Jorge Galindo-Villegas

Regulation of immunity and disease resistance by commensal microbes and chromatin modifications during zebrafish development

How fish larvae are protected from infection before the maturation of adaptive immunity, a process which may take up to several weeks in most species, has long been a matter of speculation. Using a germ-free model, we show that colonization by commensals in newly hatched zebrafish primes neutrophils and induces several genes encoding proinflammatory and antiviral mediators, increasing the resistance of larvae to viral infection. Commensal microbe recognition was found to be mediated mainly through a TLR/MyD88 signaling pathway, and professional phagocytes were identified as the source of these immune mediators. However, the induction of proinflammatory and antiviral genes, but not of antimicrobial effector genes, also required the covalent modification of histone H3 at gene promoters. Interestingly, chromatin modifications were not altered by commensal microbes or hatching. Taken together, our results demonstrate that gene-specific chromatin modifications are associated with the protection of zebrafish larvae against infectious agents before adaptive immunity has developed and prevent pathologies associated with excessive inflammation during development.

Selective Manipulation of the Gut Microbiota Improves Immune Status in Vertebrates

All animals develop in association with complex microbial communities. It is now well established that commensal microbiota is essential for the correct functionality of each organ in the host. Particularly, the commensal gastro-intestinal microbiota (CGIM) is a key factor for development, immunity and nutrient conversion, rendering them bio-available for various uses. Thus, nutritional inputs generate a positive loop in maintaining host health and are essential in shaping the composition of the CGIM communities. Probiotics, which are live exogenous microorganisms, selectively provided to the host, are a promising concept for manipulating the microbiota and thus for increasing the host health status. Nevertheless, most mechanisms induced by probiotics to fortify the immune system are still a matter of debate. Alternatively, prebiotics, which are non-digestible food ingredients, can favor the growth of specific target groups of CGIM. Several metabolites are produced by the CGIM, one of the most important are the short-chain fatty acids (SCFAs), which emerge from the fermentation of complex carbohydrates. SCFAs have been recognized as key players in triggering beneficial effects elicited by simple diffusion and by specific receptors present, thus, far only in epithelial cells of higher vertebrates at different gastro-intestinal locations. However, both strategies have shown to provide resistance against pathogens during periods of high stress. In fish, knowledge about the action of pro- and prebiotics and SCFAs is still limited. Thus, in this review, we briefly summarize the mechanisms described on this topic for higher vertebrates and discuss why many of them may operate in the fish gut representing a model for different mucosal tissues

Differential Modulation of IgT and IgM upon Parasitic, Bacterial, Viral, and Dietary Challenges in a Perciform Fish

Three different immunoglobulin (Ig) isotypes can be found in teleost fish, IgM, IgD, and the teleost-specific IgT. IgM is considered to have a systemic activity, and IgT is attributed a mucosal role, similar to mammalian IgA. In this study, the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. GCB IgM and IgT have the prototypical structure when compared to other fish Igs. The constitutive expression of sIgM was the highest overall in all tissues, whereas mIgT expression was highest in mucosal tissues, such as gills and intestine. IgM and IgT were differentially regulated upon infection. IgT was highly upregulated locally upon infection with the intestinal parasite Enteromyxum leei or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against Photobacterium damselae subsp. piscicida finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT upregulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studies.

Cxcl8-l1 and Cxcl8-l2 are required in the zebrafish defense against Salmonella Typhimurium.

In recent years zebrafish has emerged as an excellent model for studying the Cxcl8 signaling pathway in inflammation elicited upon tissue damage or infection. Zebrafish has two true homologs of mammalian CXCL8, named Cxcl8-l1 and Cxcl8-l2. Previously, we have shown that in wound-associated inflammation, these chemokines are up-regulated and are relevant for neutrophil recruitment. In infections, no such knowledge is available as most studies performed on this subject in zebrafish have mainly focused on Cxcl8-l1 even though Cxcl8-l2 shares higher homology with human CXCL8. In this study, we aimed to address the biological function of both zfCxcl8s in infection to improve our understanding of their respective roles under different inflammatory conditions. Gene expression analysis first confirmed that both Cxcl8-l1 and l2 are induced upon infection or in PAMP-elicited inflammatory processes. In addition, we also found that cxcl8-deficient larvae show higher susceptibility to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, reduced neutrophil recruitment to the infection site assayed in the line Tg(mpx:gfp), and decreased bacterial clearance. These data indicate that both zebrafish Cxcl8s play important roles in neutrophil recruitment and in the inflammatory response elicited upon infection or tissue damage, suggesting that even though the divergence of lower vertebrates and humans from a common ancestor occurred about 450 millions years ago, the basic principles of neutrophil recruitment are apparently conserved in all vertebrates.

TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity

As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca2+/TGF-β–activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response.

Role of histamine in the regulation of intestinal immunity in fish

In mammals, during the acute inflammatory response, the complex interrelationship and cross-talk among histamine and the immune system has been fairly well characterized. There is a substantial body of information on its structure, metabolism, receptors, signal transduction, physiologic and pathologic effects. However, for early vertebrates, there is little such knowledge. In the case of teleost fish, this lack of knowledge has been due to the widely held belief that histamine is not present in this phylogenetic group. However, it has been recently demonstrated, that granules of mast cells in perciforms contain biologically active histamine. More importantly, the inflammatory response was clearly demonstrated to be regulated by the direct action of histamine on professional phagocytes. Nevertheless, the molecular basis and exact role of this biogenic amine in perciforms is still a matter of speculation. Therefore, this review intends to summarize recent experimental evidence regarding fish mast cells and correlate the same with their mammalian counterparts to establish the possible role of histamine in the fish intestinal inflammatory response.

Recent findings on vertebrate developmental immunity using the zebrafish model.

To grant survival against sterile or microbe induced inflammation, all animals rely on correct immune system functioning. The development of immunity occurs in vertebrates during embryogenesis in a process called hematopoiesis, which is characterized by the formation of blood cellular components such as embryonic erythrocytes and primitive macrophages. These cells are formed in a sterile environment from a rare subset of pluripotent hematopoietic stem cells (HSC) during a brief period of the primitive hematopoietic wave. Diverse signals, like Notch, are indispensable in HSC emergence and differentiation. However, to successfully replicate the process in vitro using pluripotent precursors, the full set of required signals is still a matter of debate. Among the latest findings, proinflammatory signals produced by transient primitive myelocites in zebrafish have been seen to act as essential mediators in establishing the HSC program of the adult vertebrate hematopoietic system. In this regard, the zebrafish immune model has emerged as a feasible live vertebrate model for examining developmental immunity and related host-microbe interactions, both at the molecular and cellular level. Thus, using the zebrafish embryo, this review summarizes recent findings, on the signals required for immune development and further maturation of the system, in a context where no adaptive immune response has yet been developed.

Cytokine Intervention: A Double Edged Sword in the Nkg2d System Regulation

The natural killer group 2 members D (NKG2D) is an activating receptor which plays a major role in immune surveillance, and the detection and elimination of malignant tumors and infected cells. NKG2D acts over both arms of the vertebrate immune response, and is expressed in some human and mouse myelopoietic, γδ T, NKT and CD4+ cells, but is present in all NK and CD8+ T cells in humans and activated mouse CD8+ T cells. In humans, eight ligands which selectively bind to the NKG2D receptor have been identified. These ligands are not systemically expressed, but are triggered in response to stress and expressed only under specific pathological states. Several research results point to the importance of cytokines for increasing expression of NKG2D to restore the functionality of NK cells as well as their ligands in the target cells. However, the NKG2D system itself in an activated state, also release pro and anti-inflammatory cytokine transcripts to establish communication with other cells or for self-regulation. Additionally, type I antiviral interferon is largely produced. Such cytokine interactions could be regarded as a double edged sword. This behavior is emphasized by a discrepancy regarding the functionality of cytokines which interact with, or on the NKG2D system. Indeed, they seem to protect the host and rather can induce ligand expression, cell proliferation or dissemination of malignant tumors, generating complicated cytokine-mediated messenger loops which are far from being fully understood. Whatever the case, cytokines related to the NKG2D system could be an attractive and useful target for immunotherapeutic approaches. Thus, here we briefly review recent findings on the main aspects involved in the regulation of this system and, particularly, attempt to clarify the role played by cytokines in the activating or inhibitory function they exert over the NKG2D system in different contexts.

Correction: Cutting Edge: TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity.

Galindo-Villegas, J., A. Montalban-Arques, S. Liarte, S. de Oliveira, C. Pardo-Pastor, F. Rubio-Moscardo, J. Meseguer, M. A. Valverde, and V. Mulero. 2016. TRPV4-mediated detection of hyposmotic stress by skin keratinocytes activates developmental immunity. J . Immunol . 196: [738–749][1]. A

Exposure to Yeast Shapes the Intestinal Bacterial Community Assembly in Zebrafish Larvae

Establishment of the early-life gut microbiota has a large influence on host development and succession of microbial composition in later life stages. The effect of commensal yeasts - which are known to create a conducive environment for beneficial bacteria - on the structure and diversity of fish gut microbiota still remains unexplored. The present study examined the intestinal bacterial community of zebrafish (Danio rerio) larvae exposed to two fish-derived yeasts by sequencing the V4 hypervariable region of bacterial 16S rRNA. The first stage of the experiment (until 7 days post-fertilization) was performed in cell culture flasks under sterile and conventional conditions for germ-free (GF) and conventionally raised (CR) larvae, respectively. The second phase was carried out under standard rearing conditions, for both groups. Exposure of GF and CR zebrafish larvae to one of the yeast species Debaryomyces or Pseudozyma affected the bacterial composition. Exposure to Debaryomyces resulted in a significantly higher abundance of core bacteria. The difference was mainly due to shifts in relative abundance of taxa belonging to the phylum Proteobacteria. In Debaryomyces-exposed CR larvae, the significantly enriched taxa included beneficial bacteria such as Pediococcus and Lactococcus (Firmicutes). Furthermore, most diversity indices of bacterial communities in yeast-exposed CR zebrafish were significantly altered compared to the control group. Such alterations were not evident in GF zebrafish. The water bacterial community was distinct from the intestinal microbiota of zebrafish larvae. Our findings indicate that early exposure to commensal yeast could cause differential bacterial assemblage, including the establishment of potentially beneficial bacteria.