Activation of toll-like receptor 2/1- and toll-like receptor 2/6-heterodimers by bacterial lipopeptides

Lipoproteine sind ein wesentlicher Bestandteil bakterieller Membranen und in der Lage, als starke Stimulatoren des angeborenen Immunsystems, sowie als potente Adjuvanzien des adaptiven Immunsystems zu fungieren. Allen Lipoproteinen gemeinsam ist ein konserviertes, N-terminales Glycerylcystein, welches durch 2 oder 3 Fettsäuren acyliert ist. Als Dipeptid stellt es die minimale aktive Struktur dar, die von Zellen des Immunsystems durch den Toll like Rezeptor 2 (TLR2) erkannt wird. Bisher wurden in Säugern 13 verschiedene TLR identifiziert. Der TLR2 ist dabei einzigartig in seiner Fähigkeit, mit den Korezeptoren TLR1 oder TLR6 Heterodimere zu bilden, die der Ligandenerkennung und Signaltransduktion dienen. Die bisherigen Erkenntnisse wurden durch Studien an humanen und murinen Rezeptoren erhalten, so dass die Untersuchung einer weiteren Spezies zusätzliche Informationen über die Erkennung verschiedener Lipopeptide erbringen sollte. Dabei konnte in bovinen Zelllinien sowie in primären bovinen Zellen erstmals die vollständige Sequenz des Rinder-TLR1 nachgewiesen und kloniert werden. Funktionelle Untersuchungen mit Hilfe synthetischer Lipopeptide im HEK293-System zeigten, dass bovine TLR2/1-Heterodimere, ähnlich den humanen, nur durch triacylierte Lipopeptide mit Fettsäureketten von einer Länge von mindestens 12 C-Atomen aktiviert werden, während murine TLR2/1-Heterodimere schon Lipopeptide mit vergleichsweise kurzen Fettsäuren (6 C-Atome) erkennen. Neben definiert TLR1- oder TLR6-abhängigen Lipopeptiden gibt es auch solche, die sowohl in TLR1- als auch in TLR6-defizienten Zellen eine Signaltransduktion induzieren. Durch Generierung von TLR1- und TLR6-doppelt-reprimierten Zellen mittels RNA-Interferenz, konnte erstmals gezeigt werden, dass diese Liganden in Abwesenheit beider Korezeptoren nicht erkannt werden, sie also beide Heterodimere und nicht etwa ein mögliches TLR2 Homodimer aktivieren. In der vorliegenden Arbeit sollte ferner geklärt werden, ob TLR2/1- und TLR2/6-Heterodimere nach der Stimulation unterschiedliche oder identische Signalwege aktivieren. Durch einen Vergleich der induzierten Signalkaskaden, der involvierten Adaptermoleküle und von MAP-Kinasen konnten zum Teil nur geringe Unterschiede nachgewiesen werden. Bei weiteren umfassenden Untersuchungen lag die Arbeitshypothese zu Grunde, dass die Aktivierung unterschiedlicher Signalwege zur Induktion unterschiedlicher Genexpressionsprofile führt. Mikroarray-Analysen zeigten jedoch eine identische Genmodulation nach Aktivierung der TLR2/1- und TLR2/6-Heterodimere durch die entsprechenden Lipopeptide. Cluster-Analysen und real time PCR-Ergebnisse belegten diese Befunde. Das Vorhandensein der beiden TLR2-Heterodimere stellt somit eher einen evolutionären Mechanismus zur Vergrößerung des Ligandenspektrums dar und resultiert nicht in unterschiedlichen Immunantworten der Zelle nach Erkennung unterschiedlicher Lipopeptide von Gram-positiven und Gram-negativen Bakterien sowie von Mykoplasmen. Die induzierten Signalwege spielen aber nicht nur eine Rolle bei der Etablierung einer angeborenen Immunantwort, sondern nehmen im Zuge der adjuvanten Wirkung von Lipopeptiden auch Einfluss auf die Ausbildung einer adaptiven Immunantwort. In einem Maus-Immunisierungsmodell konnte durch Verwendung des Lieschgras-Majorallergens Phl p 1, die adjuvante Wirkung des Lipopeptids FSL-1 aufgezeigt werden. Zum einen wurde eine vermehrte Freisetzung Allergen-spezifischer Immunglobuline der Klasse IgG1 nachgewiesen. Zum anderen ist jedoch hervorzuheben, dass die adjuvante Aktivität des Lipopeptids zu einer Induktion von IgG2a führte, was ein Indiz für die Verschiebung der TH2 dominierten Immunantwort in Richtung einer TH1 Immunantwort darstellt. Beides konnte in TLR6 defizienten Tieren nicht mehr beobachtet werden, so dass die essentielle Beteiligung dieses Korezeptors auch an der FSL-1-induzierten Modulation der adaptiven Immunantwort in vivo nachgewiesen werden konnte. Die in dieser Arbeit erhaltenen Ergebnisse bezüglich der Spezies-spezifischen Ligandenerkennung, Heterodimerisierung und der resultierenden Signaltransduktion, sowie der Modulation der adaptiven Immunität durch bakterielle Lipopeptide, liefern neue Erkenntnisse über die Aktivierung der TLR2-Heterodimere und ihre Bedeutung für eine Immunantwort.Lipoproteins represent an important constituent of the bacterial cell wall and are known to be strong stimulators of the innate immune system as well as potent adjuvants of the adaptive immune system. All lipoproteins are composed of a conserved N-terminal glycerylcysteine which can be acylated with 2 or 3 fatty acids. As a dipeptide it demonstrates the minimal active structure, that is recognized by immune cells through toll-like receptor 2 (TLR2). Until now there are 13 different TLR identified in mammals. Among them, TLR2 is unique in its ability to form heterodimers with TLR1 or TLR6 to mediate ligand recognition and signal transduction. Most of our knowledge is based on studies using human or murine TLR. One aim was to investigate the TLR of another species to get further insights into the requirements for lipopeptide recognition by TLR2 and its coreceptors. Thereby the full-lenght bovine TLR1 was identified in bovine cell lines and primary cells for the first time. Subsequent cloning and functional analyses using HEK293 cells and synthetic lipopeptides revealed, that bovine TLR2/1 heterodimers recognize triacylated lipopeptides with acid chains of at least 12 carbon atoms. This observation indicated similarity to the human heterodimer while the murine TLR2/1 dimer could already be activated by lipopeptides with shorter fatty acids (6 carbon atoms). Besides lipopeptides that show a distinct TLR1 or TLR6 dependency there are others which are able to induce signal transduction in TLR1- and in TLR6-deficient cells. By using RNA interference to obtain cells diminished in both coreceptors it was demonstrate that these lipopeptides are no longer recognized, indicating that they use both TLR2 heterodimers and no putative TLR2 homodimer to mediate signal transduction. Another aim was to investigate whether activation of TLR2/1 and TLR2/6 heterodimers would induce different or identical signal transduction pathways. By comparing the induced signaling pathways, involved adaptor molecules and MAP kinases small differences could be detected. By reason that differences in the signal transduction pathways would result in different gene expression patterns microarray analyses were performed. However, stimulation of the different TLR2 heterodimers with respective lipopeptides resulted in the induction of an identical gene modulation. Cluster analyses and real-time PCR verified these results. In conclusion, heterodimerization of TLR2 with TLR1 or TLR6 has been evolutionary developed to enable the innate immune system to broaden the ligand spectrum for recognizing the variety of different lipopeptides from Gram-positive and Gram-negative bacteria as well as mycoplasma rather than to induce different immune responses to the different lipopeptides. Besides playing an important role in establishing an innate immune response, through their adjuvant properties lipopeptides are also critically involved in modulating adaptive immune responses. In a mouse immunization model using the Timothy grass pollen major allergen Phl p 1 the adjuvant activity of the synthetic lipopeptide FSL-1 could be demonstrated. FSL-1 was able to enhance the concentration of Phl p 1-specific immunoglobulin IgG1 and more importantly induced the production of IgG2a, thereby shifting allergen-mediated TH2- immune response towards a TH1-immune response. This effect could not be observed in TLR6 deficient mice thereby emphasising the importance of TLR6 in the FSL-1-induced modulation of the adaptive immune response in vivo. Taken together, the data on species-specific ligand recognition, heterodimerization and signal transduction as well as modulation of the adaptive immune system by bacterial lipopeptides provide new insights into the function of TLR2 heterodimers and their relevance for the immune system

Toll-like receptors in domestic animals

Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed belo

Identification of full length bovine TLR1 and functional characterization of lipopeptide recognition by bovine TLR2/1 heterodimer

Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides

Carcinoma cells misuse the host tissue damage response to invade the brain

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis

Neutralization, effector function and immune imprinting of Omicron variants

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain$^{1}$ (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting

The Stimulatory Gαs Protein Is Involved in Olfactory Signal Transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gαs plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO2 responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gαs also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gαs plays a role in the OR mediated signaling cascade in Drosophila

A potential test system for detecting contaminations by bacterial lipoproteins.

Biological specimens are often contaminated with bacteria-derived products such as LPS or lipoproteins (LP), which trigger unwanted inflammatory responses in hosts. Whereas a contamination by LPS can be determined by various test systems, a contamination by LP can as yet not be determined. TLR4 and TLR2 are key components of the LPS and the LP receptor complex, respectively. It was tested in this study whether HEK293 cell stably transfected with bovine TLR2 have the ability to react to low concentrations of diacylated and triacylated synthetic LP. The stable cell lines we present here recognize low concentrations of synthetic LP resembling LP of different bacteria. Therefore, these cells are suitable to detect low contaminations present in probes. For example, HEK293 cells stably transfected with bovine TLR2 recognized an egg albumin preparation as contaminated, as evidenced by copious production of IL-8. In contrast, these cells did not respond to a highly purified human serum albumin (HSA) preparation used in the clinic but responded to HSA containing small amounts of diacylated synthetic LP. The TLR4 ligand LPS is often said to activate TLR2. Here we present evidence that LP contaminations are responsible for TLR2 activity. HEK293 cells stably transfected with bovine TLR2 and TLR1 (e.g. clone 1) did not respond to ultra-pure Escherichia coli LPS preparations but acquired responsiveness when stimulated with differently purified commercial LPS. Thus, the described system involving HEK293 cells stably transfected with bovine TLR2 and TLR1 is the first test system described attempting to measure a contamination by LP

PHD2 is a regulator for glycolytic reprogramming in macrophages

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior