Auswahl der wissenschaftlichen Literatur zum Thema „Immune signalling“

Immune responses in insects include components and mechanisms that are highly evolutionarily conserved. In addition to providing insight into the insects themselves, knowledge of the conserved mechanisms involved in insect immunity can offer valuable insight that is broadly relevant to a wide variety of other species. Three aspects of insect immune cell signalling have been studied here. Cell signalling responses have been investigated in two insect cell lines following treatment with double stranded (ds) RNA, a common intermediate of viral replication. It has been established that both cell lines investigated, from the fruitfly Drosophila melanogaster and vector mosquito Aedes albopictus, do not exhibit activation of mitogen activated protein kinases (MAPKs) or NF-KB proteins as a direct response to dsRNA. Secondly, a detailed analysis of the mechanisms of transcriptional regulation has been carried out on the Drosophila drosomycin gene, a key factor both in terms of its function during the immune response and in terms of its role during previous characterisation of Drosophila immune signalling. The drosomycin promoter was found to be regulated independently by the Toll and IMD signalling pathways via distinct sequence elements. Finally, investigation of the responses of an A. albopictus cell line to treatment with bacterial cell wall components has revealed key differences in the mechanisms involved in immune-induced regulation of transcription compared with the model established in Drosophila. A role for p38 MAPK has been identified in the negative regulation of transcription of A. albopictus cecropin Ai, an inducible antimicrobial peptide gene.

Inappropriate stimulation of mast cells can trigger allergies including asthma, allergic rhinitis and eczema which, combined, affect almost 30% of the population in western societies. Mast cell activation begins with aggregation of IgE receptors in response to antigen. This then triggers a series of reactions resulting in the tyrosine phosphorylation of Syk kinase, PKC activation and ultimately both degranulation and secretion of leukotrienes and cytokines. CRAC channels are expressed on mast cells, and are essential for IgE-mediated mast cell activation. Previous work in our laboratory has shown that local Ca²⁺ influx through CRAC channels activates Ca2+-dependent phopholipase A₂, ERK and 5-lipoxygenase, resulting in LTC₄ secretion from mast cells. Therefore, I have investigated how Ca2+ microdomains through CRAC channels are detected and how they trigger cellular responses. I find that phosphorylation of Syk following antigen stimulation is enhanced by Ca²⁺ influx through CRAC channels. I also show synergy between CRAC channels and antigen in activating Syk. These findings reveal a novel positive feedback step in mast cell activation, where local Ca²⁺ entry through CRAC channels activates Syk which, in turn, supports CRAC channels. Earlier work from our group has demonstrated that in RBL cells, Ca2+ influx through CRAC channels induces expression of the gene c-fos, an important regulator of pro-inflammatory gene expression. I have discovered that local Ca²⁺ entry is sensed by the non-receptor tyrosine kinase Syk, which accumulates at the cell periphery. Syk then signals to the nucleus through recruitment of the transcription factor STAT5. The results therefore identify Syk as a new link in excitation-transcription coupling, converting local Ca²⁺ influx into expression of genes that are essential for immune cell activation. Activation of G protein-coupled cysteinyl leukotriene type I receptors by the pro-inflammatory molecule LTC₄ is tightly linked to immune cell function and the receptor is an established therapeutic target for allergies including asthma. Desensitization of cysteinyl leukotriene type I receptors arises following protein kinase C-dependent phosphorylation of three serine residues in the receptor C-terminus. Here I show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression. Physiological concentrations of LTC₄ led to repetitive cytoplasmic Ca²⁺ oscillations, which were accompanied by the opening of store-operated CRAC channels in the plasma membrane. Ca²⁺ microdomains near the open channels were relayed to the nucleus to increase expression of the transcription factor c-fos. In the absence of receptor desensitization, agonist-driven gene expression was suppressed. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged Ca²⁺ release, which led to accelerated Ca²⁺-dependent inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Rather than serving to turn off a biological response, the experiments show that reversible receptor desensitization is an ‘on’switch’, sustaining long-term signalling in the immune system.

This work investigates the mechanisms of binding of T cell receptors (TCRs) to Class I MHC-peptide complexes (pMHC). The structure of a TCR specific for the Melan-A tumour antigen bound to its cognate pMHC was solved to a resolution of 2.5 Å which gives insight into how this TCR could be mutated to optimize binding and subsequently used as a cancer vaccine. Detailed sequence and geometric analyses of all currently available structures of Class I TCR-pMHC complexes revealed that TCRs can bind to pMHC with a range of orientations, yet always focus on the central portion of the peptide and use a specific subset of six residues on the MHC helices for binding. The most striking finding was the use of aromatic residues in the TCR CDR loops to bind to residue Q155 on the MHC α2 helix. Attempts were also made to express and purify Toll-like receptors (TLRs) with the aim of solving one or more of these structures. However, despite testing of over 50 different constructs from 12 different TLRs or associated proteins, insufficient soluble protein expression was obtained for crystallization trials. Finally, a protein disorder prediction tool was developed to aid construct design for structural biology studies and improve the chances of obtaining protein crystals. This tool is based on a novel type of neural network and blind tests comparing it to 8 other disorder prediction tools showed it is one of the best in the field. It is freely available at www.strubi.ox.ac.uk/RONN. Analysis of large datasets revealed that the position of order/disorder transitions is quite precisely defined in amino-acid sequences and that transition regions have an amino acid composition distinct from that of bulk ordered and disordered sequences. There is a steady decrease in order-promoting residues on the ordered side of boundaries as well as a weak sequence signal, both of which signify the approaching disorder and may prove useful for improving existing disorder prediction tools.

NF-κB transcription factors play essential roles in regulating the expression of a large array of genes involved in immune and inflammatory responses, cell proliferation, apoptosis and oncogenesis. In recent years, ubiquitination of key components has emerged as a crucial regulatory mechanism in NF-κB signalling pathways. This can either directly modulate the activity of the target substrate or provide a scaffold for the recognition and recruitment of other signalling molecules via their respective ubiquitin binding domains. NEMO, the regulatory subunit of the IKK complex, is a prime example of a signalling component which interacts with ubiquitin chains of different topologies as well as being modified by ubiquitination. These events are thought to regulate the activation of the kinase subunits of the complex, IKKα and IKKβ, which ultimately results in liberation of NF-κB and translocation to the nucleus. NEMO itself possesses two ubiquitin-binding domains: the CoZi/UBAN domain and a C-terminal zinc finger (ZF). The work presented in this thesis shows that the synergistic action of these two domains confers specificity for K63-linked ubiquitin chains. Importantly, chain length plays a crucial role in these binding events. The discovery that NEMO becomes modified by the E3 ligase LUBAC with a novel type of ubiquitin chain, termed linear, opened up a whole new exciting area of research. So far, LUBAC is the only E3 ligase known to synthesize this type of chain. This thesis provides a first glimpse into the mechanistic determinants which allow LUBAC to enforce the formation of linear ubiquitin chains and demonstrates, for the first time, that one of its subunits, HOIL-1L, transfers ubiquitin to a substrate via a thioester intermediate.

Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), initiating a broad-spectrum defense response against pathogens, known as PRR-triggered immunity (PTI). However, immunity comes at a cost; and immune responses need to be tightly regulated. How PTI signalling is negatively regulated in plants is not fully understood. PRRs are present at the plasma membrane in dynamic kinase complexes that heavily rely on trans-phosphorylation to initiate signaling. The Arabidopsis cytoplasmic kinase BIK1 associates with different PRRs and plays a central role in the activation of downstream immune signaling. In this study, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the regulatory receptor kinase (RK) BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Notably, upon PAMP perception, PP2C38 is phosphorylated on serine 77, most likely by BIK1, and dissociates from the PRR-BIK1 complex. We suggest that this mechanism relieves the negative regulation imposed by PP2C38 to enable efficient BIK1 activation. This study uncovers an important regulatory mechanism of this central immune component, and extends our knowledge on how plant immunity is appropriately controlled.

The expression and the role of Notch receptors and ligands during the induction of an immune response were investigated. I have shown that components of the Notch pathway were present in peripheral CD4 and CD8 T cells. Upon culturing and activation <i>in vitro</i>, in many Notch components were differentially expressed, in particular targets of Notch activation. In contrast, bone marrow-derived dendritic cells (DCs) expressed only low levels of Notch targets. However, the Notch ligand Jagged1 was strongly upregulated upon maturation of DCs with TNFα or LPS. By regulating the expression of the ligands, a role for antigen presenting cells (APCs) as the “signalling cells” is likely, whereas T cells expressing Notch receptors and Notch target genes may be the “receiving cells”. To investigate this hypothesis <i>in vivo</i>, transgenic mice with inducible overexpression of Jagged1 or Delta1 in DCs were generated. An <i>in vitro</i> approach to study the role of Notch ligands on APCs was carried out using an I-A^b transfected murine fibroblast cell line (I-A^b+ L cells) with endogenous B7.1 expression. I-A^b+ L cells co-transfected with Jagged1 or Delta (Jagged1⁺ or Delta1⁺L cells, respectively) were used as APCs in a mixed lymphocyte reaction (MLR) <i>in vitro.</i> Jagged1⁺ and I-A^b+ L cells induced similar levels of allogeneic T cell proliferation, whereas Delta1⁺ L cells had a slightly increased capacity to activate T cell proliferation. There were no phenotypical differences or changes in the level of apoptosis observed between T cells activated by Jagged1⁺, Delta1⁺ or I-A^b+ L cells. However, IFNγ secretion by T cells in response to Jagged1⁺ L cells was strongly reduced, whereas Delta1⁺ and I-A^b+ L cells induced normal levels of IFNγ secretion. There were no significant differences neither in the level of intracellular IFNγ nor of IFNγ transcripts between T cells in response to Jagged1⁺, Delta1⁺ or I-A^b+ L cells. Therefore I hypothesise that Jagged1-induced Notch signalling may be involved at the level of IFNγ secretion.