Investigating the role of sphingolipids in innate immunity

Einer der Hauptakteure des angeborenen Immunsystems, ist die Gruppe der Toll-like Rezeptoren (TLRs). Diese konservierten Transmembranrezeptoren erkennen molekulare Strukturen von Pathogenen und initiieren daraufhin eine sofortige Immunantwort. Die große Gruppe der Membranproteine zusammen mit tausenden von Lipidmolekülen formen die zelluläre Membrane, deren physikalische Eigenschaften stark von der Zusammensetzung abhängen. Der Großteil der Membranlipide gehört zu den Glycerophospholipiden oder den Sphingolipiden, deren Mengen durch massenspektrometrische Analyse (Lipidomics) ermittelt werden können. Die Kombination von gezielten genetischen Störungen der Sphingolipid-modifizierenden Enzyme in murinen Macrophagen, zusammen mit der Lipidomics Analyse von 245 verschiedenen Lipidspezies in diesen Zelllinien, offenbarte umfassende Veränderungen der Lipidzusammensetzung. Mit Hilfe dieser Daten konnten wir die Koregulation der Lipide identifizieren. Bemerkenswerter Weise zeigte die Visualisierung dieser Lipid Koregulationen ein zirkuläres Netzwerk, welches den Metabolismus, Adaptierungsmechanismen und die subzelluläre Lokalisation der Lipide darstellte. Um die Konsequenzen einer gestörten Lipidzusammensetzung auf die angeborene Immunantwort zu identifizieren, charakterisierten wir funktionell die TLR-induzierte Signalweiterleitung dieser Zelllinien. Durch das Kombinieren des Netzwerkes der koregulierten Lipide mit der detaillierten Quantifizierung der TLR-abhängigen Prozesse, konnten wir Funktionen für einzelne Lipidspezies in der angeboren Immunantwort ableiten und beschreiben. Basierend auf den Veränderungen der Lipidzusammensetzung in einer weiteren Zelllinie, in der das bisher nur dürftig beschriebene Gen Smpdl3b genetisch dezimiert wurde, konnten wir mit Hilfe der vorher ermittelten Funktionen von verschiedenen Lipiden die pro-inflammatorische Immunantwort dieser Zelllinie vorhersagen. SMPDL3B wurde auch als Bindungspartner von TLR7, TLR9 und TLR4 identifiziert, und konnte durch diverse inflammatorische Stimuli induziert werden. Des Weiteren wurde die negative regulatorische Rolle dieses Proteins in einer Smpdl3b-defizienten Maus mittels eines Peritonitismodels auch in vivo bestätigt, da erhöhte Zytokinausschüttung gemessen wurde. Zusätzlich wurden Fibroblasten von verschiedenen Patienten mit einer Lipidspeichererkrankung ebenfalls mittels Lipidomics analysiert und ein Vergleich der Lipid Koregulationen zeigte, dass das zirkuläre Netzwerk zwischen murinen und humanen Zellen konserviert ist. Mit Hilfe der funktionellen Lipidbeschreibung, basierend auf den Daten der murinen Macrophagen, konnten wir die Immunantwort der humanen Zellen genau vorhersagen. Diese Prognose beruhte einzig auf den Veränderungen ihrer Lipidzusammensetzung und konnte experimentell bestätigt werden.As key regulators of the innate immune system, Toll-like receptors (TLRs) are conserved transmembrane receptors that recognize molecular structures of pathogens and initiate the immediate immune response. Together with a large panel of other proteins and thousands of lipids they comprise the cellular membranes, whose physical properties strongly depend on their lipid composition. The majority of membrane lipids belong to the glycerophospholipids or sphingolipids and their abundance can be measured using mass spectrometry-based lipidomics. The combination of shRNA-mediated genetic perturbations of lipid-modifying enzymes important for the sphingolipid metabolism in mouse macrophages, with the lipidomics analysis of 245 different lipid species, revealed broad changes in sphingolipid and glycerophospholipid levels. Using this data, we could identify lipids, which were coregulated across all perturbed cell lines. Strikingly, visualization of the lipid-lipid coregulation across all lipid species revealed a circular network of lipid coregulation, reflecting lipid metabolism, adaptation mechanisms, and subcellular localization. To further dissect the consequences of perturbed membrane lipid composition on the innate immune response we functionally characterized the TLR-mediated responses of these cell lines. By combining the network of coregulated lipids with the detailed characterization of TLR biology, we could infer functions for single lipid species in TLR signaling. These inferred functions were validated using lipid supplementation approaches. Based on changes in the lipid composition upon genetic perturbation we could predict the pro-inflammatory response of the previously unknown gene Smpdl3b (Sphingomyelin Phosphodiesterase, Acid- Like 3B) in a loss-of-function cell line. The gene product SMPDL3B was identified as a TLR4, TLR7, and TLR9 interactor, whose expression levels were induced upon diverse inflammatory stimuli. Further, in an Smpdl3b deficient mouse model the negative regulatory role of SMPDL3B was confirmed in vivo using a peritonitis model resulting in enhanced cytokine release after infection. Fibroblasts derived from a panel of patients suffering from lipid storage disorders were also analyzed using lipidomics and a comparison of the lipid coregulation revealed that the circular network was largely conserved between mouse and human cells. Using the functional lipid annotation of mouse macrophages we could correctly predict the inflammatory response of human fibroblasts solely based on their changes in lipid composition.subm. by Marielle S. KöberlinAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)171387

Functional crosstalk between membrane lipids and TLR biology

Toll-like receptors (TLRs) are important transmembrane proteins of the innate immune system that detect invading pathogens and subsequently orchestrate an immune response. The ensuing inflammatory processes are connected to lipid metabolism at multiple levels. Here, we describe different aspects of how membrane lipids can shape the response of TLRs. Recent reports have uncovered the role of individual lipid species on membrane protein function and mouse models have contributed to the understanding of how changes in lipid metabolism alter TLR signaling, endocytosis, and cytokine secretion. Finally, we discuss the importance of systematic approaches to identify the function of individual lipid species or the composition of membrane lipids in TLR-related processes

A conserved circular network of coregulated lipids modulates innate immune responses

SummaryLipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems

The lipid-modifying enzyme SMPDL3B negatively regulates innate immunity

Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity

Understanding the diversity of membrane lipid composition

Cellular membranes are formed from a chemically diverse set of lipids present in various amounts and proportions. A high lipid diversity is universal in eukaryotes and is seen from the scale of a membrane leaflet to that of a whole organism, highlighting its importance and suggesting that membrane lipids fulfil many functions. Indeed, alterations of membrane lipid homeostasis are linked to various diseases. While many of their functions remain unknown, interdisciplinary approaches have begun to reveal novel functions of lipids and their interactions. We are beginning to understand why even small changes in lipid structures and in composition can have profound effects on crucial biological functions