Microvesicles, in general, and exosomes together with their delivered content in particular, are now being widely recognized as key players in atherosclerosis. We have previously reviewed the role of ...microvesicles in atherosclerosis pathogenesis, diagnosis and therapy. Here, we focus on the roles of exosomes and discuss their emergent role in mediating activation and response to inflammation, vessel infiltration and induction of coagulation. We will finally give an outlook to discuss novel detection techniques and systems biology based data analyses to investigate exosome-mediated cell-to-cell communication.
Recent research points to a role of exosomes in delivering apoptotic and inflammatory content between blood cells and vascular cells, with a potential contribution of exosomes secreted by adipose tissue. An atheroprotective role of exosomes in response to coagulation that may contrast with the procoagulatory role of platelet-derived larger microvesicles is envisaged. New detection and separation methods and systems biology techniques are emerging.
We project that the development of novel detection, separation and analysis mechanism and systems-based analysis methods will further unravel the paracrine and endocrine 'communication protocol' between cellular players in atherosclerosis, mediating inflammation, oxidative stress and apoptosis.
The chemotherapeutic agent doxorubicin (DOX) has significantly increased survival rates of pediatric and adult cancer patients. However, 10% of pediatric cancer survivors will 10–20 years later ...develop severe dilated cardiomyopathy (DCM), whereby the exact molecular mechanisms of disease progression after this long latency time remain puzzling. We here revisit the hypothesis that elevated apoptosis signaling or its increased likelihood after DOX exposure can lead to an impairment of cardiac function and cause a cardiac dilation. Based on recent literature evidence, we first argue why a dilated phenotype can occur when little apoptosis is detected. We then review findings suggesting that mature cardiomyocytes are protected against DOX-induced apoptosis downstream, but not upstream of mitochondrial outer membrane permeabilisation (MOMP). This lack of MOMP induction is proposed to alter the metabolic phenotype, induce hypertrophic remodeling, and lead to functional cardiac impairment even in the absence of cardiomyocyte apoptosis. We discuss findings that DOX exposure can lead to increased sensitivity to further cardiomyocyte apoptosis, which may cause a gradual loss in cardiomyocytes over time and a compensatory hypertrophic remodeling after treatment, potentially explaining the long lag time in disease onset. We finally note similarities between DOX-exposed cardiomyocytes and apoptosis-primed cancer cells and propose computational system biology as a tool to predict patient individual DOX doses. In conclusion, combining recent findings in rodent hearts and cardiomyocytes exposed to DOX with insights from apoptosis signal transduction allowed us to obtain a molecularly deeper insight in this delayed and still enigmatic pathology of DCM.
Interleukin-6 is a pleiotropic cytokine with high clinical relevance and an important mediator of cellular communication, orchestrating both pro- and anti-inflammatory processes. ...Interleukin-6-induced signalling is initiated by binding of IL-6 to the IL-6 receptor alpha and subsequent binding to the signal transducing receptor subunit gp130. This active receptor complex initiates signalling through the Janus kinase/signal transducer and activator of transcription pathway. Of note, IL-6 receptor alpha exists in a soluble and a transmembrane form. Binding of IL-6 to membrane-bound IL-6 receptor alpha induces anti-inflammatory classic signalling, whereas binding of IL-6 to soluble IL-6 receptor alpha induces pro-inflammatory trans-signalling. Trans-signalling has been described to be markedly stronger than classic signalling. Understanding the molecular mechanisms that drive differences between trans- and classic signalling is important for the design of trans-signalling-specific therapies. These differences will be addressed here using a combination of dynamic mathematical modelling and molecular biology. We apply an iterative systems biology approach using set-based modelling and validation approaches combined with quantitative biochemical and cell biological analyses. The combination of experimental analyses and dynamic modelling allows to relate the observed differences between IL-6-induced trans- and classic signalling to cell-type specific differences in the expression and ratios of the individual subunits of the IL-6 receptor complex. Canonical intracellular Jak/STAT signalling is indifferent in IL-6-induced trans- and classic signalling. This study contributes to the understanding of molecular mechanisms of IL-6 signal transduction and underlines the power of combined dynamical modelling, model-based validation and biological experiments. The opposing pro- and anti-inflammatory responses initiated by IL-6 trans- and classic signalling depend solely on the expression ratios of the subunits of the entire receptor complex. By pointing out the importance of the receptor expression ratio for the strength of IL-6 signalling this study lays a foundation for future precision medicine approaches that aim to selectively block pro-inflammatory trans-signalling. Furthermore, the derived models can be used for future therapy design.
In-vitro
to
in-vivo
correlations (IVIVC), relating
in-vitro
parameters like
IC
50 to
in-vivo
drug exposure in plasma and tumour growth, are widely used in oncology for experimental design and dose ...decisions. However, they lack a deeper understanding of the underlying mechanisms. Our paper therefore focuses on linking empirical IVIVC relations for small-molecule kinase inhibitors with a semi-mechanistic tumour-growth model. We develop an approach incorporating parameters like the compound’s peak-trough ratio (
PTR
), Hill coefficient of
in-vitro
dose-response curves, and xenograft-specific properties. This leads to formulas for determining efficacious doses for tumor stasis under linear pharmacokinetics equivalent to traditional empirical IVIVC relations, but enabling more systematic analysis. Our findings reveal that
in-vivo
xenograft-specific parameters, specifically the growth rate (
g
) and decay rate (
d
), along with the average exposure, are generally more significant determinants of tumor stasis and effective dose than the compound’s peak-trough ratio. However, as the Hill coefficient increases, the dependency of tumor stasis on the
PTR
becomes more pronounced, indicating that the compound is more influenced by its maximum or trough values rather than the average exposure. Furthermore, we discuss the translation of our method to predict population dose ranges in clinical studies and propose a resistance mechanism that solely relies on specific
in-vivo
xenograft parameters instead of
IC
50 exposure coverage. In summary, our study aims to provide a more mechanistic understanding of IVIVC relations, emphasizing the importance of xenograft-specific parameters and
PTR
on tumor stasis.
Abstract
More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7–H3), a potential immune checkpoint family member. Several studies have ...shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.
Activation of the NLRP3-inflammasome pathway and production of the inflammatory cytokine IL-1B after cellular damage caused by infarct or infection is a key process in several diseases such as acute ...myocardial infarction and inflammatory bowel disease. However, while the molecular triggers of the NLRP3-pathway after cellular damage are well known, the mechanisms that sustain or confine its activity are currently under investigation. We present here an Ordinary Differential Equation-based model that investigates the mechanisms of inflammasome activation and regulation in monocytes to predict IL-1β activation kinetics upon a two-step activation by Damage-Associate-Molecular-Particles (DAMP) and extracellular ATP. Assuming both activation signals to be concomitantly present or present with a delay of 12h, the model predicted a transient IL-1β activation at different concentration levels dependent on signal synchronisation. Introducing a positive feedback loop mediated by active IL-1β resulted in a sustained IL-1β activation, hence arguing for a paracrine signalling between inflammatory cells to guarantee a temporally stable inflammatory response. We then investigate mechanisms that control termination of inflammation using two recently identified molecular intervention points in the inflammasome pathway. We found that a more upstream regulation, by attenuating production of the IL-1β-proform, was more potent in attenuating active IL-1β production than direct inhibition of the NLRP3-inflammasome. Interestingly, ablating this upstream negative feedback led to a high variability of IL-1β production in monocytes from different subjects, consistent with a recent pre-clinical study. We finally discuss the relevance and implications of our findings in disease models of acute myocardial infarction and spontaneous colitis.
•We provide a pathway model of the NLRP3-inflammasome after cellular damage.•NLRP3-inflammasome attenuation by paracrine IFN-I is more potent than by IFN-II.•Change from IFN-II to IFN-I may stop inflammation during tissue repair after infarct.•Impairment of IFN-I feedback can lead to hyperinflammation as in spontaneous colitis.•Regulation of inflammation may directly be encoded within regulatory pathways.
The effect of combination therapies in many cancers has often been shown to be superior to that of monotherapies. This success is commonly attributed to drug synergies. Combinations of two (or more) ...drugs in xenograft tumor growth inhibition (TGI) studies are typically designed at fixed doses for each compound. The available methods for assessing synergy in such study designs are based on combination indices (CI) and model-based analyses. The former methods are suitable for screening exercises but are difficult to verify in in vivo studies, while the latter incorporate drug synergy in semi-mechanistic frameworks describing disease progression and drug action but are unsuitable for screening. In the current study, we proposed the empirical radius additivity (Rad-add) score, a novel CI for synergy detection in fixed-dose xenograft TGI combination studies. The Rad-add score approximates model-based analysis performed using the semi-mechanistic constant-radius growth TGI model. The Rad-add score was compared with response additivity, defined as the addition of the two response values, and the bliss independence model in combination studies derived from the Novartis PDX dataset. The results showed that the bliss independence and response additivity models predicted synergistic interactions with high and low probabilities, respectively. The Rad-add score predicted synergistic probabilities that appeared to be between those predicted with response additivity and the Bliss model. We believe that the Rad-add score is particularly suitable for assessing synergy in the context of xenograft combination TGI studies, as it combines the advantages of CI approaches suitable for screening exercises with those of semi-mechanistic TGI models based on a mechanistic understanding of tumor growth.
Detection methods that do not rely on the amplification of DNA but can reach sensitivity, specificity and throughput of gold standard methods, such as qPCR, have been extensively explored in recent ...years. Here, we present a hydrophilic-in-hydrophobic (HIH)-microwell array platform that empowers a panel of different amplification-free DNA bioassays: digital enzyme-linked oligonucleotide assay (ELONA), ligation-assisted (LA) digital ELONA and so-called ‘analog’ bioassays. We developed all three bioassays by using magnetic beads for capturing DNA target, followed by hybridization of enzyme-labelled detection probes and sealing of the built complexes into the femtoliter HIH microwells to achieve the fluorescent readout of single DNA molecules. With the optimized digital ELONA bioassay, we successfully detected 97 and 200 nt-long ssDNA molecules down to 68 and 92 aM, respectively, demonstrating extremely high sensitivity of the bioassay and its flexibility towards targets of different lengths. Importantly, we also proved that the same bioassay concept was suited to detect substantially higher concentrations of ssDNA (up to picomolar levels) by quantifying the total fluorescent intensity rather than counting fluorescent events for digital quantification. Finally, we advanced this concept towards LA digital ELONA capable of differentiating wildtype strands from those carrying single-point mutations even when the former were constituting only 1% of the DNA mixture and were present at 2 fM concentration. In conclusion, the developed platform showed remarkably high sensitivity, specificity and versatility for amplification-free detection of DNA and as such can be valuable for numerous applications in medical diagnostics, gene analysis, food safety and environmental monitoring.
Display omitted
•A digital enzyme-linked oligonucleotide assay was developed on a microwell-array.•Versatility of the assay was proven detecting DNA strands of two different lengths.•Developed assay enabled DNA detection from attomolar to picomolar concentrations.•The assay concept was broadened towards detection of single nucleotide polymorphism.•Wildtype strands were distinguished from mutants at 100-fold lower concentration.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized ...as an attractive novel immune intervention therapy for counteracting the destructive effects of graft-
-host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs
from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation
, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between
and
functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft-
-tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of
-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.
Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both
and
, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to ...manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining
and
bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated
in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages
. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts
and
. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.
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