The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics

Система генерации отчетов САПР бортового программного обеспечения космических аппаратов

Целью работы является проектирование и реализация системы генерации отчетов в САПР БПО. В ходе работы разработана система, призванная автоматизировать создание отчетов по данным, которыми оперирует САПР БПО. В частности, система позволяет генерировать отчеты по ресурсам, отчет-Д по отображению формата кадров телеметрической информации и документ "Исходные данные на изделие". В отчетах заполняются таблицы по использованию аппаратных ресурсов спутника бортовым программным обеспечением. Отчеты формируются в качестве документов Microsoft Word и Excel с требуемым форматированием.The aim of the work is to design and implement the report generating system in CAD BS. As the result of the work, the system that automates the creation of reports was developed. Particularly, the system allows to generate resource reports, “Report-D” that shows formats of the telemetry data and the document “Satellite Source Data”. The system fills the tables describing satellite hardware resource distribution by on-board software. The reports are formed as Microsoft Word and Excel documents with required formatting

Leucine Reconstitutes Phagocytosis-Induced Cell Death in E. coli-Infected Neonatal Monocytes—Effects on Energy Metabolism and mTOR Signaling

MΦ differentiate from circulating monocytes (Mo). The reduced ability of neonatal Mo to undergo apoptosis after E. coli infection (phagocytosis-induced cell death (PICD)) could contribute to sustained inflammatory processes. The objective of our study was to investigate whether immune metabolism in Mo can be modified to gain access to pro-apoptotic signaling. To this end, we supplemented Mo from neonates and from adults with the branched amino acid leucine. In neonatal Mo, we observed increased energy production via oxidative phosphorylation (Oxphos) after E. coli infection via Seahorse assay. Leucine did not change phagocytic properties. In neonatal Mo, we detected temporal activation of the AKT and mTOR pathways, accompanied with subsequent activation of downstream targets S6 Kinase (S6K) and S6. FACS analyses showed that once mTOR activation was terminated, the level of anti-apoptotic BCL-2 family proteins (BCL-2; BCL-XL) decreased. Release of cytochrome C and cleavage of caspase-3 indicated involvement of the intrinsic apoptotic pathway. Concomitantly, the PICD of neonatal Mo was initiated, as detected by hypodiploid DNA. This process was sensitive to rapamycin and metformin, suggesting a functional link between AKT, mTOR and the control of intrinsic apoptotic signaling. These features were unique to neonatal Mo and could not be observed in adult Mo. Supplementation with leucine therefore could be beneficial to reduce sustained inflammation in septic neonates

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

Neonates are highly susceptible to microbial infections which is partially attributable to fundamental phenotypic and functional differences between effector cells of the adult and neonatal immune system. The resolution of the inflammation is essential to return to tissue homeostasis, but given that various neonatal diseases, such as periventricular leukomalacia, necrotizing enterocolitis, or bronchopulmonary dysplasia, are characterized by sustained inflammation, newborns seem predisposed to a dysregulation of the inflammatory response. Targeted apoptosis of effector cells is generally known to control the length and extent of the inflammation, and previous studies have demonstrated that phagocytosis-induced cell death (PICD), a special type of apoptosis in phagocytic immune cells, is less frequently triggered in neonatal monocytes than in adult monocytes. We concluded that a rescue of monocyte PICD could be a potential therapeutic approach to target sustained inflammation in neonates. The EGFR ligand amphiregulin (AREG) is shed in response to bacterial infection and was shown to mediate cellular apoptosis resistance. We hypothesized that AREG might contribute to the reduced PICD of neonatal monocytes by affecting apoptosis signaling. In this study, we have examined a cascade of signaling events involved in extrinsic apoptosis by using a well-established in vitro E. coli infection model in monocytes from human peripheral blood (PBMO) and cord blood (CBMO). We found that CBMO shows remarkably higher pro-AREG surface expression as well as soluble AREG levels in response to infection as compared to PBMO. AREG increases intracellular MMP-2 and MMP-9 levels and induces cleavage of membrane-bound FasL through engagement with the EGF receptor. Our results demonstrate that loss of AREG rescues PICD in CBMO to the level comparable to adult monocytes. These findings identify AREG as a potential target for the prevention of prolonged inflammation in neonates

PONE-D-16-46185_R2 cumulative data

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Metalloproteinases TACE and MMP-9 Differentially Regulate Death Factors on Adult and Neonatal Monocytes After Infection with <i>Escherichia coli</i>

Background: Cleaving ligands and receptors of the tumor necrosis factor (TNF) superfamily can critically regulate the induction of apoptosis. Matrix metalloproteinases (MMPs) such as MMP-9 and tumor necrosis factor-&#945;-converting enzyme (TACE) have been shown to cleave CD95-Ligand (CD95L) and TNF/(TNF receptor-1) TNFR1 which induce phagocytosis induced cell death (PICD) in adult monocytes. This process is reduced in neonatal monocytes. Methods: Here we tested in vitro, whether Escherichia coli infection mounts for activation of MMP-9 and TACE in monocytes and whether this process regulates PICD. Results: The surface expression of TACE was most prominent on infected adult monocytes. In contrast, surface presentation of MMP-9 was highest on infected neonatal monocytes. Selective blocking of MMP-9 decreased CD95L secretion, while inhibition of TACE left CD95L secretion unaltered. Blocking of MMP-9 increased surface CD95L (memCD95L) expression on infected neonatal monocytes to levels comparable to infected adult monocytes. Moreover, MMP-9 inhibition raised PICD of infected neonatal monocytes to levels observed for infected adult monocytes. In contrast, TACE inhibition decreased PICD in infected monocytes. Addition of extracellular TNF effectively induced memCD95L presentation and PICD of adult monocytes and less of neonatal monocytes. Conclusion: MMP-9 activity is crucial for downregulating cell-contact dependent PICD in E. coli infected neonatal monocytes. By this mechanism, MMP-9 could contribute to reducing sustained inflammation in neonates

Data from: Reduced PICD in monocytes mounts altered neonate immune response to Candida albicans

Background: Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO). Hypothesis: Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses. Methods: The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor). Results: Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-alpha which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO). Conclusion: Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO