Development and Homeostasis of Mucosal Tissues

Publications de l’équipe

Année de publication : 2019

Maximo Coronado, Camila J Solis, Pedro P Hernandez, Carmen G Feijóo (2019 Apr 20)

Soybean Meal-Induced Intestinal Inflammation in Zebrafish Is T Cell-Dependent and Has a Th17 Cytokine Profile.

Frontiers in immunology : 610 : DOI : 10.3389/fimmu.2019.00610 En savoir plus

Currently, inflammatory bowel disease (IBD) is a serious public health problem on the rise worldwide. In this work, we utilized the zebrafish to introduce a new model of intestinal inflammation triggered by food intake. Taking advantage of the translucency of the larvae and the availability of transgenic zebrafish lines with fluorescently labeled macrophages, neutrophils, or lymphocytes, we studied the behavior of these cell types during the course of inflammation. We established two feeding strategies, the first using fish that were not previously exposed to food (naïve strategy) and the second in which fish were initially exposed to normal food (developed strategy). In both strategies, we analyzed the effect of subsequent intake of a control or a soybean meal diet. Our results showed increased numbers of innate immune cells in the gut in both the naïve or developed protocols. Likewise, macrophages underwent drastic morphological changes after feeding, switching from a small and rounded contour to a larger and dendritic shape. Lymphocytes colonized the intestine as early as 5 days post fertilization and increased in numbers during the inflammatory process. Gene expression analysis indicated that lymphocytes present in the intestine correspond to T helper cells. Interestingly, control diet only induced a regulatory T cell profile in the developed model. On the contrary, soybean meal diet induced a Th17 response both in naïve and developed model. In addition, when feeding was performed in -deficient fish, intestinal inflammation was not induced indicating that inflammation induced by soybean meal is T cell-dependent.

Konrad Gronke, Pedro P Hernández, Jakob Zimmermann, Christoph S N Klose, Michael Kofoed-Branzk, Fabian Guendel, Mario Witkowski, Caroline Tizian, Lukas Amann, Fabian Schumacher, Hansruedi Glatt, Antigoni Triantafyllopoulou, Andreas Diefenbach (2019 Feb 1)

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Nature : 249-253 : DOI : 10.1038/s41586-019-0899-7 En savoir plus

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR), and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated ‘sensing’ of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.


Année de publication : 2018

Pedro P Hernández, Paulina M Strzelecka, Emmanouil I Athanasiadis, Dominic Hall, Ana F Robalo, Catherine M Collins, Pierre Boudinot, Jean-Pierre Levraud, Ana Cvejic (2018 Nov 18)

Single-cell transcriptional analysis reveals ILC-like cells in zebrafish.

Science immunology : DOI : eaau5265 En savoir plus

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates are poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and after immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of -deficient zebrafish that lack T and B cells, and discovered populations of ILC-like cells. We uncovered a -positive subset of ILCs that could express cytokines associated with type 1, 2, and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed and after exposure to inactivated bacteria or after exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.


Année de publication : 2017

Pedro Hernandez, Konrad Gronke, Andreas Diefenbach (2017 Nov 28)

A catch-22: Interleukin-22 and cancer.

European journal of immunology : 15-31 : DOI : 10.1002/eji.201747183 En savoir plus

Barrier surfaces of multicellular organisms are in constant contact with the environment and infractions to the integrity of epithelial surfaces is likely a frequent event. Interestingly, components of the immune system, that can be activated by environmental compounds such as the microbiota or nutrients, are interspersed among epithelial cells or directly underlie the epithelium. It is now appreciated that immune cells continuously receive and integrate signals from the environment. Curiously, such continuous reception of stimulation does not normally trigger an inflammatory response but mediators produced by immune cells in response to such signals seem to rather promote barrier integrity and repair. The molecular mediators involved in this process are poorly understood. In recent years, the cytokine interleukin-22, produced mainly by group 3 innate lymphoid cells (ILCs), has been studied as a paradigm for how immune cells can control various aspects of epithelial cell function because expression of its receptor is restricted to non-hematopoietic cells. We will summarize here the diverse roles of IL-22 for the malignant transformation of epithelial cells, for tumor growth, wound healing and tissue repair. Furthermore, we will discuss IL-22 as a potential therapeutic target.


Année de publication : 2015

Pedro P Hernández, Tanel Mahlakoiv, Ines Yang, Vera Schwierzeck, Nam Nguyen, Fabian Guendel, Konrad Gronke, Bernhard Ryffel, Christoph Hoelscher, Laure Dumoutier, Jean-Christophe Renauld, Sebastian Suerbaum, Peter Staeheli, Andreas Diefenbach (2015 May 26)

Interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection.

Nature immunology : 698-707 : DOI : 10.1038/ni.3180 En savoir plus

The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were ‘preferentially’ expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons.

Tanel Mahlakõiv, Pedro Hernandez, Konrad Gronke, Andreas Diefenbach, Peter Staeheli (2015 Apr 8)

Leukocyte-derived IFN-α/β and epithelial IFN-λ constitute a compartmentalized mucosal defense system that restricts enteric virus infections.

PLoS pathogens : e1004782 : DOI : 10.1371/journal.ppat.1004782 En savoir plus

Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/β) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/β, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/β receptors. Accordingly, after oral infection of IFN-α/β receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/β for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/β system which would induce exacerbated inflammation.


Année de publication : 2014

Christoph S N Klose, Katharina Blatz, Yannick d'Hargues, Pedro P Hernandez, Michael Kofoed-Nielsen, Juliane F Ripka, Karolina Ebert, Sebastian J Arnold, Andreas Diefenbach, Ed Palmer, Yakup Tanriver (2014 Aug 23)

The transcription factor T-bet is induced by IL-15 and thymic agonist selection and controls CD8αα(+) intraepithelial lymphocyte development.

Immunity : 230-43 : DOI : 10.1016/j.immuni.2014.06.018 En savoir plus

CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαβ(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.


Année de publication : 2011

Pedro P Hernandez, Cristian Undurraga, Viviana E Gallardo, Natalia Mackenzie, Miguel L Allende, Ariel E Reyes (2011 Jul 2)

Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae.

Biological research : 7-15 : DOI : 10.4067/S0716-97602011000100002 En savoir plus

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.


Année de publication : 2010

Cedric Vonarbourg, Arthur Mortha, Viet L Bui, Pedro P Hernandez, Elina A Kiss, Thomas Hoyler, Melanie Flach, Bertram Bengsch, Robert Thimme, Christoph Hölscher, Manfred Hönig, Ulrich Pannicke, Klaus Schwarz, Carl F Ware, Daniela Finke, Andreas Diefenbach (2010 Nov 25)

Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt(+) innate lymphocytes.

Immunity : 736-51 : DOI : 10.1016/j.immuni.2010.10.017 En savoir plus

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.


Année de publication : 2008

Francisco A Olivari, Pedro P Hernández, Miguel L Allende (2008 Oct 14)

Acute copper exposure induces oxidative stress and cell death in lateral line hair cells of zebrafish larvae.

Brain research : 1-12 : DOI : 10.1016/j.brainres.2008.09.050 En savoir plus

Numerous physical and chemical agents can destroy mechanosensory hair cells in the inner ear of vertebrates, a process that is irreversible in mammals. Few experimental systems allow the observation of hair cell death mechanisms in vivo, in the intact animal, one of these being the lateral line system in the zebrafish. In this work we characterize the behavior of dying lateral line hair cells in fish exposed to low doses of copper in the water. The concentration of copper used in our study kills hair cells in a few hours, but removal of the metal is followed by robust regeneration of new hair cells. We use a combination of membrane and nuclear live stains, ultrastructural analysis and measurement of reactive oxygen species to characterize the events leading to the death of hair cells under these conditions. Our results show that a combination of necrotic cell death, accompanied by apoptotic features such as rapid DNA fragmentation, lead to the loss of these cells. We also show that hair cells exposed to copper undergo oxidative stress and that antioxidants can protect these cells from the effects of the metal. The study of this process in the zebrafish lateral line allows rapid morphological analysis of hair cell death and may be used as an efficient end point for molecule screens aimed at preventing these effects.

Pedro P Hernández, Miguel L Allende (2008 Sep 10)

Zebrafish (Danio rerio) as a model for studying the genetic basis of copper toxicity, deficiency, and metabolism.

The American journal of clinical nutrition : 835S-9S En savoir plus

Unicellular eukaryotes and cultured cells from several animal species were invaluable in discovering the mechanisms that govern incorporation, handling, and excretion of copper at the cellular level. However, understanding the systemic regulation of copper availability and distribution among the different tissues of an intact multicellular organism has proven to be more challenging. This analysis is made even more difficult if the genetic variability among organisms is taken into account. The zebrafish has long been considered a powerful animal model because of its tractable genetics and embryology, but it has more recently become a player in environmental studies, pharmaceutical screening, and physiologic analysis. In particular, the use of the larvae, small enough to fit into a microtiter well, but developed enough to have full organ functionality, represents a convenient alternative for studies that aim to establish effects of environmental agents on the intact, living organism. Studies by our group and others have characterized absorption and tissue distribution of copper and have described the acute effects of the metal on larvae in terms of survival, organ stress, and functionality of sensory organs. A large body of work has shown that there is strong conservation in mechanisms and genes between fish and mammals, opening the possibility for genetic or small molecule screens or for generating fish models of human diseases related to copper metabolism. The variability within humans in terms of tolerance to copper excess or deficiency requires a genetic approach to be taken to understand the behavior of populations, because markers and vulnerabilities need to be identified. The zebrafish could represent a unique tool to move in this direction.


Année de publication : 2007

Pedro P Hernández, Francisco A Olivari, Andrés F Sarrazin, Pablo C Sandoval, Miguel L Allende (2007 Apr 20)

Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal.

Developmental neurobiology : 637-54 En savoir plus

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.


Année de publication : 2006

Pedro P Hernández, Virginia Moreno, Francisco A Olivari, Miguel L Allende (2006 Jan 3)

Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio).

Hearing research : 1-10 En savoir plus

In teleosts, the lateral line system is composed of neuromasts containing hair cells that are analogous to those present in the inner ear of all vertebrates. In the zebrafish embryo and early larva, this system is composed of the anterior lateral line (ALL), which covers the head, and the posterior lateral line (PLL), present in the trunk and tail. The mechanosensory hair cells found in neuromasts can be labeled in vivo using fluorescent dyes such as 4-di-2-Asp (DiAsp) or FM1-43. We have studied the effects of water-borne copper exposure on the function of the lateral line system in zebrafish larvae. Our results show that transient incubation of post-hatching larvae for 2h with non-lethal concentrations of copper (1-50 microM CuSO4) induces cellular damage localized to neuromasts, apoptosis, and loss of hair cell markers. This effect is specific to copper, as other metals did not show these effects. Since hair cells in fish can regenerate, we followed the reappearance of viable hair cells in neuromasts after copper removal. In the PLL, we determined that there is a threshold concentration of copper above which regeneration does not occur, whereas, at lower concentrations, the length of time it takes for viable hair cells to reappear is dependent on the amount of copper used during the treatment. The ALL behaves differently though, as regeneration can occur even after treatments with concentrations of copper an order of magnitude higher than the one that irreversibly affects the PLL. Regeneration of hair cells is dependent on cell division within the neuromasts as damage that precludes proliferation prevents reappearance of this cell type.