Psoriasis is an inflammatory skin disease with strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37. LL37 in complex with DNA and RNA is thought to initiate disease ...exacerbation via plasmacytoid dendritic cells. However, the source of nucleic acids supposed to start this initial inflammatory event remains unknown. We show here that primary murine and human PMNs mount a fulminant and self-propagating neutrophil extracellular trap (NET) and cytokine response, but independently of the canonical NET component, DNA. Unexpectedly, RNA, which is abundant in NETs and psoriatic but not healthy skin, in complex with LL37 triggered TLR8/TLR13-mediated cytokine and NET release by PMNs in vitro and in vivo. Transfer of NETs to naive human PMNs prompts additional NET release, promoting further inflammation. Our study thus uncovers a self-propagating vicious cycle contributing to chronic inflammation in psoriasis, and NET-associated RNA (naRNA) as a physiologically relevant NET component.
The application of human pluripotent stem cells (hPSCs) in vitro is of high value for therapeutic and industrial applications. Perfusion was shown to result in a more homogeneous culture environment ...and enabled 47% higher cell yields compared with conventional repeated batch culture. Furthermore, results show the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development.
The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high‐quality cells by defined processes. Culturing hPSCs as cell‐only aggregates in (three‐dimensional 3D) suspension has the potential to overcome numerous limitations of conventional surface‐adherent (two‐dimensional 2D) cultivation. Utilizing single‐use instrumented stirred‐tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse‐transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix‐free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture‐induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP‐compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.
Significance
Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single‐use, clinically compliant bioreactor technology to develop advanced, matrix‐free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.
There is evidence for a relevant role of inflammation in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene represent the most frequent genetic cause for autosomal dominant PD. ...LRRK2 is highly expressed in macrophages and microglia suggesting an involvement in inflammatory pathways. The objectives are to test (1) whether idiopathic PD and LRRK2-associated PD share common inflammatory pathways or present distinct profiles and (2) whether non-manifesting LRRK2 mutation carriers present with similar aspects of inflammatory profiles as seen in PD-affected patients.
We assessed serum profiles of 23 immune-associated markers and the brain-derived neurotrophic factor in 534 individuals from the MJFF LRRK2 consortium.
A large proportion of inflammatory markers were gender-dependent. Both PD-affected cohorts showed increased levels of the pro-inflammatory marker fatty-acid-binding protein. Additionally, idiopathic PD but not LRRK2-associated PD patients showed increased levels of the pro-inflammatory marker interleukin-12-p40 as well as the anti-inflammatory species interleukin-10, brain-derived neurotrophic factor, and stem cell factor. Non-manifesting LRRK2 mutation carriers including those with prodromal characteristics of PD presented with control-like inflammatory profiles.
Concomitant inflammation seems to be associated with idiopathic and LRRK2-associated PD. Identifying PD patients in whom inflammatory processes play a major role in their pathophysiology might offer a new therapeutic window at least for a subgroup of patients. Since non-manifesting LRRK2 mutation carriers with symptoms of the prodromal phase of PD did not show inflammatory profiles, activation of the immune system seems not an early event in the disease cascade.
Inflammation modifies the incidence and progression of Parkinson's disease (PD). By using 30 inflammatory markers in CSF in 498 people with PD and 67 people with dementia with Lewy bodies (DLB) we ...show that: (1) levels of ICAM-1, Interleukin-8, MCP-1, MIP-1 beta, SCF and VEGF were associated with clinical scores and neurodegenerative CSF biomarkers (Aβ1-42, t-Tau, p181-Tau, NFL and α-synuclein). (2) PD patients with GBA mutations show similar levels of inflammatory markers compared to PD patients without GBA mutations, even when stratified by mutation severity. (3) PD patients who longitudinally developed cognitive impairment during the study had higher levels of TNF-alpha at baseline compared to patients without the development of cognitive impairment. (4) Higher levels of VEGF and MIP-1 beta were associated with a longer duration until the development of cognitive impairment. We conclude that the majority of inflammatory markers is limited in robustly predicting longitudinal trajectories of developing cognitive impairment.
Blood levels of immune markers have been proposed to discriminate patients with Parkinson's disease (PD) from controls. However, differences between clinical PD subgroups regarding these markers ...still need to be identified.
To investigate whether clinical phenotypes can be predicted by the assessment of immune marker profiles in the serum of PD patients.
Phenotypes of clinical PD from Tübingen, Germany (
= 145) and Toronto, Canada (
= 90) were defined regarding clinical subtype, disease onset, severity, and progression as well as presence of cognitive and/or autonomic dysfunction. A panel of serum immune markers was assessed using principal component analysis (PCA) and regression models to define the marker(s) that were associated with clinical phenotypes after adjusting for potential confounders. Findings of both centers were compared for validation. Further, a 18F FEPPA-PET was performed in a group of patients with high and low values of candidate markers for the assessment of
brain microglial activation.
Overall, serum immune markers did not cluster to define a pro/anti-inflammatory profile in PCA. Out of 25 markers only IL-12p40 showed a trend to discriminate between PD subgroups in both cohorts which could not be replicated by 18F FEPPA-PET.
Assessment of cytokines in serum does not reliably differentiate clinical PD subtypes. Accompanying subtype-irrelevant inflammation in PD, dual activity, and lack of specificity of the immune markers, the complex function of microglia, probable effects of treatment, disease stage, and progression on inflammation as well as current technical limitations may limit the usefulness of serum immune markers for the differentiation of subtypes.
Nuclear receptors mediate the hepatic induction of drug-metabolizing enzymes by xenobiotics. Not much is known about enzyme induction in liver tumors. Here, we treated tumor-bearing mice with ...phenobarbital, an activator of the constitutive androstane receptor (CAR), to analyze the response of chemically induced
- and
mutated mouse liver adenoma to CAR activation in vivo. Both tumor subpopulations possess almost identical gene expression profiles. CAR target gene induction in the tumors was studied at the mRNA and protein levels, and a reverse-phase protein microarray approach was chosen to characterize important signaling cascades. CAR target gene induction was pronounced in
-mutated but not in
-mutated tumors. Phosphoproteomic profiling revealed that phosphorylation-activated extracellular signal-regulated kinase (ERK) 1/2 was more abundant in
-mutated than in
-mutated tumors. ERK activation in tumor tissue was negatively correlated with CAR target induction. ERK activation is known to inhibit CAR-dependent transcription. In summary, profound differences exist between the two closely related tumor subpopulations with respect to the activation of mitogenic signaling cascades, and these dissimilarities might explain the differences in xenobiotic induction of CAR target genes.
ABSTRACT
The first ATP‐competitive p38a MAPK/MAPK14 inhibitor with excellent in vivo efficacy and selectivity, skepinone‐L, is now available. We investigated the impact of selective p38a MAPK/MAPK14 ...inhibition on enzymatically modified LDL (eLDL) stimulated human monocytes with its implications for atherosclerosis. Among the different p38 MAPK isoforms, p38a/MAPK14 was the predominantly expressed and activated isoform in isolated human peripheral blood monocytes. Moreover, eLDL colocalized with macrophages positive for p38a MAPK/ MAPK14 in human carotid endarterectomy specimens. Using the human leukemia cell line THP‐1 and/or primary monocyte‐derived macrophages, skepinone‐L inhibited eLDL‐induced activation of the p38 MAPK pathway, inhibited eLDL induced expression of both cluster of differentiation 36 (CD36) and ATP‐binding cassette, subfamily A, member 1 (ABCA1), without a net effect on foam cell formation, had a cell‐and time‐dependent effect on eLDLtriggered apoptosis, and inhibited eLDL‐stimulated secretion of IL‐8 and MIP‐1b/CCL4 (macrophage inflammatory protein‐1b/chemokine, CC motif, ligand 4). Inhibition of a key signaling molecule of the p38 MAPK pathway, p38a MAPK/MAPK14, by selective inhibitors like skepinone‐L, conclusively facilitates elucidation of the impact of the complex network of p38 MAPK signaling on atherogenesis and might provide a promising therapeutic tool to prevent inflammatory cascades in atherosclerosis.—Cheng, F., Twardowski, L., Fehr, S., Aner, C., Schaeffeler, E., Joos, T., Knorpp, T., Dorweiler, B., Laufer, S., Schwab, M., Torzewski, M. Selective p38a MAP kinase/MAPK14 inhibition in enzymatically modified LDL‐stimulated human monocytes: implications for atherosclerosis. FASEB J. 31, 674–686 (2017). www.fasebj.org
As electron microscopy did in the past, super-resolution microscopy, or nanoscopy, provides the ability to see details of cellular and even macromolecular structure that were not possible to see ...before. Notably, however, nanoscopy is compatible with live cells and has the capability for multiplex labeling with high molecular specificity.
Aberrant signaling through the Wnt/β-catenin pathway is a critical determinant in human and rodent liver carcinogenesis and generally accepted to be a potent driver of proliferation. Xenobiotic ...agonists of the constitutive androstane receptor (CAR) induce massive acute hyperplasia of mouse liver and facilitate the outgrowth of hepatocellular carcinomas with activated β-catenin. In the present study, the interplay of β-catenin-dependent and CAR-dependent signaling in the liver and its effect on hepatocyte proliferation were analyzed in transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) following treatment with two CAR agonists, 1,4-bis2-(3,5-dichloropyridyloxy)-benzene (TCPOBOP) and phenobarbital. Hepatocyte-specific knockout of β-catenin inhibited CAR agonists-induced hepatocyte proliferation in male mice. By contrast, the proliferative effect of CAR agonists was strongly augmented in female β-catenin knockout animals. This was due to prolonged proliferation of the knockout hepatocytes. CAR-mediated hepatocyte proliferation was, at least in part, dependent on estrogen signaling and was associated with enhanced expression of FoxM1 and elevated activity of the PDK1/p90RSK pathway. In conclusion, our study shows that gender-specific factors determine whether β-catenin signaling plays a pro- or an antiproliferative role in the regulation of mouse hepatocyte proliferation induced by CAR agonists.
Common in vitro cell culture systems for testing implant material immune compatibility either rely on immortal human leukocyte cell lines or isolated primary cells. Compared to in vivo conditions, ...this generates an environment of substantially reduced complexity, often lacking important immune cell types, such as neutrophil granulocytes and others. The aim of this study was to establish a reliable test system for in vitro testing of implant materials under in vivo-like conditions.
Test materials were incubated in closed, CO
-independent, tube-based culture vessels containing a proprietary cell culture medium and human whole blood in either a static or occasionally rotating system. Multiplex cytokine analysis was used to analyze immune cell reactions.
To demonstrate the applicability of the test system to implant materials, three commercially available barrier membranes (polytetrafluoroethylene (PTFE), polycaprolactone (PCL) and collagen) used for dental, trauma and maxillofacial surgery, were investigated for their potential interactions with immune cells. The results showed characteristic differences between the static and rotated incubation methods and in the overall activity profiles with very low immune cell responses to PTFE, intermediate ones to collagen and strong reactions to PCL.
This in vitro human whole blood model, using a complex organotypic matrix, is an excellent, easily standardized tool for categorizing immune cell responses to implant materials. Compared to in vitro cell culture systems used for materials research, this new assay system provides a far more detailed picture of response patterns the immune system can develop when interacting with different types of materials and surfaces.
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