Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

Anti-parasitic and anti-viral immune responses in insects

Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.

Pathogenic role of IL-33-mediated eosinophil infiltration and function in experimental inflammatory bowel disease

IL-33/ST2 axis is known to promote Th2 immune responses and has been linked to several autoimmune and inflammatory disorders, including inflammatory bowel disease (IBD), and recent evidences show that it can regulate eosinophils (EOS) infiltration and function. Based also on the well documented relationship between EOS and IBD, we assessed the role of IL-33-mediated eosinophilia and ileal inflammation in SAMP1/YitFc (SAMP) murine model of Th1/Th2 chronic enteritis, and we found that IL-33 is related to inflammation progression and EOS infiltration as well as IL-5 and eotaxins increase. Administering IL-33 to SAMP and AKR mice augmented eosinophilia, eotaxins mRNA expression and Th2 molecules production, whereas blockade of ST2 and/or typical EOS molecules, such as IL-5 and CCR3, resulted in a marked decrease of inflammation, EOS infiltration, IL-5 and eotaxins mRNA expression and Th2 cytokines production. Human data supported mice’s showing an increased colocalization of IL-33 and EOS in the colon mucosa of UC patients, as well as an augmented IL-5 and eotaxins mRNA expression, when compared to non-UC. Lastly we analyzed SAMP raised in germ free (GF) condition to see the microbiota effect on IL-33 expression and Th2 responses leading to chronic intestinal inflammation. We found a remarkable decrease in ileal IL-33 and Th2 cytokines mRNA expression as well as EOS infiltration in GF versus normal SAMP with comparable inflammatory scores. Moreover, EOS depletion in normal SAMP didn’t affect IL-33 mRNA expression. These data demonstrate a pathogenic role of IL-33-mediated eosinophilia in chronic intestinal inflammation, and that blockade of IL-33 and/or downstream EOS activation may represent a novel therapeutic modality to treat patients with IBD. Also they highlight the gut microbiota role in IL-33 production, and the following EOS infiltration in the intestinal mucosa, confirming that the microbiota is essential in mounting potent Th2 response leading to chronic ileitis in SAMP.

Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH-terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Lentivirus-mediated knockdown of Galectin-10 and Foxp3 in human immune cells

Die Suppression von autoreaktiven T-Zellen ist eine Funktion von CD4+CD25+ regulatorischen T-Zellen (CD4+CD25+ Tregs). CD4+CD25+ Tregs unterdrücken autoaggressive Immunantworten. Galectin-10 und Foxp3 sind wichtige Proteine, die an dem supprimierenden Mechanismus der Tregs beteiligt sind. Galectin-10 ist eines der ältesten bekannten humanen Proteine, die nicht in anderen Spezies gefunden worden sind. Foxp3 ist ein Transkriptionsfaktor, der in menschlichen CD4+CD25+ Tregs und in CD4+CD25- T-Effektor-Zellen nach Aktivierung exprimiert wird. Ein siRNA-vermittelter Knockdown dieses intrazellulären löslichen Proteins hebt die supprimierende Funktion der humanen CD4+CD25+ Tregs auf.rnDiese Arbeit beinhaltet in vitro durchgeführte Untersuchungen zur Ermöglichung eines Knockdown von Galectin-10 und/oder Foxp3 in humanisierten Mäusen. Es war möglich, ein Verfahren für die Produktion von lentiviralen Partikeln zu etablierten, die sich als effizientes Vehikel für den Gentransfer in humane Stammzellen und verschiedene Tumor- und Immunzellen erwiesen. Nach der Transduktion von AML14.3D10 Tumorzellen mit GFP-codierenden lentiviralen Partikeln konnte eine langfristige Expression von GFP erreicht werden. Außerdem war es möglich lentivirale Partikel zu erzeugen, die mit shRNA gegen Galectin-10 codiert waren. Die erzeugten Partikel erwiesen sich als funktionell, indem sie eine deutliche Herunterregulation von Galectin-10 in konstitutiv Galectin-10 exprimierenden AML14.3D10 Tumorzellen bewirkten. Unsere Studie präsentierte außerdem eine erstmalige Untersuchung zum Nachweis von Galectin-10-Protein in Eosinophilen aus humanen CD34+ hämatopoetischen Stammzellen (HSC). Diese stabile in vitro Galectin-10-Expression bietet ein alternatives Untersuchungsmodell zu CD4+CD25+ Tregs, die nicht aus CD34+ HSC differenziert werden können. Der zusätzliche Einbau des GFP-Gens in die mit shRNA gegen Galectin-10 codierende lentivirale Partikel war ein wichtiger Schritt zur Markierung von Zellen, die einen Galectin-10-Knockdown aufwiesen. Die neuen bicistronischen lentiviralen Partikel erwiesen sich sowohl in aus CD34+ HSC differenzierten Eosinophilen als auch in AML14.3D10 Zellen, die einen eosinophilen Phänotyp aufweisen, als funktionell. Schließlich konnte mit den bicistronischen lentiviralen Partikeln, die mit GFP und shRNA gegen Foxp3 codiert waren, eine Herunterregulation von Foxp3 in CD4+CD25- T-Effektor-Zellen erreicht werden, was erneut die erfolgreiche Herstellung von funktionellen lentiviralen Partikeln bewies.rn

Regulatory T cell-mediated suppression of Th9 cell development and effector function

T helper (Th) 9 cells are an important subpopulation of the CD4+ T helper cells. Due to their ability to secrete Interleukin-(IL-)9, Th9 cells essentially contribute to the expulsion of parasitic helminths from the intestinal tract but they play also an immunopathological role in the course of asthma. Recently, a beneficial function of Th9 cells in anti-tumor immune responses was published. In a murine melanoma tumor model Th9 cells were shown to enhance the anti-melanoma immune response via the recruitment of CD8+ T cells, dendritic cells and mast cells. In contrast to Th9 effector cells regulatory T cells (Tregs) are able to control an immune response with the aid of different suppressive mechanisms. Based on their ability to suppress an immune response Tregs are believed to be beneficial in asthma by diminishing excessive allergic reactions. However, concerning cancer they can have a detrimental function because Tregs inhibit an effective anti-tumor immune reaction. Thus, the analysis of Th9 suppression by Tregs is of central importance concerning the development of therapeutic strategies for the treatment of cancer and allergic diseases and was therefore the main objective of this PhD thesis.rnIn general it could be demonstrated that the development of Th9 cells can be inhibited by Tregs in vitro. The production of the lineage-specific cytokine IL-9 by developing Th9 cells was completely suppressed at a Treg/Th9 ratio of 1:1 on the transcriptional (qRT-PCR) as well as on the translational level (ELISA). In contrast, the expression of IRF4 that was found to strongly promote Th9 development was not reduced in the presence of Tregs, suggesting that IRF4 requires additional transcription factors to induce the differentiation of Th9 cells. In order to identify such factors, which regulate Th9 development and therefore represent potential targets for Treg-mediated suppressive mechanisms, a transcriptome analysis using “next-generation sequencing” was performed. The expression of some genes which were found to be up- or downregulated in Th9 cells in the presence of Tregs was validated with qRT-PCR. Time limitations prevented a detailed functional analysis of these candidate genes. Nevertheless, the analysis of the suppressive mechanisms revealed that Tregs probably suppress Th9 cells via the increase of the intracellular cAMP concentration. In contrast, IL-9 production by differentiated Th9 cells was only marginally affected by Tregs in vitro and in vivo analysis (asthma, melanoma model). Hence, Tregs represent very effective inhibitors of Th9 development whereas they have only a minimal suppressive influence on differentiated Th9 cells.rn

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Portal levels of latent transforming growth factor-β are related to liver function in patients with liver cirrhosis

Transforming growth factor-β1 (TGFβ1) is a short-lived immune suppressive and profibrotic protein. Its latent precursor is relatively stable and may even protect from fibrosis. Latent TGFβ1 is synthesized by various tissues including the liver and portal, hepatic, and systemic concentrations of latent TGFβ1 were determined in patients with liver cirrhosis and patients with normal liver function to find out whether circulating levels are affected by liver disease.

Multiple drug hypersensitivity: normal Treg cell function but enhanced in vivo activation of drug-specific T cells

Up to 10% of patients with severe immune-mediated drug hypersensitivity reactions have tendencies to develop multiple drug hypersensitivities (MDH). The reason why certain individuals develop MDH and the underlying pathomechanism are unclear. We investigated different T cell subpopulations in MDH patients and compared them with patients allergic to a single drug and with healthy controls (HC).

Immune Activation Reduces Sperm Quality in the Great Tit

Mounting an immune response against pathogens incurs costs to organisms by its effects on important life-history traits, such as reproductive investment and survival. As shown recently, immune activation produces large amounts of reactive species and is suggested to induce oxidative stress. Sperm are highly susceptible to oxidative stress, which can negatively impact sperm function and ultimately male fertilizing efficiency. Here we address the question as to whether mounting an immune response affects sperm quality through the damaging effects of oxidative stress. It has been demonstrated recently in birds that carotenoid-based ornaments can be reliable signals of a male's ability to protect sperm from oxidative damage. In a full-factorial design, we immune-challenged great tit males while simultaneously increasing their vitamin E availability, and assessed the effect on sperm quality and oxidative damage. We conducted this experiment in a natural population and tested the males' response to the experimental treatment in relation to their carotenoid-based breast coloration, a condition-dependent trait. Immune activation induced a steeper decline in sperm swimming velocity, thus highlighting the potential costs of an induced immune response on sperm competitive ability and fertilizing efficiency. We found sperm oxidative damage to be negatively correlated with sperm swimming velocity. However, blood resistance to a free-radical attack (a measure of somatic antioxidant capacity) as well as plasma and sperm levels of oxidative damage (lipid peroxidation) remained unaffected, thus suggesting that the observed effect did not arise through oxidative stress. Towards the end of their breeding cycle, swimming velocity of sperm of more intensely colored males was higher, which has important implications for the evolution of mate choice and multiple mating in females because females may accrue both direct and indirect benefits by mating with males having better quality sperm.

Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo

Proteases of Staphylococcus aureus have long been considered to function as important virulence factors, although direct evidence of the role of particular enzymes remains incomplete and elusive. Here, we sought to provide a collective view of the prevalence of extracellular protease genes in genomes of commensal and pathogenic strains of S. aureus and their expression in the course of human and mouse infection. Data on V8 protease, staphopains A and B, aureolysin, and the recently described and poorly characterized group of six Spl proteases are provided. A phylogenetically diverse collection of 167 clinical isolates was analyzed, resulting in the comprehensive genetic survey of the prevalence of protease-encoding genes. No correlation between identified gene patterns with specific infections was established. Humoral response against the proteases of interest was examined in the sera derived from human patients and from a model mouse infection. The analysis suggests that at least some, if not all, tested proteases are expressed and secreted during the course of infection. Overall, the results presented in this study support the hypothesis that the secretory proteases as a group may contribute to the virulence of S. aureus.

DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses

To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.