Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

Rapid disruption of cortical activity and loss of cerebral blood flow in a mouse model of mild traumatic brain injury

Every year 2.8 million Americans suffer a traumatic brain injury (TBI). Despite the prevalence and debilitating consequences of TBI, effective treatment options are scarce due to the limited understanding of the neurobiological effects of injury, especially in acute phases when the cellular processes leading to neuropathology are first initiated. To identify changes in neural function and cerebral blood flow (CBF) that might account for TBI-induced cognitive and sensory deficits, we took a multidisciplinary approach, examining synaptic function, cortical activity patterns, and microvascular hemodynamics. we used a weight drop model in mice to induce mild TBI, the most common form in humans, and focused on responses within the first hours of injury where existing data are particularly limited. For synaptic function, we measured excitatory and inhibitory input onto pyramidal cells in the piriform cortex with whole-cell recordings in acute brain slices. Increased excitation appeared at one hour but excitatory-inhibitory balance was reestablished by 48 hours, highlighting the importance of studying rapid-onset injury responses. We also compared neural activity before and after TBI using in vivo two-photon calcium imaging of pyramidal cells in visual cortex. While neural activity substantially decreased in most cells one hour after injury, a minority of cells showed hyperactivation or prolonged increases in intracellular calcium, again indicating major physiological disturbances during immediate post-injury phases. Finally, we measured in vivo changes in CBF throughout the cortical microvasculature with laser speckle contrast imaging and optical coherence tomography, tracking injury effects up to three weeks after TBI. CBF and capillary flow were dramatically reduced within minutes and remained suppressed for over one hour. As neurons’ high energetic needs require a constant supply of glucose and oxygen from local vasculature, decreased CBF likely contributes to altered neural activity and loss of ion homeostasis and thus potentially cognitive and sensory deficits after TBI. Our results reveal that even mild injury creates rapid, pronounced, and heterogeneous alterations in neural activity and capillary flow. The transient nature of these effects suggests that the first two hours after injury may be a key window for delivering interventions, and that restoring CBF may reduce damage due to metabolic stress

In Vivo Imaging to Characterize Dynamic Tissue Responses after Neural Electrode Implantation

Implantable neural electrodes are promising technologies to restore motor, sensory, and cognitive function in many neural pathologies through brain-computer interfacing (BCI). Many BCI applications require electrode implantation within neural tissue to resolve and/or modulate the physiological activity of individual neurons via electrical recording and stimulation. This invasive implantation leads to acute and long-term deterioration of both the electrode device as well as the neurons surrounding the device. Ultimately, damage to the electrode and neural tissue results in electrode recording failure within the first years after implantation. Many strategies to improve BCI longevity focus on mitigating tissue damage through improving neuronal survival or reducing inflammatory activity around implants. Despite incremental improvements, electrode failure persists as an obstacle to wide-spread clinical deployment of BCIs. This can be partly attributed to an incomplete understanding of the biological correlates of recording performance. These correlates have largely been identified through post-mortem histological staining, which cannot capture dynamic changes in cellular physiology and morphology. In the following dissertation, we use longitudinal two-photon in vivo imaging to quantify how neurons, microglia, and meningeal immune cells are affected by an intracortical electrode during and after implantation in mouse cortex. We go beyond conventional histological techniques to show the time-course of neuronal injury and microglial recruitment after implantation. Neuronal injury occurs instantaneously, with prolonged, high calcium levels evident in neurons within 100 µm of implants. Microglial activation occurs within minutes of implantation and subsequent microglial encapsulation of electrodes can be modulated by bioactive surface coatings. Within the first day post-implant, there is high trafficking of peripheral immune cells through venules at the surface of the brain as well as along the electrode’s shank at the surface of the brain. Over the next month, calcium activity in neurons increases while the collagenous meningeal tissues at the surface of the brain thicken. We further show that meningeal thickening can have profound implications for devices implanted into non-human primates as well. In sum, these results define new potential therapeutic targets and windows that could improve the longevity of implantable neural electrodes

NON-LINEAR OPTICAL METHODS TO UNDERSTAND PATHOPHYSIOLOGY OF SMALL BLEEDS

Cerebral microhemorrhages (CMBs) are small hemorrhagic strokes found in the brain, also known as silent stroke since they do not illicit noticeable symptoms. Recently, due to the development of various imaging modalities and aging population in the western world, increasing number of CMBs are detected. Clinical studies have shown that aging and hypertension significantly increases the chance of such bleeds and the National Institute of Health recognizes CMBs as a major factor in Alzheimer disease pathology. Independent events of CMBs are also a risk factor for subsequent larger intracerebral hemorrhages, ischemic stroke, Binswager’s disease and Alzheimer’s disease. However, studies in cellular level are lacking, partially due to inadequate animal model that allow both detection and follow up analysis of such small bleeds. We used tightly focused femtosecond laser pulses to injure single penetrating arterioles in the cortex of live anesthetized rodents and used multi-photon excited fluorescence imaging to quantify inflammatory responses over long periods of time. The work presented in this dissertation provides comprehensive spatial and temporal pathological consequences after micro scale hemorrhagic injury to a single blood vessel in the brain

Functional roles of the chemokine CCL17 in skin and brain immunity

The chemokines CCL17 and CCL22 represent ligands of CCR4 and are mainly produced by dendritic cells (DCs) and macrophages (Mφs) in the immune system. CCL17 was found to promote various inflammatory and allergic diseases, whereas CCL22 has more often been associated with an immunosuppressive environment. These differential functions are reflected by preferential recruitment of distinct subsets of immune cells to sites of inflammation. Whereas CCL17 induces chemotaxis of effector T cells and facilitates T cell-DC interactions, CCL22 appears to be involved in the recruitment of regulatory T cells. In addition, CCL22 induces a more rapid desensitization and internalization of CCR4 than CCL17, implying biased agonism of CCL17 and CCL22. In this thesis, newly generated CCL17/22-double-deficient (CCL17E/E/22-/-) mice were used to further explore the differential function of CCL17 and CCL22. In agreement with previous reports in the literature, CCR4-deficient mice displayed an exaggerated contact hypersensitivity (CHS) response. In contrast, CCL17E/E/22-/- and CCL17-single deficient (CCL17E/E) mice were protected from CHS. Thus, the opposing phenotypes of CCR4KO- versus CCL17E/E mice cannot be explained by residual CCL22 signaling in CCL17E/E mice. Furthermore, intravital microscopy (IVM) and flow cytometry were performed to characterize CCL17+ cells in the skin of CCL17-EGFP reporter (CCL17E/+) mice in a wild-type (WT) and GM-CSF-deficient background. Whereas expression of CCL17 in skin DCs was GM-CSF-dependent, transcription of CCL17 in skin Mφs occurred independently of GM-CSF. In line, two distinct CCL17+ cell types could be identified in the skin by IVM as judged by their motility: a population of sessile CCL17+ cells in close proximity to dermal blood vessels, presumably representing perivascular Mφs, and a migratory cell population resembling DCs in the interstitium. To develop novel strategies for treatment of contact allergy, two RNA aptamers were validated in vitro and in vivo for their capability to neutralize CCL17. The two aptamers effectively inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 towards CCL17 in a dose-dependent manner. In the CHS model, systemic application of either one of the aptamers significantly prevented the ear swelling response and reduced T cell infiltration into the ears. These experiments provide proof-of-principle that CCL17-specific aptamers may potentially be used therapeutically in humans to treat allergies and perhaps other inflammatory diseases. In the second part of the thesis, the expression and function of CCL17 in the murine brain was investigated. CCL17/EGFP+ neurons were primarily detected in in a subset of hippocampal CA1 neurons, whereas only few cortical neurons stained positive for CCL17/EGFP. The basal Ccl17 expression in hippocampal neurons strongly increased by peripheral challenge with lipopolysaccharide (LPS) in a tumor necrosis factor (TNF) dependent manner. In addition, Ccl22 was also detected in the hippocampus, but its LPS-dependent upregulation required GM-CSF. Analysis of brains from CCL17E/E mice revealed a diminished microglia density in the hippocampus under homeostatic and inflammatory conditions. A combination of confocal microscopy and computer-assisted morphological analyses demonstrated that microglia from naïve CCL17E/E mice displayed a reduced cellular volume and a more polarized process tree compared to WT controls. Furthermore, overall branching, cell surface area and total tree length of microglia from naïve CCL17E/E mice were similar to that of microglia from LPS-treated WT mice. In addition, electrophysiological recordings of acute slices from naïve WT and CCL17E/E mice indicated a downmodulation of basal synaptic transmission at CA3-CA1 Schaffer collaterals through CCL17. In conclusion, the work presented in this thesis identifies CCL17 as a homeostatic and inducible neuromodulatory chemokine which affects the abundance and morphologic appearance of microglia as well as synaptic transmission in the hippocampus.Funktionen des Chemokins CCL17 im Immunsystem der Haut und des Gehirns Die Chemokine CCL17 und CCL22 sind Liganden von CCR4 und werden hauptsächlich von dendritischen Zellen (DCs) und Makrophagen produziert. Für CCL17 wurde gezeigt, dass es verschiedene entzündliche und allergische Erkrankungen fördert. Im Gegensatz dazu, wird CCL22 eher mit einer immunsuppressiven Wirkung assoziiert. Diese gegenläufigen Funktionen spiegeln sich ganz besonders in der Fähigkeit wider, nur bestimmte Immunzellen zu Entzündungsherden zu rekrutieren. Während CCL17 die Chemotaxis von Effektor-T-Zellen induziert und eine Interaktion von T-Zellen und DCs erleichtert, wird CCL22 hauptsächlich mit der Rekrutierung regulatorischer T-Zellen, z.B. in das Tumormikromilieu, in Verbindung gebracht. Im Vergleich zu CCL17 führt CCL22 außerdem zu einer schnelleren Desensibilisierung und Internalisierung von CCR4, was eine gewisse funktionelle Selektivität (engl. biased agonism) von CCL17 und CCL22 für CCR4 impliziert. In der vorliegenden Arbeit wurden neu generierte CCL17/22-doppelt defiziente Mäuse (CCL17E/E/22-/-) dazu verwendet, die differentielle Funktion von CCL17 und CCL22 weitergehend zu untersuchen. Interessanterweise entwickelten CCL17E/E/22-/- Mäuse genau wie CCL17-defiziente (CCL17E/E) Mäuse eine deutlich reduzierte Kontakthypersensitivitäts-(CHS)-Reaktion im Vergleich zu wildtypischen (WT) Kontrollmäusen, während CCR4-/- Mäuse eine verstärkte allergische Reaktion ausbildeten. Somit konnte gezeigt werden, dass der schon bekannte Unterschied zwischen CCR4-/- und CCL17E/E-Mäusen im CHS Modell nicht durch die in CCL17E/E Mäusen verbleibende Wirkung von CCL22 erklärt werden kann. Darüber hinaus wurden intravitale Mikroskopie (IVM) und Durchflusszytometrie angewandt, um CCL17-positive Zellen in der Haut von CCL17-EGFP Reporter (CCL17E/+) Mäusen in der An- bzw. Abwesenheit von GM-CSF zu charakterisieren. Hier konnte eine GM-CSF-abhängige Expression von CCL17 in DCs der Haut gezeigt werden, wohingegen die Regulation von CCL17 in Makrophagen unabhängig von GM-CSF war. Ferner konnten mittels IVM zwei verschiedene CCL17-positive Zelltypen in der Haut nachgewiesen werden. Neben einer sessilen CCL17-positiven Zellpopulation, welche in der Nähe von dermalen Blutgefäßen lokalisiert war und möglicherweise zu den perivaskulären Makrophagen gehört, wurde eine zweite, durch das Interstitium wandernde CCL17-positive Zellpopulation beobachtet , bei der es sich wahrscheinlich um DCs handelt. Um neue Möglichkeiten zur Behandlung von Allergien zu entwickeln, wurden zwei neuartige RNA-Aptamere auf ihre Fähigkeit hin getestet, CCL17 in vitro und in vivo zu neutralisieren. Mithilfe eines Zell-Migrationstests konnte gezeigt werden, dass beide Aptamere die gerichtete Migration der CCR4+-Lymphom-Zelllinie BW5147.3 entlang eines CCL17-Gradienten dosisabhängig hemmen. Außerdem konnte in Aptamer-behandelten WT Mäusen eine deutlich reduzierte T-Zell-Infiltration und eine verringerte Ohrschwellung gemessen werden. Des Weiteren konnte in Inhibitionsexperimenten gezeigt werden, dass CCL17 eine vielversprechende Zielstruktur zur Behandlung von allergischen und möglicherweise auch anderen entzündlichen Krankheiten darstellt. Im zweiten Teil der Arbeit wurde die Expression und Funktion von CCL17 im murinen Gehirn untersucht. CCL17-exprimierende Neuronen konnten vor allem in der hippocampalen CA1 Region identifiziert werden, während im Kortex nur wenige CCL17-produzierende Neuronen nachgewiesen wurden. Systemische Gabe von Lipopolysaccharid (LPS) führte zu einer stark erhöhten Expression von Ccl17 und Ccl22 im Hippocampus. Interessanterweise war die LPS-induzierte Expression von Ccl17 abhängig von lokal produziertem Tumornekrosefaktor (TNF), während GM-CSF die Expression von Ccl22 regulierte. Eine genaue Untersuchung der Gehirne von LPS-behandelten CCL17E/E- und WT-Mäusen und entsprechenden Kontrolltieren ergab eine stark reduzierte Anzahl von Mikroglia in Hippocampi von CCL17E/E Mäusen. Des Weiteren konnte mittels konfokaler Mikroskopie und einer computergestützten morphologischen Analyse gezeigt werden, dass Mikroglia in naiven CCL17E/E Mäusen, im Vergleich zu WT Mäusen, ein deutlich reduziertes Zellvolumen und einen stärker polarisierten Prozessbaum aufweisen. Außerdem ähnelten die Gesamtverzweigung (engl. ramification), die Zelloberfläche und die Gesamtbaumlänge der Mikroglia von naiven CCL17E/E-Mäusen denen der Mikroglia von LPS-behandelten WT-Mäusen. Des Weiteren wiesen elektrophysiologische Messungen an akuten Gehirnschnitten aus naiven WT- und CCL17E/E-Mäusen darauf hin, dass CCL17 die basale synaptische Übertragung zwischen den Schaffer-Kollateralen der CA3-CA1 Region reprimiert. Damit konnte CCL17 erstmalig als ein neues, homöostatisches und induzierbares neuromodulatorisches Chemokin identifiziert werden, welches sowohl die Häufigkeit und Morphologie von Mikroglia als auch die synaptische Übertragung im Hippocampus beeinflusst

Thrombospondin-1 and Cd47 Mediate Peripheral Microvascular Dysfunction Following Pulmonary Exposure to Multi-Walled Carbon Nanotubes

Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) has been shown to disrupt endothelium-dependent arteriolar dilation in the peripheral microcirculation. The molecular mechanisms behind these arteriolar disruptions have yet to be fully elucidated. The secreted matricellular matrix protein thrombospondin-1 (TSP-1) is capable of moderating arteriolar vasodilation by inhibiting NO signaling at several points, including the inhibition soluble guanylate cyclase activity and eNOS activation. The central hypothesis was that TSP-1, following pulmonary exposure to MWCNT, mediates peripheral changes in dilatory capacity. To test this hypothesis, wild-type C57B6J (WT), TSP-1 knockout (TSP-1 KO) and CD47 knockout (CD47 KO) mice were exposed via lung aspiration to 50 microg MWCNT or a sham dispersion medium control. Following exposure (24hrs), arteriolar characteristics and reactivity were measured in the gluteus maximus muscle using intravital microscopy (IVM) coupled with microiontophoretic delivery of acetylcholine (ACh) or sodium nitroprusside (SNP). In WT mice exposed to MWCNT, skeletal muscle TSP-1 protein increased (p \u3c 0.05) 517.9 +/- 112.5 % compared to sham exposed, and exhibited a 38.5 +/- 2.5 % and 47.9 +/- 7.3 % decrease (p \u3c 0.05) in endothelium-dependent and independent vasodilation, respectively. In contrast, TSP-1 protein was not increased following MWCNT exposure in TSP-1 KO mice and KO were protected from losses in dilatory capacity. Microvascular leukocyte-endothelium interactions were measured by leukocyte adhesion and rolling activity in third order venules. The WT+MWCNT group demonstrated 223.8 +/- 7.6 % higher (p \u3c 0.05) leukocyte rolling compared to WT+SHAM controls. TSP-1 KO and CD47 animals exposed to MWCNT showed no differences from WT+SHAM control. Total tissue total nitrite (NO-2) and nitrate (NO-3) a measure of nitric oxide status, was decreased (p \u3c 0.05) in WT+MWCNT lung by 47.4 +/- 13.8 % and plasma by 32.3 +/- 4.0 %, while not different from sham controls in the KO groups. Finally, several inflammatory cytokines, as quantified by Meso Scale Discovery, were upregulated in tissues of both TSP-1 and CD47 KO animals, but not in either of the WT groups. Taken together, these data provide the first evidence that TSP-1 is likely a central mediator of the systemic microvascular dysfunction that follows pulmonary MWCNT exposure

Collective phenomena in blood suspensions

This work was carried out in collaboration between I.R.P.H.E. (Institut de Recherche sur les Phénomènes Hors Équilibre), research unit of Aix-Marseille University and University of Saarland, Faculty of Experimental Physics (Naturwissenschaftlich-Technische Fakultät der Universität des Saarlandes) and aims to investigate microcirculatory hydrodynamics of blood in vitro. The study is dedicated to better understanding of complex collective phenomena that take place in microcirculation of blood through microfluidic in vitro experiments. It mainly focuses rigidity based margination in suspension of RBCs. For this purpose, model experiment was developed to examine margination caused exclusively by contrast of deformability between two sub-populations of RBCs.Diese Arbeit wurde durch die Zusammenarbeit zwischen dem I.R.P.H.E. (Institut de Recherche sur les Phénomènes Hors Équilibre), der Forschungseinheit der Aix-Marseille Universität und der Universität des Saarlandes, Fakultät für Experimentalphysik, ermöglicht und zielt auf die Erforschung der Mikrozirkulations-Hydrodynamik von Blut in vitro. Im Speziellen soll diese Studie dem besseren Verständnis komplexer, kollektiver Phänomene dienen, welche in der Mikrozirkulation von Blut in mikrofluiden in vitro Experimente entehen. Ein Hauptpunkt der Studie ist die steifigkeitsbedingte Margination in Suspensionen roter Blutzellen. Zu diesem Zweck wurde ein Modellversuch entwickelt, um die Margination zu untersuchen, welche lediglich durch den Unterschied der Verformbarkeit zwischen zwei Subpopulationen roter Blutzellen entsteht

The role of Preimplantation Factor (PIF) on leukocyte recruitment in vivo

Throughout pregnancy, immune cells infiltrate and colonize the placenta to ensure fetal development and successful birth. Thereby, they regulate tissue remodeling and protect the unborn from invading pathogens. At the same time, immune cells within the placenta require tight regulation in order to prevent recognition of the embryo as a ’semi-allograft’. Extra-embryonic tissue actively modulates immune cell functions by expressing growth factors and cytokines. Preimplanation factor, a 15 amino acid small peptide, is produced by trophoblast cells and continuously secreted into maternal circulation. It has been shown to interfere with immune cell functions in autoimmune disease models, but underlying molecular mechanisms remain unclear. This work investigated the function of PIF in acute inflammatory scenarios, reflecting its role within maternal serum. Analysis of leukocyte recruitment in postcapillary venules of TNF-α stimulated cremaster muscles in the mouse revealed that (i) leukocyte rolling, (ii) leukocyte adhesion and (iii) neutrophil extravasation is impaired in the presence of PIF. With the help of several ex vivo and in vitro assays, reduced leukocyte rolling could be linked to effects of PIF on the endothelial compartment. Impaired leukocyte adhesion and reduced extravasation in turn could be attributed to a direct effect of PIF on neutrophils. PIF inhibits K+ efflux via the voltage gated potassium channel KV1.3 on neutrophils, thereby reducing sustained calcium influx into the cells. Decreased intracellular Ca2+ concentrations impair post-arrest modification steps, namely neutrophil spreading and adhesion-strengthening, resulting in increased susceptibility to physiological shear forces and in reduced adhesion and extravasation. Taken together, this work demonstrates that PIF modulates neutrophil function during immune responses, offering therapeutic potential beyond pregnancy to protect patients from exuberant inflammation and excess neutrophil recruitment