Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Changes to transcript expression in two liver progenitor cell lines following GLI inhibition.

<p>Heat map representing changes to gene transcript levels in BMOL1.2 (<i>n</i> = 3) or BMOL-TAT (<i>n</i> = 3) lines following GANT61 (10 μM) 8 h treatment. Log₂ intensity scale shown. Downregulated genes (blue) are shown above the yellow dashed line, with upregulated genes (red) below. Grey indicates no data.</p

Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.

<p>Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.</p

SHH expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (<i>n</i> = 1)] were screened for SHH (C-terminus) by immunofluorescence. 5x objective. Representative images shown. <b>(B)</b> qRT-PCR for <i>SHH</i> in human donor and ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, *<i>p</i><0.05). <b>(C)</b> SHH expression (red) by EpCAM⁺ LPCs (green) in donor liver. 63x objective. <b>(D, E)</b> Comprehensive characterisation of SHH expression by liver cell populations in ALD. Images obtained using 63x objective. The majority of SHH is produced by hepatocytes. <b>(D)</b> SHH (red) is expressed by EpCAM⁺ LPCs (green). Co-localisation in merged images indicated by yellow. A subset of CD31⁺ ECs (green) at the cirrhotic interface express SHH. <b>(E)</b> SHH (red) is not expressed by CD45⁺ leukocytes (green). Minimal SHH is expressed by vimentin⁺ ECs (solid arrows), with negligible SHH expressed by vimentin⁺ myofibroblasts (dashed arrows). DAPI, blue.</p

A very minimal population of human vimentin⁺ HSCs/myofibroblasts express a primary cilium, with none detected on CD31⁺ endothelial cells.

<p>Human ALD liver tissue was examined for the expression of primary cilia (α-acetylated tubulin, green; γ-tubulin, red) by vimentin⁺ (grey) HSCs/myofibroblasts <b>(A)</b> or CD31+ (grey) ECs <b>(C)</b>. <b>(A)</b> The majority of vimentin⁺ cells were Pc^-ve in the tissues examined. Representative image shown, displaying absence of Pc on vimentin⁺ cells. To confirm this result, ciliary protein Arl13b (green) was co-stained with vimentin (grey). Rare Arl13b ciliary structures (arrow) co-localised with vimentin⁺ cells. Final panel in A illustrates rare Pc⁺ (α-acetylated tubulin, green; γ-tubulin, red) vimentin⁺ (grey) HSCs/myofibroblasts, at the cirrhotic interface. <b>(B)</b> Number of vimentin⁺ Pc⁺ cells or vimentin⁺ Pc^neg cells per FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample). <b>(C)</b> No Pc were detected on CD31⁺ cells in the tissues examined (ALD <i>n</i> = 3, 8 FOV/sample). Representative image shown. All images obtained using confocal microscopy, 63x objective. DAPI, blue. White arrows illustrate Pc. * Non-specific liver autofluorescence.</p

Widespread GLI expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (n = 1)] were screened for GLI2 (red) expression by immunofluorescence. Representative images taken at 5x or 40x (insets) objective shown. DAPI, blue. <b>(B)</b> qRT-PCR for <i>GLI1</i> and <i>GLI3</i> transcript in human donor or ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, **<i>p</i><0.005). Western blot for full-length GLI1 protein (>150 kDa) in donor (Don) or ALD patient samples. Densitometry analysis with GLI1 normalised to GAPDH (Image J). Mean±S.E.M; **<i>p</i> = 0.0093 (Two-sided student t-test). <b>(C)</b> Nuclear GLI2 (green) expression in EpCAM⁺ (red) LPCs in donor, ALD, PBC and NASH liver. <b>(D)</b> Nuclear GLI2 (green) expression demonstrated within CD31⁺ (red) ECs, CK18⁺ (red) hepatocytes, CD45⁺ (red) leukocytes and vimentin⁺ (red) HSCs/myofibroblasts, in ALD. 63x objective. <b>(E)</b> Maximum intensity projection illustrating close physical association between EpCAM⁺ LPCs (green) and vimentin⁺ HSCs/myofibroblasts (red), both of which express GLI2 (grey), in ALD tissue. Arrows indicate myofibroblasts directly contacting LPCs. Confocal microscopy, 63x objective. Quantitation (%) of EpCAM⁺ GLI2⁺ cells and vimentin⁺ GLI2⁺ cells within the same FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample).</p

Gene changes in BMOL1.2 cells following GANT61 treatment.

<p>Gene changes in BMOL1.2 cells following GANT61 treatment.</p