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- Medizinische Klinik und Poliklinik I (11)
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The role of serum amyloid A (SAA) proteins, which are ligands for toll-like receptors, was analyzed in human bone marrow-derived mesenchymal stem cells (hMSCs) and their osteogenic offspring with a focus on senescence, differentiation andmineralization. In vitro aged hMSC developed a senescence-associated secretory phenotype (SASP), resulting in enhanced SAA1/2, TLR2/4 and proinflammatory cytokine (IL6, IL8, IL1\(\beta\), CXCL1, CXCL2) expression before entering replicative senescence. Recombinant human SAA1 (rhSAA1) induced SASP-related genes and proteins in MSC, which could be abolished by cotreatment with the TLR4-inhibitor CLI-095. The same pattern of SASP-resembling genes was stimulated upon induction of osteogenic differentiation, which is accompanied by autocrine SAA1/2 expression. In this context additional rhSAA1 enhanced the SASP-like phenotype, accelerated the proinflammatory phase of osteogenic differentiation and enhanced mineralization. Autocrine/paracrine and rhSAA1 via TLR4 stimulate a proinflammatory phenotype that is both part of the early phase of osteogenic differentiation and the development of senescence. This signaling cascade is tightly involved in bone formation and mineralization, but may also propagate pathological extraosseous calcification conditions such as calcifying inflammation and atherosclerosis.
PURPOSE:
To evaluate the heart and the Tc-99m-sestamibi biodistribution after statin pretreatment in a rat model of abdominal sepsis.
METHODS:
Twenty-four Wistar rats were randomly distributed into four groups (n=6 per group): 1) sepsis with simvastatin treatment, 2) sepsis with vehicle, 3) sham control with simvastatin and 4) sham control with vehicle. 24 hours after cecal ligation and puncture rats received 1.0MBq of Tc-99m-sestamibi i.v. 30min after, animals were euthanized for ex-vivo tissue counting and myocardium histological analysis.
RESULTS:
Myocardial histologic alterations were not detected 24 hours post-sepsis. There was significantly increased cardiac Tc-99m-sestamibi activity in the sepsis group with simvastatin treatment (1.9\(\pm\)0.3%ID/g, p<0.001) in comparison to the sepsis group+vehicle (1.0\(\pm\)0.2% ID/g), control sham group+ simvastatin (1.2\(\pm\)0.3% ID/g) and control sham group (1.3\(\pm\)0.2% ID/g). Significant Tc-99m-sestamibi activity in liver, kidney and lungs was also detected in the sepsis group treated with simvastatinin comparison to the other groups.
CONCLUSIONS:
Statin treatment altered the biodistribution of Tc-99m-sestamibi with increased cardiac and solid organ activity in rats with abdominal sepsis, while no impact on controls. Increased myocardial tracer activity may be a result of a possible protection effect due to increased tissue perfusion mediated by statins.
Background
Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.
Aim
The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4\(^+\) lymphocytes.
Methods
Purified human CD4\(^+\) T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.
Results
Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4\(^+\) lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4\(^+\) lymphocytes.
Conclusion
For the first time, the immunomodulatory capacity of CHF5633 on CD4\(^+\) lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4\(^+\) cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.
The aim is to evaluate the effect of modifying poly[(L-lactide)-co-(epsilon-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1 alpha 2, and ANGPT1 are signifi cantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates infl ammation while lowering the dose of BMP-2 to a relatively safe and economical level.
Background
Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network.
Methods and Results
In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response.
Conclusion and Significance
Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.
NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells
(2016)
Epicutaneous application of Aldara cream containing the TLR7 agonist imiquimod (IMQ) to mice induces skin inflammation that exhibits many aspects of psoriasis, an inflammatory human skin disease. Here we show that mice depleted of B cells or bearing interleukin (IL)-10-deficient B cells show a fulminant inflammation upon IMQ exposure, whereas ablation of NFATc1 in B cells results in a suppression of Aldara-induced inflammation. In vitro, IMQ induces the proliferation and IL-10 expression by B cells that is blocked by BCR signals inducing NFATc1. By binding to HDAC1, a transcriptional repressor, and to an intronic site of the Il10 gene, NFATc1 suppresses IL-10 expression that dampens the production of tumour necrosis factor-α and IL-17 by T cells. These data indicate a close link between NFATc1 and IL-10 expression in B cells and suggest NFATc1 and, in particular, its inducible short isoform, NFATc1/αA, as a potential target to treat human psoriasis.
Background: In infarcted heart, improper clearance of dying cells by activated neighboring phagocytes may precipitate the transition to heart failure. We analyzed the coordinated role of 2 major mediators of efferocytosis, the myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8), in directing cardiac remodeling by skewing the inflammatory response after myocardial infarction.
Methods and Results: We generated double-deficient mice for Mertk and Mfge8 (Mertk\(^{-/-}\)/Mfge8\(^{-/-}\)) and challenged them with acute coronary ligature. Compared with wild-type, Mertk-deficient (Mertk\(^{-/-}\)), or Mfge8-deficient (Mfge8\(^{-/-}\)) animals, Mertk\(^{-/-}\)/Mfge8\(^{-/-}\) mice displayed greater alteration in cardiac function and remodeling. Mertk and Mfge8 were expressed mainly by cardiac Ly6C\(^{High and Low}\) monocytes and macrophages. In parallel, Mertk\(^{-/-}\)/Mfge8\(^{-/-}\) bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis. We found that the abrogation of efferocytosis affected neither the ability of circulating monocytes to infiltrate cardiac tissue nor the number of resident Ly6C\(^{High}\) and Ly6C\(^{Low}\) monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6C\(^{High}\)/Ly6C\(^{Low}\) monocytes/macrophages either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. Using LysMCre\(^+\)/VEGFA\(^{fl/fl}\) mice, we further identified an important role for myeloid-derived VEGFA in improving cardiac function and angiogenesis.
Conclusions: After myocardial infarction, Mertk- and Mfge8-expressing monocyte/macrophages synergistically engage the clearance of injured cardiomyocytes, favoring the secretion of VEGFA to locally repair the dysfunctional heart.
Mechanical thrombectomy is a novel treatment option for patients with acute ischemic stroke (AIS). Only a few studies have previously suggested strategies to categorize retrieved clots according to their histologic composition. However, these reports did not analyze potential biomarkers that are of importance in stroke-related inflammation. We therefore histopathologically investigated 37 intracerebral thrombi mechanically retrieved from patients with AIS, and focused on the composition of immune cells and platelets. We also conducted correlation analyses of distinctive morphologic patterns (erythrocytic, serpentine, layered, red, white, mixed appearance) with clinical parameters. Most T cells and monocytes were detected in erythrocytic and red clots, in which the distribution of these cells was random. In contrast, von Willebrand factor (vWF)-positive areas co-localized with regions of fibrin and collagen. While clots with huge amounts of vWF seem to be associated with a high National Institute of Health Stroke Scale score at admission, histologic findings could not predict the clinical outcome at discharge. In summary, we provide the first histologic description of mechanically retrieved intracerebral thrombi regarding biomarkers relevant for inflammation in ischemic stroke.
Toll like receptor (TLR) signaling has been suggested to play an important role in the inflammatory microenvironment of solid tumors and through this inflammation-mediated tumor growth. Here, we studied the role of tumor cells in their process of self-maintaining TLR expression independent of inflammatory cells and cytokine milieu for autoregulative tumor growth signaling in pancreatic cancer. We analyzed the expression of TLR2, -4, and -9 in primary human cancers and their impact on tumor growth via induced activation in several established pancreatic cancers. TLR-stimulated pancreatic cancer cells were specifically investigated for activated signaling pathways of VEGF/PDGF and anti-apoptotic Bcl-xL expression as well as tumor cell growth. The primary pancreatic cancers and cell lines expressed TLR2, -4, and -9. TLR-specific stimulation resulted in activated MAP-kinase signaling, most likely via autoregulative stimulation of demonstrated TLR-induced VEGF and PDGF expression. Moreover, TLR activation prompted the expression of Bcl-xL and has been demonstrated for the first time to induce tumor cell proliferation in pancreatic cancer. These findings strongly suggest that pancreatic cancer cells use specific Toll like receptor signaling to promote tumor cell proliferation and emphasize the particular role of TLR2, -4, and -9 in this autoregulative process of tumor cell activation and proliferation in pancreatic cancer.
Objectives: Chronic recurrent multifocal osteomyelitis (CRMO), the most severe form of chronic nonbacterial osteomyelitis (CNO), is an autoinflammatory bone disorder. In the absence of diagnostic criteria or biomarkers, CNO/CRMO remains a diagnosis of exclusion. The aim of this study was to identify biomarkers for diagnosing multifocal disease (CRMO).
Study design: Sera from 71 pediatric CRMO patients, 11 patients with osteoarticular infections, 62 patients with juvenile idiopathic arthritis (JIA), 7 patients with para-infectious or reactive arthritis, and 43 patients with acute leukemia or lymphoma, as well as 59 healthy individuals were collected. Multiplex analysis of 18 inflammation- and/or bone remodeling-associated serum proteins was performed. Statistical analysis included univariate ANOVA, discriminant analysis, univariate receiver operating characteristic (ROC) analysis, and logistic regression analyses.
Results: For 14 of 18 blood serum proteins, significant differences were determined between CRMO patients, at least one alternative diagnosis, or healthy controls. Multi-component discriminant analysis delivered five biomarkers (IL-6, CCL11/eotaxin, CCL5/RANTES, collagen Iα, sIL-2R) for the diagnosis of CRMO. ROC analysis allowed further reduction to a core set of 2 biomarkers (CCL11/eotaxin, IL-6) that are sufficient to discern between CRMO, healthy controls, and alternative diagnoses.
Conclusion: Serum biomarkers CCL11/eotaxin and IL-6 differentiate between patients with CRMO, healthy controls, and alternative diagnoses (leukemia and lymphoma, osteoarticular infections, para-infectious arthritis, and JIA). Easily accessible biomarkers may aid in diagnosing CRMO. Further studies testing biomarkers in larger unrelated cohorts are warranted.
Macrophages predominate the inflammatory landscape within multiple sclerosis (MS) lesions, not only regarding cellularity but also with respect to the diverse functions this cell fraction provides during disease progression and remission. Researchers have been well aware of the fact that the macrophage pool during central nervous system (CNS) autoimmunity consists of a mixture of myeloid cells. Yet, separating these populations to define their unique contribution to disease pathology has long been challenging due to their similar marker expression. Sophisticated lineage tracing approaches as well as comprehensive transcriptome analysis have elevated our insight into macrophage biology to a new level enabling scientists to dissect the roles of resident (microglia and non-parenchymal macrophages) and infiltrating macrophages with unprecedented precision. To do so in an accurate way, researchers have to know their toolbox, which has been filled with diverse, discriminating approaches from decades of studying neuroinflammation in animal models. Every method has its own strengths and weaknesses, which will be addressed in this review. The focus will be on tools to manipulate and/or identify different macrophage subgroups within the injured murine CNS.
TNF receptor type 2 (TNFR2) has gained attention as a costimulatory receptor for T cells and as critical factor for the development of regulatory T cells (Treg) and myeloid suppressor cells. Using the TNFR2-specific agonist TNCscTNF80, direct effects of TNFR2 activation on myeloid cells and T cells were investigated in mice. \(In\) \(vitro\), TNCscTNF80 induced T cell proliferation in a costimulatory fashion, and also supported \(in\) \(vitro\) expansion of Treg cells. In addition, activation of TNFR2 retarded differentiation of bone marrow-derived immature myeloid cells in culture and reduced their suppressor function. \(In\) \(vivo\) application of TNCscTNF80-induced mild myelopoiesis in naïve mice without affecting the immune cell composition. Already a single application expanded Treg cells and improved suppression of CD4 T cells in mice with chronic inflammation. By contrast, multiple applications of the TNFR2 agonist were required to expand Treg cells in naïve mice. Improved suppression of T cell proliferation depended on expression of TNFR2 by T cells in mice repeatedly treated with TNCscTNF80, without a major contribution of TNFR2 on myeloid cells. Thus, TNFR2 activation on T cells in naïve mice can lead to immune suppression \(in\) \(vivo\). These findings support the important role of TNFR2 for Treg cells in immune regulation.
Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund’s adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.
Fullerenols, water-soluble C60-fullerene derivatives, have been shown to exert neuroprotective effects in vitro and in vivo, most likely due to their capability to scavenge free radicals. However, little is known about the effects of fullerenols on the blood–brain barrier (BBB), especially on cerebral endothelial cells under inflammatory conditions. Here, we investigated whether the treatment of primary mouse brain microvascular endothelial cells with fullerenols impacts basal and inflammatory blood–brain barrier (BBB) properties in vitro. While fullerenols (1, 10, and 100 µg/mL) did not change transendothelial electrical resistance under basal and inflammatory conditions, 100 µg/mL of fullerenol significantly reduced erk1/2 activation and resulted in an activation of NFκB in an inflammatory milieu. Our findings suggest that fullerenols might counteract oxidative stress via the erk1/2 and NFκB pathways, and thus are able to protect microvascular endothelial cells under inflammatory conditions.
Background:
Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation.
Methods:
Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4.
Results:
Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1β and IL-8 in term neonatal and adult monocytes (p < 0.01 and p < 0.05). Intracellular protein expression of TNF-α, IL-1β and IL-8 in Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1β in adult monocytes (Uu8: p < 0.05). Remarkably, ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1β/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p < 0.01, vs. LPS). In contrast to LPS, both isolates induced TLR2 mRNA in neonatal and adult cells (p < 0.001 and p < 0.05) and suppressed TLR4 mRNA in adult monocytes (p < 0.05). Upon co-stimulation, Uu8 and Up3 inhibited LPS-induced intracellular IL-1β (p < 0.001 and p < 0.05) and IL-8 in adult monocytes (p < 0.01), while LPS-induced neonatal cytokines were maintained or aggravated (p < 0.05).
Conclusion:
Our data demonstrate a considerable pro-inflammatory capacity of Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of TLR2 and TLR4 expression may shape host susceptibility to inflammation.
Despite growing effort to advance materials towards a low fibrotic progression, all implants elicit adverse tissue responses. Pre-clinical biomaterial assessment relies on animals testing, which can be complemented by in vitro tests to address the Russell and Burch’s 3R aspect of reducing animal burden. However, a poor correlation between in vitro and in vivo biomaterial assessments confirms a need for suitable in vitro biomaterial tests. The aim of the study was to identify a test setting, which is predictive and might be time- and cost-efficient. We demonstrated how sensitive in vitro biomaterial assessment based on human primary macrophages depends on test conditions. Moreover, possible clinical scenarios such as lipopolysaccharide contamination, contact to autologous blood plasma, and presence of IL-4 in an immune niche influence the outcome of a biomaterial ranking. Nevertheless, by using glass, titanium, polytetrafluorethylene, silicone, and polyethylene representing a specific material-induced fibrotic response and by comparison to literature data, we were able to identify a test condition that provides a high correlation to state-of-the-art in vivo studies. Most important, biomaterial ranking obtained under native plasma test conditions showed a high predictive accuracy compared to in vivo assessments, strengthening a biomimetic three-dimensional in vitro test platform.
Background
Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology.
Methods and results
We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age.
Conclusion
Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.
Aims: Although mortality rate is very high, diagnosis of acute myocarditis remains challenging with conventional tests. We aimed to elucidate the potential role of longitudinal 2-Deoxy-2-\(^{18}\)F-fluoro-D-glucose (\(^{18}\)F-FDG) positron emission tomography (PET) inflammation monitoring in a rat model of experimental autoimmune myocarditis.
Methods and results: Autoimmune myocarditis was induced in Lewis rats by immunizing with porcine cardiac myosin emulsified in complete Freund’s adjuvant. Time course of disease was assessed by longitudinal \(^{18}\)F-FDG PET imaging. A correlative analysis between in- and ex vivo \(^{18}\)F-FDG signalling and macrophage infiltration using CD68 staining was conducted. Finally, immunohistochemistry analysis of the cell-adhesion markers CD34 and CD44 was performed at different disease stages determined by longitudinal \(^{18}\)F-FDG PET imaging. After immunization, myocarditis rats revealed a temporal increase in 18F-FDG uptake (peaked at week 3), which was followed by a rapid decline thereafter. Localization of CD68 positive cells was well correlated with in vivo \(^{18}\)F-FDG PET signalling (R\(^2\) = 0.92) as well as with ex vivo 18F-FDG autoradiography (R\(^2\) = 0.9, P < 0.001, respectively). CD44 positivity was primarily observed at tissue samples obtained at acute phase (i.e. at peak 18F-FDG uptake), while CD34-positive staining areas were predominantly identified in samples harvested at both sub-acute and chronic phases (i.e. at \(^{18}\)F-FDG decrease).
Conclusion: \(^{18}\)F-FDG PET imaging can provide non-invasive serial monitoring of cardiac inflammation in a rat model of acute myocarditis.
T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca2+ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca2+ entry (SOCE) mediated by the Ca2+ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE.
Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1\(^{-/-}\) mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1\(^{-/-}\) mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy.
Importance:
The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease.