intravital-imaging-of-the-mouse-thymus-using-2-photon-microscopy

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Die experimentelle autoimmune Enzephalomyelitis kann verhindert werden und durch eine Infektion mit Trypanosoma cruzi geheilt werden.

Trypanosoma cruzi is an intracellular parasite that induces a strong Th1-type response and immunosuppression during the acute phase of infection. To study how the infection with T. cruzi would modulate the development of an autoimmune disease, we immunized C57BL/6 mice and IL-10 or iNOS knock-out mice of the same background with the encephalitogenic MOG 35-55 peptide and infected them with T. cruzi. Our results demonstrate that infection with T. cruzi completely prevents EAE development and furthermore induces complete and lasting remission in mice that were infected with this parasite after they had developed clinical EAE. Nitric oxide and IL-10 participate in triggering the mechanisms associated with EAE suppression by the infection. Decreased lymphoproliferation and increased frequencies of Annexin-positive cells and of T cells bearing CD95, CD95L or CTLA-4 were observed in the spleen from immunized/infected mice, as well as lower IL-2 and increased TGF-beta production in comparison with only immunized mice. Our results indicate that several effector and regulatory mechanisms of the immune response that arise during the acute phase of T. cruzi infection lastingly affect the expansion and/or effector functions of encephalitogenic cells, preventing the onset or inducing complete remission of EAE.

Experimental autoimmune encephalomyelitis can be prevented and cured by infection with Trypanosoma cruzi.

IL-12 is one of the main cytokines driving the immune response to a resistant phenotype in leishmaniasis and in several other diseases involving intracellular microbes. In this study, we investigated IL-12 production by mononuclear phagocytes at several developmental stages when stimulated with Leishmania major, L. amazonensis or L. chagasi. Bone marrow cells were cultured for 4-6 days in vitro in the presence of M-CSF, GM-CSF or IL-3. After density separation, only cells banding at the 40-50% Percoll interface, but not those at 20-40% or 50-80% interfaces, produced large amounts of IL-12 p40 when stimulated with LPS or live Leishmania promastigotes. However, only low levels of IL-12 p70 were produced under these conditions. The high IL-12 p40-producing cells could be similarly derived from mouse strains with different susceptibility to Leishmania. Quantitative analysis of monocyte/macrophage lineage marker expression, in combination with positive and negative selection, led to the conclusion that the high IL-12 p40-producing cells are macrophages at an intermediate stage of maturation between immature and fully differentiated cells, expressing ER-HR3 but only low levels of the mature markers, scavenger receptor and CD11b/Mac-1. They do not express any of the precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 or ER-MP58. Because recruitment of monocytes to an infection site and its draining lymph node is a general phenomenon, the notion that, developing from these monocytes, a population of mononuclear phagocytes at an intermediate maturation stage has the capacity to synthesize large amounts of IL-12 p40 has significant bearing on our understanding of immune regulation in leishmaniasis and also in infections by other pathogens.

Macrophages at intermediate stage of maturation produce high levels of IL-12 p40 upon stimulation with Leishmania.

Makrophagen bei der Zwischenstufe der Reifung hohe Konzentrationen von IL-12 p40 nach Stimulation mit Leishmania.

Recruitment of lymphocytes into the retina and to the vitreous during the development of experimental autoimmune uveitis (EAU) is governed by factors such as the state of activation of inflammatory cells and the repertoire of adhesion molecules expressed by the local vascular endothelia. alpha4 Integrins and their receptors play an important role during homing of cells to the inflammatory site. In the present study, the effect of alpha4-integrin inhibitor on the development of EAU was investigated.

Administration of a peptide inhibitor of alpha4-integrin inhibits the development of experimental autoimmune uveitis.

Die Verabreichung einer Peptid-Inhibitor alpha4-Integrin hemmt die Entwicklung der experimentellen autoimmunen Uveitis.

Hom&ouml;ostatische Proliferation von T-Zellen f&uuml;hrt zu der Generation von Effektor/Speicherzellen, die haben das Potenzial, den Host zu Schaden. Die Rolle der Tregs bei der Kontrolle der hom&ouml;ostatischen Verbreitung ist unklar. In dieser Studie benutzt wir M&auml;use, die entweder Hafen oder Tregs als Empf&auml;nger der monoklonale oder polyklonalen T-Zellen fehlen. Wir beobachteten, dass w&auml;hrend Tregs vollst&auml;ndig der Zellteilung verhindert T Zellen anzeigen geringe Affinit&auml;t f&uuml;r selbst Liganden, sie hatten eine weniger markiert, wenn auch erheblich, Auswirkungen auf Zellzyklus-Eintrag von T-Zellen, die angezeigte h&ouml;here Affinit&auml;t. Das Vorhandensein von Tregs ergab eine niedrigere Ansammlung von T Zellen, verst&auml;rkte Apoptose und gest&ouml;rter Differenzierung ein Zytokin produzierender Staat. Wir schlussfolgern, dass Tregs spielen eine wichtige Rolle bei der Kontrolle der hom&ouml;ostatische Proliferation.

Kontrolle der hom&ouml;ostatische Proliferation von regulatorischen T-Zellen.

Homeostatic proliferation of T cells leads to the generation of effector/memory cells, which have the potential to cause harm to the host. The role of Tregs in the control of homeostatic proliferation is unclear. In this study we utilized mice that either harbor or lack Tregs as recipients of monoclonal or polyclonal T cells. We observed that while Tregs completely prevented cell division of T cells displaying low affinity for self ligands, they had a less marked, albeit significant, effect on cell cycle entry of T cells displaying higher affinity. The presence of Tregs resulted in a lower accumulation of T cells, enhanced apoptosis, and impaired differentiation to a cytokine-producing state. We conclude that Tregs play a major role in the control of homeostatic proliferation.

Control of homeostatic proliferation by regulatory T cells.

Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs.

Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.

Regulatorische T (T-Reg)-Zellen &uuml;ben starke unten-modulierende Wirkung auf Immunantworten, aber es ist nicht bekannt, wie sie in-vivo handeln. Mit intravital zwei-Photonen-Laser-scanning-Mikroskopie haben wir festgestellt, dass in Ermangelung T-Reg-Zellen, die Fortbewegung des Autoantigen-spezifische T-Zellen in Lymphknoten verringert wird und die Kontakte zwischen T-Zellen und Antigen belasteter dendritische Zellen (DCs) von l&auml;ngerer Dauer sind. T-Reg-Zellen k&ouml;nnen so die fr&uuml;he Beeinflussung der Immunantworten aus&uuml;ben, durch die Herstellung von stabilen Kontakten w&auml;hrend der Grundierung der naive T-Zellen durch DCs abgeschw&auml;cht.

Regulatorische T-Zellen hemmen stabile Kontakte zwischen CD4 + T-Zellen und Makrophagen, die in-vivo.

IL-17-producing T lymphocytes have been recently shown to comprise a distinct lineage of proinflammatory T helper cells, termed Th17 cells, that are major contributors to autoimmune disease. We show here that the orphan nuclear receptor RORgammat is the key transcription factor that orchestrates the differentiation of this effector cell lineage. RORgammat induces transcription of the genes encoding IL-17 and the related cytokine IL-17F in naÃ¯ve CD4(+) T helper cells and is required for their expression in response to IL-6 and TGF-beta, the cytokines known to induce IL-17. Th17 cells are constitutively present throughout the intestinal lamina propria, express RORgammat, and are absent in mice deficient for RORgammat or IL-6. Mice with RORgammat-deficient T cells have attenuated autoimmune disease and lack tissue-infiltrating Th17 cells. Together, these studies suggest that RORgammat is a key regulator of immune homeostasis and highlight its potential as a therapeutic target in inflammatory diseases.

The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.

T-Lymphozyten Produktion von IL-17 wurden vor kurzem gezeigt, umfassen eine deutliche Linie der Proinflammatory T-Helferzellen, nannte Th17-Zellen, die Hauptverursacher Autoimmunerkrankung sind. Wir zeigen hier, dass die verwaiste Kernempf&auml;nger RORgammat wichtigen Transkriptionsfaktors ist, die die Differenzierung dieser Effektor-Zelle-Linie orchestriert. RORgammat induziert die Transkription der Gene IL-17 und die damit verbundenen Cytokine IL-17F in naiven CD4(+) T-Helferzellen-Codierung und ihren Ausdruck als Reaktion auf IL-6 und TGF-Beta, die Zytokine IL-17 induzieren bekannterma&szlig;en erforderlich ist. Th17-Zellen sind konstitutiv vorhanden w&auml;hrend der intestinalen Lamina Propria, express RORgammat und fehlen in den M&auml;usen unzul&auml;nglich f&uuml;r RORgammat oder IL-6. M&auml;use mit RORgammat-defizienten T-Zellen haben Autoimmun-Krankheit abgeschw&auml;cht und fehlende Gewebe-infiltrierende Th17-Zellen. Diese Studien schlagen zusammen, RORgammat ist ein wichtiger Regulator der immun-Hom&ouml;ostase und weisen Sie ihr Potenzial als ein therapeutisches Ziel bei entz&uuml;ndlichen Erkrankungen.

Die verwaiste Kernempf&auml;nger RORgammat leitet das Programm Differenzierung der Proinflammatory IL-17 + T-Helfer-Zellen.

Many studies have positioned Notch signaling at various critical junctions during T-cell development. There is, however, debate regarding the role of Notch in the CD4 versus CD8 lineage commitment. Because there are 4 Notch receptors and RBP-Jkappa-independent Notch signaling has been reported, we decided to eliminate gamma-secretase activity once its activity is required for all forms of Notch signaling. T-cell-specific elimination of gamma-secretase was carried out by crossing presenilin-1 (PS1) floxed mice with CD4-Cre mice and PS2 KO mice, generating PS KO mice. Thymic CD4+CD8+ double-positive (DP) cells from these mice were strikingly resistant to apoptosis by anti-CD3 treatment in vivo and expressed more Bcl-X(L) than control thymocytes, and deletion of only one allele of Bcl-X(L) gene restored wild-type levels of sensitivity to apoptosis. In addition, these PS KO animals displayed a significant decrease in the number of CD8+ T cells in the periphery, and these cells had higher level of phosphorylated p38 than cells from control littermates. Our results show that ablation of presenilins results in deficiency of CD8 cells in the periphery and a dramatic change in the physiology of thymocytes, bringing to our attention the potential side effects of presenilin inhibitors in ongoing clinical trials.

Effect of presenilins in the apoptosis of thymocytes and homeostasis of CD8+ T cells.

Viele Studien haben Notch Signalisierung an verschiedenen kritischen Knotenpunkten w&auml;hrend der T-Zell-Entwicklung positioniert. Jedoch gibt es eine Debatte &uuml;ber die Rolle der Kerbe in die CD4 versus CD8 Lineage Engagement. Da gibt es 4 Notch Rezeptoren und RBP-Jkappa unabh&auml;ngig Notch Signalisierung gemeldet wurde, beschlossen wir, die Gamma-Sekretase-Aktivit&auml;t zu beseitigen, sobald seine Aktivit&auml;t f&uuml;r alle Formen von Notch Signalisierung erforderlich ist. T-Zelle-spezifische Eliminierung von Gamma-Sekretase wurde durch die Kreuzung von Pr&auml;senilin-1 (PS1) Floxed M&auml;use mit CD4-Cre-M&auml;use und PS2 KO M&auml;usen, Generieren von PS KO M&auml;usen durchgef&uuml;hrt. Thymom CD4 + CD8 + Doppel-positivem (DP) Zellen von diesen M&auml;usen waren auffallend resistent gegen Apoptose durch in-vivo Anti-CD3-Behandlung und mehr Bcl-X(L) als Steuerelement Thymozyten ausgedr&uuml;ckt und L&ouml;schung nur ein Allel des Bcl-X(L)-Gens Wildtyp Ebenen der Empfindlichkeit in Apoptosis wiederhergestellt. Dar&uuml;ber hinaus diese PS KO Tiere angezeigt eine signifikante Abnahme der Zahl von CD8 + T-Zellen in der Peripherie, und diese Zellen hatten h&ouml;here phosphorylierten p38 als Zellen vom Steuerelement Wurfgeschwister. Unsere Ergebnisse zeigen, dass Ablation von Preseniline Ergebnisse bei Mangel von CD8-Zellen in der Peripherie und eine dramatische Ver&auml;nderung in der Physiologie der Thymozyten, dass unsere Aufmerksamkeit die m&ouml;glichen Nebenwirkungen von Presenilin-Hemmer in klinischen Studien.

Wirkung von Preseniline in die Apoptose von Thymozyten und Hom&ouml;ostase von CD8 + T-Zellen.

The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP(+) microglial cells in CX3CR1(gfp/+) mice. We next demonstrate a distinct rostrocaudal migration of GFP(+) immune cells in the spinal cord of CXCR6(gfp/+) mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP(+) cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells.

Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.

Das Maus-R&uuml;ckenmark ist ein wichtiger Standort f&uuml;r autoimmune und Verletzungen-Modelle. Sch&auml;del Ausd&uuml;nnung Chirurgie bietet eine minimal-invasive Fenster f&uuml;r die Mikroskopie der Gro&szlig;hirnrinde Maus, aber es gibt keine parallelen Verfahren f&uuml;r das R&uuml;ckenmark. Wir f&uuml;hren einen Roman, einfache und kosteng&uuml;nstige Methode f&uuml;r die zwei-Photonen-Laser-Rastermikroskopie des R&uuml;ckenmarks intakt in die Maus unter Ausnutzung der nat&uuml;rlich zug&auml;nglich Foramina Raum. Hierbei handelt es sich um leistungsf&auml;hige Methoden in Kombination mit Gen-bezogene M&auml;use in der endogene Immunzellen mit gr&uuml;n fluoreszierendes Protein (GFP) beschriftet sind. Zuerst zeigen wir, dass die Generation der Foramina Fenster eine Reaktion der GFP(+) Microglial Zellen bei CX3CR1(gfp/+) M&auml;usen nicht entlocken wird. Als N&auml;chstes zeigen wir eine unterschiedliche Rostrocaudal Migration von GFP(+) Immunzellen im R&uuml;ckenmark von M&auml;usen CXCR6(gfp/+) w&auml;hrend der aktiven experimentellen autoimmunen Enzephalomyelitis (EAE). Interessanterweise Infiltration der Gro&szlig;hirnrinde von GFP(+) Zellen in diesen M&auml;usen drei Voraussetzungen erforderlich: EAE Induktion, kortikale Verletzungen und Berechnung der CXCR6 auf Immunzellen.

Zwei-Photonen-Laser-scanning-Mikroskopie-Bildgebung der intakte R&uuml;ckenmark und Gro&szlig;hirnrinde zeigt f&uuml;r die CXCR6 und Neuroinflammation in Immunzellen Infiltration der kortikalen Verletzung Websites.

Article	Year
Experimental autoimmune encephalomyelitis can be prevented and cured by infection with Trypanosoma cruzi. Journal of autoimmunity\| PubMed ID: 15324929	2004
Macrophages at intermediate stage of maturation produce high levels of IL-12 p40 upon stimulation with Leishmania. Microbes and infection / Institut Pasteur\| PubMed ID: 15725387	2005
Administration of a peptide inhibitor of alpha4-integrin inhibits the development of experimental autoimmune uveitis. Investigative ophthalmology & visual science\| PubMed ID: 15914623	2005
Control of homeostatic proliferation by regulatory T cells. The Journal of clinical investigation\| PubMed ID: 16294223	2005
Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. The Journal of experimental medicine\| PubMed ID: 16533880	2006
The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell\| PubMed ID: 16990136	2006
Effect of presenilins in the apoptosis of thymocytes and homeostasis of CD8+ T cells. Blood\| PubMed ID: 17626841	2007
Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury site... Journal of immunological methods\| PubMed ID: 19800886	2010

Carlos E. Tadokoro

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