Tina Leuenberger

Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.

Differential immune cell dynamics in the CNS cause CD4+ T cell compartmentalization

In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4(+) T cell subsets in contrast to random motility of CD8(+) T cells, indicating a dominant role of the perivascular area primarily for CD4(+) T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4(+) T-cell motility within the healthy and diseased CNS.

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

BackgroundTwo-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.MethodsWe applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.ResultsBy applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.ConclusionsThe activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.

The Role of CD8+ T Cells and Their Local Interaction with CD4+ T Cells in Myelin Oligodendrocyte Glycoprotein35–55–Induced Experimental Autoimmune Encephalomyelitis

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4+ T cells it is well established that they contribute to the disease, less is known about the role of CD8+ T cells. Our aim was to determine the individual contribution of CD4+ and CD8+ T cells in myelin oligodendrocyte glycoprotein (MOG)35–55–induced EAE. We investigated MOG35–55–activated CD8+ T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8+ T cells and their interaction with CD4+ T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4+ T cells did not develop relevant clinical signs of disease, although CD8+ T cells were present in the CNS of these mice. These CD8+ T cells displayed reduced motility compared with those in the presence of CD4+ T cells. In mice that harbored CD4+ and CD8+ T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4+ T cells. Furthermore, the dynamic motility and viability of CD4+ T cells were not disturbed by CD8+ T cells in the lesions of these mice. Therefore, we conclude that in MOG35–55–induced EAE, CD8+ T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4+ T cells, the latter being the true inducers of the disease.

MAPK3 deficiency drives autoimmunity via DC arming

DC are professional APC that instruct T cells during the inflammatory course of EAE. We have previously shown that MAPK3 (Erk1) is important for the induction of T‐cell anergy. Our goal was to determine the influence of MAPK3 on the capacity of DC to arm T‐cell responses in autoimmunity. We report that DC from Mapk3−/− mice have a significantly higher membrane expression of CD86 and MHC‐II and – when loaded with the myelin oligodendrocyte glycoprotein – show a superior capacity to prime naïve T cells towards an inflammatory phenotype than Mapk3+/+ DC. Nonetheless and as previously described, Mapk3−/− mice were only slightly but not significantly more susceptible to myelin oligodendrocyte glycoprotein‐induced EAE than WT littermate mice. However, Mapk3+/+ mice engrafted with Mapk3−/− BM (KO→WT) developed a severe form of EAE, in direct contrast to WT→KO mice, which were even less sick than control WT→WT mice. An infiltration of DC and accumulation of Th17 cells was also observed in the CNS of KO→WT mice. Therefore, triggering of MAPK3 in the periphery might be a therapeutic option for the treatment of neuroinflammation since absence of this kinase in the immune system leads to severe EAE.

Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.

Cross-Recognition of a Myelin Peptide by CD8+ T Cells in the CNS Is Not Sufficient to Promote Neuronal Damage

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8+ T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8+ T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40–54 (MOG40–54) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8+ T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8+ T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8+ T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4+ Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8+ T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury.

A new light source for multimodal multiphoton microscopy including CARS

We will present a new flexible laser source for multimodal Multiphoton excitation microscopy including CARS. It consists of a tuneable femtosecond-Ti:Sapphire laser and an optical parametric oscillator (OPO). The new OPO-design allows for high flexibility in pump- and output wavelengths giving rise to for instance image EGFP with the Ti:Sapphire and tdRFP with the OPO simultaneously. This is presented on living mouse brain tissue. The minimum energy difference between Ti:Sapphire and OPO-wavelengths achievable is 2500cm-1. Thus CARS imaging of lipids is possible. Due to synchronous pumping of the OPO the pump- and OPO pulses are intrinsically locked in time to each other thus they can be brought to perfect overlap of pump and stokes pulses. Uncaging multiphoton microscopy is also possible with this system due to the low minimum OPO pump wavelength of 730nm.

Role of extracellular signal-related kinase (Erk) 1 in the regulation of neuroinflammation

We have previously shown that the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HMGCRI) atorvastatin is therapeutic in experimental autoimmune encephalomyelitis (EAE) while also inducing a sustained phosphorylation of the MAPK Erk1 that is important for inducing T cell anergy. However it is also known, that HMGCRI also influence the antigen presenting cell compartment including dendritic cells (DC). This led us to investigate the role of Erk1 in DC biology in more detail. Indeed bone-marrow derived dendritic cells from Erk1 deficient mice had an increased migratory capacity when compared with DC isolated from wildtype littermate mice. As a likely consequence to cytoskeletal regulation, Erk1-/- DC had an increased surface expression of costimulatory molecules and were more potent to prime T cells in vivo. To investigate the implications of these findings in an inflammatory scenario, we induced EAE (experimental autoimmune encephalomyelitis) in Erk1-/- and Erk1+/+ mice with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) and could show that a deficiency of this MAPK results in a moderate increase in disease severity. To differentiate the role of Erk1 between peripheral immune system and the brain compartment we induced EAE in Erk1+/+ mice harboring Erk1-/- immune cells by applying bone marrow chimeras (Erk1-/- --> Erk1+/+). We report that Erk1 has an important regulatory function in the immune system as shown by pronounced disease severity in Erk1-/- --> Erk1+/+ bone marrow chimeras. All together these results indicate the significance of Erk1 in regulating DC functions that are relevant for T cell priming and neuroinflammation, and thus signal the importance of therapeutically targeting this MAPK for the treatment of autoimmune diseases.