Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. CONCLUSION: Using a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88).

Proteomic analysis of altered extracellular matrix turnover in bleomycin-induced pulmonary fibrosis.

Fibrotic disease is characterized by the pathological accumulation of extracellular matrix (ECM) proteins. Surprisingly, very little is known about the synthesis and degradation rates of the many proteins and proteoglycans that constitute healthy or pathological extracellular matrix. A comprehensive understanding of altered ECM protein synthesis and degradation during the onset and progression of fibrotic disease would be immensely valuable. We have developed a dynamic proteomics platform that quantifies the fractional synthesis rates of large numbers of proteins via stable isotope labeling and LC/MS-based mass isotopomer analysis. Here, we present the first broad analysis of ECM protein kinetics during the onset of experimental pulmonary fibrosis. Mice were labeled with heavy water for up to 21 days following the induction of lung fibrosis with bleomycin. Lung tissue was subjected to sequential protein extraction to fractionate cellular, guanidine-soluble ECM proteins and residual insoluble ECM proteins. Fractional synthesis rates were calculated for 34 ECM proteins or protein subunits, including collagens, proteoglycans, and microfibrillar proteins. Overall, fractional synthesis rates of guanidine-soluble ECM proteins were faster than those of insoluble ECM proteins, suggesting that the insoluble fraction reflected older, more mature matrix components. This was confirmed through the quantitation of pyridinoline cross-links in each protein fraction. In fibrotic lung tissue, there was a significant increase in the fractional synthesis of unique sets of matrix proteins during early (pre-1 week) and late (post-1 week) fibrotic response. Furthermore, we isolated fast turnover subpopulations of several ECM proteins (e.g. type I collagen) based on guanidine solubility, allowing for accelerated detection of increased synthesis of typically slow-turnover protein populations. This establishes the presence of multiple kinetic pools of pulmonary collagen in vivo with altered turnover rates during evolving fibrosis. These data demonstrate the utility of dynamic proteomics in analyzing changes in ECM protein turnover associated with the onset and progression of fibrotic disease.

Mannose receptor 2 attenuates renal fibrosis.

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3(-/-) mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3(-/-) mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway.

Differential in vivo effects on target pathways of a novel arylpyrazole glucocorticoid receptor modulator compared with prednisolone.

Glucocorticoids are widely prescribed to treat autoimmune and inflammatory diseases. Although they are extremely potent, their utility in clinical practice is limited by a variety of adverse side effects. Development of compounds that retain the potent immunomodulating and anti-inflammatory properties of classic glucocorticoids while exhibiting reduced adverse actions is therefore a priority. Using heavy water labeling and mass spectrometry to measure fluxes through multiple glucocorticoid-responsive, disease-relevant target pathways in vivo in mice, we compared the effects of a classic glucocorticoid receptor (GR) ligand, prednisolone, with those of a novel arylpyrazole-based compound, L5 {[1-(4-fluorophenyl)-4a-methyl-5,6,7,8-tetrahydro-4H-benzo[f]indazol-5-yl]-[4-(trifluoromethyl)phenyl]methanol}. We show for the first time that L5 exhibits clearly selective actions on disease-relevant pathways compared with prednisolone. Prednisolone reduced bone collagen synthesis, skin collagen synthesis, muscle protein synthesis, and splenic lymphocyte counts, proliferation, and cell death, whereas L5 had none of those actions. In contrast, L5 was a more rapid and potent inhibitor of hippocampal neurogenesis than prednisolone, and L5 and prednisolone induced insulin resistance equally. Administration of prednisolone or L5 increased expression comparably for one GR-regulated gene involved in protein degradation in skeletal muscle (Murf1) and one GR-regulated gluconeogenic gene in liver (PEPCK). In summary, L5 dissociates the pleiotropic effects of the GR ligand prednisolone in intact animals in ways that neither gene expression nor cell-based models were able to fully capture or predict. Because multiple actions can be measured concurrently in a single animal, this method is a powerful systems approach for characterizing and differentiating the effects of ligands that bind nuclear receptors.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease.

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2-0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.

Extracellular matrix proteins: a positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation. Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0-10 ng/ml transforming growth factor (TGF)ß1. mRNA expression was analyzed by quantitative real-time PCR. New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFß1 strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen. The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.

Novel combination of collagen dynamics analysis and transcriptional profiling reveals fibrosis-relevant genes and pathways.

Collagen deposition is a key process during idiopathic pulmonary fibrosis; however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analyses do not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labeled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation. Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labeled with deuterium by providing mice with deuterated drinking water. After sacrifice, we collected lung tissue for microarray analysis, determination of new collagen formation, and histology. Furthermore, we measured in vitro the expression of selected genes after transforming growth factor (TGF) ß1-induced myofibroblast differentiation. Deuterated water labeling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data allowed us to create a gene expression signature of fibrosis within the lung and revealed fibrosis-specific processes, among which proliferation. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation in a separate experiment. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, Wnt-1 inducible signaling pathway protein 1, tenascin C, lysyl oxidase, and type V collagen. Gene expression of these genes was upregulated in vitro in fibroblasts stimulated with TGFß1. Together, these data demonstrate, using a novel combination of technologies, that the core process of fibrosis, i.e. the formation of new collagen, correlates not only with a wide range of genes involved in general extracellular matrix production and modification but also with cell proliferation. The observation that the large majority of the genes which correlated with new collagen formation also were upregulated during TGFß1-induced myofibroblast differentiation provides further evidence for their involvement in fibrosis.

A pilot study demonstrating a non-invasive method for the measurement of protein turnover in skin disorders: application to psoriasis.

BACKGROUND: Previous studies of epidermal kinetics in psoriasis have relied on invasive biopsy procedures or the use of radioactive labels. We previously developed a non-invasive method for measuring keratin synthesis in human skin using deuterated water labeling, serial collection of tape strips and measurement of deuterium enrichment in protein by mass spectrometry. This powerful method can be applied to measure other skin proteins and lipids collected by tape stripping. Here, for the first time, we apply this technique to investigate the epidermal kinetics of psoriasis, the first step in defining a kinetic profile for normal skin versus activated or quiescent psoriatic skin. METHODS: Psoriatic subjects were given (2)H2O orally as twice-daily doses for 16-38 days. Affected and unaffected skin was sampled by tape stripping and washing (modified Pachtman method). Proteins were isolated from the tape strips by a method that enriches for keratin. Turnover times were determined by gas chromatography/mass spectrometry. Kinetic data were compared to transepidermal water loss (TEWL). RESULTS: Deuterium-labeled protein from lesional psoriatic skin appeared at the skin surface within 3-8 days of label administration, whereas labeled protein from non-lesional skin requires 10-20 days to appear. Psoriatic skin had similar rate of growth despite varying anatomic location. Proteins recovered from tape strips were identified by nanoscale liquid chromatography/tandem mass spectrometry. Isolated peptides were >98% from keratin in uninvolved skin and >72% keratin in psoriatic skin. Revealing that one-quarter of all newly synthesized proteins in psoriatic skin are antimicrobial defense and other immune-related proteins. TEWL values were greater in lesional than non-lesional skin, suggesting barrier compromise in psoriatic skin despite increased clinical thickness. CONCLUSIONS: This simple, elegant, and non-invasive method for measuring epidermal protein synthesis, which can also be adapted to measure epidermal lipids, provides a metric that may reveal new insights into the mechanisms and dynamic processes underlying psoriasis and may also provide an objective scale for determining response to therapeutic agents in pre-clinical and clinical trials. This opens a pathway to the non-invasive study of kinetics of protein formation in psoriasis or other skin diseases.

Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages.

Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8-/- mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8-/- macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.

IL-4 induces characteristic Th2 responses even in the combined absence of IL-5, IL-9, and IL-13.

Functional redundancy is highly prevalent among the Th2 interleukins (IL)-4, IL-5, IL-9, and IL-13. To define the critical functions of these cytokines, we have generated a novel panel of compound Th2 cytokine-deficient mice (from single to quadruple cytokine knockouts). We find that these Th2 cytokines are not essential for fetal survival even during allogeneic pregnancy. Using intestinal parasite infection and a pulmonary granuloma model, we demonstrate cryptic roles for IL-4, IL-5, IL-9, and IL-13 in these responses. Significantly, although IL-5, IL-9, and IL-13 add to the speed and magnitude of the response, a threshold is reached at which IL-4 alone can activate all Th2 effector functions. These mice reveal distinct spatial, temporal, and hierarchical cytokine requirements in immune function.