Abstract
Background
Bacterial meningitis is still a cause of severe neurological disability. The brain is protected from penetrating pathogens by the blood-brain barrier and the innate immune system. ...The invading pathogens are recognized by pattern recognition receptors including the G-protein-coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. FPRs show a broad spectrum of ligands, including pro- and anti-inflammatory ones. Here, we investigated the effects of the annexin A1 mimetic peptide Ac2-26 in a mouse model of pneumococcal meningitis.
Methods
Wildtype (WT) and
Fpr1
- and
Fpr2
-deficient mice were intrathecally infected with
Streptococcus pneumoniae
D39 (type 2). Subsequently, the different mice groups were treated by intraperitoneal injections of Ac2-26 (1 mg/kg body weight) 2, 8, and 24 h post-infection. The extent of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR) 30 h post-infection.
Results
Ac2-26-treated WT mice showed less severe neutrophil infiltration, paralleled by a reduced induction of pro-inflammatory glial cell responses in the hippocampal formation and cortex. While meningitis was ameliorated in Ac2-26-treated
Fpr1
-deficient mice, this protective effect was not observed in
Fpr2
-deficient mice. Irrespective of Ac2-26 treatment, inflammation was more severe in
Fpr2
-deficient compared to
Fpr1
-deficient mice.
Conclusions
In summary, this study demonstrates anti-inflammatory properties of Ac2-26 in a model of bacterial meningitis, which are mediated via FPR2, but not FPR1. Ac2-26 and other FPR2 modulators might be promising targets for the development of novel therapies for
Streptococcus pneumoniae
-induced meningitis.
Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for ...more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM–Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.
Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role ...in the inflammatory response involved in neurodegenerative disorders such as Alzheimer's disease (AD).Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1-42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains.
We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy.
The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.
Intra-neuronal misfolding of monomeric tau protein to toxic β-sheet rich neurofibrillary tangles is a hallmark of Alzheimer’s disease (AD). Tau pathology correlates not only with progressive dementia ...but also with microglia-mediated inflammation in AD. Amyloid-beta (Aβ), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1β (IL1β) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1β and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer’s PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.
Our research explores the immunomodulatory effects of sulforaphane (SFN), a well-known nuclear factor erythroid 2-related factor 2 (Nrf2) pathway agonist, on the sterile inflammation of and ...ischemia-reperfusion injuries to the liver after hemorrhagic shock (HS) followed by resuscitation (R). Male C57/BL6 wild-type and transgenic ARE-
mice were exposed to mean arterial pressure-controlled HS. Fluid resuscitation was performed after 90 min of HS, and SFN was administrated intraperitoneally after that. The animals were sacrificed at 6 h, 24 h, and 72 h after resuscitation, and their livers were extracted to perform H&E staining and myeloperoxidase (MPO) activity analysis. The Kupffer cells were isolated for cytokines profile measurements and Nrf2 immunofluorescence staining. Further, the ARE-
mice were used to assess hepatic Nrf2 activity
. We identified that SFN-activated Kupffer cells' Nrf2 pathway and modulated its cytokines expression, including TNF-α, MCP-1, KC/CXCL1, IL-6, and IL-10. Furthermore, SFN mitigated liver ischemia-reperfusion injury, as evidenced by the downregulation of the Suzuki score and the enhanced hepatic Nrf2 activity. The
SFN treatment decreased neutrophils infiltration, as shown by the decreased MPO levels. Our study shows that SFN can decrease HS/R-induced hepatic ischemia-reperfusion injury and modulate the activity of Kupffer cells
an Nrf2-dependent pathway.
An important hallmark of Alzheimer's disease (AD) is the increase of Aβ1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia ...cell function represents an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aβ1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model.
To address this question, APP/PS1 double-transgenic AD mice were treated for 20 weeks with either the pro-inflammatory FPR agonist fMLF, the FPR1/2 antagonist Boc2 or the anti-inflammatory FPR2 agonist Ac2-26. Spatial learning and memory were evaluated using a Morris water maze test. Immunohistological staining, gene expression studies, and flow cytometry analyses were performed to study neuronal loss, gliosis, and Aß-load in the hippocampus and cortex, respectively.
FPR antagonism by Boc2-treatment significantly improved spatial memory performance, reduced neuronal pathology, induced the expression of homeostatic growth factors, and ameliorated microglia, but not astrocyte, reactivity. Furthermore, the elevated levels of amyloid plaques in the hippocampus were reduced by Boc2-treatment, presumably by an induction of amyloid degradation.
We suggest that the modulation of FPR signaling cascades might be considered as a promising therapeutic approach for alleviating the cognitive deficits associated with early AD. Additional studies are now needed to address the downstream effectors as well as the safety profile of Boc2.
There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an ...anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid β (Aβ)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD.
The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aβ burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze.
Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aβ deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice.
In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.
Bone regeneration is a complex process that is influenced by tissue interactions, inflammatory responses, and progenitor cells. Diseases, lifestyle, or multiple trauma can disturb fracture healing, ...which might result in prolonged healing duration or even failure. The current gold standard therapy in these cases are bone grafts. However, they are associated with several disadvantages, e.g., donor site morbidity and availability of appropriate material. Bone tissue engineering has been proposed as a promising alternative. The success of bone-tissue engineering depends on the administered cells, osteogenic differentiation, and secretome. Different stem cell types offer advantages and drawbacks in this field, while adipose-derived stem or stromal cells (ASCs) are in particular promising. They show high osteogenic potential, osteoinductive ability, and immunomodulation properties. Furthermore, they can be harvested through a noninvasive process in high numbers. ASCs can be induced into osteogenic lineage through bioactive molecules, i.e., growth factors and cytokines. Moreover, their secretome, in particular extracellular vesicles, has been linked to fracture healing. The aim of this review is a comprehensive overview of ASCs for bone regeneration and bone tissue engineering.
The effects of mechanical stress on cells and their extracellular matrix, especially in gliding sections of tendon, are still poorly understood. This study sought to compare the effects of uniaxial ...stretching on both gliding and traction areas in the same tendon. Flexor digitorum longus muscle tendons explanted from rats were subjected to stretching in a bioreactor for 6, 24, or 48 h, respectively, at 1 Hz and an amplitude of 2.5%. After stimulation, marker expression was quantified by histological and immunohistochemical staining in both gliding and traction areas. We observed a heightened intensity of scleraxis after 6 and 24 h of stimulation in both tendon types, though it had declined again 48 h after stimulation. We observed induced matrix metalloproteinase-1 and -13 protein expression in both tendon types. The bioreactor produced an increase in the mechanical structural strength of the tendon during the first half of the loading time and a decrease during the latter half. Uniaxial stretching of flexor tendon in our set-up can serve as an overloading model. A combination of mechanical and histological data allows us to improve the conditions for cultivating tendon tissues.
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