Studying the Tumor Microenvironment in Zebrafish.

The tumor microenvironment significantly contributes to tumor initiation, progression, neo-angiogenesis, and metastasis, and a better understanding of the role of the different cellular players would facilitate the development of novel therapeutic strategies for cancer treatment. Towards this goal, intravital imaging is a powerful method to unravel interaction partners of tumor cells. Among vertebrate model organisms, zebrafish is uniquely suited for in vivo imaging studies. In recent years zebrafish has also become a valuable model in cancer research. In this chapter, we will summarize, how zebrafish has been used to characterize cells of the tumor microenvironment. We will cover both genetically engineered cancer models and xenograft models in zebrafish. The majority of work has been done on the role of innate immune cells and their role during tumor initiation and metastasis, but we will also cover studies focusing on adipocytes, fibroblasts, and endothelial cells. Taken together, we will highlight the versatile use of the zebrafish model for in vivo tumor microenvironment studies.

A pH-sensitive Macromolecular Prodrug as TLR7/8 Targeting Immune Response Modifier.

The chemical synthesis and biological activity of novel functionalized imidazoquinoline derivatives (ImQ) to generate Toll-like receptor (TLR) 7/8 specific prodrugs are presented. In vivo activity of ImQs to induce inflammation was confirmed in zebrafish larvae. After covalent ligation to fully biodegradable polyphosphazenes (ImQ-polymer), the macromolecular prodrugs were designed to undergo intracellular pH-sensitive release of ImQs to induce inflammation through binding to endosomal TLR7/8 (danger signal). We showed ImQ dissociation from prodrugs at a pH 5 pointing towards endosomal prodrug degradability. ImQ-polymers strongly activated ovalbumin-specific T cells in murine splenocytes as shown by increased proliferation and expression of the IL-2 receptor (CD25) on CD8+ T cells accompanied by strong IFN-γ release. ImQ prodrugs presented here are suggested to form the basis of novel nanovaccines, for example, for intravenous or intratumoral cancer immunotherapeutic applications to trigger physiological antitumor immune responses.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Non-destructive characterization of adult zebrafish models using Jones matrix optical coherence tomography.

The zebrafish is a valuable vertebrate animal model in pre-clinical cancer research. A Jones matrix optical coherence tomography (JM-OCT) prototype operating at 1310 nm and an intensity-based spectral-domain OCT setup at 840 nm were utilized to investigate adult wildtype and a tumor-developing zebrafish model. Various anatomical features were characterized based on their inherent scattering and polarization signature. A motorized translation stage in combination with the JM-OCT prototype enabled large field-of-view imaging to investigate adult zebrafish in a non-destructive way. The diseased animals exhibited tumor-related abnormalities in the brain and near the eye region. The scatter intensity, the attenuation coefficients and local polarization parameters such as the birefringence and the degree of polarization uniformity were analyzed to quantify differences in tumor versus control regions. The proof-of-concept study in a limited number of animals revealed a significant decrease in birefringence in tumors found in the brain and near the eye compared to control regions. The presented work showed the potential of OCT and JM-OCT as non-destructive, high-resolution, and real-time imaging modalities for pre-clinical research based on zebrafish.

Human rhinoviruses induce IL-35-producing Treg via induction of B7-H1 (CD274) and sialoadhesin (CD169) on DC.

IL-35 is a heterodimer of EBV-induced gene 3 and of the p35 subunit of IL-12, and recently identified as an inhibitory cytokine produced by natural Treg in mice, but not in humans. Here we demonstrate that DC activated by human rhinoviruses (R-DC) induce IL-35 production and release, as well as a suppressor function in CD4(+) and CD8(+) T cells derived from human peripheral blood but not in naïve T cells from cord blood. The induction of IL-35-producing T cells by R-DC was FOXP3-independent, but blocking of B7-H1 (CD274) and sialoadhesin (CD169) on R-DC with mAb against both receptors prevented the induction of IL-35. Thus, the combinatorial signal delivered by R-DC to T cells via B7-H1 and sialoadhesin is crucial for the induction of human IL-35(+) Treg. These results demonstrate a novel pathway and its components for the induction of immune-inhibitory T cells.

Epigenetic regulation of dendritic cell differentiation and function by oxidized phospholipids.

Dendritic cells (DCs) are the key cell type in the regulation of an adaptive immune response. Under inflammatory conditions monocytes can give rise to immunostimulatory DCs, depending on microenvironmental stimuli. Here we show that oxidized phospholipids (Ox-Pls), which are generated during inflammatory reactions, dysregulate the differentiation of DCs. DCs generated in the presence of Ox-Pls up-regulated the typical DC marker DC-SIGN but did not express CD1a, CD1b, and CD1c. These DCs generated in the presence of Ox-Pls had a substantially diminished T cell-stimulating capacity after stimulation with Toll-like receptor ligands. Toll-like receptor ligand-induced production of interleukin-12 also was strongly diminished, whereas induction of CD83 was not altered. In addition, we found that Ox-Pls strongly inhibit inflammatory stimuli-induced phosphorylation of histone H3, a key step of interleukin-12 production, yet leaving activation of nuclear factor-kappaB unaltered. Taken together, Ox-Pls present during differentiation yielded DCs with a reduced capacity to become immunostimulatory mature DCs. Furthermore, the presence of Ox-Pls blocked histone modifications required for full activation of DCs. Therefore, inflammation-derived Ox-Pls control DC functions in part by epigenetic mechanisms.

The ssRNA genome of human rhinovirus induces a type I IFN response but fails to induce maturation in human monocyte-derived dendritic cells.

Dendritic cells (DCs) use pattern recognition receptors to sense invading viruses and triggering of these receptors induces a maturation program. Human rhinoviruses (HRVs) belong to the family of Picornaviridae, which have a single-stranded, coding RNA genome. Because HRV does not replicate in DCs, we used genomic RNA from HRV in this study to analyze the impact of natural occurring viral ssRNA on DC function. We found that transfection of human monocyte-derived DCs with viral ssRNA induced type I IFN production but failed to activate the NF-kappaB pathway in DCs. In line with this observation, the up-regulation of typical maturation markers such as CD83 or the production of the proinflammatory cytokines IL-12p40, IL-6, and TNF-alpha was not detectable. Most importantly, the T cell stimulatory capacity of viral ssRNA-treated DCs was not enhanced and remained at the level of immature DCs. Taken together, our results demonstrate that viral ssRNA efficiently activates the innate defense arm of DCs, whereas it is insufficient to activate the stimulatory capacity of DCs for the adaptive defense responses.

The cytoplasmic tail of CD45 is released from activated phagocytes and can act as an inhibitory messenger for T cells.

CD45 is the prototypic transmembrane protein tyrosine phosphatase (PTP), which is expressed on all nucleated hematopoietic cells and plays a central role in the integration of environmental signals into immune cell responses. Here we report an alternative function for the intracellular domain of CD45. We dis-covered that CD45 is sequentially cleaved by serine/metalloproteinases and gamma-secretases during activation of human monocytes and granulocytes by fungal stimuli or phorbol 12-myristate 13-acetate but not by other microbial stimuli. Proteolytic processing of CD45 occurred upon activation of monocytes or granulocytes but not of T cells, B cells, or dendritic cells and resulted in a 95-kDa fragment of the cytoplasmic tail of CD45 (ct-CD45). ct-CD45 was released from monocytes and granulocytes upon activation-induced cell death. Binding studies with ct-CD45 revealed a counter-receptor on preactivated T cells. Moreover, T-cell proliferation induced by dendritic cells or CD3 antibodies was inhibited in the presence of ct-CD45. Taken together, the results of our study demonstrate that fragments of the intracellular domain of CD45 from human phagocytes can function as intercellular regulators of T-cell activation.

The oxidation state of phospholipids controls the oxidative burst in neutrophil granulocytes.

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.

Quo natas, Danio?-Recent Progress in Modeling Cancer in Zebrafish.

Over the last decade, zebrafish has proven to be a powerful model in cancer research. Zebrafish form tumors that histologically and genetically resemble human cancers. The live imaging and cost-effective compound screening possible with zebrafish especially complement classic mouse cancer models. Here, we report recent progress in the field, including genetically engineered zebrafish cancer models, xenotransplantation of human cancer cells into zebrafish, promising approaches toward live investigation of the tumor microenvironment, and identification of therapeutic strategies by performing compound screens on zebrafish cancer models. Given the recent advances in genome editing, personalized zebrafish cancer models are now a realistic possibility. In addition, ongoing automation will soon allow high-throughput compound screening using zebrafish cancer models to be part of preclinical precision medicine approaches.

Engagement of ICAM-1 by major group rhinoviruses activates the LFA-1/ICAM-3 cell adhesion pathway in mononuclear phagocytes.

Cell-cell interactions are critical at key points of immune responses and are mediated by a complex array of adhesion receptors. One of the most important adhesion molecules on leukocytes is intercellular adhesion molecule 1 (ICAM-1, CD54). Here we demonstrate that engagement of ICAM-1 with human major group rhinoviruses (HRV) enhances adhesiveness and homotypic aggregation of human monocytes and monocyte-derived dendritic cells (DC). Cluster formation upon engagement of ICAM-1 with HRV14 represents an active process. It is temperature and energy dependent, requires divalent cations, an intact cytoskeleton and protein de novo synthesis. Homotypic interaction between monocytes induced by HRV14 can be inhibited with blocking mAbs against LFA-1 (CD11a/CD18) and ICAM-3 (CD50) as well as with a mAb against the first immunoglobulin (Ig)-domain of PECAM-1 (CD31). Induction of enhanced cytoadhesiveness by HRV14 was not accompanied with an upregulation of LFA-1, ICAM-3 or PECAM-1 expression. Binding studies with recombinant PECAM-1 proteins indicated, however, that monocyte clustering upon engagement of ICAM-1 with HRV was accompanied with increased homophilic PECAM-1 interactions. Taken together the results of our study demonstrate that signalling via ICAM-1 induces adhesiveness of mononuclear phagocytes, which critically involves PECAM-1 and is mediated via LFA-1/ICAM-3.

A detailed proteomic profiling of plasma membrane from zebrafish brain.

Zebrafish (Danio rerio) is a well-established model organism in developmental biology and disease modeling. In recent years, an increasing amount of studies used zebrafish to analyze the genetic changes underlying various neurological disorders. The brain plasma membrane proteome represents the major subsets of signaling proteins and promising drug targets, but is often understudied due to traditional experimental difficulties including problems with solubility, detergent removal, or low abundance. Here, we report a comprehensive dataset of the proteins identified in the enriched plasma membrane of the zebrafish brain by applying sequential trypsin/chymotrypsin digestion with multidimensional LC-MS/MS. A total number of 97 017 peptide groups corresponding to 9201 proteins were identified. These were annotated in various molecular functions or neurological disorders. The dataset of the current study provides a useful data source for further utilizing zebrafish in basic and clinical neuroscience.

Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model.

Patients with inflammatory bowel disease (IBD) have an increased risk of colon cancer. However, the immune cells and cytokines that mediate the transition from intestinal inflammation to cancer are poorly understood. We show that bacteria-induced colon cancer is accompanied by differential accumulation of IL-17(+)IL-22(+) colonic innate lymphoid cells (cILCs), which are phenotypically distinct from LTi and NK-22 cells, and that their depletion in mice with dysplastic inflammation blocks the development of invasive colon cancer. Analysis of the functional role of distinct Type 17 cytokines shows that although blockade of IL-17 inhibits some parameters of intestinal inflammation, reduction in dysplasia and colorectal cancer (CRC) requires neutralization of IL-22 indicating a unique role for IL-22 in the maintenance of cancer in this model. Mechanistic analyses showed that IL-22 selectively acts on epithelial cells to induce Stat3 phosphorylation and proliferation. Importantly, we could detect IL-22(+)CD3(+) and IL-22(+)CD3(−) cells in human CRC. Our results describe a new activity of IL-22 in the colon as a nonredundant mediator of the inflammatory cascade required for perpetuation of CRC, highlighting the IL-22 axis as a novel therapeutic target in colon cancer.

Identification of a genetic locus controlling bacteria-driven colitis and associated cancer through effects on innate inflammation.

Chronic inflammation of the intestine has been associated with an elevated risk of developing colorectal cancer. Recent association studies have highlighted the role of genetic predisposition in the etiology of colitis and started to unravel its complexity. However, the genetic factors influencing the progression from colon inflammation to tumorigenesis are not known. We report the identification of a genetic interval Hiccs that regulates Helicobacter hepaticus-induced colitis and associated cancer susceptibility in a 129.RAG(-/-) mouse model. The 1.7-Mb congenic interval on chromosome 3, containing eight genes and five microRNAs, renders susceptible mice resistant to colitis and reduces tumor incidence and multiplicity. Bone marrow chimera experiments showed that resistance is conferred by the hematopoietic compartment. Moreover, the Hiccs locus controls the induction of the innate inflammatory response by regulating cytokine expression and granulocyte recruitment by Thy1(+) innate lymphoid cells. Using a tumor-promoting model combining chronic Helicobacter hepaticus infection and the carcinogen azoxymethane, we found that Hiccs also regulates the frequency of colitis-associated neoplasia. Our study highlights the importance of innate immune cells and their genetic configuration in driving progression from inflammation toward cancer and opens the door for analysis of these pathways in human inflammatory disorders and associated cancers.

Oxidized phospholipids induce anergy in human peripheral blood T cells.

Lipids are key regulators of immune responses. In this study we investigated the direct impact of oxidized phospholipids (ox-PL) on T cell activation and function. We could demonstrate that ox-PL strongly inhibit proliferation of purified human T cells induced with anti-CD3/CD28 or anti-CD3/CD63 mAb, whereas proliferation of naive T cells from human cord blood was not affected by ox-PL. Unoxidized phospholipids showed no such effect. Inhibition of T cell proliferation by ox-PL was not due to cell death. Moreover, T cell proliferation triggered by PMA/ionomycin activation was not diminished by ox-PL. T cells activated in the presence of ox-PL produced and released low amounts of IFN-gamma and IL-2, whereas IL-4 was only slightly diminished. Ox-PL prevented the expression of de novo synthesized activation markers (CD25, MHC class II) but not expression of CD63 or CD69. We further observed that T cells stimulated in the presence of ox-PL are poorly cytotoxic T cells. Most importantly, T cells activated in the presence of ox-PL failed to proliferate in response to restimulation. This hypo-proliferative state was accompanied with an up-regulation of early growth response gene 3 and Casitas B-lineage lymphoma protein B. Taken together, our results demonstrate that ox-PL are potent and specific regulators of T cell activation and function.

Modulation of the immune system by human rhinoviruses.

Human rhinoviruses (HRV) are the major cause of the common cold, one of the most frequent infectious diseases in humans. Though HRV infections of the upper respiratory tract are usually rather harmless, there is increasing evidence that HRV sets the stage for more dangerous pathogens, elicits asthmatic exacerbations, severe diseases in the lower respiratory tract and even autoimmunity. The pathogenic mechanisms of HRV infections leading to such complications are still poorly understood. It is a common strategy of pathogens to manipulate our immune system in order to evade an efficient immune response. A major characteristic of HRV is a high degree of species specificity. Thus, analyzing the potential immune evasion mechanisms used by HRV will be helpful for a better understanding of the pathogenesis of the common cold and may contribute to a better understanding of the human immune system as well. In this review we want to illuminate what is known about potential immune escape mechanisms used by HRV and discuss how such disturbances might lead to a suppressed and dysregulated immune competence in man.

Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40.

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression.

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.