C-C chemokine receptor type 7 (CCR7) regulates hepatic CD8 + T cell homeostasis and response to acute liver injury.

BACKGROUND AND AIMS: Acute liver failure (ALF) is a rare but life-threatening condition, and DILI, particularly acetaminophen toxicity, is the leading cause of ALF. Innate immune mechanisms further perpetuate liver injury, while the role of the adaptive immune system in DILI-related ALF is unclear. APPROACH AND RESULTS: We analyzed liver tissue from 2 independent patient cohorts with ALF and identified hepatic T cell infiltration as a prominent feature in human ALF. CD8 + T cells were characterized by zonation toward necrotic regions and an activated gene expression signature. In murine acetaminophen-induced liver injury, intravital microscopy revealed zonation of CD8 + but not CD4 + T cells at necrotic areas. Gene expression analysis exposed upregulated C-C chemokine receptor 7 (CCR7) and its ligand CCL21 in the liver as well as a broadly activated phenotype of hepatic CD8 + T cells. In 2 mouse models of ALF, Ccr7-/- mice had significantly aggravated early-phase liver damage. Functionally, CCR7 was not involved in the recruitment of CD8 + T cells, but regulated their activation profile potentially through egress to lymphatics. Ccr7-/- CD8 + T cells were characterized by elevated expression of activation, effector, and exhaustion profiles. Adoptive transfer revealed preferential homing of CCR7-deficient CD8 + T cells to the liver, and depletion of CD8 + T cells attenuated liver damage in mice. CONCLUSIONS: Our study demonstrates the involvement of the adaptive immune system in ALF in humans and mice. We identify the CCR7-CCL21 axis as an important regulatory pathway, providing downstream protection against T cell-mediated liver injury.

Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease.

UNLABELLED: Pathogen- and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver-derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild-type (WT) and Hrg(-/-) mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti-inflammatory M2 subtype in Hrg(-/-) mice. Upon chronic liver damage induced by CCl4 or methionine-choline-deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg(-/-) , compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg(-/-) mice. Moreover, HRG-deficient mice showed significantly enhanced hepatic vascularization by micro-computed tomography and histology, corroborating proangiogenic activities of M2-polarized liver macrophages. Purified HRG protein induced, but HRG-deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg(-/-) mice transplanted with Hrg(+/+) bone marrow, but not Hrg(-/-) -transplanted Hrg(+/+) mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up-regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. CONCLUSIONS: Liver-derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases.

Cyclic adenosine monophosphate-responsive element modulator alpha overexpression impairs function of hepatic myeloid-derived suppressor cells and aggravates immune-mediated hepatitis in mice.

UNLABELLED: Molecular factors driving immune-mediated inflammation in the liver are incompletely understood. The transcription factor, cyclic adenosine monophosphate-responsive element modulator alpha (CREMα) can endorse differentiation of T lymphocytes toward T-helper (Th)17 cells, thereby promoting autoimmunity in systemic lupus erythematosus or lung inflammation. To investigate the role of CREMα in liver disease, we subjected transgenic (Tg) mice overexpressing CREMα under control of the CD2 promoter (cremtg mice), which restrains expression mainly to lymphocytes (T, natural killer [NK], and NKT cells), to acute and chronic liver injury models. Already in steady state, Tg CREMα overexpression broadly reduced hepatic immune cell numbers by decreasing their viability, but did not affect immune cell migration or the fibrogenic response to chronic liver injury. Strikingly, cremtg mice developed more severe immune-mediated hepatitis with a higher mortality rate, compared to wild-type (wt) mice, upon concanavalin A (ConA) administration. Unlike in T cells from spleen, CREMα overexpression did not induce a predominant Th17 response in intrahepatic T cells, given that hepatic cremtg CD4+ T cells expressed less interleukin (IL)-17 than wt T cells. Reconstitution of Rag1-/- mice with Crem-/- T cells did not ameliorate ConA hepatitis. Overexpression of CREMα did not influence NK and NKT-cell effector functions either. Interestingly, a subset of monocytic myeloid-derived suppressor cells (MDSCs) also expressed CD2 and CREMα. Cremtg MDSCs isolated from liver expressed reduced inducible nitric oxide synthase and arginase 1 and displayed a reduced T-cell suppressive activity. The adoptive transfer of wt MDSCs was capable of reducing the fulminant immune-mediated liver damage in cremtg mice to wt level. CONCLUSION: These results suggest compartmental differences of T cell activation pathways between liver and other organs in autoimmunity and define a functional role of CREMα in hepatic monocytic MDSCs for the pathogenesis of immune-mediated liver disease.

Liposomal encapsulation of dexamethasone modulates cytotoxicity, inflammatory cytokine response, and migratory properties of primary human macrophages.

The encapsulation of drugs into liposomes aims to enhance their efficacy and reduce their toxicity. Corticosteroid-loaded liposomes are currently being evaluated in patients suffering from rheumatoid arthritis, atherosclerosis, colitis, and cancer. Here, using several different fluorophore-labeled formulations, we comprehensively studied the impact of liposome encapsulation of the prototypic corticosteroid dexamethasone on various primary human cells in vitro. Liposomal dexamethasone targeted several primary cell types in a dose and time-dependent manner, but specifically reduced cytotoxicity against human fibroblasts and macrophages in comparison to the solute drug. Furthermore, macrophage maturation and polarization markers were altered. Interestingly, liposomal dexamethasone induced proinflammatory cytokine secretion (specifically TNF, IL1ß, IL6) in unstimulated cells, but reduced this response under inflammatory conditions. Monocyte and macrophage migration was significantly inhibited by dexamethasone-loaded liposomes. The findings indicate that the encapsulation of dexamethasone into liposomes modulates their cellular mechanism of action, and provides important indications for follow-up in vivo investigations. FROM THE CLINICAL EDITOR: This study investigates mechanism of action of liposomal dexamethason in the treatment of inflammatory conditions. It is concluded that liposomal dexamethasone actually induces proinflammatory cytokine secretion in unstimulated cells, but reduces the same response under inflammatory conditions. Monocyte and macrophage migration was also inhibited. The findings indicate that liposomal dexamethasone may have different mechanisms of action than its native counterpart.

Roles of CCR2 and CCR5 for Hepatic Macrophage Polarization in Mice With Liver Parenchymal Cell-Specific NEMO Deletion.

BACKGROUND & AIMS: Macrophages are key regulators of inflammation and cancer promotion in the liver, and their recruitment and activation is linked to chemokine receptor signaling. However, the exact roles of the chemokine receptors CCR2 and CCR5 for macrophage functions in the liver is obscure. METHODS: To study CCR2 and CCR5 in inflammatory liver injury, we used mice with a hepatocyte-specific knock-out of the nuclear factor κB (NF-κB) essential modulator (NEMO), termed NEMOLPC-KO mice, and generated NEMOLPC-KOCcr2-/- and NEMOLPC-KOCcr5-/- mice. NEMOLPC-KO mice develop hepatitis and fibrosis after two and liver tumors after six months. RESULTS: We found that both CCR2 and CCR5 deficiency led to reduced fibrosis, while CCR5 deficiency increased steatosis and tumor burden in NEMOLPC-KO mice. CCR2 was required for recruitment of hepatic macrophages, whereas CCR5 promoted stellate cell activation. The reduction of monocytes and macrophages by either anti-Gr1 antibody or clodronate-loaded liposomes (CLL), but not of CD8+ T cells or NK cells, significantly aggravated liver injury in NEMOLPC-KO mice and was further increased in NEMOLPC-KOCcr5-/- mice. CLL-induced liver injury was dampened by the adoptive transfer of ex vivo generated macrophages, whereas the adoptive transfer of control CD115+ immature monocytes or B cells did not reduce liver injury. CONCLUSIONS: Although CCR2 and CCR5 principally promote liver fibrosis, they exert differential functions on hepatic macrophages during liver disease progression in NEMOLPC-KO mice. While CCR2 controls the recruitment of monocytes to injured livers, CCR5-dependent functions of liver macrophages limit hepatic injury, thereby reducing steatosis and hepatocarcinogenesis.

Differential Gene Expression in Circulating CD14+ Monocytes Indicates the Prognosis of Critically Ill Patients with Sepsis.

Critical illness and sepsis are characterized by drastic changes in the systemic innate immune response, particularly involving monocytes. The exact monocyte activation profile during sepsis, however, has remained obscure. Therefore, we prospectively analyzed the gene expression profile of circulating CD14+ monocytes from healthy volunteers (n = 54) and intensive care unit (ICU) patients (n = 76), of which n = 36 had sepsis. RNA sequencing of selected samples revealed that monocytes from septic ICU patients display a peculiar activation pattern, which resembles characteristic functional stages of monocyte-derived macrophages and is distinct from controls or non-sepsis ICU patients. Focusing on 55 highly variable genes selected for further investigation, arachidonate 5-lipoxygenase-activating protein (ALOX5AP) was highly upregulated in monocytes of ICU patients and only normalized during 7 days in the ICU in non-sepsis patients. Strikingly, low monocytic guanine nucleotide exchange factor 10-like protein (ARHGEF10L) mRNA expression was associated with the disease severity and mortality of ICU patients. Collectively, our comprehensive analysis of circulating monocytes in critically ill patients revealed a distinct activation pattern, particularly in ICU patients with sepsis. The association with disease severity, the longitudinal recovery or lack thereof during the ICU stay, and the association with prognosis indicate the clinical relevance of monocytic gene expression profiles during sepsis.

Correction to: Neutrophils are a main source of circulating suPAR predicting outcome in critical illness.

[This corrects the article DOI: 10.1186/s40560-019-0381-5.].

Neutrophils are a main source of circulating suPAR predicting outcome in critical illness.

BACKGROUND: Circulating levels of soluble urokinase plasminogen activation receptor (suPAR) have been proposed as a prognostic biomarker in patients with critical illness and sepsis. However, the origin of suPAR in sepsis has remained obscure. We investigated the potential cellular sources of suPAR by analyzing membrane-bound urokinase plasminogen activator receptor (uPAR, CD87) and evaluated its clinical relevance in critically ill patients. METHODS: We studied 87 critically ill patients (44 with sepsis, 43 without sepsis) from the medical intensive care unit (ICU) in comparison to 48 standard care patients with infections and 27 healthy controls in a prospective single-center non-interventional cohort study. Cellular uPAR expression of different immune cell subsets (by flow cytometry from peripheral blood) and corresponding serum suPAR concentrations were determined upon ICU admission and at day 3. Furthermore, we analyzed the effects of serum from sepsis patients on the activation and uPAR cleavage of primary human neutrophils and macrophages in vitro. RESULTS: In healthy controls, uPAR (CD87) expression was detected on nearly all blood neutrophils and monocytes, but only scarcely on lymphocytes. While uPAR expression on monocytes was maintained in ICU patients, only 58% of neutrophils from critically ill patients expressed uPAR, which was significantly lower than in healthy controls or standard care patients. Concomitantly, serum suPAR levels were significantly increased in ICU patients. We noted a clear inverse correlation between low neutrophilic uPAR and high serum suPAR in standard care and ICU patients, indicating that shedding of uPAR from activated neutrophils represents a main source of suPAR in systemic inflammation. Both low uPAR and high suPAR were closely associated with mortality in critically ill patients. Furthermore, serum from sepsis patients induced uPAR protein expression and subsequent receptor shedding on isolated primary neutrophils, but not on macrophages, in vitro. CONCLUSIONS: The inverse correlation between low uPAR surface expression on neutrophils and high serum suPAR in critically ill patients supports that neutrophils are a main source of shed suPAR proteins in systemic inflammation. Furthermore, high suPAR levels and low neutrophilic uPAR expression predict mortality in ICU patients.

Erratum: Hohlstein, P. et al. Prognostic Relevance of Altered Lymphocyte Subpopulations in Critical Illness and Sepsis. Journal of Clinical Medicine 2019, 8, 353.

The authors wish to make the following corrections to this paper [...].

Prognostic Relevance of Altered Lymphocyte Subpopulations in Critical Illness and Sepsis.

Lymphopenia and functional defects in lymphocytes may impact the prognosis in patients with critical illness or sepsis. Therefore, we prospectively analyzed peripheral blood leukocytes from 63 healthy volunteers, 50 non-critically ill standard care (SC) patients with infections, and 105 intensive care unit (ICU) patients (52 with sepsis, 53 without sepsis) using flow cytometry. Compared to healthy volunteers, SC and ICU patients showed significant leukocytosis, especially in sepsis, while lymphocyte numbers were significantly decreased. All major lymphocyte populations (B, T, and natural killer (NK) cells) decreased in ICU patients. However, we observed a relative reduction of T cells, alongside decreased CD8+ T cells, in critically ill patients, independent of sepsis. High absolute T cell counts (>0.36/nL) at ICU admission were associated with a significantly reduced mortality, independent of patient's age. Moreover, patients that survived ICU treatment showed dynamic changes within 48 h towards restoration of lymphopenia and T cell depletion, while non-surviving patients failed to restore lymphocyte counts. In conclusion, the flow-cytometric analysis of peripheral blood revealed striking changes in circulating lymphocyte subsets in critically ill patients, independent of sepsis. Lymphopenia and T cell depletion at ICU admission were associated with increased mortality, supporting their relevance as predictive biomarkers and potential therapeutic targets in intensive care medicine.

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/ß-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

Targeting and Modulation of Liver Myeloid Immune Cells by Hard-Shell Microbubbles.

Poly n-butylcyanoacrylate (PBCA)-based hard-shell microbubbles (MB) have manifold biomedical applications, including targeted drug delivery or contrast agents for ultrasound (US)-based liver imaging. MB and their fragments accumulate in phagocytes, especially in the liver, but it is unclear if MB affect the function of these immune cells. We herein show that human primary monocytes internalize different PBCA-MB by phagocytosis, which transiently inhibits monocyte migration in vertical chemotaxis assays and renders monocytes susceptible to cytotoxic effects of MB during US-guided destruction. Conversely, human macrophage viability and function, including cytokine release and polarization, remain unaffected after MB uptake. After i.v. injection in mice, MB predominantly accumulate in liver, especially in hepatic phagocytes (monocytes and Kupffer cells). Despite efficiently targeting myeloid immune cells in liver, MB or MB after US-elicited burst do not cause overt hepatotoxicity or inflammation. Furthermore, MB application with or without US-guided burst does not aggravate the course of experimental liver injury in mice or the inflammatory response to liver injury in vivo. In conclusion, PBCA-MB have immunomodulatory effects on primary human myeloid cells in vitro, but do not provoke hepatotoxicity, inflammation or altered response to liver injury in vivo, suggesting the safety of these MB for diagnostic and therapeutic purposes.

Gold Nanocarriers for Macrophage-Targeted Therapy of Human Immunodeficiency Virus.

The human immunodeficiency virus (HIV) continues to be a global pandemic and there is an urgent need for innovative treatment. Immune cells represent a major target of virus infection, but are also therapeutic targets. Currently, no antiretroviral therapy targets macrophages, which function as portal of entry and as major long-term deposit of HIV. It has been shown before that human macrophages efficiently internalize gold nanoparticles, a fact which might be used to target them with drug-nanoparticle conjugates. Here, the authors use gold nanocarriers to facilitate delivery of stavudine, a widely used antiretroviral drug, to primary human macrophages. Using an ease-of-use coupling method, a striking potentiation of stavudine intake by macrophages using gold nanocarriers is shown. Further, the carriers induce a specific subtype of proinflammatory activation indicative for antiviral activity of macrophages, which suggests promising novel treatment options for HIV.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases.

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

Molecular response of liver sinusoidal endothelial cells on hydrogels.

There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

Isolation and time lapse microscopy of highly pure hepatic stellate cells.

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60-95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

Therapeutic targeting of liver inflammation and fibrosis by nanomedicine.

Nanomedicine constitutes the emerging field of medical applications for nanotechnology such as nanomaterial-based drug delivery systems. This technology may hold exceptional potential for novel therapeutic approaches to liver diseases. The specific and unspecific targeting of macrophages, hepatic stellate cells (HSC), hepatocytes, and liver sinusoidal endothelial cells (LSEC) using nanomedicine has been developed and tested in preclinical settings. These four major cell types in the liver are crucially involved in the complex sequence of events that occurs during the initiation and maintenance of liver inflammation and fibrosis. Targeting different cell types can be based on their capacity to ingest surrounding material, endocytosis, and specificity for a single cell type can be achieved by targeting characteristic structures such as receptors, sugar moieties or peptide sequences. Macrophages and especially the liver-resident Kupffer cells are in the focus of nanomedicine due to their highly efficient and unspecific uptake of most nanomaterials as well as due to their critical pathogenic functions during inflammation and fibrogenesis. The mannose receptor enables targeting macrophages in liver disease, but macrophages can also become activated by certain nanomaterials, such as peptide-modified gold nanorods (AuNRs) that render them proinflammatory. HSC, the main collagen-producing cells during fibrosis, are currently targeted using nanoconstructs that recognize the mannose 6-phosphate and insulin-like growth factor II, peroxisome proliferator activated receptor 1, platelet-derived growth factor (PDGF) receptor ß, or integrins. Targeting of the major liver parenchymal cell, the hepatocyte, has only recently been achieved with high specificity by mimicking apolipoproteins, naturally occurring nanoparticles of the body. LSEC were found to be targeted most efficiently using carboxy-modified micelles and their integrin receptors. This review will summarize important functions of these cell types in healthy and diseased livers and discuss current strategies of cell-specific targeting for liver diseases by nanomedicine.