Korbinian Brand

During early pregnancy, maternal blood surrounds the embryo before the placenta is fully developed, requiring tight regulation of maternal blood flow into the placental vasculature. We identify placental microthrombi (PMTs) as essential structures guiding this process. PMTs contain platelets, coagulation factors, and complement proteins, and their formation depends on maternal platelet activation by thrombin through the protease-activated receptor 4 (PAR4). Deficiency of PAR4 abolished PMTs and caused excessive bleeding at the implantation site. C3 deficiency also led to increased bleeding events, indicating that complement activation contributes to thrombosis in the placental circulation. Conversely, dysregulated complement activation in CMP–sialic acid synthase–deficient ( Cmas ^–/– ) mice led to widespread thrombosis and failed placental development. Strikingly, platelet activation via PAR4 was necessary to localize complement activation to trophoblast surfaces, thereby coupling coagulation and complement in PMT formation. Depletion of maternal platelets mitigated complement-driven thromboinflammation in Cmas ^–/– pregnancies, restoring placental growth. These findings uncover a critical cooperation between platelets, coagulation, and complement in establishing maternal blood flow to the placenta. Successful pregnancy therefore requires not only activation but also tight regulation of these systems to balance necessary PMT formation with the prevention of pathological thrombosis.

Keywords: Interferons, cytokines, mutual regulation, pro-inflammatory signaling, interferon-14 stimulated genes, inflammation, autoimmunity, interferon-associated diseases 15 16Interferons (IFNs) are crucial regulators of the immune response mediating both pro-and anti-17 inflammatory effects (1, 2). To date, the human IFN family comprises type I (IFN-I: IFN-α, -β, -ε, -κ, 18 and -ω), II (IFN-γ), and III IFNs (IFN-λ). Following binding to their receptors, they activate intracellular 19 signaling, predominantly the Jak-STAT pathway, and induce the expression of a broad range of 20 interferon-stimulated genes (3). However, due to the variety of potentially activated cascades, shared 21 target promoter elements, and the specific characteristics of interferon-responsive cells (among 22 other things), different (types of) interferons may have distinct as well as overlapping functions. While 23 their canonical function is the mediation of anti-viral activity and the defense against microbial 24 infections, IFNs further proved to be involved in anti-tumor immunity, autoimmunity, cell 25 development, tissue protection, homeostasis, and metabolism (1,4).Under both physiological and pathophysiological conditions, however, interferons do not act 27 on target cells alone, but are part of a complex cocktail of cytokines, chemokines, growth factors, and 28 other mediators, whose interaction defines the reaction of the cells to the specific stimulatory 29 situation (5). Alterations in the proper orchestration of this immunomodulatory network may result 30 in the dysregulations of the respective response, thus enabling infectious, inflammatory, or malignant 31 diseases. Li et al., for instance, examine this aspect in the field of osteoimmunology, an 32 interdisciplinary area studying the crosstalk between the immune system and the skeletal system. In 33 their review, they focus on the intricate and in part contradictory impact of IFN-γ on bone remodeling 34 during osteoporosis, a systemic disease characterized by low bone mass and an increased risk of 35 fracture (6). The authors recognize in detail the multifaceted contribution of IFN-γ to osteoblast differentiation and the limitation of osteoclast formation, but also consider its capacity to indirectly 37 increase the bone-resorbing activity of mature osteoclasts. Thus, IFN-γ exhibits both osteoprotective 38 and osteodestructive properties. In the case of osteoporosis, however, IFN-γ appears to account for 39 a net loss of bone mass, although its effects may be complex and disease stage-specific. 40The As illustrated, IFNs are of decisive importance in the development of (patho-)physiological 58 states associated with inflammation, but their impact cannot be understood without knowledge on 59 the events regulating their expression (1) or the interaction with other mediators (5). Therefore, Yu 60 and colleagues have a closer look on interferon regulatory factor (IRF)5, a transcription factor that -61 due to the functional differentiation from its relatives -contributes to the expression of both IFN-I 62 and pro-inflammatory cytokines/chemokines. The authors characterize IRF5 as a potent player in 63 antiviral immunity, but also as a driver of inflammatory and autoimmune diseases such as 64 inflammatory bowel disease, SLE, or rheumatoid arthritis. Interestingly, IRF5 appears to fan 65 inflammation-associated malignancies, while it may adopt the role of a tumor suppressor in other 66 forms of cancer, depending on the type of affected cells and tissues. Due to its involvement in these 67 and other diseases, IRF5 can be regarded as a therapeutic target, and influence on TNF expression or the symptomatic of the infection, carriers of the rs179008 major allele 78 exhibited increased IFN-α amounts, while the minor allele of rs3853839 was associated with higher 79 TLR7 levels. Thus, the rs179008 major and the rs3853839 minor allele may have protective effects by 80 supporting antiviral defense. Finally, Kaminiów and co-workers investigate the association of two 81SNPs located in introns of the IFN-γ gene, rs2430561 (T/A) and rs1861494 (A/G), with the serological 82 response in early treated syphilis patients. In both cases, homozygous carriers of the major alleles 83 showed higher IFN-γ levels in combination with serological cure. In contrast, the homozygous minor 84 genotype was associated with low IFN-γ levels and a (persisting) serofast state, an observation 85 suggesting a predisposition for the development of serofast syphilis.In summary, this article collection adds a few more mosaic tiles to a complex and still 87 expanding topic and thus complements the picture we have of IFNs, their properties, and their 88 interrelationship with other factors. The manuscripts included thus enhance our understanding of 89 the molecular mechanisms underlying the diverse effects of IFNs under multiple conditions. 90 91

In contrast to other common anticoagulants such as citrate and low-molecular-weight heparin (LMWH), high-molecular-weight heparin (HMWH) induces the expression of matrix metalloproteinase (MMP)-9, which is also measured as a biomarker for stroke in blood samples. Mechanistically, HMWH-stimulated T cells produce cytokines that induce monocytic MMP-9 expression. Here, the influence of further anticoagulants (Fondaparinux, Hirudin, and Alteplase) and the heparin-contaminating glycosaminoglycans (GAG) hyaluronic acid (HA), dermatan sulfate (DS), chondroitin sulfate (CS), and over-sulfated CS (OSCS) on MMP-9 was analyzed to assess its suitability as a biomarker under various conditions. Therefore, starved Jurkat T cells were stimulated with anticoagulants/contaminants. Subsequently, starved monocytic THP-1 cells were incubated with the conditioned Jurkat supernatant, and MMP-9 mRNA levels were monitored (quantitative (q)PCR). Jurkat-derived mediators secreted in response to anticoagulants/contaminants were also assessed (proteome profiler array). The supernatants of HMWH-, Hirudin-, CS-, and OSCS-treated Jurkat cells comprised combinations of activating mediators and led to a significant (in the case of OSCS, dramatic) MMP-9 induction in THP-1. HA induced MMP-9 only in high concentrations, while LMWH, Fondaparinux, Alteplase, and DS had no effect. This indicates that depending on molecular weight and charge (but independent of anticoagulant activity), anticoagulants/contaminants provoke the expression of T-cell-derived cytokines/chemokines that induce monocytic MMP-9 expression, thus potentially impairing the diagnostic validity of MMP-9.

Nowadays, GSK3 is accepted as an enzyme strongly involved in the regulation of inflammation by balancing the pro- and anti-inflammatory responses of cells and organisms, thus influencing the initiation, progression, and resolution of inflammatory processes at multiple levels. Disturbances within its broad functional scope, either intrinsically or extrinsically induced, harbor the risk of profound disruptions to the regular course of the immune response, including the formation of severe inflammation-related diseases. Therefore, this review aims at summarizing and contextualizing the current knowledge derived from animal models to further shape our understanding of GSK3α and β and their roles in the inflammatory process and the occurrence of tissue/organ damage. Following a short recapitulation of structure, function, and regulation of GSK3, we will focus on the lessons learned from GSK3α/β knock-out and knock-in/overexpression models, both conventional and conditional, as well as a variety of (predominantly rodent) disease models reflecting defined pathologic conditions with a significant proportion of inflammation and inflammation-related tissue injury. In summary, the literature suggests that GSK3 acts as a crucial switch driving pro-inflammatory and destructive processes and thus contributes significantly to the pathogenesis of inflammation-associated diseases.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS’s functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCβ KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCβ KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.

Background: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context. Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays. Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects. Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.

Anti-infective treatment of pulmonary exacerbations is a major issue in people with cystic fibrosis (CF). Individualized dosing strategies and adaptation of infusion times are important concepts to optimize anti-infective therapy. In this prospective non-randomized controlled open-label trial, we compared pharmacokinetics of meropenem in 12 people with CF experiencing a pulmonary exacerbation, of whom six received parenteral meropenem 2 g tid as short infusion over 30 min and six extended infusion over 120 min. We measured blood concentrations of meropenem at five predetermined time points over 240 min and calculated differences in the percentages of the time above the minimal inhibitory concentration (fT > MIC) for meropenem concentrations >16 and >32 mg/L, respectively. Mean percentages of fT > 16 and fT > 32 mg/L were higher in the extended compared to the short infusion group (83 and 56% vs. 59% and 34%), with a statistically significant prolongation of the fT > 32 mg/L (mean 134 vs. 82 min; p = 0.037). Our results demonstrate that, in people with CF, longer fT > MIC can be achieved with a simple modification of meropenem dosing. Further studies are needed to clarify if this may translate into improved microbiological and clinical outcomes, in particular in adults with difficult-to-treat chronic infection by carbapenem-resistant Pseudomonas aeruginosa.

Background: In recent years, treatment‐adherence gained increasing attention in nearly every area of medicine including transplant medicine. Medication adherence following solid organ transplantation is known to be indispensable for a satisfactory allograft survival.Methods: We examined 60 patients between the ages of four months and 20 years who underwent kidney transplantation at Hannover Medical School between January 2011 and August 2017. Age at transplantation varied from 4 months to 20 years. 12 patients (20%) already underwent their second solid organ transplantation. 5 patients (8.3%) had a combined kidney‐liver‐transplantation.We used two different methods for rating adherence: An objective one based on the coefficient of variation (CoV%) of immunosuppressant trough levels, and a subjective questionnaire answered by the patients themselves, their parents or legal custodians, the treating pediatrician, as well as by the attending psychologist.Results: The CoV% in our study was by‐trend higher in those patients who suffered from a biopsy‐proven rejection (x̅_CoV% = 35.7, σ _CoV% = 30.1 in patients with rejection vs. x̅ _CoV% = 26.0, σ _CoV% = 10.5 in patients without rejection). Furthermore, the psychologist's assessment correlated significantly both with rejections as well as with the formation of de novo donor‐specific antibodies (dnDSA) while the pediatrician's rating showed no correlation (P_rejections = 0.005 and P_dnDSA = 0.03 for psychologist's rating vs. P_rejections = 0.50 and P_dnDSA = 0.50 for pediatrician).Conclusions: Apart from underlining the importance of medication adherence, the present research stresses the role of a multi‐disciplinary treatment approach to support pediatric renal transplant recipients and their families.