Nuclease activity and protein A release of Staphylococcus aureus clinical isolates determine the virulence in a murine model of acute lung infection.

Staphylococcus aureus is a common cause of hospital-acquired pneumonia associated with high mortality. Adequate clinical treatment is impeded by increasing occurrence of antibiotic resistances. Understanding the underlying mechanisms of its virulence during infections is a prerequisite to finding alternative treatments. Here, we demonstrated that an increased nuclease activity of a S. aureus isolate from a person with cystic fibrosis confers a growth advantage in a model of acute lung infection compared to the isogenic strain with low nuclease activity. Comparing these CF-isolates with a common MRSA-USA300 strain with similarly high nuclease activity but significantly elevated levels of Staphylococcal Protein A (SpA) revealed that infection with USA300 resulted in a significantly increased bacterial burden in a model of murine lung infection. Replenishment with the cell wall-bound SpA of S. aureus, which can also be secreted into the environment and binds to tumor necrosis factor receptor -1 (TNFR-1) to the CF-isolates abrogated these differences. In vitro experiments confirmed significant differences in spa-expression between USA300 compared to CF-isolates, thereby influencing TNFR-1 shedding, L-selectin shedding, and production of reactive oxygen species through activation of ADAM17.

Bioactive Nanogels Mimicking the Antithrombogenic Nitric Oxide-Release Function of the Endothelium.

Nitric oxide (NO) plays a significant role in controlling the physiology and pathophysiology of the body, including the endothelial antiplatelet function and therefore, antithrombogenic property of the blood vessels. This property of NO can be exploited to prevent thrombus formation on artificial surfaces like extracorporeal membrane oxygenators, which when come into contact with blood lead to protein adsorption and thereby platelet activation causing thrombus formation. However, NO is extremely reactive and has a very short biological half-life in blood, so only endogenous generation of NO from the blood contacting material can result into a stable and kinetically controllable local delivery of NO. In this regards, highly hydrophilic bioactive nanogels are presented which can endogenously generate NO in blood plasma from endogenous NO-donors thereby maintaining a physiological NO flux. It is shown that NO releasing nanogels could initiate cGMP-dependent protein kinase signaling followed by phosphorylation of vasodilator-stimulated phosphoprotein in platelets. This prevents platelet activation and aggregation even in presence of highly potent platelet activators like thrombin, adenosine 5'-diphosphate, and U46619 (thromboxane A2 mimetic).

Role of S100A8/A9 in Platelet-Neutrophil Complex Formation during Acute Inflammation.

Acute respiratory distress syndrome (ARDS) due to pulmonary infections is associated with high morbidity and mortality. Upon inflammation, the alarmin S100A8/A9 is released and stimulates neutrophil recruitment mainly via binding to Toll-like receptor 4 (TLR4). TLR4 is also expressed on platelets, which modulate the immune response through direct interaction with leukocytes. In a murine model of Klebsiella pneumoniae-induced pulmonary inflammation, global S100A9 deficiency resulted in diminished neutrophil recruitment into the lung alveoli and neutrophil accumulation in the intravascular space, indicating an impaired neutrophil migration. A lack of TLR4 on platelets resulted in reduced neutrophil counts in the whole lung, emphasising the impact of TLR4-mediated platelet activity on neutrophil behaviour. Flow cytometry-based analysis indicated elevated numbers of platelet-neutrophil complexes in the blood of S100A9-/- mice. Intravital microscopy of the murine cremaster muscle confirmed these findings and further indicated a significant increase in neutrophil-platelet complex formation in S100A9-/- mice, which was reversed by administration of the S100A8/A9 tetramer. An in vitro bilayer assay simulated the murine alveolar capillary barrier during inflammation and validated significant differences in transmigration behaviour between wild-type and S100A9-/- neutrophils. This study demonstrates the role of S100A8/A9 during platelet-neutrophil interactions and neutrophil recruitment during pulmonary inflammation.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Platelets at the Crossroads of Pro-Inflammatory and Resolution Pathways during Inflammation.

Platelets are among the most abundant cells in the mammalian circulation. Classical platelet functions in hemostasis and wound healing have been intensively explored and are generally accepted. During the past decades, the research focus broadened towards their participation in immune-modulatory events, including pro-inflammatory and, more recently, inflammatory resolution processes. Platelets are equipped with a variety of abilities enabling active participation in immunological processes. Toll-like receptors mediate the recognition of pathogens, while the release of granule contents and microvesicles promotes direct pathogen defense and an interaction with leukocytes. Platelets communicate and physically interact with neutrophils, monocytes and a subset of lymphocytes via soluble mediators and surface adhesion receptors. This interaction promotes leukocyte recruitment, migration and extravasation, as well as the initiation of effector functions, such as the release of extracellular traps by neutrophils. Platelet-derived prostaglandin E2, C-type lectin-like receptor 2 and transforming growth factor ß modulate inflammatory resolution processes by promoting the synthesis of pro-resolving mediators while reducing pro-inflammatory ones. Furthermore, platelets promote the differentiation of CD4+ T cells in T helper and regulatory T cells, which affects macrophage polarization. These abilities make platelets key players in inflammatory diseases such as pneumonia and the acute respiratory distress syndrome, including the pandemic coronavirus disease 2019. This review focuses on recent findings in platelet-mediated immunity during acute inflammation.

C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9.

The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but are completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of S100a8 and S100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout (KO)-based screening approach in immortalized murine monocytes, we identified the transcription factor C/EBPδ as a central regulator of S100a8 and S100a9 expression. We showed that S100A8/A9 expression and thereby neutrophil recruitment and cytokine release were decreased in C/EBPδ KO mice in a mouse model of acute lung inflammation. S100a8 and S100a9 expression was further controlled by the C/EBPδ antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within S100a8 and S100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100a8 and S100a9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterized by S100a8 and S100a9 overexpression.

The Pathogenesis of Ischemia-Reperfusion Induced Acute Kidney Injury Depends on Renal Neutrophil Recruitment Whereas Sepsis-Induced AKI Does Not.

Acute kidney injury (AKI) may be induced by different causes, including renal ischemia-reperfusion injury and sepsis, which represent the most common reasons for AKI in hospitalized patients. AKI is defined by reduced urine production and/or increased plasma creatinine. However, this definition does not address the molecular mechanisms of different AKI entities, and uncertainties remain regarding distinct pathophysiological events causing kidney injury in the first place. In particular, sepsis-induced AKI is considered not to be associated with leukocyte infiltration into the kidney, but a direct investigation of this process is missing to this date. In this study, we used two murine AKI models induced by either renal ischemia-reperfusion injury (IRI) or cecal ligation and puncture (CLP) to investigate the contribution of neutrophils to tissue injury and kidney function. By using VEC-Y731F mice, in which neutrophil recruitment is impaired, we analyzed the specific contribution of neutrophil recruitment to the pathogenesis of IRI- and CLP-induced AKI. We observed that the degree of renal injury evaluated by plasma creatinine, urinary biomarkers and histological analyses, following IRI-induction was dependent on neutrophil migration into the kidney, whereas the pathogenesis of CLP-induced AKI was independent of neutrophil recruitment. Furthermore, plasma transfer experiments suggest that the pathogenesis of CLP-induced AKI relies on circulating inflammatory mediators. These results extend our knowledge of the AKI pathogenesis and may help in the development of prophylactic and therapeutic treatments for AKI patients.

DNase I functional microgels for neutrophil extracellular trap disruption.

Neutrophil extracellular traps (NETs) are web-like chromatin structures produced and liberated by neutrophils under inflammatory conditions which also promote the activation of the coagulation cascade and thrombus formation. The formation of NETs is quite prominent when blood comes in contact with artificial surfaces like extracorporeal circuits, oxygenator membranes, or intravascular grafts. DNase I as a factor of the host defense system, digests the DNA backbone of NETs, which points out its treatment potential for NET-mediated thrombosis. However, the low serum stability of DNase I restricts its clinical/therapeutic applications. To improve the bioavailability of the enzyme, DNase I was conjugated to the microgels (DNase I MG) synthesized from highly hydrophilic N-(2-hydroxypropyl) methacrylamide (HPMA) and zwitterionic carboxybetaine methacrylamide (CBMAA). The enzyme was successfully conjugated to the microgels without any alternation to its secondary structure. The Km value representing the enzymatic activity of the conjugated DNase I was calculated to be 0.063 µM demonstrating a high enzyme-substrate affinity. The DNase I MGs were protein repellant and were able to digest NETs more efficiently compared to free DNase in a biological media, remarkably even after long-term exposure to the stimulated neutrophils continuously releasing NETs. Overall, the conjugation of DNase I to a non-fouling microgel provides a novel biohybrid platform that can be exploited as non-thrombogenic active microgel-based coatings for blood-contacting surfaces to reduce the NET-mediated inflammation and microthrombi formation.

Systemic Inflammatory Response Syndrome After Surgery: Mechanisms and Protection.

The immune system is an evolutionary hallmark of higher organisms that defends the host against invading pathogens and exogenous infections. This defense includes the recruitment of immune cells to the site of infection and the initiation of an inflammatory response to contain and eliminate pathogens. However, an inflammatory response may also be triggered by noninfectious stimuli such as major surgery, and, in case of an overshooting, still not comprehensively understood reaction, lead to tissue destruction and organ dysfunction. Unfortunately, in some cases, the immune system may not effectively distinguish between stimuli elicited by major surgery, which ideally should only require a modest inflammatory response, and those elicited by trauma or pathogenic infection. Surgical procedures thus represent a potential trigger for systemic inflammation that causes the secretion of proinflammatory cytokines, endothelial dysfunction, glycocalyx damage, activation of neutrophils, and ultimately tissue and multisystem organ destruction. In this review, we discuss and summarize currently available mechanistic knowledge on surgery-associated systemic inflammation, demarcation toward other inflammatory complications, and possible therapeutic options. These options depend on uncovering the underlying mechanisms and could include pharmacologic agents, remote ischemic preconditioning protocols, cytokine blockade or clearance, and optimization of surgical procedures, anesthetic regimens, and perioperative inflammatory diagnostic assessment. Currently, a large gap between basic science and clinically confirmed data exists due to a limited evidence base of translational studies. We thus summarize important steps toward the understanding of the precise time- and space-regulated processes in systemic perioperative inflammation.

ArhGAP15, a RacGAP, Acts as a Temporal Signaling Regulator of Mac-1 Affinity in Sterile Inflammation.

During inflammation, leukocyte recruitment has to be tightly controlled to prevent overwhelming leukocyte infiltration, activation, and, consequently, organ damage. A central regulator of leukocyte recruitment is Rac1. In this study, we analyzed the effects of the RacGAP ArhGAP15 on leukocyte recruitment. Using ArhGAP15-deficient mice, reduced neutrophil adhesion and transmigration in the TNF-α-inflamed cremaster muscle and a prolongation of chemokine-dependent leukocyte adhesion could be observed. In a murine model of sterile kidney injury, reduced neutrophil infiltration, and serum creatinine levels were apparent. Further in vitro and in vivo analyses revealed a defective intravascular crawling capacity, resulting from increased affinity of the ß2-integrin Mac-1 after prolonged chemokine stimulation of neutrophils. LFA-1 activity regulation was not affected. Summarizing, ArhGAP15 specifically regulates Mac-1, but not LFA-1, and affects leukocyte recruitment by controlling postadhesion strengthening and intravascular crawling in a Mac-1-dependent manner. In conclusion, ArhGAP15 is involved in the time-dependent regulation of leukocyte postadhesion in sterile inflammation.

The integrin-linked kinase is required for chemokine-triggered high-affinity conformation of the neutrophil ß2-integrin LFA-1.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the ß2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of ß2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1.