STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22–dependent mucosal wound healing

Effects of deltamethrin and ADP-ribosylation of C3 exoenzymes in the development of hippocampal neurons in culture

Hippokampale Primärkulturen der Maus stellen ein häufig benutztes Modellsystem zur Untersuchung zellbiologischer und degenerativer Prozesse dar. Im ersten Teil der Arbeit wurde die Wirkung von Deltamethrin als ausgewähltem Vertreter der Insektizide auf die neuronale Entwicklung hippokampaler Zellkulturen untersucht. Dabei wurde die Überlebensfähigkeit der hippokampalen Neurone nach Gabe von Deltamethrin sowie die Effekte von Deltamethrin auf die Synthese synaptischer Proteine und das Verteilungsmuster von Kaliumkanälen untersucht. Wiederholte Gabe von Deltamethrin (2-2000nM) reduziert die Anzahl hippokampaler Neurone um 16-40%. Neuronale Degeneration wurde von der Abnahme der synaptischen Proteine (21-35%) und synaptischen Kontakte (ca. 60%) begleitet. Alle untersuchten synaptischen Proteine (Synaptophysin, Synaptobrevin, Synapsin und SNAP25) sowie Kv Kanal alpha-Untereinheiten kommen in Deltamethrin behandelten Kulturen reduziert vor. Während Synaptophysin gegenüber Deltamethrin-Behandlung relativ resistent (25% Abnahme) ist, reagieren die Kv Kanal alpha-Untereinheiten, speziell Kv1.1 (64% Abnahme) sehr sensibel. Trotz hoher Deltamethrinkonzentration überlebten eine große Anzahl der Neurone (ca. 70%), welche typische neuronale Strukturen aufwiesen. Somit scheint es, dass in hippokampalen Kulturen Subpopulationen von Neuronen vorzukommen, die eine unterschiedliche Sensitivität gegenüber Deltamethrin aufweisen. Die Deltamethrin-Behandlung reduzierte die Zahl an Calbindin- positiven Neuronen um 50%, vornehm-lich der Körnerzellen sowie somatostatinergen Neurone um 60%, jedoch stieg die Anzahl der NPY-haltiger Neurone um 250% an. Im zweiten Teil der Arbeit wurde die Wirkung von C3 ADP- Ribosyltransferasen, die von verschiedenen Bakterienstämmen wie Clostridium botulinum (C3bot), Clostridium limosum (C3lim) und Staphylococcus aureus (C3stau2) produziert werden, auf die sich entwickelnde hippokampalen Neuronen untersucht. Alle diese Exoenzyme besitzen die ADP-Ribosyltransferase Aktivität, die die Rho Proteine A, B und C durch ADP-ribosy-lierung inaktivieren. Die mit C3-Exoenzymen aus Clostridium botulinum (C3bot) im nanomolaren Bereich behandelten Neurone zeigten eine hochsignifikante Erhöhung der Axonlänge (ca. 70%) und Axonverzweigung (ca. 30%). C3 -Isoformen wie C3stau2 und C3lim wiesen diese wachstumsfördernde Eigenschaft nicht auf. Die Enzym-defiziente C3bot Mutanten zeigten ebenfalls signifikant erhöhtes axonales Wachstum (Länge und Verzweigung). Diese C3bot Mutanten ohne ADP- Ribosyltransferase Aktivität können Rho nicht ADP-ribosylieren. Daher können die neurotropischen Effekte der Mutanten C3bot Proteine nicht durch die Inaktivierung der Rho Proteine vermittelt worden sein. Somit besitzen C3bot Proteine eine bisher unbekannte neurotrophe Wirkung, die nicht an seine enzymatische Aktivität gekoppelt ist. C3bot Proteine binden wahrscheinlich wie einem neurotrophen Faktor an einem Rezeptor der neuronalen Zellmembran, wodurch die Signale dann weitergeleitet werden. Die mit enzymatisch aktiven C3bot und C3stau2 transfizierten Neurone zeigten eine signifikante Reduzierung der Axonlänge (ca. 30%) und Axonverzweigungen (ca. 40%). Die intrazelluläre Expression der C3 Proteine (C3bot und C3stau2 ) reduziert axonales Wachstum (Länge und Verzweigung). Da die Expression von enzym-defizientem C3 Mutanten keine Wirkung auf Axone zeigen, wurden die Wirkung von enzymdefizientem C3 Mutanten extrazellär vermittelt. Durch die ultrastrukturellen Aufnahmen konnte die wachstumsfördernde Wirkung von C3bot (enzymdefizient und enzymaktiv) bestätigt werden. Die signifikante Erhöhung der Fortsatzlänge und Anzahl der Verzweigungen bis zu 300% belegen die trophische Wirkung der C3 Exoenzyme von Clostridium botulinum (C3bot) auch bei Astrozyten. Diese als Stellation bezeichnete morphologische Veränderung wurde bei enzymdefizienten C3 Exoenzymen nicht nachgewiesen. Astrozyten scheinen ebenfalls über eine sehr affine Bindungsstelle zu verfügen, über die enzymatisch aktives C3bot in die Zelle gelangt.Hippocampal primary cultures are a widely used model system for studying cell biology and degenerative processes. In the first part, the effect of deltamethrin on the development of hippocampal neuronal cell cultures was investigated. Repeated applications of deltamethrin (between 2 nM and 2000 nM) decreased the number of neurons by 16-40%, respectively. Neuronal death was accompanied by an overall decrease of synaptic proteins. A considerable number of neurons survived treatment with high concentrations of deltamethrin (200-2000 nM) and still displayed characteristics of mature neurons such as synaptic contacts or the expression of members of the Kv1 channel family. When analyzing subtypes of neurons calbindin- as well as somatostatin-positive neurons decreased by 50% after repeated treatment with 2 nM deltamethrin. Under the same conditions neuropeptide Y-positive neurons were up-regulated by 250%. Deltamethrin at concentrations relevant in human toxicology differentially affects survival of neuronal subtypes by exerting either deleterious or supportive effects. In the second part of the study, the effect of C3 ADP-ribosyltransferases, such as Clostridium botulinum (C3bot), Clostridium limosum (C3lim) and Staphylococcus aureus (C3stau2), were studied on the developing hippocampal neurons. Using hippocampal neurones at defined states of differentiation, i have dissected the function of RhoA in axonal and dendritic growth. Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3lim) or Staphylococcus aureus (C3stau2), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3bot) or enzymatically dead C3bot significantly increased axon growth and axon branching. Extracellular application of enzymatically active or dead C3bot exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity. Furthermore, low nanomolar concentrations of C3 isoforms (C3bot or enzymatically dead C3bot) significantly promoted process outgrowth and increased process branching in astrocytes

Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function

Attenuation of immune activation in patients with multiple sclerosis on a wheat-reduced diet: a pilot crossover trial

Background: Western lifestyle has been associated with an increase in relapsing–remitting multiple sclerosis (RRMS). In mice, dietary wheat amylase–trypsin inhibitors (ATIs) activate intestinal myeloid cells and augment T cell-mediated systemic inflammation. Objective: The aim of this study was to assess whether a wheat- and thus ATI-reduced diet might exert beneficial effects in RRMS patients with modest disease activity. Methods: In this 6-month, crossover, open-label, bicentric proof-of-concept trial, 16 RRMS patients with stable disease course were randomized to either 3 months of a standard wheat-containing diet with consecutive switch to a > 90% wheat-reduced diet, or vice versa. Results: The primary endpoint was negative, as the frequency of circulating pro-inflammatory T cells did not decrease during the ATI-reduced diet. We did, however, observe decreased frequencies of CD14 + CD16 ++ monocytes and a concomitant increase in CD14 ++ CD16 − monocytes during the wheat-reduced diet interval. This was accompanied by an improvement in pain-related quality of life in health-related quality of life assessed (SF-36). Conclusion: Our results suggest that the wheat- and thus ATI-reduced diet was associated with changes in monocyte subsets and improved pain-related quality of life in RRMS patients. Thus, a wheat (ATI)-reduced diet might be a complementary approach accompanying immunotherapy for some patients. Registration: German Clinical Trial Register (No. DRKS00027967)