Macrophages and Development of Immunity - CNRS-URA 2578  

  HEADDr Philippe HERBOMEL /
TAUZIN Muriel, PhD student / LE GUYADER Dorothée, PhD student / DEMY Doris-Lou, PhD student
FRETAUD Maxence, ingénieur / BRIOLAT Valérie, technician / VIVIER Catherine, technician
ALEXANDRE Jacqueline

  Annual Report

We focus on the successive emergence and functional traits of the first innate immune cell types in vertebrate development, using the assets of a small tropical freshwater fish, the zebrafish, especially the full transparency of its embryos and swimming larvae, which allows us to image cell behaviours in vivo at high resolution in a totally non invasive manner. This also led us to investigate the transition from primitive to definitive hematopoiesis, including the origin and migrations of hematopoietic stem cells, using in vivo cell tracing tools unavailable in mammals. Finally we use the zebrafish model to study innate responses to infections in the live animal at high resolution.

Macrophages, granulocytes, and developmental hematopoiesis

In all vertebrate embryos, the first leukocytes to arise are the 'primitive macrophages', which we showed to originate from a distinct anterior mesoderm territory adjacent to the cardiac field. These cells differentiate in the yolk sac, then quickly invade the still unvascularized embryonic tissues, notably the brain and retina where they become primitive microglia. Neutrophilic granulocytes - the other main type of phagocytes involved in innate immunity - appear a day later in development, throughout the embryo, surprisingly barely in the blood, but mostly wandering in the mesenchyme and epidermis, like the macrophages. Using an in vivo photoactivatable cell tracer, we found that these granulocytes arise from two origins: i) the progenitors of the primitive macrophages, which we thus discovered to be bipotent; ii) a previously undescribed caudal hematopoietic tissue (CHT), which we found to be a transitional site for the homing, expansion and differentiation of definitive hematopoietic stem cells (HSCs), before their migration to the final hematopoietic organs (the thymus and kidney in fish). More recently we could image and analyze in real-time the emergence of HSCs in the embryo, and the subsequent routes of their progeny to colonize the nascent thymus.

Within the european Integrated Project "Zebrafish Models for Human Development and Disease", we performed two large-scale screens for mutants affected in macrophage or granulocyte differentiation, or their capacity to colonize tissues. We found many, and are now analyzing further the phenotypes and associated mutations of the most promising ones.

Innate host responses to infections (J.-P. Levraud, E. Colucci-Guyon)

We also study in vivo the respective roles and behaviour of macrophages and granulocytes in fighting bacterial infections, and found that they differ widely depending on the particular tissue microenvironment where the infection occurs.

Mycobacterium marinum, the agent of fish tuberculosis, is very close to its human counterpart M. tuberculosis. The complex glycolipids of the cell wall of mycobacteria are thought to be a major factor of their pathogenicity. We inject these components purified from M. marinum in zebrafish larvae, either as such or coupled to fluorescent beads, and assess their effects on phagocytosis, cell recruitment, granuloma formation, or induction of host genes (collaboration with L. Kremer, CNRS Montpellier, and Y. Guérardel, CNRS Lille).

Finally, we set up an experimental system of viral infection of zebrafish embryos, which makes it possible to exploit antisense (morpholino) technology to assess the anti-viral effects of any virus-induced host gene (collaboration with G. Lutfalla, CNRS Montpellier and P. Boudinot, INRA Jouy-en-Josas). This already allowed us to identify functionally the two chains of the fish interferon (IFN) receptor among all class II helical cytokine receptor genes, which led us to conclude that the ancestral innate IFN system is neither IFN-α nor -ß, but IFN lambda.

Keywords: development, macrophages, granulocytes, hematopoiesis, stem cells, cell migration, zebrafish, in vivo imaging, infection, viral infection, interferon


Figure: A wild-type fish larva has Sudan Black stained granulocytes (black dots) dispersed in the tissues; a NL144 mutant larva has none.


Murayama E., Kissa K., Zapata A., Mordelet E., Briolat V., Lin HF., Handin R.I., Herbomel P. (2006). Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity 25, 963-975. PMID: 17157041

Levraud JP, Boudinot P, Colin I, Benmansour A, Peyrieras N, Herbomel P., Lutfalla G. (2007). Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system. J. Immunol. 178, 4385-4394. PMID: 17371995

Levraud J.P., Colucci-Guyon E., Redd M.J., Lutfalla G., Herbomel P. (2008) In vivo analysis of zebrafish innate immunity. Methods Mol. Biol. 415, 337-64. PMID: 18370164

Le Guyader D, Redd MJ, Colucci-Guyon E, Murayama E, Kissa K, Briolat V, Mordelet E, Zapata A, Shinomiya H, Herbomel P. (2007) Origins and unconventional behavior of neutrophils in developing zebrafish. Blood111, 132-141. PMID: 17875807

Kissa K., Murayama E., Zapata A, Cortes A, Perret E, Machu C, Herbomel P. (2007) Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood 111, 1147-1156. PMID: 17934068

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