Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Antagonistic effects of the cytotoxic molecules granzyme B and TRAIL in the immunopathogenesis of sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis. We showed an elevated interferon γ response in patients with primary sclerosing cholangitis and in multidrug resistance protein 2-deficient ( Mdr2-/- ) mice developing sclerosing cholangitis. Interferon γ induced expression of the cytotoxic molecules granzyme B (GzmB) and TRAIL in hepatic lymphocytes and mediated liver fibrosis in sclerosing cholangitis. APPROACH AND RESULTS: In patient samples and Mdr2-/- mice, we identified lymphocyte clusters with a cytotoxic gene expression profile using single-cell RNA-seq and cellular indexing of transcriptomes and epitopes by sequencing analyses combined with multi-parameter flow cytometry. CD8 + T cells and NK cells showed increased expression of GzmB and TRAIL in sclerosing cholangitis. Depletion of CD8 + T cells ameliorated disease severity in Mdr2-/- mice. By using Mdr2-/- × Gzmb-/- and Mdr2-/- × Tnfsf10-/- mice, we investigated the significance of GzmB and TRAIL for disease progression in sclerosing cholangitis. Interestingly, the lack of GzmB resulted in reduced cholangiocyte apoptosis, liver injury, and fibrosis. In contrast, sclerosing cholangitis was aggravated in the absence of TRAIL. This correlated with elevated GzmB and interferon γ expression by CD8 + T cells and NK cells enhanced T-cell survival, and increased apoptosis and expansion of cholangiocytes. CONCLUSIONS: GzmB induces apoptosis and fibrosis in sclerosing cholangitis, whereas TRAIL regulates inflammatory and cytotoxic immune responses, subsequently leading to reduced liver injury and fibrosis.

Conventional NK Cells and Type 1 Innate Lymphoid Cells Do Not Influence Pathogenesis of Experimental Glomerulonephritis.

Innate lymphoid cells (ILCs) that express NK cell receptors (NCRs) and the transcription factor T-bet populate nonlymphoid tissues and are crucial in immune responses against viral infections and malignancies. Recent studies highlighted the heterogeneity of this ILC population and extended their functional spectrum to include important roles in tissue homeostasis and autoimmunity. In this article, we provide detailed profiling of NCR+T-bet+ ILC populations in the murine kidney, identifying conventional NK (cNK) cells and type 1 ILCs (ILC1s) as the two major subsets. Induction of renal inflammation in a mouse model of glomerulonephritis did not substantially influence abundance or phenotype of cNK cells or ILC1s in the kidney. For functional analyses in this model, widely used depletion strategies for total NCR+ ILCs (anti-NK1.1 Ab application) and cNK cells (anti-asialoGM1 serum application) were unreliable tools, because they were accompanied by significant off-target depletion of kidney NKT cells and CD8+ T cells, respectively. However, neither depletion of cNK cells and ILC1s in NKT cell-deficient mice nor specific genetic deletion of cNK cells in Ncr1 Cre/wt × Eomes fl/fl mice altered the clinical course of experimental glomerulonephritis. In summary, we show in this article that cNK cells and ILC1s are dispensable for initiation and progression of immune-mediated glomerular disease and advise caution in the use of standard Ab depletion methods to study NCR+ ILC function in mouse models.

Transcriptional and Clonal Characterization of Cytotoxic T Cells in Crescentic Glomerulonephritis.

SIGNIFICANCE STATEMENT: T-cell infiltration is a hallmark of crescentic GN (cGN), often caused by ANCA-associated vasculitis. Pathogenic T-cell subsets, their clonality, and downstream effector mechanisms leading to kidney injury remain to be fully elucidated. Single-cell RNA sequencing and T-cell receptor sequencing revealed activated, clonally expanded cytotoxic CD4 + and CD8 + T cells in kidneys from patients with ANCA-associated cGN. In experimental cGN, kidney-infiltrating CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), which induced apoptosis in renal tissue cells by activation of procaspase-3, and aggravated disease pathology. These findings describe a pathogenic function of (clonally expanded) cytotoxic T cells in cGN and identify GzmB as a mediator and potential therapeutic target in immune-mediated kidney disease. BACKGROUND: Crescentic GN (cGN) is an aggressive form of immune-mediated kidney disease that is an important cause of end stage renal failure. Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis is a common cause. T cells infiltrate the kidney in cGN, but their precise role in autoimmunity is not known. METHODS: Combined single-cell RNA sequencing and single-cell T-cell receptor sequencing were conducted on CD3 + T cells isolated from renal biopsies and blood of patients with ANCA-associated cGN and from kidneys of mice with experimental cGN. Functional and histopathological analyses were performed with Cd8a-/- and GzmB-/- mice. RESULTS: Single-cell analyses identified activated, clonally expanded CD8 + and CD4 + T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Clonally expanded CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), in the mouse model of cGN. Deficiency of CD8 + T cells or GzmB ameliorated the course of cGN. CD8 + T cells promoted macrophage infiltration and GzmB activated procaspase-3 in renal tissue cells, thereby increasing kidney injury. CONCLUSIONS: Clonally expanded cytotoxic T cells have a pathogenic function in immune-mediated kidney disease.

Hemophagocytic lymphohistiocytosis-like hyperinflammation due to a de novo mutation in DPP9.

BACKGROUND: Genetic defects in components of inflammasomes can cause autoinflammation. Biallelic loss-of-function mutations in dipeptidyl peptidase 9 (DPP9), a negative regulator of the NLRP1 and CARD8 inflammasomes, have recently been shown to cause an inborn error of immunity characterized by pancytopenia, skin manifestations, and increased susceptibility to infections. OBJECTIVE: We sought to study the molecular basis of autoinflammation in a patient with severe infancy-onset hyperinflammation associated with signs of fulminant hemophagocytic lymphohistiocytosis. METHODS: Using heterologous cell models as well as patient cells, we performed genetic, immunologic, and molecular investigations to identify the genetic cause and to assess the impact of the identified mutation on inflammasome activation. RESULTS: The patient exhibited pancytopenia with decreased neutrophils and T, B, and natural killer cells, and markedly elevated levels of lactate dehydrogenase, ferritin, soluble IL-2 receptor, and triglycerides. In addition, serum levels of IL-1ß and IL-18 were massively increased, consistent with inflammasome activation. Genetic analysis revealed a previously undescribed de novo mutation in DPP9 (c.755G>C, p.Arg252Pro) affecting a highly conserved amino acid residue. The mutation led to destabilization of the DPP9 protein as shown in transiently transfected HEK293T cells and in patient-derived induced pluripotent stem cells. Using functional inflammasome assays in HEK293T cells, we demonstrated that mutant DPP9 failed to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation. These findings suggest that the Arg252Pro DPP9 mutation acts in a dominant-negative manner. CONCLUSIONS: A de novo mutation in DPP9 leads to severe infancy-onset autoinflammation because of unleashed inflammasome activation.

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase.

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.

The Amphiregulin/EGFR axis protects from lupus nephritis via downregulation of pathogenic CD4+ T helper cell responses.

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immuno-pathogenesis. Lupus nephritis (LN) is a frequent and difficult to treat complication, which causes high morbidity and mortality. The multifunctional cytokine amphiregulin (AREG) has been implicated in SLE pathogenesis, but its function in LN currently remains unknown. We thus studied the model of pristane-induced LN and found increasing renal and systemic AREG expression during the course of disease. Importantly, renal injury was significantly aggravated in the absence of AREG, revealing a net anti-inflammatory role. Analyses of immune responses showed dual effects. On the one hand, AREG enhanced activation of pro-inflammatory myeloid cells, which however did not play a major role for the course of LN. More importantly, on the other hand, AREG strongly suppressed pathogenic cytokine production by T helper effector cells. This effect was more general in nature and could be reproduced in response to antigen immunization. Since AREG has been postulated to downregulate T cell responses via enhancing Treg suppressive capacity, we followed up on this aspect. Interestingly, however, in vitro studies revealed potential direct and Treg independent effects of AREG on T helper effector cells. In favor of this notion, we found significantly enhanced T cell responses and consecutive aggravation of LN, only if epidermal growth factor receptor (EGFR) signaling was abrogated in total T cells, but not if the EGFR was absent on Tregs alone. Finally, we also found enhanced AREG expression in plasma and renal biopsies of patients with LN, supporting the relevance of our findings for human disease. In summary, our data identify AREG as an anti-inflammatory mediator of LN via broad downregulation of pathogenic T cell immunity. These findings further highlight the AREG/EGFR axis as a potential therapeutic target.

Comparison of cerebral oxygen desaturation events between children under general anesthesia and chloral hydrate sedation - a randomized controlled trial.

BACKGROUND: During pediatric general anesthesia (GA) and sedation, clinicians aim to maintain physiological parameters within normal ranges. Accordingly, regional cerebral oxygen saturation (rScO2) should not drop below preintervention baselines. Our study compared rScO2 desaturation events in children undergoing GA or chloral hydrate sedation (CHS). METHODS: Ninety-two children undergoing long auditory assessments were randomly assigned to two study arms: CHS (n = 40) and GA (n = 52). Data of 81 children (mean age 13.8 months, range 1-36 months) were analyzed. In the GA group, we followed a predefined 10 N concept (no fear, no pain, normovolemia, normotension, normocardia, normoxemia, normocapnia, normonatremia, normoglycemia, and normothermia). In this group, ENT surgeons performed minor interventions in 29 patients based on intraprocedural microscopic ear examinations. In the CHS group, recommendations for monitoring and treatment of children undergoing moderate sedation were met. Furthermore, children received a double-barreled nasal oxygen cannula to measure end-tidal carbon dioxide (etCO2) and allow oxygen administration. Chloral hydrate was administered in the parent's presence. Children had no intravenous access which is an advantage of sedation techniques. In both groups, recommendations for fasting were followed and an experienced anesthesiologist was present during the entire procedure. Adverse event (AE) was a decline in cerebral oxygenation to below 50% or below 20% from the baseline for ≥1 min. The primary endpoint was the number of children with AE across the study arms. Secondary variables were: fraction of inspired oxygen (FIO2), oxygen saturation (SpO2), etCO2, systolic and mean blood pressure (BP), and heart rate (HR); these variables were analyzed for their association with drop in rScO2 to below baseline (%drop_rScO2). RESULTS: The incidence of AE across groups was not different. The analysis of secondary endpoints showed evidence that %drop_rScO2 is more dependent on HR and FIO2 than on BP and etCO2. CONCLUSIONS: This study highlights the strong association between HR and rScO2 in children aged < 3 years, whereas previous studies had primarily discussed the role of BP and etCO2. Prompt HR correction may result in shorter periods of cerebral desaturation. TRIAL REGISTRATION: The study was retrospectively registered with the German Clinical Trials Registry (DRKS00024362, 04/02/2021).

Immune regulation in renal inflammation.

Renal inflammation, induced by autoantigen recognition or toxic drugs, leads to renal tissue injury and decline in kidney function. Recent studies have demonstrated the crucial role for regulatory T cells in suppressing pathogenic adaptive but also innate immune responses in the inflamed kidney. However, there is also evidence for other immune cell populations with immunosuppressive function in renal inflammation. This review summarizes mechanisms of immune cell regulation in immune-mediated glomerulonephritis and acute and chronic nephrotoxicity.

Amphiregulin Aggravates Glomerulonephritis via Recruitment and Activation of Myeloid Cells.

BACKGROUND: Recent studies have identified the EGF receptor (EGFR) ligand amphiregulin (AREG) as an important mediator of inflammatory diseases. Both pro- and anti-inflammatory functions have been described, but the role of AREG in GN remains unknown. METHODS: The nephrotoxic nephritis model of GN was studied in AREG-/- mice after bone marrow transplantation, and in mice with myeloid cell-specific EGFR deficiency. Therapeutic utility of AREG neutralization was assessed. Furthermore, AREG's effects on renal cells and monocytes/macrophages (M/M) were analyzed. Finally, we evaluated AREG expression in human renal biopsies. RESULTS: Renal AREG mRNA was strongly upregulated in murine GN. Renal resident cells were the most functionally relevant source of AREG. Importantly, the observation that knockout mice showed significant amelioration of disease indicates that AREG is pathogenic in GN. AREG enhanced myeloid cell responses via inducing chemokine and colony stimulating factor 2 (CSF2) expression in kidney resident cells. Furthermore, AREG directly skewed M/M to a proinflammatory M1 phenotype and protected them from apoptosis. Consequently, anti-AREG antibody treatment dose-dependently ameliorated GN. Notably, selective abrogation of EGFR signaling in myeloid cells was sufficient to protect against nephritis. Finally, strong upregulation of AREG expression was also detected in kidneys of patients with two forms of crescentic GN. CONCLUSIONS: AREG is a proinflammatory mediator of GN via (1) enhancing renal pathogenic myeloid cell infiltration and (2) direct effects on M/M polarization, proliferation, and cytokine secretion. The AREG/EGFR axis is a potential therapeutic target for acute GN.

The validity of the Acoustic Breathiness Index in the evaluation of breathy voice quality: A Meta-Analysis.

BACKGROUND: The evaluation of voice quality with acoustic measurements is useful to objectify the diagnostic process. Particularly, breathiness was highly evaluated and the Acoustic Breathiness Index (ABI) might have promising features. OBJECTIVE OF REVIEW: The goal of the present meta-analysis is to quantify, from existing cross-validation studies, the evidence for the diagnostic accuracy of ABI, including its sensitivity and specificity. TYPE OF REVIEW: Meta-analysis. SEARCH STRATEGY: We searched in MEDLINE, Google Scholar and Science Citation Index, and as manual search for the term Acoustic Breathiness Index from inception to February 2020. Studies were included that used equal proportion of continuous speech and sustained vowel segments, a recording hardware with a sufficient standard for voice signal analyses, the software Praat for signal processing and the customised Praat script, and two groups of subjects (vocally healthy and voice-disordered). Furthermore, the diagnostic accuracy of ABI was measured. EVALUATION METHOD: The primary outcome variable was ABI. The score ranged from 0 to 10 with varying thresholds according to different languages to determine the absence or presence of breathiness. A meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses of diagnostic test accuracy study guidelines. Data were extracted, and the risk of bias was assessed using the QUADAS-2 tool. The pooled sensitivity and specificity of ABI were determined using a summary receiver operating characteristic (SROC) approach to calculate also a weighted threshold value of ABI with its sensitivity and specificity. RESULTS: A total of 34 unique citations were screened, and 10 full-text articles were reviewed, including six studies. In total, 3603 voice samples were considered for further analysis separating into 467 vocally healthy and 3136 voice-disordered voice samples. The pooled sensitivity was 0.84 (95% CI, 0.83-0.85), and the pooled specificity was 0.92 (95% CI, 0.89-0.94). The area under the curve of the SROC curve of this analysis showed an excellent value of 0.94. The weighted ABI threshold was determined at 3.40 (sensitivity: 0.86, 95% CI, 0.84-0.87.; specificity: 0.90, 95% CI 0.88-0.92). CONCLUSIONS: The results confirm the ABI as robust and valid objective measure for evaluating breathiness.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.

The effectiveness of voice therapy on voice-related handicap: A network meta-analysis.

BACKGROUND: Treatment approaches for voice therapy are diverse, yet their differential effects are not well understood. Evaluations of treatment effects across approaches are important for clinical guidance and evidence-based practice. OBJECTIVE OF REVIEW: To quantify the evidence of treatment effectiveness on the outcome measure Voice Handicap Index with the 30-items (VHI-30) from existing randomised controlled/clinical trials (RCT) of voice therapy using the statistical approach of a network meta-analysis (NMA) with a random effects model. TYPE OF REVIEW: Meta-analysis. SEARCH STRATEGY: We searched in MEDLINE (PubMed, 1950 to 2019), Embase (1974 to 2019) and Science Citation Index (1994 to 2019) using five key terms. The inclusion criteria were reports of randomised controlled/clinical trials (RCTs) published in English or German which evaluated the effectiveness of a specific voice therapy treatment using VHI-30 as an outcome measure in adult participants with non-organic or organic voice disorders. Studies were excluded if participants had been diagnosed with neurological motor speech disorders or who were vocally healthy. Furthermore, no medical, pharmacological or instrumental (eg voice amplification) treatments were considered. EVALUATION METHOD: The primary outcome variable was VHI-30 with a score from 0 to 120. The pre-post treatment change in VHI-30 scores was an average score of 13 points related to various VHI-30 test-retest results. RESULTS: We retrieved 464 publications (ie with duplicates) and included 13 RCTs, which evaluated nine interventions, in the final analysis. The most effective intervention with a significant and clinically relevant effect was Stretch-and-Flow Phonation (SFP) (mean pre-post difference -28.37, 95% confidence interval [CI], -43.05 to-13.68). Resonant Voice (RV), the Comprehensive Voice Rehabilitation Program (CVRP) and Vocal Function Exercises (VFE) also demonstrated significant improvements. CONCLUSIONS: Of the nine voice interventions identified with the present NMA, SFP, RVT, CVRP, and VFE effectively improved VHI-30 scores from pre- to post-treatment. SFP proved to be the most significant and clinically relevant treatment. Further contributions of high-quality intervention studies are needed to support evidence-based practice in vocology.

Renal proximal tubular epithelial cells exert immunomodulatory function by driving inflammatory CD4+ T cell responses.

In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4+ T cells accumulate within the tubulointerstitial compartment in close contact to proximal and distal tubular epithelial cells and drive renal inflammation and tissue damage. However, whether renal epithelial cell populations play a role in the pathogenesis of cGN by modulating CD4+ T cell responses is less clear. In the present study, we aimed to investigate the potential of renal epithelial cells to function as antigen-presenting cells, thereby stimulating CD4+ T cell responses. Using a FACS-based protocol that allowed comparative analysis of cortical epithelial cell populations, we showed that particularly proximal tubular epithelial cells (PTECs) express molecules linked with antigen-presenting cell function, including major histocompatibility complex class II (MHCII), CD74, CD80, and CD86 in homeostasis and nephrotoxic nephritis, a murine model of cGN. Protein expression was visualized at the PTEC single cell level by imaging flow cytometry. Interestingly, we found inflammation-dependent regulation of epithelium-expressed CD74, CD80, and CD86, whereas MHCII expression was not altered. Antigen-specific stimulation of CD4+ T cells by PTECs in vitro supported CD4+ T cell survival and induced CD4+ T cell activation, proliferation, and inflammatory cytokine production. In patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis, MHCII and CD74 were expressed by both proximal and distal tubules, whereas CD86 was predominantly expressed by proximal tubules. Thus, particularly PTECs have the potential to induce an inflammatory phenotype in CD4+ T cells in vitro, which might also play a role in the pathology of immune-mediated kidney disease.

Interferon-γ-dependent immune responses contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. METHODS: Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2-/- x Rag1-/- mice, which lack T and B cells, and following depletion of CD90.2+ or natural killer (NK)p46+ cells in Mdr2-/- mice. Liver pathology was also determined in Mdr2-/- x Ifng-/- mice and following anti-IFNγ antibody treatment of Mdr2-/- mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. RESULTS: Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56bright NK cells. In Mdr2-/- mice, hepatic CD8+ T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2+ cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2-/- x Rag1-/- mice. Depletion of NK cells resulted in reduced CD8+ T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2-/-x Ifng-/- mice reduced NK cell and CD8+ T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2-/- mice. CONCLUSION: IFNγ changed the phenotype of hepatic CD8+ T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis, whose current medical treatment is hardly effective. We observed an increased interferon (IFN)-γ response in patients with PSC and in a mouse model of sclerosing cholangitis. IFNγ changed the phenotype of hepatic CD8+ T lymphocytes and NK cells towards increased cytotoxicity, and its absence decreased liver cell death, reduced frequencies of inflammatory macrophages in the liver and attenuated liver fibrosis. Therefore, IFNγ-dependent immune responses may disclose checkpoints for future therapeutic intervention strategies in sclerosing cholangitis.

Carcinoembryonic antigen-related cell adhesion molecule 1 controls IL-2-dependent regulatory T-cell induction in immune-mediated hepatitis in mice.

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4+ T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1-/- mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic forkhead box protein P3+ (Foxp3+ )CD4+ Treg numbers. CEACAM1-/- CD4+ T cells showed impaired IL-2-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation, which correlated with a failure of naïve CEACAM1-/- CD4+ T cells to convert into Tregs in vitro. Furthermore, CEACAM1-/- Tregs expressed reduced levels of Foxp3, CD25, and B-cell lymphoma 2. Adoptive transfer experiments demonstrated that hepatic Treg expansion and suppressive activity required CEACAM1 expression on both CD4+ T cells and Tregs. We identified predominant CEACAM1S expression on hepatic CD4+ T cells and Tregs from mice with acute liver injury and expression of both isoforms in liver-derived CD4+ T-cell clones from patients with liver injury. CONCLUSION: Our data suggest that CEACAM1S expression in CD4+ T cells augments IL-2 production and STAT5 phosphorylation leading to enhanced Treg induction and stability, which, ultimately, confers protection from T-cell-mediated liver injury. (Hepatology 2018;68:200-214).

A Proinflammatory Role of Type 2 Innate Lymphoid Cells in Murine Immune-Mediated Hepatitis.

Type 2 innate lymphoid cells (ILC2) mediate inflammatory immune responses in the context of diseases triggered by the alarmin IL-33. In recent years, IL-33 has been implicated in the pathogenesis of immune-mediated liver diseases. However, the immunoregulatory function of ILC2s in the inflamed liver remains elusive. Using the murine model of Con A-induced immune-mediated hepatitis, we showed that selective expansion of ILC2s in the liver was associated with highly elevated hepatic IL-33 expression, severe liver inflammation, and infiltration of eosinophils. CD4+ T cell-mediated tissue damage and subsequent IL-33 release were responsible for the activation of hepatic ILC2s that produced the type 2 cytokines IL-5 and IL-13 during liver inflammation. Interestingly, ILC2 depletion correlated with less severe hepatitis and reduced accumulation of eosinophils in the liver, whereas adoptive transfer of hepatic ILC2s aggravated liver inflammation and tissue damage. We further showed that, despite expansion of hepatic ILC2s, 3-d IL-33 treatment before Con A challenge potently suppressed development of immune-mediated hepatitis. We found that IL-33 not only activated hepatic ILC2s but also expanded CD4+ Foxp3+ regulatory T cells (Treg) expressing the IL-33 receptor ST2 in the liver. This Treg subset also accumulated in the liver during resolution of immune-mediated hepatitis. In summary, hepatic ILC2s are poised to respond to the release of IL-33 upon liver tissue damage through expression of type 2 cytokines thereby participating in the pathogenesis of immune-mediated hepatitis. Inflammatory activity of ILC2s might be regulated by IL-33-elicited ST2+ Tregs that also arise in immune-mediated hepatitis.

Type 2 Innate Lymphoid Cells in Liver and Gut: From Current Knowledge to Future Perspectives.

Innate lymphoid cells (ILCs) represent a heterogeneous population of recently discovered immune cells that mirror the functions of adaptive T lymphocytes. However, ILCs are devoid of specific antigen receptors and cellular activation depends on environmental cytokines, rendering them as early regulators of immune responses. Type 2 innate lymphoid cells (ILC2s) respond to alarmins, such as interleukin-25 and -33 and shape Th2-associated immunity by expressing IL-5 and IL-13 in a GATA3-dependent manner. In addition, ILC2s express the epidermal growth factor-like molecule Amphiregulin thereby promoting regeneration of injured tissue during inflammation. The gut and liver confer nutrient metabolism and bidirectional exchange of products, known as the gut-liver axis. Accordingly, both organs are continuously exposed to a large variety of harmless antigens. This requires avoidance of immunity, which is established by a tolerogenic environment in the gut and liver. However, dysregulations within the one organ are assumed to influence vitality of the other and frequently promote chronic inflammatory settings with poor prognosis. Intensive research within the last years has revealed that ILC2s are involved in acute and chronic inflammatory settings of gut and liver. Here, we highlight the roles of ILC2s in intestinal and hepatic inflammation and discuss a regulatory potential.

Music complexity prediction for cochlear implant listeners based on a feature-based linear regression model.

This paper presents a model for predicting music complexity as perceived by cochlear implant (CI) users. To this end, 10 CI users and 19 normal-hearing (NH) listeners rated 12 selected music pieces on a bipolar music complexity scale and 5 other perception-related scales. The results indicate statistically significant differences in the ratings between CI and NH listeners. In particular, the ratings among different scales were significantly correlated for CI users, which hints at a common, hidden scale. The median complexity ratings by CI listeners and features accounting for high-frequency energy, spectral center of gravity, spectral bandwidth, and roughness were used to train a linear principal component regression model for an average CI user. The model was evaluated by means of cross-validation and using an independent database of processed chamber music signals for which music preferences scores by CI users were available. The predictions indicate a clear linear relationship with the preference scores, confirming the negative correlation between music complexity and music preference for CI users found in previous studies. The proposed model is a first step toward an instrumental evaluation procedure in the emerging field of music processing for CIs.