Die Rolle des Transkriptionsfaktors NF-kappaB für die Entwicklung und Funktion natürlich vorkommender regulatorischer T-Zellen

Naturally occurring regulatory T cells (Treg cells) develop in the thymus due to strong T cell receptor (TCR) stimulation and represent 5-10% of the peripheral CD4+ T cell repertoire. Treg cells play a crucial role in maintaining peripheral self-tolerance as they are capable of suppressing the proliferation and cytokine secretion of effector T cells. Treg cell development as well as homeostasis was selectively impaired in mice lacking components of proximal TCR signalling leading to NF-kappaB activation, as well as NF-kappaB-proteins themselves. Here our aim was to investigate whether NF-kappaB activation is required in a Treg cell-intrinsic manner for differentiation, survival or function of developing and mature Tregs. To address these questions we used transgenic mice with a thymocyte-specific overexpression of a mutated IkappaBalpha-protein (IkappaBalpha-SR). Due to the markedly reduced NF-kappaB activity, total cell numbers of thymic Treg precursor cells as well as mature Treg cells were diminished in IkappaBalpha-SR transgenic mice. The administration of exogenous IL-2 almost normalized the numbers of mature thymic and splenic Treg cells in IkappaBalpha-SR transgenic mice, but displayed no influence on thymic Treg precursors. Mixed bone marrow chimeras unravelled a Treg cell-intrinsic requirement for NF-kappaB activation during thymic maturation. In the periphery, however, NF-kappaB activation appeared to be preferentially important to promote IL-2 secretion by T helper cells, whereas Treg cell-intrinsic NF-kappaB activity was dispensable for mature Treg cell survival This conclusion is further supported by the observation that in the presence of exogenous IL-2 as well as in mixed bone marrow chimeras, peripheral Treg cell frequencies almost reached wild-type levels. Beyond that, peripheral Treg cells from IkappaBalpha-SR transgenic mice were able to suppress the proliferation of T effector cells to a similar extent as wild-type Treg cells suggesting that classical NF-kappaB activation is not necessary for mediating Treg functions. Moreover, pharmacological IKKbeta-inhibition selectively impaired thymic Treg cell development without altering the maturation of other T and B cell subpopulations. Together, these data show that cell-intrinsic NF-kappaB activation is crucial for thymic Treg cell differentiation, and suggest pharmacological IKKbeta-inhibition as potential therapeutic approach for manipulating this process. Finally, we wanted to establish a Knockin-mouse (Foxp3rtTAKI) expressing a modified reverse tetracyclin-transactivator (rtTA2S-M2) under control of the endogenous foxp3-promoter specifically in Foxp3+ Treg cells. So far we have generated eight correctly targeted embryonic stem cell clones, whereas two of them have already generated highly chimeric offspring after blastocyst injection. After crossing Foxp3rtTAKI-mice with mouse lines expressing genes of interest under the control of a tetracycline-regulated promoter these mice will enable us to analyze in detail the function of NF-kappaB as well as other factors and pathways in a Treg cell-specific and inducible manner.Natürlich vorkommende regulatorische T-Zellen (Tregs) reifen im Thymus infolge starker Stimulation ihres T-Zellrezeptors (TZR) und bilden etwa 5-10% des peripheren CD4+ T-Zellrepertoires. Tregs spielen eine zentrale Rolle bei der Aufrechterhaltung der peripheren Toleranz, da sie nach ihrer Aktivierung in der Lage sind, sowohl die Proliferation als auch die Zytokinsekretion von Effektor-T-Zellen zu unterdrücken. Anhand verschiedener genetisch modifizierter Mauslinien wurde gezeigt, dass das Fehlen proximaler TZR-Signalmoleküle, die verstärkt zur klassischen Aktivierung von NF-kappaB führen sowie das Fehlen bestimmter NF-kappaB/Rel-Proteine selbst, selektiv die Reifung und Homöostase von Tregs beeinträchtigt. Im Rahmen dieser Arbeit sollte untersucht werden, welche Rolle der Transkriptionsfaktor NF-kappaB für die Differenzierung, das Überleben und/oder die Funktion von Tregs einnimmt. Anhand eines transgenen Mausmodells, das Thymozyten-spezifisch ein mutiertes IkappaBalpha-Protein überexprimiert (IkappaBalpha-SR), konnten wir zeigen, dass infolge der deutlich verringerten NF-kappaB-Aktivität sowohl thymische Treg-Vorläuferzellen als auch reife Tregs deutlich reduziert waren. Die Applikation von IL-2 konnte zwar die Zahl reifer Tregs sowohl im Thymus als auch in der Milz signifikant steigern, nicht aber die Zahl der intrathymischen Treg-Vorläuferzellen. Mithilfe eines adoptiven Knochenmarkstransfers, wobei Knochenmarksstammzellen von Wildtyp und IkappaBalpha-SR transgenen Mäusen im gleichen Verhältnis übertragen worden waren, konnten wir zeigen, dass eine Treg-Zell-intrinsische NF-kappaB-Aktivierung für deren intrathymische Entwicklung essentiell ist, und insbesondere in den Treg-Vorläufern nicht durch exogenes IL-2 ersetzt werden kann. In der Peripherie hingegen scheint NF-kappaB verstärkt Treg-Zell-extrinsisch für die IL-2-Sekretion durch T-Helferzellen wichtig zu sein, da die prozentualen Anteile peripherer Tregs in der Milz von IkappaBalpha-SR transgenen Mäusen nur leicht verringert waren, und nach IL-2 Gabe bzw. in gemischt chimären Tieren sogar Wildtyp-Niveau erreichten. Abgesehen davon hemmten periphere Tregs aus IkappaBalpha-SR transgenen Mäusen die Proliferation von T-Helferzellen in vitro vergleichbar gut, weshalb eine klassische NF-kappaB-Aktivierung nach TZR-Stimulation nicht an der Vermittlung der suppressiven Eigenschaften von Tregs beteiligt zu sein scheint. Schließlich konnte durch einen spezifischen IKKbeta-Inhibitor die thymische Reifung von Tregs selektiv vermindert werden, wobei die Entwicklung anderer T- und B-Zellsubpopulationen unbeeinträchtigt blieb. Somit zeigen diese Daten, dass eine Treg-Zell-intrinsische NF-kappaB-Aktivierung für die Differenzierung von Tregs wichtig ist, nicht aber für Überleben und Funktion reifer Tregs. Eine pharmakologische Modulation der NF-kappaB-Aktivität, z. B. durch IKKbeta-Inhibition, dürfte einen neuen Ansatzpunkt darstellen, gezielt die Treg-Reifung zu beeinflussen. In einem dritten Teilprojekt sollte eine KnockIn-Mauslinie (Foxp3rtTAKI) etabliert werden, die einen modifizierten reversen Tetrazyklin-Transaktivator (rtTA2S-M2) unter der Kontrolle des endogenen Foxp3-Promotors exprimiert. Hierfür wurden mehrere homolog rekombinierte Stammzellklone generiert, wovon bisher zwei nach Blastozysteninjektion hochchimäre Nachkommen hervorgebracht haben. Foxp3rtTAKI-Mäuse ermöglichen uns nach ihrer Kreuzung mit Mauslinien, die bestimmte Gene unter Kontrolle eines Tetrazyklin-regulierten Promotors exprimieren, die Untersuchung der biologischen Relevanz nicht nur von NF-kappaB, sondern auch von anderen Proteinen induzierbar und selektiv in Tregs

Cell-Intrinsic NF-κB Activation Is Critical for the Development of Natural Regulatory T Cells in Mice

regulatory T (Treg) cells develop in the thymus and represent a mature T cell subpopulation critically involved in maintaining peripheral tolerance. The differentiation of Treg cells in the thymus requires T cell receptor (TCR)/CD28 stimulation along with cytokine-promoted Foxp3 induction. TCR-mediated nuclear factor kappa B (NF-κB) activation seems to be involved in differentiation of Treg cells because deletion of components of the NF-κB signaling pathway, as well as of NF-κB transcription factors, leads to markedly decreased Treg cell numbers in thymus and periphery. thymic Treg precursors and their further differentiation into mature Treg cells. Treg cell development could neither be completely rescued by the addition of exogenous Interleukin 2 (IL-2) nor by the presence of wild-type derived cells in adoptive transfer experiments. However, peripheral NF-κB activation appears to be required for IL-2 production by conventional T cells, thereby participating in Treg cell homeostasis. Moreover, pharmacological NF-κB inhibition via the IκB kinase β (IKKβ) inhibitor AS602868 led to markedly diminished thymic and peripheral Treg cell frequencies.Our results indicate that Treg cell-intrinsic NF-κB activation is essential for thymic Treg cell differentiation, and further suggest pharmacological NF-κB inhibition as a potential therapeutic approach for manipulating this process

Proteasome Inhibition Aggravates Tumor Necrosis Factor-Mediated Bone Resorption in a Mouse Model of Inflammatory Arthritis

Objective. The proteasome inhibitor bortezomib has potent anti-myeloma and bone-protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis. Methods. Heterozygous human tumor necrosis factor ␣ (hTNF␣)-transgenic mice and their wild-type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF-transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes as well as apoptosis and ubiquitination. Results. Bortezomib-treated hTNF-transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF-transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor؊associated factor 6, c-Fos, and nuclear factor of activated T cells c1 in osteoclast precursors. Conclusion. In TNF-mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone-resorptive effect via stimulation of osteoclastogenesis during chronic arthritis

Tumor progression locus 2 reduces severe allergic airway inflammation by inhibiting Ccl24 production in dendritic cells

BackgroundThe molecular and cellular pathways driving the pathogenesis of severe asthma are poorly defined. Tumor progression locus 2 (TPL-2) (COT, MAP3K8) kinase activates the MEK1/2-extracellular-signal regulated kinase 1/2 MAP kinase signaling pathway following Toll-like receptor, TNFR1, and IL-1R stimulation.ObjectiveTPL-2 has been widely described as a critical regulator of inflammation, and we sought to investigate the role of TPL-2 in house dust mite (HDM)-mediated allergic airway inflammation.MethodsA comparative analysis of wild-type and Map3k8−/− mice was conducted. Mixed bone marrow chimeras, conditional knockout mice, and adoptive transfer models were also used. Differential cell counts were performed on the bronchoalveolar lavage fluid, followed by histological analysis of lung sections. Flow cytometry and quantitative PCR was used to measure type 2 cytokines. ELISA was used to assess the production of IgE, type 2 cytokines, and Ccl24. RNA sequencing was used to characterize dendritic cell (DC) transcripts.ResultsTPL-2 deficiency led to exacerbated HDM-induced airway allergy, with increased airway and tissue eosinophilia, lung inflammation, and IL-4, IL-5, IL-13, and IgE production. Increased airway allergic responses in Map3k8−/− mice were not due to a cell-intrinsic role for TPL-2 in T cells, B cells, or LysM+ cells but due to a regulatory role for TPL-2 in DCs. TPL-2 inhibited Ccl24 expression in lung DCs, and blockade of Ccl24 prevented the exaggerated airway eosinophilia and lung inflammation in mice given HDM-pulsed Map3k8−/− DCs.ConclusionsTPL-2 regulates DC-derived Ccl24 production to prevent severe type 2 airway allergy in mice