Periodic Reporting for period 1 - ZF-ILC (Characterization of zebrafish innate lymphoid cells and IL-22)
Période du rapport: 2017-09-01 au 2019-08-31
The gastrointestinal system represents one of the largest surfaces continuously exposed to elements from the outside world such as dietary nutrients and several mutualistic as well as pathogenic microorganisms. This constant exposure represents a unique challenge for the mucosal immune network, which remains tolerant of commensal bacteria while at the same time being able to effectively respond to pathogens. Within the intestines there are three main compartments: the epithelial layer, which forms a barrier to the outside environment; the immune system, which protects the body from invading organisms; and the microorganisms that live in our intestines, known as the gut microbiota. Disruption of the composition of any of these compartments or inappropriate interactions between them can provoke diseases that impair the function of the gut and affect many other organs. These abnormalities can lead to severe diseases such as diabetes, obesity inflammatory bowel disease (IBD) and intestinal cancer. These diseases constitute an important public health and economic issue. Thus, understanding the mechanisms regulating the composition of and interaction between these 3 elements is a challenge that, once comprehended, promises an immense improvement for human health.
Innate lymphoid cells (ILCs) are the most recently discovered constituent of the immune system and have been shown to be crucial mediators of intestinal immune responses and epithelial homeostasis. Most functions of ILCs are achieved via secretion of signals that target several cell types including epithelial cells. One important signal produced by ILCs is the cytokine interleukin-22 (IL-22), which has been shown to safeguard intestinal epithelial integrity during inflammation. ILCs have been only described in mammals, being the mouse the main experimental animal model used so far. Although important advances on the development and function of these cells have been made, it is not fully understood how they are integrated in the complex immune network, the role of ILC subtypes in different diseases, and details about their appearance in the intestine during early life.
The zebrafish is an emerging experimental animal model that offers important advantages such as ex utero and rapid development, transparency, large progeny, and simple genetic manipulation, while at the same time having 84% of genes associated with human disease. We aimed to identify the existence and diversity of ILCs in zebrafish and study in detail the cellular sources and function of zebrafish IL-22, with special focus on early developmental stages.
Innate lymphoid cells (ILCs) are the most recently discovered constituent of the immune system and have been shown to be crucial mediators of intestinal immune responses and epithelial homeostasis. Most functions of ILCs are achieved via secretion of signals that target several cell types including epithelial cells. One important signal produced by ILCs is the cytokine interleukin-22 (IL-22), which has been shown to safeguard intestinal epithelial integrity during inflammation. ILCs have been only described in mammals, being the mouse the main experimental animal model used so far. Although important advances on the development and function of these cells have been made, it is not fully understood how they are integrated in the complex immune network, the role of ILC subtypes in different diseases, and details about their appearance in the intestine during early life.
The zebrafish is an emerging experimental animal model that offers important advantages such as ex utero and rapid development, transparency, large progeny, and simple genetic manipulation, while at the same time having 84% of genes associated with human disease. We aimed to identify the existence and diversity of ILCs in zebrafish and study in detail the cellular sources and function of zebrafish IL-22, with special focus on early developmental stages.
In order to identify ILCs in zebrafish we established several new tools. We implemented methods to study intestinal lymphocytes and established inflammatory assays that activate different types of immune responses. We revealed evolutionary conservation of specific inflammatory responses against different types of pathogens between fish and mammals. These assays were then applied to zebrafish lines labeling lymphocytes in vivo, allowing purification of putative ILCs after stimulation of fish. We combined these assays with single-cell RNA sequencing allowing us to prove the existence and diversity of zebrafish ILCs, which showed high similarity to human ILCs. This work has been published in an international high impact scientific journal (Hernandez et al, Science Immunol, 2018) and has been presented in several conferences including the 2018 ILC meeting held in Tokyo; the 2017 Symposium of Innovative Models in Immunity and Infection, Lille, France; the 1st (2016) and 2nd (2019) Young Scientists Symposium in Mucosal Biology, University of Bern, Switzerland; and in seminars in Institutions such as Karolinska Institutet, the Charité Universitätsmedizin Berlin, the University of Chile, the Francis Crick Institute UK, and in several scientific meetings within France.
Understanding how the different members of the immune system work together to protect us against disease, and how deregulated immune responses lead to pathology have a strong impact in public health. Intense research over the last years has increased the understanding of the genetic as well as environmental factors determining the development and function of the different ILC subsets. The important role of ILCs at the initiation of immune response offers the possibility of new approaches for immunotherapy, in particular targeting pathologies at mucosal surfaces. For instance, by enhancing the function of specific ILC subsets participating in type 1 immunity to viruses, intracellular bacteria and tumors, type 2 immunity to helminths, also promoting tissue repair and regulating metabolism of adipose tissue, or type 3 immunity to extracellular microbes could be strengthened. Various already available drugs that target inducer or effector cytokines of type 1, 2 or 3 immune responses such as recombinant IFN-γ, GM-CSF and IL-22 proteins as well as neutralizing antibodies against most effector cytokines could be used locally and early during immune responses to primarily affect ILCs or block their activity. However, the principal challenge is the identification of specific activators and suppressors of the different ILC subsets and compounds avoiding systemic deregulation of immune function during homeostasis and disease. In this regard, characterization of zebrafish ILCs will allow exploiting the advantages of this model (small size, cheap maintenance, large offspring, fast grow rate, transparency of embryos and ex utero development) to perform screening for molecules, genes and gene regulatory regions controlling ILC function. In addition the simplicity of targeted genome editing using the CRISPR/Cas9 system in zebrafish will make possible to quickly generate zebrafish lines affecting ILC function.
Studies about the pathogenesis of numerous diseases indicate that enhanced antimicrobial defense, resistance against damage and tissue regeneration might be beneficial in several disorders. In this regard, it has been proposed that reinforcement of the activity of IL-22 might have a favorable impact in inflammatory bowel disease, graft‐versus‐host disease (GvHD), pancreas and alcoholic liver injury, and organ transplantation. On the other hand, attenuation of IL-22 action would be beneficial in psoriasis and certain cancers, where differentiation, proliferation and inflammation play a detrimental role. As IL‐22 does not target immune cells, its modulation might not lead to immune‐related side effects occurring when targeting cytokines such as type I interferons (IFNs) or tumor necrosis factors (TNFs). Therefore, producing short and long term modulators of IL-22 function, which could be used locally or systemically depending on the pathological context, will be beneficial for the treatment of several diseases. In this regard, we are currently collaborating to use our il22:mCherry reporter zebrafish line to perform screening of genes and molecules modulating intestinal inflammation and to study IBD-risk genes with unknown function.
Studies about the pathogenesis of numerous diseases indicate that enhanced antimicrobial defense, resistance against damage and tissue regeneration might be beneficial in several disorders. In this regard, it has been proposed that reinforcement of the activity of IL-22 might have a favorable impact in inflammatory bowel disease, graft‐versus‐host disease (GvHD), pancreas and alcoholic liver injury, and organ transplantation. On the other hand, attenuation of IL-22 action would be beneficial in psoriasis and certain cancers, where differentiation, proliferation and inflammation play a detrimental role. As IL‐22 does not target immune cells, its modulation might not lead to immune‐related side effects occurring when targeting cytokines such as type I interferons (IFNs) or tumor necrosis factors (TNFs). Therefore, producing short and long term modulators of IL-22 function, which could be used locally or systemically depending on the pathological context, will be beneficial for the treatment of several diseases. In this regard, we are currently collaborating to use our il22:mCherry reporter zebrafish line to perform screening of genes and molecules modulating intestinal inflammation and to study IBD-risk genes with unknown function.