine receptor CX3CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes.

UNLABELLED: Chemokines modulate inflammatory responses that are prerequisites for organ fibrosis upon liver injury. Monocyte-derived hepatic macrophages are critical for the development, maintenance, and resolution of hepatic fibrosis. The specific role of monocyte-associated chemokine (C-X3-C motif) receptor 1 (CX3CR1) and its cognate ligand fractalkine [chemokine (C-X3-C motif) ligand 1)] in liver inflammation and fibrosis is currently unknown. We examined 169 patients with chronic liver diseases and 84 healthy controls; we found that CX3CL1 is significantly up-regulated in the circulation upon disease progression, whereas CX3CR1 is down-regulated intrahepatically in patients with advanced liver fibrosis or cirrhosis. To analyze the functional relevance of this pathway, two models of experimental liver fibrosis were applied to wild-type (WT) and CX3CR1-deficient mice. Fractalkine expression was induced upon liver injury in mice, primarily in hepatocytes and hepatic stellate cells. CX3CR1(-/-) animals developed greater hepatic fibrosis than WT animals with carbon tetrachloride-induced and bile duct ligation-induced fibrosis. CX3CR1(-/-) mice displayed significantly increased numbers of monocyte-derived macrophages within the injured liver. Chimeric animals that underwent bone marrow transplantation revealed that CX3CR1 restricts hepatic fibrosis progression and monocyte accumulation through mechanisms exerted by infiltrating immune cells. In the absence of CX3CR1, intrahepatic monocytes develop preferentially into proinflammatory tumor necrosis factor-producing and inducible nitric oxide synthase-producing macrophages. CX3CR1 represents an essential survival signal for hepatic monocyte-derived macrophages by activating antiapoptotic bcl2 expression. Monocytes/macrophages lacking CX3CR1 undergo increased cell death after liver injury, which then perpetuates inflammation, promotes prolonged inflammatory monocyte infiltration into the liver, and results in enhanced liver fibrosis. CONCLUSION: CX3CR1 limits liver fibrosis in vivo by controlling the differentiation and survival of intrahepatic monocytes. The opposing regulation of CX3CR1 and fractalkine in patients suggests that pharmacological augmentation of this pathway may represent a possible therapeutic antifibrotic strategy.

Corrigendum: Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

[This corrects the article DOI: 10.3389/fimmu.2021.651399.].

Grandmaternal cells in cord blood.

BACKGROUND: During pregnancy a feto-maternal exchange of cells through the placenta conducts to maternal microchimerism (Mc) in the child and fetal Mc in the mother. Because of this bidirectional traffic of cells, pregnant women have also acquired maternal cells in utero from their mother and could transfer grandmaternal (GdM) cells to their child through the maternal bloodstream during pregnancy. Thus, cord blood (CB) samples could theoretically carry GdMMc. Nevertheless this has never been demonstrated. METHODS: Using Human Leukocyte Antigen (HLA)-specific quantitative PCR assays on three-generation families, we were able to test 28 CB samples from healthy primigravid women for GdMMc in whole blood (WB) and isolated cells (PBMC, T, B, granulocytes, stem cells). FINDINGS: Five CB samples (18%) had GdMMc which could not be confounded with maternal source, with quantities 100 fold lower than maternal Mc in WB and PBMC. Risk of aneuploidies and/or related invasive prenatal procedures significantly correlated with the presence of GdMMc in CB (p=0.024). Significantly decreased HLA compatibility was observed in three-generation families from CB samples carrying GdMMc (p=0.019). INTERPRETATION: Transgenerational transfer of cells could have implications in immunology and evolution. Further analyses will be necessary to evaluate whether GdMMc in CB is a passive or immunologically active transfer and whether invasive prenatal procedures could trigger GdMMc. FUNDING: Provence-Alpes-Côte d'Azur APEX grant # 2012_06549E, 2012_11786F and 2014_03978) and the Foundation for Medical Research (FRM Grant #ING20140129045).

Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

Background: Cord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition. Patients and Methods: Here we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status. Results: MMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or -DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc. Conclusions: We have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.

Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.

UNLABELLED: In addition to liver-resident Kupffer cells, infiltrating immune cells have recently been linked to the development of liver fibrosis. Blood monocytes are circulating precursors of tissue macrophages and can be divided into two functionally distinct subpopulations in mice: Gr1(hi) (Ly6C(hi)) and Gr1(lo) (Ly6C(lo)) monocytes. The role of these monocyte subsets in hepatic fibrosis and the mechanisms of their differential recruitment into the injured liver are unknown. We therefore characterized subpopulations of infiltrating monocytes in acute and chronic carbon tetrachloride (CCl(4))-induced liver injury in mice using flow cytometry and immunohistochemistry. Inflammatory Gr1(hi) but not Gr1(lo) monocytes are massively recruited into the liver upon toxic injury constituting an up to 10-fold increase in CD11b(+)F4/80(+) intrahepatic macrophages. Comparing wild-type with C-C chemokine receptor (CCR2)-deficient and CCR2/CCR6-deficient mice revealed that CCR2 critically controls intrahepatic Gr1(hi) monocyte accumulation by mediating their egress from bone marrow. During chronic liver damage, intrahepatic CD11b(+)F4/80(+)Gr1(+) monocyte-derived cells differentiate preferentially into inducible nitric oxide synthase-producing macrophages exerting proinflammatory and profibrogenic actions, such as promoting hepatic stellate cell (HSC) activation, T helper 1-T cell differentiation and transforming growth factor beta (TGF-beta) release. Impaired monocyte subset recruitment in Ccr2(-/-) and Ccr2(-/-)Ccr6(-/-) mice results in reduced HSC activation and diminished liver fibrosis. Moreover, adoptively transferred Gr1(hi) monocytes traffic into the injured liver and promote fibrosis progression in wild-type and Ccr2(-/-)Ccr6(-/-) mice, which are otherwise protected from hepatic fibrosis. Intrahepatic CD11b(+)F4/80(+)Gr1(+) monocyte-derived macrophages purified from CCl(4)-treated animals, but not naïve bone marrow monocytes or control lymphocytes, directly activate HSCs in a TGF-beta-dependent manner in vitro. CONCLUSION: Inflammatory Gr1(+) monocytes, recruited into the injured liver via CCR2-dependent bone marrow egress, promote the progression of liver fibrosis. Thus, they may represent an interesting novel target for antifibrotic strategies.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5'URR/3'UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5'URR/3'UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes -716 (G/T), -201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.

Activation of ectopic Oct-4 and Rex-1 promoters in human amniotic fluid cells.

Recently, amniotic fluid was suggested as a new source for stem-cell research and tissue engineering approaches. In order to enable isolation of stem cells and establishment of lines of such cells with an undifferentiated phenotype we have introduced green fluorescent protein regulated by the promoters of the stem cell-specific genes, Oct-4 or Rex-1, into human amniotic fluid cells. For the introduction of DNA into human amniotic fluid cells, we have optimized a specific transfection protocol. We found that human amniotic fluid contains cell populations which are able to activate these promoters. These undifferentiated cells expressing green fluorescent protein can be analysed on a flow cytometer. In addition, we have introduced a plasmid harboring a neomycin-resistance gene under the control of the Oct-4 promoter. G418 selection allowed the isolation of undifferentiated stem cells expressing Oct-4 protein out of human amniotic fluid samples. Our findings confirm the existence of stem cells within amniotic fluid. In addition, the ability to transfect human amniotic fluid cells and to isolate stem-cell marker-positive cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.

Chemokine-directed immune cell infiltration in acute and chronic liver disease.

The infiltration of various immune cell populations, including monocytes/macrophages, natural killer (NK), NKT cells and T cells, is a central pathogenic feature following acute- and chronic liver injury. Chemotactic cytokines, chemokines, are small-protein mediators that direct the migration of immune cells. Several hepatic cell populations, including hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells, can secrete chemokines upon activation. Samples from liver-disease patients and animal models of experimental injury highlight multiple activated chemokine pathways during initiation, maintenance or resolution of liver pathology. Monocyte chemoattractant protein-1 (Chemokine [C-C motif] ligand [CCL]2) can attract monocytes via CCR2. Infiltrating monocytes probably have functions in both disease progression and resolution of damage. RANTES (CCL5) may promote infiltration of NK (via CCR1) and T cells (via CCR5). Dissecting the exact functional contribution of immune cell subsets, chemokines and chemokine-receptor pathways in liver injury will hopefully identify novel targets for the treatment of acute liver failure, liver fibrosis or cirrhosis.

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.