Robust collection and processing for label-free single voxel proteomics.

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of ∼900 and ∼4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 µm × 20 µm × 10 µm (close to single cells) and 200 µm × 200 µm × 10 µm (∼100 cells), respectively. 100s-1000s of protein signatures with differential expression levels were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. Antibody-based CODEX imaging was used to validate label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer. SIGNIFICANCE: This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.

Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis.

Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

Machine learning-assisted elucidation of CD81-CD44 interactions in promoting cancer stemness and extracellular vesicle integrity.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Predictors of impaired SARS-CoV-2 immunity in healthcare workers after vaccination with BNT162b2.

Healthcare workers are at substantially increased risk for infection with SARS-CoV-2. Successful vaccination constitutes a crucial prerequisite to protect this group during the pandemic. Since post vaccination antibody monitoring is not standard of care in all healthcare institutions, data on risk factors of impaired vaccine induced immune response are urgently required. Moreover, there are no data on cellular immune responses in humoral low responders so far. Anti-SARS-CoV-2 spike IgG was assessed after vaccination with BNT162b2 in 1386 employees of three hospitals of a German healthcare provider. Concentrations were compared to those of 45 convalescent employees. Vaccine-induced cellular immunity was measured in employees with reduced humoral response by assessment of frequencies of SARS-CoV-2-reactive CD4+ and CD8+ T cell. Anti-SARS-CoV-2 spike IgG were detected in 99.9% of 1386 healthcare workers after completed vaccination. The median antibody concentration was significantly higher after vaccination than after infection with SARS-CoV-2 (p = 0.0001). 10 subjects (0.7%) generated an IgG concentration < 100 IU/ml, and only two persons (0.1%, solid organ recipients) did not produce detectable antibodies at all. T cell responses of those subjects with submaximal or lacking humoral response were comparable to employees with maximal antibody titers. 50% of those individuals with impaired or lacking humoral immune response were on immunosuppression. Vaccination to SARS-CoV-2 with BNT162b2 is very effective in healthcare workers yielding a seroconversion rate of 99.9%. Immunosuppression is the most important risk factor of an impaired immune response. There was no case of vaccination failure without immunosuppression. Thus, post vaccination antibody monitoring is highly recommendable in those employees with immunosuppression.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer.

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Multiplexed Tissue Tomography.

Multiplexed tissue tomography enables comprehensive spatial analysis of markers within a whole tissue or thick tissue section. Clearing agents are often used to make tissue transparent and facilitate deep tissue imaging. Many methods of clearing and tissue tomography are currently used in a variety of tissue types. Here we detail a workflow known as transparent tissue tomography (T3), which builds upon previous methods and can be applied to difficult to clear tissues such as tumors.

Pharmacokinetics and safety of TCMCB07, a melanocortin-4 antagonist peptide in dogs.

The melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 was developed for the treatment of cachexia. The objectives of this study were to examine pharmacokinetics and safety of TCMCB07 administered subcutaneously to healthy dogs. Dogs were treated with high- (2.25 mg kg-1 ) (n = 5) and low-dose TCMCB07 (0.75 mg kg-1 ) (n = 5) once daily for 28 days with a 14-day washout period between groups. Histamine levels, complete blood count, chemistry panel, blood pressure, 24-hour Holter recording, and pharmacokinetic parameters were monitored in the high-dose group. Physical examination changes were limited to weight gain and darkening of the coat color. There was no elevation of plasma histamine within 24 hours of injection but there was a significant elevation of plasma histamine across time. An approximately doubled eosinophil count and an approximately 25% increase, and then 25% decrease back to pre-treatment plasma phosphorous were also found, although both remained within the reference interval. Serial blood pressure and 24-hour Holter monitors revealed no clinically relevant changes. A difference was found in the AUC between dosing groups and a significant effect of dose, time, and interaction was noted for Vd . Low-dose TCMCB07 had a Cmax of 2.1 ug ml-1 at day 28, compared to high-dose TCMCB07 which had a Cmax 3.6 ug ml-1 at day 28. Once-daily subcutaneous administration of TCMCB07 was well-tolerated for up to 28 days in dogs when administered at doses one and three times (0.75 mg kg-1 and 2.25 mg kg-1 ) the predicted therapeutic dose and pharmacokinetic parameters are described. SIGNIFICANCE STATEMENT: Melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 is safe at both low and high doses in dogs. Therapy was tolerated well as determined by physical examination, clinical pathology, and cardiovascular parameters; darkening of the coat was noted with treatment and resolved with discontinuation. Pharmacokinetics are described and further study in the naturally occurring canine model is warranted.

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-ß-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for 'all-in-one' one-pot sample preparation. This 'all-in-one' method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

Heterozygous carriage of the alpha1-antitrypsin Pi*Z variant increases the risk to develop liver cirrhosis.

OBJECTIVE: Homozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants ('Pi*Z' and 'Pi*S'), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse. DESIGN: We analysed multicentric case-control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed. RESULTS: The Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (p<0.0001). Accordingly, the Pi*Z variant increased the risk of NAFLD subjects to develop cirrhosis (adjusted OR=7.3 (95% CI 2.2 to 24.8)). Likewise, the Pi*Z variant presented in 6.2% of alcohol misusers with cirrhosis but only in 2.2% of alcohol misusers without significant liver injury (p<0.0001). Correspondingly, alcohol misusers carrying the Pi*Z variant were prone to develop cirrhosis (adjusted OR=5.8 (95% CI 2.9 to 11.7)). In contrast, the Pi*S variant was not associated with NAFLD-related cirrhosis and only borderline with alcohol-related cirrhosis (adjusted OR=1.47 (95% CI 0.99 to 2.19)). CONCLUSION: The Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%-4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

Endoglin in human liver disease and murine models of liver fibrosis-A protective factor against liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor-ß (TGF-ß) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF-ß type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC-specific endoglin deletion. METHODS: Eng expression was measured in human and murine samples of liver injury. After generating GFAPCre(+) EngΔHSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Engflox/flox HSCs were infected with Cre-expressing virus to deplete Endoglin and fibrogenic responses were analysed. RESULTS: Endoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L-Eng splice variant. Comparing GFAPCre(-) Engf/f to GFAPCre(+) EngΔHSC mice in toxic liver injury, livers of GFAPCre(+) EngΔHSC mice showed 39.9% (P < .01) higher Hydroxyproline content compared to GFAPCre(-) Engf/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P < .05) more Collagen deposition in livers of GFAPCre(+) EngΔHSC mice. Similar results were obtained in mice subjected to cholestatic injury. CONCLUSION: Endoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α-SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF-ß signalling.

Contribution of bone marrow-derived fibrocytes to liver fibrosis.

Since the discovery of fibrocytes in 1994 by Dr. Bucala and colleagues, these bone marrow (BM)-derived collagen Type I producing CD45(+) cells remain the most fascinating cells of the hematopoietic system. Despite recent reports on the emerging contribution of fibrocytes to fibrosis of parenchymal and non-parenchymal organs and tissues, fibrocytes remain the most understudied pro-fibrogenic cellular population. In the past years fibrocytes were implicated in the pathogenesis of liver, skin, lung, and kidney fibrosis by giving rise to collagen type I producing cells/myofibroblasts. Hence, the role of fibrocytes in fibrosis is not well defined since different studies often contain controversial results on the number of fibrocytes recruited to the site of injury versus the number of fibrocyte-derived myofibroblasts in the same fibrotic organ. Furthermore, many studies were based on the in vitro characterization of fibrocytes formed after outgrowth of BM and/or peripheral blood cultures. Therefore, the fibrocyte function(s) still remain(s) lack of understanding, mostly due to (I) the lack of mouse models that can provide complimentary in vivo real-time and cell fate mapping studies of the dynamic differentiation of fibrocytes and their progeny into collagen type I producing cells (and/or possibly, other cell types of the hematopoietic system); (II) the complexity of hematopoietic cell differentiation pathways in response to various stimuli; (III) the high plasticity of hematopoietic cells. Here we summarize the current understanding of the role of CD45(+) collagen type I(+) BM-derived cells in the pathogenesis of liver injury. Based on data obtained from various organs undergoing fibrogenesis or other type of chronic injury, here we also discuss the most recent evidence supporting the critical role of fibrocytes in the mediation of pro-fibrogenic and/or pro-inflammatory responses.

Endoglin in liver fibrogenesis: Bridging basic science and clinical practice.

Endoglin, also known as cluster of differentiation CD105, was originally identified 25 years ago as a novel marker of endothelial cells. Later it was shown that endoglin is also expressed in pro-fibrogenic cells including mesangial cells, cardiac and scleroderma fibroblasts, and hepatic stellate cells. It is an integral membrane-bound disulfide-linked 180 kDa homodimeric receptor that acts as a transforming growth factor-ß (TGF-ß) auxiliary co-receptor. In humans, several hundreds of mutations of the endoglin gene are known that give rise to an autosomal dominant bleeding disorder that is characterized by localized angiodysplasia and arteriovenous malformation. This disease is termed hereditary hemorrhagic telangiectasia type I and induces various vascular lesions, mainly on the face, lips, hands and gastrointestinal mucosa. Two variants of endoglin (i.e., S- and L-endoglin) are formed by alternative splicing that distinguishes from each other in the length of their cytoplasmic tails. Moreover, a soluble form of endoglin, i.e., sol-Eng, is shedded by the matrix metalloprotease-14 that cleaves within the extracellular juxtamembrane region. Endoglin interacts with the TGF-ß signaling receptors and influences Smad-dependent and -independent effects. Recent work has demonstrated that endoglin is a crucial mediator during liver fibrogenesis that critically controls the activity of the different Smad branches. In the present review, we summarize the present knowledge of endoglin expression and function, its involvement in fibrogenic Smad signaling, current models to investigate endoglin function, and the diagnostic value of endoglin in liver disease.

Experimental liver fibrosis research: update on animal models, legal issues and translational aspects.

Liver fibrosis is defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requires in vitro and in vivo experimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

The chemokine CCL3 promotes experimental liver fibrosis in mice.

Liver fibrosis is associated with infiltrating immune cells and activation of hepatic stellate cells. We here aimed to investigate the effects of the CC chemokine CCL3, also known as macrophage inflammatory protein-1α, in two different fibrosis models. To this end, we treated mice either with carbon tetrachloride or with a methionine- and choline-deficient diet to induce fibrosis in CCL3 deficient and wild-type mice. The results show that the protein expression of CCL3 is increased in wild-type mice after chronic liver injury. Deletion of CCL3 exhibited reduced liver fibrosis compared to their wild-type counterparts. We could validate these results by treating the two mouse groups with either carbon tetrachloride or by feeding a methionine- and choline-deficient diet. In these models, lack of CCL3 is functionally associated with reduced stellate cell activation and liver immune cell infiltration. In vitro, we show that CCL3 leads to increased proliferation and migration of hepatic stellate cells. In conclusion, our results define the chemokine CCL3 as a mediator of experimental liver fibrosis. Thus, therapeutic modulation of CCL3 might be a promising target for chronic liver diseases.

Circulating microRNA-150 serum levels predict survival in patients with critical illness and sepsis.

BACKGROUND AND AIMS: Down-regulation of miR-150 was recently linked to inflammation and bacterial infection. Furthermore, reduced serum levels of miR-150 were reported from a small cohort of patients with sepsis. We thus aimed at evaluating the diagnostic and prognostic value of miR-150 serum levels in patients with critically illness and sepsis. METHODS: miR-150 serum levels were analyzed in a cohort of 223 critically ill patients of which 138 fulfilled sepsis criteria and compared to 76 healthy controls. Results were correlated with clinical data and extensive sets of routine and experimental biomarkers. RESULTS: Measurements of miR-150 serum concentrations revealed only slightly reduced miR-150 serum levels in critically ill patients compared to healthy controls. Furthermore miR-150 levels did not significantly differ in critically ill patients with our without sepsis, indicating that miR-150 serum levels are not suitable for diagnostic establishment of sepsis. However, serum levels of miR-150 correlated with hepatic or renal dysfunction. Low miR-150 serum levels were associated with an unfavorable prognosis of patients, since low miR-150 serum levels predicted mortality with high diagnostic accuracy compared with established clinical scores and biomarkers. CONCLUSION: Reduced miR-150 serum concentrations are associated with an unfavorable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of miR-150 in critical illness, our study indicates a potential use of circulating miRNAs as a prognostic rather than diagnostic marker in critically ill patients.

Overexpression of endoglin modulates TGF-ß1-signalling pathways in a novel immortalized mouse hepatic stellate cell line.

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-ß1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-ß1 did not significantly induce GFP expression contrasting the TGF-ß1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-ß-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

Origin of myofibroblasts in liver fibrosis.

Most chronic liver diseases of all etiologies result in progressive liver fibrosis. Myofibroblasts produce the extracellular matrix, including type I collagen, which constitutes the fibrous scar in liver fibrosis. Normal liver has little type I collagen and no detectable myofibroblasts, but myofibroblasts appear early in experimental and clinical liver injury. The origin of the myofibroblast in liver fibrosis is still unresolved. The possibilities include activation of endogenous mesenchymal cells including fibroblasts and hepatic stellate cells, recruitment from the bone marrow, and transformation of epithelial or endothelial cells to myofibroblasts. In fact, the origin of myofibroblasts may be different for different types of chronic liver diseases, such as cholestatic liver disease or hepatotoxic liver disease. This review will examine our current understanding of the liver myofibroblast.

Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.

BACKGROUND & AIMS: Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice. METHODS: Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice). RESULTS: In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis. CONCLUSIONS: IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis.