Final Report Summary - INFLAM (Regulation of Inflammation)
In an effort to extend the applicability of the zebrafish model for biomedical research related to inflammatory diseases we focused during the second and final period of this project on several aspects of zebrafish innate immunity as well as the generation of new tools.
Neutrophil granulocytes are pivotal cells within the first line of host defense of the innate immune system. We thus explored the proteome of primary marrow neutrophils from adult zebrafish. The identified proteins originated from all major cellular compartments. Gene ontology analysis revealed significant association of proteins with different immune-related network and pathway maps. 75% of proteins identified in neutrophils were identified as enriched in neutrophils when compared to neutrophil-free brain tissue. Moreover, cross-species comparison with human peripheral blood neutrophils showed partial conservation of immune-related proteins between human and zebrafish. To characterize the molecular changes on the protein level in neutrophils during sterile inflammation we established the chemically-induced inflammation assay (ChIn) in adult zebrafish and investigated the proteome dynamics within neutrophils of adult zebrafish upon inflammation. We identified 48 proteins that were differentially regulated during inflammation. Gene ontology analysis revealed that these proteins were associated with cell cycle, nitric oxide signalling, regulation of cytoskeleton rearrangement and intermediate filaments as well as immune-related processes such as antigen presentation, leukocyte chemotaxis and IL-6 signalling. Comparison of protein expression dynamics with transcript expression dynamics suggests the existence of regulatory mechanisms confined to the protein level for some genes. Our data represent the first analysis of protein dynamics in adult zebrafish neutrophils upon chemically-induced inflammation providing a valuable reference for future studies using zebrafish inflammation models.
A compounds screen using the ChIn assay resulted in a number of abti-inflammatory hits. One of the identified anti-inflammatory compounds was a nitric oxide synthase (NOS) 1 specific inhibitor. Follow-up experiments revealed that NO is a universal wound signal required for efficient initiation of inflammation in both, epithelial inflammation and neuroinflammation. NO serves as a calcium wave promoting or sustaining signal during inflammation and therefore can be considered as a feed-forward signal promoting the generation and/or release of calcium-dependent secondary mediators such as ATP.
Reactive oxygen species are critical mediators of inflammation. Therefore, high-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. We contributed to the generation of HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrated this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.
Neutrophil granulocytes are pivotal cells within the first line of host defense of the innate immune system. We thus explored the proteome of primary marrow neutrophils from adult zebrafish. The identified proteins originated from all major cellular compartments. Gene ontology analysis revealed significant association of proteins with different immune-related network and pathway maps. 75% of proteins identified in neutrophils were identified as enriched in neutrophils when compared to neutrophil-free brain tissue. Moreover, cross-species comparison with human peripheral blood neutrophils showed partial conservation of immune-related proteins between human and zebrafish. To characterize the molecular changes on the protein level in neutrophils during sterile inflammation we established the chemically-induced inflammation assay (ChIn) in adult zebrafish and investigated the proteome dynamics within neutrophils of adult zebrafish upon inflammation. We identified 48 proteins that were differentially regulated during inflammation. Gene ontology analysis revealed that these proteins were associated with cell cycle, nitric oxide signalling, regulation of cytoskeleton rearrangement and intermediate filaments as well as immune-related processes such as antigen presentation, leukocyte chemotaxis and IL-6 signalling. Comparison of protein expression dynamics with transcript expression dynamics suggests the existence of regulatory mechanisms confined to the protein level for some genes. Our data represent the first analysis of protein dynamics in adult zebrafish neutrophils upon chemically-induced inflammation providing a valuable reference for future studies using zebrafish inflammation models.
A compounds screen using the ChIn assay resulted in a number of abti-inflammatory hits. One of the identified anti-inflammatory compounds was a nitric oxide synthase (NOS) 1 specific inhibitor. Follow-up experiments revealed that NO is a universal wound signal required for efficient initiation of inflammation in both, epithelial inflammation and neuroinflammation. NO serves as a calcium wave promoting or sustaining signal during inflammation and therefore can be considered as a feed-forward signal promoting the generation and/or release of calcium-dependent secondary mediators such as ATP.
Reactive oxygen species are critical mediators of inflammation. Therefore, high-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. We contributed to the generation of HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrated this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.