TNFα Impairs Rhabdoviral Clearance by Inhibiting the Host Autophagic Antiviral Response.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections

Caracterización funcional de los receptores de TNF de pez cebra : Functional characterization of TNF receptors in cebrafish.

OBJETIVOS 1. Caracterización de los receptores de Tnf (Tnfr1 y Tnfr2) en la homeostasis vascular durante el desarrollo en pez cebra. 2. Caracterización de las rutas de señalización de Tnfr1 y Tnfr2 involucradas en el desarrollo de las células endoteliales y su homeostasis. 3. Caracterización del papel que juegan Tnfrs y sus ligandos (Tnfa y Lta) en la ola primitiva de hematopoyesis en el pez cebra. 4. Caracterización de los Tnfrs y sus ligandos (Tnfa y Lta) en la especificación y mantenimiento de las HSCs en el embrión de pez cebra. METODOLOGÍA Para la presente tesis doctoral, se ha utilizado el pez cebra (Danio rerio) como modelo de experimentación animal. Además, para la experimentación in vitro se han usado varias líneas celulares y técnicas de cultivo celulares. En cuanto a las técnicas de biología molecular, podemos destacar la microinyección en estadio de huevo de RNAm, morfolinos y DNA; RT-qPCR e hibridación in situ. También se han usado técnicas de microscopía tales como la microscopía confocal y de fluorescencia; técnicas inmunohistoquímicas y TUNEL para la detección de apoptosis; y técnicas de citometría y aislamiento físico de células mediante fluorescencia (FACS). RESULTADOS Durante la presente tesis doctoral, hemos caracterizado el papel que desempeñan el factor de necrosis tumoral (Tnfa) y sus receptores (receptores del factor de necrosis tumoral, Tnfrs) en el desarrollo de las células endoteliales y del sistema hematopoyético. Para ello hemos usado el pez cebra (Danio rerio), como modelo animal de vertebrados. Por un lado, nuestros resultados nos han llevado a concluir que la deficiencia en el gen de tnfr2 en embriones de pez cebra conlleva la inducción en células endoteliales de un programa apoptótico que depende de caspasa-8, caspasa-2 y P53, pero no caspasa-3. Además, la deficiencia simultánea de Tnfr1 o la activación de NF-ĸB, rescata la apoptosis de las células endoteliales, lo que indica que debe haber un equilibrio entre los dos receptores de Tnf para la correcta homeostasis vascular de este tipo celular. De forma similar, el Tnfa promueve la apoptosis de las células endoteliales humanas a través de Tnfr1, desencadenando la activación de caspasa-2 y P53. Por otro lado, en este trabajo demostramos que la señalización de Tnfa a través de Tnfr2 se necesita de forma intrínseca por las células madre hematopoyéticas (HSCs) para su mantenimiento y expansión. La deficiencia genética de Tnfa o Tnfr2, pero no de linfotoxina (Lta) o Tnfr1, conlleva la apoptosis de las HSCs cuando se especifican en el embrión. CONCLUSIONES 1. El silenciamiento génico dirigido contra Tnfr2 resulta en la inducción de un programa apoptótico dependiente de caspasa-8 en células endoteliales. Esta apoptosis se rescata mediante la eliminación de Tnfr1, lo que indica que se requiere un balance adecuado entre los Tnfrs para la integridad de las células endoteliales y la homeostasis vascular. 2. En las células endoteliales, el Tnfr1 señaliza apoptosis a través de la formación del complejo II y la consecuente activación de caspasa-8 y caspasa-2, mientras que Tnfr2 señaliza supervivencia mediante el comple I y la activación de NF-ĸB. 3. El programa apoptótico inducido por Tnfr1, en el cual participa caspasa-8, caspasa-2 y P53, se encuentra conservado evolutivamente en células endoteliales humanas. 4. La señalización de Tnfrs es dispensable para la hematopoyesis primitiva en el embrión de pez cebra. 5. La inhibición génica de Tnfa o Tnfr2, pero no Tnfr1 o Lta, resulta en la apoptosis de las HSCs cuando éstas emergen de la aorta dorsal. 6. La señalización Tnfa/Tnfr2 se requiere de forma intrínseca para la supervivencia de las HSCs a partir del endotelio hemogénico. OBJECTIVES 1. Characterization of Tnfa receptors (Tnfr1 and Tnfr2) in vascular homeostasis during the zebrafish embryo development. 2. Characterization of the Tnfr1 and Tnfr2 signaling pathways involved in endothelial cell development and homeostasis. 3. Characterization of the role played by Tnfrs and their ligands (Tnfa and Lta) in the primitive wave of hematopoiesis in the zebrafish embryo. 4. Characterization of Tnfrs and their ligands (Tnfa and Lta) in HSCs specification and maintenance in the zebrafish embryo. METHODOLOGY In this Doctoral Thesis, zebrafish (Danio rerio) has been utilized as research animal model. In addition, several cell lines and tissue culture techniques have been used for the in vitro experimentation. Related to the molecular biology techniques utilized, the injection at one-cell stage with mRNA, morpholinos and DNA; RT-qPCR and in situ hybridization can be emphasized. Moreover, other techniques used have been used such as confocal microscopy and fluorescence microscopy; immunohystochemistry techniques and TUNEL for apoptosis detection; as well as cytometry-related assays as fluorescence activated cell sorting (FACS). RESULTS During this Doctoral Thesis, we have characterized the role of Tnfa and Tnf receptors (Tnfrs) during endothelial and hematopoiesis development in the embryo using zebrafish (Danio rerio) as a vertebrate model. Targeted gene knockdown of Tnfr2 in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of Tnfr1 or the activation of NF-ĸB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. Similarly, Tnfa promotes the apoptosis of human endothelial cells through Tnfr1 and triggers caspase-2 and P53 activation. On the other hand, we show that Tnfa signaling through Tnfr2 is intrinsically required for hematopoietic stem cells (HSCs) maintenance and expansion. The genetic inhibition of Tnfa or Tnfr2, but not of lymphotoxin α (Lta) or Tnfr1, results in the apoptosis of HSCs soon after their emergence. CONCLUSIONS 1. Target gene silencing of Tnfr2 results in the induction of a caspase-8-dependent apoptotic program in endothelial cells. This apoptosis can be rescued by depletion of Tnfr1, indicating that an appropriate signaling balance between both Tnfrs is required for endothelial cell integrity and vascular homeostasis. 2. In endothelial cells, Tnfr1 signals apoptosis through complex II formation and caspase-8 and caspase-2 activation, while Tnfr2 signals survival via complex I and NF-ĸB activation. 3. The apoptotic program induced by Tnfr1, which involves caspase-8, caspase-2 and P53, is evolutionary conserved in human endothelial cells. 4. Tnfrs signaling is dispensable for primitive hematopoiesis in the zebrafish embryo. 5. Genetic inhibition of Tnfa or Tnfr2, but not of Tnfr1 or Lta, results in HSC apoptosis soon after their emergence from the floor of the dorsal aorta. 6. Tnfa/Tnfr2 signaling is intrinsically required for HSC survival after their emergence from the hemogenic endothelium

Md1 and Rp105 regulate innate immunity and viral resistance in zebrafish

11 páginas, 7 figuras, 2 tablasTLR4 was the first TLR family member identified in mammals and is responsible for the activation of the immune response by bacterial LPS. Later, MD1 and RP105 were shown to form complexes that directly interact with the MD2-TLR4 complex, acting as physiological negative regulators of LPS signaling. Despite the general conservation of various TLR families from fish to mammals, several differences can be appreciated, such as the high tolerance of fish to LPS, the absence of the crucial accessory molecules Md2 and Cd14 for Tlr4 signaling in fish, the absence of Tlr4 in some fish species, and the confirmation that LPS does not signal through Tlr4 in zebrafish. The present study has identified the Rp105 and Md1 homologs in zebrafish, confirming (i) Rp105 and Tlr4 evolved from a common ancestor before the divergence between fish and tetrapods and (ii) the presence of Md1 in teleost fish and the lack of Md2, suggesting that the divergence of these accessory molecules occurred in the tetrapod lineage. Biochemical and functional studies indicate that Md1 binds both Rp105 and Tlr4 in zebrafish. Genetic inhibition of zebrafish Md1 and Rp105 reveals that Md1 or Rp105 deficiency impairs the expression of genes encoding pro-inflammatory and antiviral molecules, leading to increased susceptibility to viral infection. These results shed light on the evolutionary history of Md1 and Rp105 and uncover a previously unappreciated function of these molecules in the regulation of innate immunityThis work was supported by the Spanish Ministry of Science and Innovation (grants BIO2011-23400 and CSD2007-00002 to VM, and PhD fellowship to SC, all co-funded with Fondos Europeos de Desarrollo Regional/European Regional Development Funds), the Fundación Séneca-Murcia (PhD fellowship to RE-P), the European 7th Framework Initial Training Network FishForPharma (PhD fellowship to SDT, PITG-GA-2011-289209), and Fundação para a Ciência e Tecnologia (PhD fellowship to SdO, SFRH/BD/62674/2009)Peer reviewe

Proinflammatory Signaling Regulates Hematopoietic Stem Cell Emergence

Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNF? activates the Notch and NF-?B signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNF?, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system

A zebrafish model of granulin deficiency reveals essential roles in myeloid cell differentiation

Granulin is a pleiotropic protein involved in inflammation, wound healing, neurodegenerative disease, and tumorigenesis. These roles in human health have prompted research efforts to use granulin to treat rheumatoid arthritis and frontotemporal dementia and to enhance wound healing. But how granulin contributes to each of these diverse biological functions remains largely unknown. Here, we have uncovered a new role for granulin during myeloid cell differentiation. We have taken advantage of the tissue-specific segregation of the zebrafish granulin paralogues to assess the functional role of granulin in hematopoiesis without perturbing other tissues. By using our zebrafish model of granulin deficiency, we revealed that during normal and emergency myelopoiesis, myeloid progenitors are unable to terminally differentiate into neutrophils and macrophages in the absence of granulin a (grna), failing to express the myeloid-specific genes cebpa, rgs2, lyz, mpx, mpeg1, mfap4, and apoeb. Functionally, macrophages fail to recruit to the wound, resulting in abnormal healing. Our CUT&RUN experiments identify Pu.1, which together with Irf8, positively regulates grna expression. In vivo imaging and RNA sequencing experiments show that grna inhibits the expression of gata1, leading to the repression of the erythroid program. Importantly, we demonstrated functional conservation between the mammalian granulin and the zebrafish ortholog grna. Our findings uncover a previously unrecognized role for granulin during myeloid cell differentiation, which opens a new field of study that can potentially have an impact on different aspects of human health and expand the therapeutic options for treating myeloid disorders such as neutropenia or myeloid leukemia

Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells

All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/β-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes

Tnfr2 mediates the Tnfa-triggered susceptibility of zebrafish to SVCV.

<p>(A) Workflow of the experimental design followed in (B-E). Std (Control), Tnfr1 or Tnfr2 mos (B-D) or antisense or DN-Tnfr2 RNAs (E) were injected in zebrafish embryos at one-cell-stage of development. At 3 dpf, these larvae were immerse in RPMI containing inactivated SVCV (control) or intact SVCV for subsequently analysis of survival (B, E) or qPCR analysis at 48 hours post-infection (hpi) (C, D). Percentage of survival of Tnfr-depleted (B) and DN-Tnfr2 overexpressing (E) zebrafish larvae exposed to 10⁹ TCID₅₀/ml SVCV. (C,D) The mRNA levels of the gene coding for the SVCV N protein as an estimation of the viral replication (C), and the the RNA- levels of G protein (D) were determined in the infected larvae by qPCR in 10 pooled larvae at 48 hpi (5 dpf). The gene expression was normalized against <i>rps11</i> and multiplied by 10⁵ for N protein. Bars represents mean ± S.E.M. of triplicate readings from pooled larvae and the data are representative of two independent experiments. *p<0.1; ***p<0.001.</p

Tnfa inhibits autophagy.

<p>(A) ZF4 cells were treated with 0.1 μg of Tnfa or inactivated Tnfa (CTnfa), 1μM of Rapa, 10 mM of 3-methyladenine (3MA) or remain untreated. After 4 hours cells were fixed and then incubated with an antibody anti-LC3. Cells were finally stained with a fluorophore-conjugated secondary antibody (red fluorescence, LC3) and DAPI (blue, cell nuclei). The number of cells with LC3 puncta (n = 10) was determined (left panel). Bars represent mean ± S.E.M. Images are representative of the results obtained in 3 independent experiments (right panel). (B-D) Whole cell lysates were obtained from cells treated with Tnfa, CTnfa, Rapa or 3MA (B) or pre-treated with Rapa for 4 hours and then Tnfa or CTnfa was added for another 4 hours (C, D). LC3-I and LC3-II bands were visualized by WB using an anti-LC3 antibody and the protein content of the stained bands estimated by densitometry. The densitometry values were used to calculate LC3-II/ LC3-I ratios. Actin bands were detected as a protein load internal control using an anti-actin antibody. Data are shown as the mean±S.E.M. of 3 independent experiments. *p<0.05. **p<0.01.</p

Tnfa inhibits the autophagy in zebrafish larvae.

<p>(A) Workflow representing the experimental design. B) Zebrafish GFP-LC3 transgenic embryos were injected with Tnfa or Std mos at the one-cell-stage of development. After 48 hours, a group of larvae injected with Std-mo was immersed in a bath with 1 μM Rapa and was freshly added every 24 h. The remaining larvae were divided in two and challenged by bath immersion with 10⁹ TCID₅₀/ml SVCV SVCV or RPMI alone. After 72 hours of infection (5 dpf), larvae were collected, anesthetized with 0.16 mg/ml tricaine, mounted in 1% low melting point agarose supplemented with 0.16 mg/ml tricaine and images of the whole larvae taken using a Leica MZ16F fluorescence stereo microscope. Numbers in pictures represent the animals with the shown phenotype per total analyzed animals. (C) Zebrafish larvae were injected with morpholino (mo) Tnfa (Tnfa-MO) or Std (Std-mo) 1 hour post fertilization (hpf). After 36 hours, a group of larvae injected with Std-mo was immersed in a bath with Rapamycin and the remaining larvae (72 each group) were then divided in two and challenged by bath immersion with SVCV as above. After 48 hours of infection samples were recollected and LC3-I and LC3-II were detected by western-blot using an anti-LC3 antibody and the densitometry values were used to calculate LC3-II/ LC3-I ratios, represented as black bar graphs. Actin bands were detected as a protein load internal control using an anti-actin antibody. Data are shown as the mean±S.E.M. of 3 independent experiments. p<0.05.</p

Tnfa enhances SVCV infection in zebrafish.

<p>(A) Workflow of the experimental design followed in (B). Recombinant zebrafish Tnfa or interferon 1 (Ifn1) were added to ZF4 cells growing in monolayer at 80% confluence and incubated for 4 hours. Subsequently, the medium was washed out and fresh medium containing SVCV was added. After 24 hours of incubation with the virus, the cells were harvested for qPCR analysis. (B) N protein mRNA expression levels assessed by qPCR relative to the housekeeping gene <i>rps11</i> and multiplied by 10⁵. Bars represent mean ± S.E.M. of indicated gene expression from one representative experiment. (C) Workflow of the experimental design followed in (D-G). Std (Control) or Tnfa mos (D-F) or antisense or Tnfa RNAs (G) were injected in zebrafish embryos at one-cell-stage of development. At 3 dpf, these larvae were immerse in RPMI containing inactivated SVCV (control) or intact SVCV for subsequently analysis of survival (D, G) or qPCR analysis at 48 hours post-infection (hpi) (E, F). Percentage of survival of Tnfa-depleted (D) and overexpressing (G) zebrafish larvae exposed to 10⁹ TCID₅₀/ml SVCV. (E, F) The mRNA levels of the gene coding for the SVCV N protein as an estimation of the viral replication (E), and the RNA- levels of G protein (F) were determined in the infected larvae by qPCR in 10 pooled larvae at 48 hpi (5 dpf). The gene expression was normalized against <i>rps11</i> and multiplied by 10⁵ for N protein. Bars represent mean ± S.E.M. of triplicate readings from pooled larvae and the data are representative of two independent experiments. ***p<0.001. ND, not detected.</p