Immune cell populations and cytokine production in spleen and mesenteric lymph nodes after laparoscopic surgery versus conventional laparotomy in mice

Purpose: There is evidence that open as well as minimally invasive abdominal surgery impair post-operative innate and acquired immune function. To compare the impact of these approaches as well as the one of different peritoneal gas exposures on immune function, we investigated cellular as well as cytokine-based immune parameters in mesenteric lymph nodes and the spleen postoperatively. Methods: Mice (n=26) were randomly assigned to the 4 study groups: (1) sham controls undergoing anesthesia alone, (2) laparotomy, and (3) air, or (4) carbon dioxide pneumoperitoneum. Mice were sacrificed 48h after the intervention, and their spleens and mesenteric lymph nodes were harvested. Cytokine production (TNF-α, IL-6, IL-10, and IFN-γ), splenic T cell subpopulations (cytotoxic T cells, T helper cells, and regulatory T cells) were analyzed. Results: TNF-α production of splenocytes 16h after ex vivo lipopolysaccharides (LPS) stimulation was significantly increased in the laparotomy group compared to all other groups. In contrast, TNF-α production of lymph node cells and IL-6 production of splenocytes after ex vivo LPS stimulation did not differ significantly between the groups. The numbers of regulatory T cells (Treg) in the spleen differed between groups. A significant reduction in Treg cell frequency was detected in the CO2 insufflation group compared to the laparotomy and the air insufflation group. Conclusion: Our findings demonstrate a distinct difference in immune effector functions and cellular composition of the spleen with regard to splenic TNF-α production and increased numbers of Treg cells in the spleen. These findings are in line with a higher peritoneal inflammatory status consequent to peritoneal air rather than CO2 exposure. Treg turned out to be key modulators of postoperative dysfunction of acquired immunit

Immune cell populations and cytokine production in spleen and mesenteric lymph nodes after laparoscopic surgery versus conventional laparotomy in mice

PURPOSE: There is evidence that open as well as minimally invasive abdominal surgery impair post-operative innate and acquired immune function. To compare the impact of these approaches as well as the one of different peritoneal gas exposures on immune function, we investigated cellular as well as cytokine-based immune parameters in mesenteric lymph nodes and the spleen postoperatively. METHODS: Mice (n = 26) were randomly assigned to the 4 study groups: (1) sham controls undergoing anesthesia alone, (2) laparotomy, and (3) air, or (4) carbon dioxide pneumoperitoneum. Mice were sacrificed 48 h after the intervention, and their spleens and mesenteric lymph nodes were harvested. Cytokine production (TNF-α, IL-6, IL-10, and IFN-γ), splenic T cell subpopulations (cytotoxic T cells, T helper cells, and regulatory T cells) were analyzed. RESULTS: TNF-α production of splenocytes 16 h after ex vivo lipopolysaccharides (LPS) stimulation was significantly increased in the laparotomy group compared to all other groups. In contrast, TNF-α production of lymph node cells and IL-6 production of splenocytes after ex vivo LPS stimulation did not differ significantly between the groups. The numbers of regulatory T cells (Treg) in the spleen differed between groups. A significant reduction in Treg cell frequency was detected in the CO(2) insufflation group compared to the laparotomy and the air insufflation group. CONCLUSION: Our findings demonstrate a distinct difference in immune effector functions and cellular composition of the spleen with regard to splenic TNF-α production and increased numbers of Treg cells in the spleen. These findings are in line with a higher peritoneal inflammatory status consequent to peritoneal air rather than CO(2) exposure. Treg turned out to be key modulators of postoperative dysfunction of acquired immunity

Identifizierung und Charakterisierung von Beta-Defensin 14 der Maus

Beta-Defensine sind eine Familie kleiner, kationischer und amphiphiler Peptide mit einem Molekulargewicht von ca. 3-6 kDa. Sie werden hauptsächlich von Epithelzellen exprimiert und weisen antimikrobielle Aktivität gegen Bakterien, Viren und Pilze auf. Zudem aktivieren sie die Immunantwort, indem sie mittels CC-Chemokinrezeptor CCR6-abhängiger Chemotaxis Leukozyten rekrutieren. Die Beta-Defensinfamilie der Maus umfasst eine große Zahl an bisher meist nur auf genomischer Ebene identifizierter Gene. Erst wenige dieser Beta-Defensine wurden bisher synthetisiert oder rekombinant exprimiert und bezüglich ihrer biologischen Funktion untersucht. Im Rahmen dieser Arbeit konnte nun erstmals das neue Beta-Defensin 14 der Maus (mBD14) als Ortholog des humanen Beta-Defensin 3 (hBD3) beschrieben werden. Entsprechend der Expression von hBD3 wurde die mRNA-Expression von mBD14 erstmals in verschiedenen Organen der Maus, darunter Trachaea, Lunge, Magen und Darm sowie in BMDCs nachgewiesen. Diese Expression wird durch die Stimulierung verschiedener Pattern-recognition-Rezeptoren wie z. B. Toll-like-Rezeptoren reguliert. Als weiterer intrazellulärer Pattern-recognition-Rezeptor wurde NOD2/CARD15 untersucht. Reportergen-Analysen zeigten, dass NOD2/CARD15-Aktivierung nach Stimulierung mit dessen Ligand MDP, einem Bestandteil des Peptidoglykans bakterieller Zellwände, zur Induktion der mBD14-Expression führt. Das für weitere funktionelle Untersuchungen hergestellte mBD14:Ig-Fusionsprotein zeigte in antimikrobiellen Assays die für die Beta-Defensin-Familie charakteristische antimikrobielle Aktivität gegen Gram-positive und Gram-negative Bakterien. Überdies wurde der Chemokinrezeptor CCR6 der Maus als potenzieller Rezeptor für mBD14 in HEK-293 Zellen exprimiert und anschließend die Bindung von mBD14:Ig an mCCR6 mittels durchflusszytometrischer Analysen nachgewiesen. In Chemotaxisversuchen mit hCCR6- oder mCCR6-exprimierenden HEK-293 Zellen sowie humanen PBMCs und residenten Peritonealzellen der Maus wurde eine speziesspezifische Interaktion von mBD14 und von mBD4 mit mCCR6 festgestellt. Im Gegensatz dazu zeigte sich, dass hBD2 und hBD3 sowohl mit mCCR6 als auch mit hCCR6 interagieren können. Zudem wurde die Bindung von mBD14:Ig an B-Lymphozyten, die endogen CCR6 exprimieren, nachgewiesen. Außerdem wurde gezeigt, dass auf Monozyten, die keinen funktionellen CCR6-Rezeptor exprimieren, noch ein weiterer chemotaktischer Rezeptor für Beta-Defensine exprimiert werden muss. Im BFS-1-Fibrosarkom-Tumormodell konnte nachgewiesen werden, dass die intratumorale mBD14:Ig-Expression das Tumorwachstum fördert, ohne jedoch das Wachstumsverhalten der BFS-1-Fibrosarkomzellen in vitro zu verändern. In Korrelation zu den Versuchen zur Interaktion von mBD14:Ig mit mCCR6 konnte gezeigt werden, dass der prozentuale Anteil an CCR6+ B220+ B-Lymphozyten in mBD14:Ig exprimierenden Tumoren, gemessen an der Gesamtheit tumorinfiltrierender Zellen, signifikant erhöht war. Anhand immunhistochemischer Untersuchungen wurde in mBD14:Ig-exprimierenden Tumoren ein erhöhter Anteil vaskulärer Gefäßstrukturen nachgewiesen, die eine Voraussetzung für das beobachtete verbesserte Tumorwachstum darstellen. Schließlich konnte mBD14 als neues Mitglied der Beta-Defensinfamilie identifiziert und charakterisiert werden. In weiteren Experimenten zeigte sich, dass mBD14 nicht nur als antimikrobielles Peptid im Rahmen der angeborenen Immunabwehr eine wichtige Rolle spielt, sondern, dass mBD14 ebenso Einfluss nimmt auf antigenpräsentierende Zellen sowie auf Lymphozyten des adaptiven Immunsystems und somit immunmodulatorisch wirken kann

Specific Binding and Chemotactic Activity of mBD4 and Its Functional Orthologue hBD2 to CCR6-expressing Cells*

β-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the β-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG1 to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these β-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the β-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the β-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and β-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the β-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific β-defensin receptor interactions

Immune cell populations and cytokine production in spleen and mesenteric lymph nodes after laparoscopic surgery versus conventional laparotomy in mice

PURPOSE: There is evidence that open as well as minimally invasive abdominal surgery impair post-operative innate and acquired immune function. To compare the impact of these approaches as well as the one of different peritoneal gas exposures on immune function, we investigated cellular as well as cytokine-based immune parameters in mesenteric lymph nodes and the spleen postoperatively. METHODS: Mice (n = 26) were randomly assigned to the 4 study groups: (1) sham controls undergoing anesthesia alone, (2) laparotomy, and (3) air, or (4) carbon dioxide pneumoperitoneum. Mice were sacrificed 48 h after the intervention, and their spleens and mesenteric lymph nodes were harvested. Cytokine production (TNF-α, IL-6, IL-10, and IFN-γ), splenic T cell subpopulations (cytotoxic T cells, T helper cells, and regulatory T cells) were analyzed. RESULTS: TNF-α production of splenocytes 16 h after ex vivo lipopolysaccharides (LPS) stimulation was significantly increased in the laparotomy group compared to all other groups. In contrast, TNF-α production of lymph node cells and IL-6 production of splenocytes after ex vivo LPS stimulation did not differ significantly between the groups. The numbers of regulatory T cells (Treg) in the spleen differed between groups. A significant reduction in Treg cell frequency was detected in the CO(2) insufflation group compared to the laparotomy and the air insufflation group. CONCLUSION: Our findings demonstrate a distinct difference in immune effector functions and cellular composition of the spleen with regard to splenic TNF-α production and increased numbers of Treg cells in the spleen. These findings are in line with a higher peritoneal inflammatory status consequent to peritoneal air rather than CO(2) exposure. Treg turned out to be key modulators of postoperative dysfunction of acquired immunity