Redefining IL11 as a regeneration-limiting hepatotoxin and therapeutic target in acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 (Il11ra1) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage.

Sources of variability in quantifying circulating thymosin beta-4: literature review and recommendations.

INTRODUCTION: Thymosin beta-4 (TB4) is an endogenous peptide with protective and regenerative effects in models of cellular and organ injury. TB4 is increasingly measured as a potential plasma or serum biomarker in human cardiovascular, liver, infectious, and autoimmune disease. AREAS COVERED: The focus of this review is the quantification of TB4 in clinical cohort studies and whether reported TB4 concentrations differ with respect to method of sample preparation. We survey current literature for studies measuring TB4 in human serum or plasma and compare reported concentrations in healthy controls. EXPERT OPINION: We find substantial intra- and inter- study variability in healthy controls, and a lack of protocol standardization. We further highlight three factors that may confound TB4 clinical measurements and should be considered in future study design: 1) residual platelets remaining in suspension after centrifugation, 2) TB4 release following ex vivo platelet activation, and 3) specificity of assays towards posttranslational modifications. Accordingly, we put forth our recommendations to minimize residual and activated platelets during sample collection, and to cross-validate TB4 measurements using both antibody-based and mass spectrometry-based methods.

Thymosin Beta-4 Is Elevated in Women With Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Thymosin beta-4 (TB4) is an X-linked gene product with cardioprotective properties. Little is known about plasma concentration of TB4 in heart failure (HF), and its relationship with other cardiovascular biomarkers. We sought to evaluate circulating TB4 in HF patients with preserved (HFpEF) or reduced (HFrEF) ejection fraction compared to non-HF controls. METHODS AND RESULTS: TB4 was measured using a liquid chromatography and mass spectrometry assay in age- and sex-matched HFpEF (n=219), HFrEF (n=219) patients, and controls (n=219) from a prospective nationwide study. Additionally, a 92-marker multiplex proximity extension assay was measured to identify biomarker covariates. Compared with controls, plasma TB4 was elevated in HFpEF (985 [421-1723] ng/mL versus 1401 [720-2379] ng/mL, P<0.001), but not in HFrEF (1106 [556-1955] ng/mL, P=0.642). Stratifying by sex, only women (1623 [1040-2625] ng/mL versus 942 [386-1891] ng/mL, P<0.001), but not men (1238.5 [586-1967] ng/mL versus 1004 [451-1538] ng/mL, P=1.0), had significantly elevated TB4 in the setting of HFpEF. Adjusted for New York Heart Association class, N-terminal pro B-type natriuretic peptide, age, and myocardial infarction, hazard ratio to all-cause mortality is significantly higher in women with elevated TB4 (1.668, P=0.036), but not in men (0.791, P=0.456) with HF. TB4 is strongly correlated with a cluster of 7 markers from the proximity extension assay panel, which are either X-linked, regulated by sex hormones, or involved with NF-κB signaling. CONCLUSIONS: We show that plasma TB4 is elevated in women with HFpEF and has prognostic information. Because TB4 can preserve EF in animal studies of cardiac injury, the relation of endogenous, circulating TB4 to X chromosome biology and differential outcomes in female heart disease warrants further study.

Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus.

Based on recent analytical and enzymological studies, a topological model for the role of alpha-D-mannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man(2)-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [(3)H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man(2)-DAG and LM at the nonpermissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man(2)-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man(2)-DAG, membranes from this mutant efficiently converted purified, exogenous [(3)H]Man(2)-DAG to [(3)H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [(3)H]Man(2)-DAG to [(3)H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man(2)-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [(3)H]Man(2)-DAG was converted to [(3)H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man(2)-DAG as the lipid anchor precursor for LM, and suggest that Man(2)-DAG may be essential for the normal growth of M. luteus cells.

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus.

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.