The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in the p38/TNF-α Pathway of Systemic and Cutaneous Inflammation

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-α, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-α levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-α and IL-1β levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-α neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-α production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-α, has a rather elusive role in T-cell-dependent cutaneous inflammation

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow

Mechanism of inhibition of inflammatory IFN-γ production in memory T cells

T-Helfer-(TH)Lymphozyten spielen eine zentrale Rolle bei der adaptiven Immunität und steuern über die Expression von Zytokinen die Immunantwort gegen pathogene Erreger. Interferon-g (IFN-g-exprimierende TH1-Zellen und Interleukin-17 (IL-17)-exprimierende TH17-Zellen sind entzündungsfördernd wirkende Subpopulationen der TH-Lymphozyten und spielen eine bedeutsame Rolle bei der Pathogenese von chronischen Entzündungskrankheiten. Interleukin-10 (IL-10) ist ein wichtiges immunregulatorisches Zytokin, welches die Induktion TH-Zell-vermittelter Immunantworten – durch indirekte Wirkung auf Antigen-präsentierende Zellen (APC) – inhibiert. Therapeutische Studien mit IL-10 bei chronischen Entzündungen zeigten jedoch nur einen geringen Erfolg. Daher wurde im Rahmen dieser Arbeit der direkte Effekt von IL-10 auf die IFN-g- und IL-17-Expression in humanen TH-Lymphozyten im peripheren Blut untersucht. Hierbei sollte insbesondere analysiert werden, inwieweit IL-10 eine bereits etablierte Gedächtnis-T-Zellantwort hemmen kann. Wir konnten nachweisen, dass IL-10 eine direkte hemmende Wirkung auf die T-Zell-Rezeptor-vermittelte IFN-g-Produktion in CD4-positiven T-Zellen, wie CD45RO-positiven-Gedächtnis-T-Zellen und in CD28-negativen Effektor-Gedächtnis-T-Zellen, hat. Dieser Effekt war unabhängig von Haupthistokompatibilitätskomplex (MHC)-II-exprimierenden APCs. Wir konnten auch beobachten, dass die IFN-g-Inhibierung unabhängig von bereits beschriebenen Wirkungen von IL-10 auf TH-Lymphozyten - wie die Hemmung der Proliferation oder die Inhibierung des CD28-Signalweges - war. Im Gegensatz dazu beeinflusste IL-10 die T-Zell-Rezeptor-induzierte IL-17-Expression in TH-Lymphozyten trotz intakter IL-10-Signaltransduktion nicht. Die Ergebnisse unserer Studie legen nahe, dass IL-10 unterschiedliche Wirkungen auf verschiedene TH-Zell-Subpopulationen aufweist. Dies könnte möglicherweise erklären, warum IL-10 bei der Therapie TH17-vermittelter chronischer Entzündungserkrankungen wenig Wirkung zeigt. Weiterführend wurde der Einfluss von der Mitogen-aktivierten Proteinkinase-aktivierte Proteinkinase 2 (MK2) – eine nachgeschaltete Kinase von p38 – welche die Expression entzündungsfördernder Zytokine reguliert, in verschiedenen Entzündungsmodellen der Haut untersucht. Wir konnten in-vitro darlegen, dass die Produktion des entzündungsfördernden Zytokins Tumornekrosefaktor-α nach Stimulation des Toll-like-Rezeptors in MK2-defizienten Milzzellen inhibiert war. Im Gegensatz dazu zeigte die Aktivierung des T-Zell-Rezeptors keine Beeinträchtigung in der Zytokinproduktion. Diese Beobachtungen implizieren, dass der Erfolg einer MK2-inhibierenden Therapie bei T-Zell-abhängigen Entzündungserkrankungen eingeschränkt sein kann. Des Weiteren entwickelten wir eine neuartige mikroendoskopische Methode, bei der Gewebebereiche im tiefen Knochenmark lebender Mäuse über einen langen Zeitraum analysiert werden können. Das Knochenmark ist ein wichtiger Ort für die Aufrechterhaltung von Gedächtnis-T-Lymphozyten. Mit Hilfe der intravitalen Multiphotonen-Mikroskopie konnten wir eine strukturelle Plastizität der Blutgefäße und eine zelluläre Dynamik während der Gewebehomöostase beobachten. Die kontinuierliche Reorganisation der Knochenmarkgefäße deutet darauf hin, dass das benötigte Blutgefäßnetz für die Migration von Zellen und die Versorgung langlebiger Gedächtniszellen, variable und anpassungsfähige Merkmale aufweisen. Diese neue Methode eröffnet unter anderem die Möglichkeit, die Überlebensfaktoren, die zur Aufrechterhaltung des immunologischen Gedächtnisses beitragen, zu charakterisieren.T helper (Th) lymphocytes play a central role in adaptive immunity and control the immune response against pathogens by the expression of cytokines. Interferon-g (IFN-g)-expressing Th1 cells and interleukin-17 (IL-17)-expressing Th17 cells are pro-inflammatory subpopulations of Th lymphocytes and play a significant role in the pathogenesis of chronic inflammatory diseases. Interleukin-10 (IL-10) is an important immunoregulatory cytokine that inhibits the induction of T cell-mediated immune responses through indirect effects on antigen-presenting cells (APC). However, therapeutic trials with IL-10 to dampen an ongoing chronic inflammation have been largely disappointing. Therefore, the direct effect of IL-10 on IFN-g- and IL-17 expression in human Th lymphocytes in peripheral blood was investigated in this study. In particular, we wanted to analyze whether IL-10 is able to inhibit an already established memory T cell response. We could demonstrate that IL-10 has a direct inhibitory effect on T cell receptor-mediated IFN-g production in CD4 positive T cells, such as CD45RO positive memory T cells and CD28 negative effector memory T cells. This effect was independent of major histocompatibility complex (MHC) class-II-expressing APCs. We furthermore showed that inhibition of IFN-g production was independent of previously described effects of IL-10 on Th lymphocytes, such as inhibition of T cell proliferation and inhibition of CD28-signaling. In contrast, IL-10 did not affect T cell receptor-induced IL-17 expression in Th lymphocytes despite an intact IL-10-signaling pathway. The results of our study suggest that IL-10 has different effects on different Th cell subpopulations. This may explain that IL-10 has its limitation in inhibiting an already established Th17-mediated chronic inflammation. Further, the influence of mitogen-activated protein-kinase-activated protein-kinase 2 (MK2) - a downstream kinase of p38 - which regulates the expression of pro-inflammatory cytokines, was investigated in different inflammatory skin models. We could demonstrate in-vitro that production of the pro-inflammatory cytokine tumor necrosis factor-α in MK2-deficient spleencells was strongly diminished after toll-like receptor stimulation. In contrast, activation of T cell receptor did not show any impairment in cytokine production. These observations imply that the success of MK2-inhibiting therapy in T cell-dependent inflammatory diseases may be limited. Furthermore, we developed a novel microendoscopic method to analyze tissue areas in deep bone marrow of living mice over a long period of time. The bone marrow is an important site for the maintenance of memory Th lymphocytes. Using intravital multiphoton microscopy, we observed structural plasticity of blood vessels and cellular dynamics during tissue homeostasis. The continuous reorganization of the bone marrow vessels indicates that the blood vessel network required for cell migration and the supply of long-lived memory cells has variable and adaptive characteristics. This new method opens the possibility to characterize survival factors that contribute to the maintenance of immunological memory

IL-10 interferes directly with TCR-induced IFN-c but not IL-17 production in memory T cells.

IL-10 is a potent immunoregulatory and anti-inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL-10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL-10 on IL-17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL-10 on T cells. We demonstrate here that IL-10 can directly interfere with TCR-induced IFN-c production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL-10 on T cells, namely inhibition of IL-2 production and inhibition of CD28 signaling. In contrast, IL-10 did not affect anti-CD3/anti-CD28-induced IL-17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

At present, it is not clear how memory B lymphocytes are maintained over time, and whetheronly as circulating cells or also residing in particular tissues. Here we describe distinctpopulations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bonemarrow, identified according to individual transcriptional signature and B cell receptorrepertoire. A population of marginal zone-like cells is located exclusively in the spleen, while apopulation of quiescent Bsm is found only in the bone marrow. Three further residentpopulations, present in spleen and bone marrow, represent transitional and follicular B cellsand B1 cells, respectively. A population representing 10-20% of spleen and bone marrowmemory B cells is the only one qualifying as circulating. In the bone marrow, all cells individuallydock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasmacells, are void of activation, proliferation and mobility.Peer Reviewe