NASH triggers cardiometabolic HFpEF in aging mice.

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68+ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.

Fate of Chiral Symmetries in the Quark-Gluon Plasma from an Instanton-Based Random Matrix Model of QCD.

We propose a new way of understanding how chiral symmetry is realized in the high temperature phase of QCD. Based on the finding that a simple free instanton gas precisely describes the details of the lowest part of the spectrum of the lattice overlap Dirac operator, we propose an instanton-based random matrix model of QCD with dynamical quarks. Simulations of this model reveal that even for small quark mass the Dirac spectral density has a singularity at the origin, caused by a dilute gas of free instantons. Even though the interaction, mediated by light dynamical quarks, creates small instanton-anti-instanton molecules, those do not influence the singular part of the spectrum, and this singular part is shown to dominate Banks-Casher type sums in the chiral limit. By generalizing the Banks-Casher formula for the singular spectrum, we show that in the chiral limit the chiral condensate vanishes if there are at least two massless flavors. Our model also indicates a possible way of resolving a long-standing debate, as it suggests that for two massless quark flavors the U(1)_{A} symmetry is likely to remain broken up to arbitrarily high finite temperatures.

A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV 1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

Comparative study of the inhalation parameters of COPD patients through NEXThaler® and Ellipta® dry powder inhalers.

The deposition of dry powder aerosol drugs depends on the inhalation parameters of the patients through the inhaler. These data are not directly measured in clinical practice. Their prediction based on the routinely measured spirometric data could help in choosing the appropriate device and optimizing the therapy. The aim of this study was to perform inhalation experiments to find correlations between inhalation parameters of COPD patients through two DPI devices and their native spirometric data, gender, age and disease severity. Another goal was to establish relationships between peak inspiratory flows through NEXThaler® and Ellipta® inhalers and their statistical determinants. Breathing parameters of 113 COPD patients were measured by normal spirometry and while inhaling through the two DPIs. Statistical analysis of the measured data was performed. The average values of peak inspiratory flow through the devices (PIFdev) were 68.4 L/min and 78.0 L/min for NEXThaler® and Ellipta®, respectively. PIFdev values were significantly higher for males than for females, but differences upon age, BMI and disease severity group were not significant. PIFdev values correlated best with their native spirometric counterparts (PIF) and linear relationships between them were revealed. Current results may be used in the future to predict the success of inhalation of COPD patients through DPI devices, which may help in the inhaler choice. By choosing the appropriate device-drug pair for each patient the lung dose can be increased and the efficiency of the therapy improved. Further results of the clinical study will be the subject of a next publication.

Depletion of muscularis macrophages ameliorates inflammation-driven dysmotility in murine colitis model.

Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (L-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent L-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent L-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Effect of the Lipid Landscape on the Efficacy of Cell-Penetrating Peptides.

Every cell biological textbook teaches us that the main role of the plasma membrane is to separate cells from their neighborhood to allow for a controlled composition of the intracellular space. The mostly hydrophobic nature of the cell membrane presents an impenetrable barrier for most hydrophilic molecules larger than 1 kDa. On the other hand, cell-penetrating peptides (CPPs) are capable of traversing this barrier without compromising membrane integrity, and they can do so on their own or coupled to cargos. Coupling biologically and medically relevant cargos to CPPs holds great promise of delivering membrane-impermeable drugs into cells. If the cargo is able to interact with certain cell types, uptake of the CPP-drug complex can be tailored to be cell-type-specific. Besides outlining the major membrane penetration pathways of CPPs, this review is aimed at deciphering how properties of the membrane influence the uptake mechanisms of CPPs. By summarizing an extensive body of experimental evidence, we argue that a more ordered, less flexible membrane structure, often present in the very diseases planned to be treated with CPPs, decreases their cellular uptake. These correlations are not only relevant for understanding the cellular biology of CPPs, but also for rationally improving their value in translational or clinical applications.

Comparison of flexible nasogastric tube and semi-rigid catheter during less invasive surfactant administration.

BACKGROUND: Various flexible and semi-rigid catheter techniques have been reported for surfactant delivery during less invasive surfactant administration (LISA) in preterm infants. Data on the effect of catheter selection on procedural success rates and adverse events are limited. Our objective was to compare the rates of success and adverse events of LISA performed with nasogastric tube and semi-rigid catheter. METHODS: This was a post-hoc analysis of data from a quality improvement project. LISA was performed according to the standardized local protocol. Baseline characteristics, data on performance of LISA, degree of difficulty in laryngoscopy and vital parameters after the initiation of LISA were collected and outcomes were compared between groups. RESULTS: Fifty-six infants were included (21 with nasogastric tube, and 35 with semi-rigid catheter). Procedure success rate (defined as a single LISA attempt resulting in intratracheal administration of the planned dose of surfactant), incidence of adverse events, heart rate and oxygen saturation values and outcomes did not differ significantly between the two groups. When using a nasogastric tube for LISA, a significantly higher fraction of inspired oxygen was needed in the 3rd (0.62 vs. 0.48, P=0.024), 4th (0.61 vs. 0.37, P<0.001) and 5th minute (0.48 vs. 0.37, P=0.001) to maintain normal oxygen saturations. CONCLUSIONS: Use of the semi-rigid catheter was associated with better oxygenation during and shortly after the procedure. Our results may help neonatal units to develop local guidelines.

Medium-Term Results of Staged Laparoscopic Traction Orchiopexy for Intra-abdominal Testes: A Multicenter Analysis.

BACKGROUND: Staged laparoscopic traction orchiopexy (SLTO) is a novel technique for the intra-abdominal testis (IAT) based on elongation of the testicular vessels without separating them. This multicenter study evaluated the medium-term results of this technique. METHODS: Data of SLTO performed in three pediatric surgical centers between 2013 and 2020 were analyzed retrospectively. In 2021, physical and Doppler ultrasound examinations were performed to determine the position and viability of testes. Success was defined as an intra-scrotal testicle without atrophy. RESULTS: SLTO was performed on 48 cases (55 testes, 7 bilateral). Mean age at first stage was 2.9 (0.8-12.6) years. High intra-abdominal testes were found in 16.4% and in 60% morphological abnormalities were observed. To fix the testes to the abdominal wall monofilament suture was used in 67.3%, braided in 29.1%. Mean time between the two stages was 16.4 weeks; three testes required redo traction. Perioperative complications occurred in 21 patients (38.2%) including insufficient fixation (11), testicular atrophy (4), wound complications (4), adhesion of the spermatic cords (1) and hydrocele (1). In case of insufficient fixation monofilament sutures were used in 90.9%. In 2021 38 patients (43 testes) had physical and 36 patients (41 testes) had ultrasound examinations. Mean follow-up was 2.7 (0.34-7.9) years. Altogether five atrophies were identified, and three testicular ascents (7.0%) occurred. The overall success rate was 82.2%. CONCLUSIONS: SLTO may be a feasible alternative to conventional treatments of IATs. Additionally, braided suture seems to be a better option to fix the testicle to the abdominal wall. LEVEL OF EVIDENCE: LEVEL IV.

Non-small cell lung cancer patients treated with Anti-PD1 immunotherapy show distinct microbial signatures and metabolic pathways according to progression-free survival and PD-L1 status.

Due to the high variance in response rates concerning anti-PD1 immunotherapy (IT), there is an unmet need to discover innovative biomarkers to predict immune checkpoint inhibitor (ICI)-efficacy. Our study included 62 Caucasian advanced-stage non-small cell lung cancer (NSCLC) patients treated with anti-PD1 ICI. Gut bacterial signatures were evaluated by metagenomic sequencing and correlated with progression-free survival (PFS), PD-L1 expression and other clinicopathological parameters. We confirmed the predictive role of PFS-related key bacteria with multivariate statistical models (Lasso- and Cox-regression) and validated on an additional patient cohort (n = 60). We find that alpha-diversity showed no significant difference in any comparison. However, there was a significant difference in beta-diversity between patients with long- (>6 months) vs. short (≤6 months) PFS and between chemotherapy (CHT)-treated vs. CHT-naive cases. Short PFS was associated with increased abundance of Firmicutes (F) and Actinobacteria phyla, whereas elevated abundance of Euryarchaeota was specific for low PD-L1 expression. F/Bacteroides (F/B) ratio was significantly increased in patients with short PFS. Multivariate analysis revealed an association between Alistipes shahii, Alistipes finegoldii, Barnesiella visceriola, and long PFS. In contrast, Streptococcus salivarius, Streptococcus vestibularis, and Bifidobacterium breve were associated with short PFS. Using Random Forest machine learning approach, we find that taxonomic profiles performed superiorly in predicting PFS (AUC = 0.74), while metabolic pathways including Amino Acid Synthesis and Fermentation were better predictors of PD-L1 expression (AUC = 0.87). We conclude that specific metagenomic features of the gut microbiome, including bacterial taxonomy and metabolic pathways might be suggestive of ICI efficacy and PD-L1 expression in NSCLC patients.

CXCR4 and CXCL12 signaling regulates the development of extrinsic innervation to the colorectum.

The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.

Virulence Characteristics and Molecular Typing of Carbapenem-Resistant ST15 Klebsiella pneumoniae Clinical Isolates, Possessing the K24 Capsular Type.

Klebsiella pneumoniae is an opportunistic pathogen that frequently causes nosocomial and community-acquired (CA) infections. Until now, a limited number of studies has been focused on the analyses of changes affecting the virulence attributes. Genotypic and phenotypic methods were used to characterise the 39 clinical K. pneumoniae isolates; all belonged to the pan-drug resistant, widespread clone ST 15 and expressed the K24 capsule. PFGE has revealed that the isolates could be divided into three distinct genomic clusters. All isolates possessed allS and uge genes, known to contribute to the virulence of K. pneumoniae and 10.25% of the isolates showed hypermucoviscosity, 94.87% produced type 1 fimbriae, 92.3% produced type 3 fimbriae, and 92.3% were able to produce biofilm. In vivo persistence could be supported by serum resistance 46.15%, enterobactin (94.87%) and aerobactin (5.12%) production and invasion of the INT407 and T24 cell lines. Sequence analysis of the whole genomes of the four representative strains 11/3, 50/1, 53/2 and 53/3 has revealed high sequence homology to the reference K. pneumoniae strain HS11286. Our results represent the divergence of virulence attributes among the isolates derived from a common ancestor clone ST 15, in an evolutionary process that occurred both in the hospital and in the community.

Maximum likelihood-based estimation of diffusion coefficient is quick and reliable method for analyzing estradiol actions on surface receptor movements.

The rapid effects of estradiol on membrane receptors are in the focus of the estradiol research field, however, the molecular mechanisms of these non-classical estradiol actions are poorly understood. Since the lateral diffusion of membrane receptors is an important indicator of their function, a deeper understanding of the underlying mechanisms of non-classical estradiol actions can be achieved by investigating receptor dynamics. Diffusion coefficient is a crucial and widely used parameter to characterize the movement of receptors in the cell membrane. The aim of this study was to investigate the differences between maximum likelihood-based estimation (MLE) and mean square displacement (MSD) based calculation of diffusion coefficients. In this work we applied both MSD and MLE to calculate diffusion coefficients. Single particle trajectories were extracted from simulation as well as from α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor tracking in live estradiol-treated differentiated PC12 (dPC12) cells. The comparison of the obtained diffusion coefficients revealed the superiority of MLE over the generally used MSD analysis. Our results suggest the use of the MLE of diffusion coefficients because as it has a better performance, especially for large localization errors or slow receptor movements.

Characterization of a Lytic Bacteriophage and Demonstration of Its Combined Lytic Effect with a K2 Depolymerase on the Hypervirulent Klebsiella pneumoniae Strain 52145.

Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

Synchronized intermittent mandatory ventilation with volume guarantee and pressure support in neonates: Detailed analysis of ventilator parameters.

OBJECTIVE: To analyse the relationship between peak inflating pressure, expired tidal volume, respiratory rate, and inspiratory time of volume-guaranteed ventilator inflations and pressure-supported spontaneous breaths during synchronized intermittent positive pressure mode with volume guarantee and pressure support (SIMV-VG-PS) in neonates. METHODS: Ventilator parameters were downloaded every second from 16 babies ventilated with SIMV-VG-PS mode using Dräger Babylog VN500 ventilators over 137 days. Transcutaneous carbon dioxide (tcCO2 ) data were also collected. Data were computationally analysed using Python. The average of each ventilator parameter was determined during each minute separately for ventilator inflations and for spontaneous breaths. These values were compared and their effect on tcCO2 levels was also analysed. RESULTS: The relationship between the peak inflating pressure of the volume guaranteed inflations (PIPVG ) and pressure-supported spontaneous breaths (PIPPS ) was highly variable. The PIPPS /PIPVG ratio differed significantly from the value (0.66) targeted by clinicians (group median: 0.80, range: 0.50-1.00). PIPPS frequently exceeded PIPVG . When PIPPS /PIPVG was >0.66, the expired tidal volume and the rate of the pressure-supported spontaneous breaths were also significantly (p < 0.0001) higher, but there was no difference in tcCO2 levels. The flow-cycled spontaneous breaths had significantly shorter inspiratory times than ventilator inflations. CONCLUSIONS: During SIMV-VG-PS it is difficult to ensure a pressure support level proportionate to the inflating pressure of ventilator inflations and to achieve the stability of tidal volumes.

Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-ß-cyclodextrin content.

BACKGROUND AND PURPOSE: Despite its contradictory clinical performance, remdesivir (Veklury®) has a pivotal role in COVID-19 therapy. Possible contributions of the vehicle, sulfobutylether-ß-cyclodextrin (SBECD) to Veklury® effects have been overlooked. The powder and solution formulations of Veklury® are treated equivalently despite their different vehicle content. Our objective was to study Veklury® effects on initial membrane-coupled events of SARS-CoV-2 infection focusing on the cholesterol depletion-mediated role of SBECD. EXPERIMENTAL APPROACH: Using time-correlated flow cytometry and quantitative three-dimensional confocal microscopy, we studied early molecular events of SARS-CoV-2-host cell membrane interactions. KEY RESULTS: Veklury® and different cholesterol-depleting cyclodextrins (CDs) reduced binding of the spike receptor-binding domain (RBD) to ACE2 and spike trimer internalization for Wuhan-Hu-1, Delta and Omicron variants. Correlations of these effects with cholesterol-dependent changes in membrane structure and decreased lipid raft-dependent ACE2-TMPRSS2 interaction establish that SBECD is not simply a vehicle but also an effector along with remdesivir due to its cholesterol-depleting potential. Veklury® solution inhibited RBD binding more efficiently due to its twice higher SBECD content. The CD-induced inhibitory effects were more prominent at lower RBD concentrations and in cells with lower endogenous ACE2 expression, indicating that the supportive CD actions can be even more pronounced during in vivo infection when viral load and ACE expression are typically low. CONCLUSION AND IMPLICATIONS: Our findings call for the differentiation of Veklury® formulations in meta-analyses of clinical trials, potentially revealing neglected benefits of the solution formulation, and also raise the possibility of adjuvant cyclodextrin (CD) therapy, even at higher doses, in COVID-19.

Football movement profile analysis and creatine kinase relationships in youth national team players.

Objective: Creatine kinase (CK) is widely used as a monitoring tool to make inferences on fatigue and readiness in elite soccer. Previous studies have examined the relationship between CK and GPS parameters, however these metrics may not accurately describe the players' load during soccer-specific movements. Football Movement Profile (FMP) monitoring is a viable option for such purposes, providing solely inertial sensor-based data and categorizing movements according to intensity (very low, low, medium, high) and movement type (running-linear locomotive, dynamic - change of direction or speed). Methods: We investigated the relationship between the FMP distribution of youth (U16-U21) national team soccer players and the absolute day-to-day change in CK. We applied Spearman's correlations, principal component analysis and K-means clustering to classify players' CK responses according to their specific FMP. Results: Moderate to large negative associations were found between very low intensity FMP parameters and CK change (r = -0.43 ± 0.12) while large positive associations were identified between CK change and other FMP metrics (r = 0.62 ± 0.12). Best fitting clustering methods were used to group players depending on their CK sensitivity to FMP values. Principal component analysis explained 83.0% of the variation with a Silhouette score of 0.61 for the 4 clusters. Conclusions: Our results suggest that soccer players can be clustered based on the relationship between FMP measures and the CK change. These findings can help to plan soccer training or recovery sessions according to the desired load on skeletal muscle, as FMP monitoring might bridge the limitations of GPS telemetry.

IL-1ß neutralization prevents diastolic dysfunction development, but lacks hepatoprotective effect in an aged mouse model of NASH.

Interleukin-1ß (IL-1ß) is a key mediator of non-alcoholic steatohepatitis (NASH), a chronic liver disease, and of systemic inflammation-driven aging. IL-1ß contributes to cardio-metabolic decline, and may promote hepatic oncogenic transformation. Therefore, IL-1ß is a potential therapeutic target in these pathologies. We aimed to investigate the hepatic and cardiac effects of an IL-1ß targeting monoclonal antibody in an aged mouse model of NASH. 24 months old male C57Bl/6J mice were fed with control or choline deficient (CDAA) diet and were treated with isotype control or anti-IL-1ß Mab for 8 weeks. Cardiac functions were assessed by conventional-and 2D speckle tracking echocardiography. Liver samples were analyzed by immunohistochemistry and qRT-PCR. Echocardiography revealed improved cardiac diastolic function in anti-IL-1ß treated mice with NASH. Marked hepatic fibrosis developed in CDAA-fed group, but IL-1ß inhibition affected fibrosis only at transcriptomic level. Hepatic inflammation was not affected by the IL-1ß inhibitor. PCNA staining revealed intensive hepatocyte proliferation in CDAA-fed animals, which was not influenced by neutralization of IL-1ß. IL-1ß inhibition increased hepatic expression of Pd-1 and Ctla4, while Pd-l1 expression increased in NASH. In conclusion, IL-1ß inhibition improved cardiac diastolic function, but did not ameliorate features of NASH; moreover, even promoted hepatic immune checkpoint expression, with concomitant NASH-related hepatocellular proliferation.

Characterization of immune checkpoint inhibitor-induced cardiotoxicity reveals interleukin-17A as a driver of cardiac dysfunction after anti-PD-1 treatment.

BACKGROUND AND PURPOSE: Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. EXPERIMENTAL APPROACH: C57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody. Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. KEY RESULTS: Anti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. CONCLUSIONS: PD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.