Die Rolle von TNFα in der Blaseninfektion

Makrophagen und neutrophile Granulozyten sind bedeutsame Phagozyten des innaten Immunsystems, die in Zusammenarbeit eine Vielzahl von bakteriellen Infektionen auflösen. Die funktionelle Spezialisierung dieser Zellen und der genaue Ablauf der innaten Abwehr konnte allerdings bislang nicht vollständig aufgeklärt werden. Mit dieser Arbeit ist es gelungen, einen weiteren Teilprozess der innaten Immunantwort zu entschlüsseln, indem eine Helfer-Funktion durch inflammatorische Makrophagen während der Zystitis aufgedeckt wurde. Im Speziellen konnte nachgewiesen werden, dass zur Auflösung einer bakteriellen Harnwegsinfektion die Kooperation zwischen drei verschiedenen Zelltypen des innaten Immunsystems notwendig ist: Ly6C- Makrophagen fungieren als residente Wächterzellen, die zirkulierende Ly6C+ Makrophagen und neutrophile Granulozyten in das infizierte Gewebe rekrutieren. Ly6C+ Makrophagen sind innate Helferzellen, die nach ihrer Rekrutierung das Zytokin TNFα produzieren, das in Ly6C- Makrophagen die Produktion von CXCL2 bewirkt. Dieses Chemokin induziert anschließend in Granulozyten die Sekretion der Metallomatrixprotease MMP9, welche die epitheliale Basalmembran degradiert und so dem Granulozyten die Wanderung in das Uroepithel ermöglicht. Im Epithel angelangt, phagozytieren und eliminieren Granulozyten anschließend die Bakterien. In dieser Arbeit konnte somit ein vorher unbekannter Helfer-Mechanismus durch Ly6C+ Makrophagen im innaten Immunsystem aufgeklärt werden, wodurch residente Makrophagen in der Koordination der epithelialen Migration von neutrophilen Granulozyten, unterstützt werden. Auf diese Weise wird letztendlich eine erfolgreiche Eliminierung der Bakterien in der Harnblase gewährleistet. In diesem Zusammenhang konnte gleichzeitig TNFα als zentraler Mediator identifiziert werden, der diese Regulation der innaten Immunantwort induziert. Es konnten weiterhin erste in vitro Daten erhoben werden, die darauf hinweisen, dass TNFα zusammen mit CXCL2 die Migrationsgeschwindigkeit von Granulozyten steuert. Für das Chemoattraktant fMLP konnten bereits Start- und Stoppsignale für Immunzellen, mit den MAP – Kinasen p38 und ERK, ermittelt werden. Mit dem Zytokin TNFα könnte somit ein potentieller Regulator für die Chemokin vermittelte Start- und Stopp – Funktion gefunden worden sein. Diese Annahme muss allerdings in Zukunft noch durch weitere Untersuchungen geklärt werden

T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therap

T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therap

Structural and functional studies on ø29 DNA polymerase

The Bac i l lus subtilis phage 029 DNA polymerase, involved in protein-primed viral DNA repli- cation, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Us- ing site-directed mutagenesis, we have studied the func- tional significance o f a C-terminal conserved region, rep- resented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have be en constructed and the corresponding proteins have been overproduced and characterized. Measurements o f the activity o f the mutant proteins indi- cated that the invariant Lys and Tyr residues play a criti- cal role in DNA polymerization. Interestingly, substitu- tion o f the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respec- tively, capability to use a protein as primer, an intrinsic property o f TP-priming DNA polymerases. On the other hand, the viral protein p6, whi ch stimulates initiation o f 029 DNA replication by formation o f a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity o f 029 DNA polymerase during the formation o f the TP-dAMP initia- tion complex. We propose a mode l in whi ch the special strategy to maintain the integrity o f the 029 DNA ends, by means o f a "sliding-back" mechanism, could also con- tribute to increase the fidelity o f 029 DNA replication.This investigation has been aided byreseareh grant 5R01 GM27242-13 from the National Institutes o f Health, by grant n ~ PB90-0091 from Direccibn General de Investigacibn Cientifica y T6c- nica, by grant BlOT CT 91-0268 from European Economic Commani- ty, and by an institutional grant from Fundaci6n Ram6n Areces. M.A.B., J.A.E. and J.M., were predoctoral fellows from Ministerio de Educaci6n y Ciencia.Peer reviewe

Crosstalk between sentinel and helper macrophages permits neutrophil migration into infected uroepithelium

The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C(-) macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C(+) macrophages. Such Ly6C(+) macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C(-) macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.publisher: Elsevier articletitle: Crosstalk between Sentinel and Helper Macrophages Permits Neutrophil Migration into Infected Uroepithelium journaltitle: Cell articlelink: http://dx.doi.org/10.1016/j.cell.2014.01.006 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe