Ricardo Villares

The achaete-scute gene complex of D. melanogaster: Conserved Domains in a subset of genes required for neurogenesis and their homology to myc

We have determined the nucleotide sequence of two transcription units of the AS-C and shown that their potential protein coding regions share two principal domains of homology. Both domains are conserved within the myc protein family, and one of them is highly homologous with the consensus for protein tyrosine kinase substrates. We show in addition that at least one of these domains is shared with other transcription units within the AS-C, some of which were not previously known. We suggest that the AS-C encodes several homologous polypeptides, which represent a subset of a larger gene family. We propose that each member of the family functions at an equivalent stage of a unique morphogenetic operation involving the segregation of individual neural lineages from the epidermal anlage.

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyers patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T-cell recruitment to target tissues.

The T‐cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4+ T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4+ T‐cell‐mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6−/− mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3+ cell frequency characterized CNS tissues from CCR6−/− compared with CCR6+/+ mice during the disease effector phase. Transfer of CCR6+/+ Treg to CCR6−/− mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3+ cells and recovery of the cytokine balance in CCR6−/− mouse CNS. Competitive assays between CCR6+/+ and CCR6−/− Treg adoptively transferred to CCR6−/− mice showed impaired ability of CCR6−/− Treg to infiltrate CNS tissues in EAE‐affected mice. Our data indicate a CCR6 requirement by CD4+ Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.

Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.

Identification on Mouse Chromosome 8 of New β-Defensin Genes with Regionally Specific Expression in the Male Reproductive Organ

Defensins are important elements in innate immunity that can also trigger adaptive immune responses. The defensins form a family of small cationic antimicrobial peptides with six characteristic cysteine residues, whose pairing pattern in forming three intramolecular disulfide bonds defines the α- and β-defensin subfamilies. In a search for new β-defensin genes, we performed computational analysis using the Celera mouse genome data base and found exons encoding 23 different β-defensins, including the eight previously characterized members of this family. Among the new β-defensins, nine of them form two groups of phylogenetically related sequences that were characterized in greater detail. Northern blot, reverse transcription PCR, and in situ hybridization analysis showed that expression of these genes is restricted to the epididymis, with a specific regional expression pattern. One of the new β-defensins (Defb38) was chemically synthesized; in in vitro assays on Gram-positive and -negative bacterial strains, Defb38 showed the characteristic salt-dependent antimicrobial activity of β-defensins. The results demonstrate the existence of a relatively large number of β-defensins with specific expression in distinct regions of the murine epididymis and suggest complex roles for these proteins in host defense and other physiological processes of the male reproductive tract.

Growth hormone prevents the development of autoimmune diabetes

Significance Although the relationship between endocrine and immune systems is well documented, few studies have been performed on autoimmune disorders other than those that are sex hormone-related. We studied a murine model of autoimmune diabetes, showing that growth hormone (GH) modifies the immune response to render diabetic mice resistant to disease development. The mechanism involves a GH-mediated effect on β-cell survival and/or proliferation and a direct effect on immune cells. GH triggers a cytokine environment that promotes anti-inflammatory macrophage polarization, maintains the activity of the suppressor T cells, and limits Th17 cell plasticity. This study provides evidence of the importance of endocrine control of immune functions and indicates that therapies based on GH analogs should be considered for treatment of autoimmune diabetes. Evidence supports a relationship between the neuroendocrine and the immune systems. Data from mice that overexpress or are deficient in growth hormone (GH) indicate that GH stimulates T and B-cell proliferation and Ig synthesis, and enhances maturation of myeloid progenitor cells. The effect of GH on autoimmune pathologies has nonetheless been little studied. Using a murine model of type 1 diabetes, a T-cell–mediated autoimmune disease characterized by immune cell infiltration of pancreatic islets and destruction of insulin-producing β-cells, we observed that sustained GH expression reduced prodromal disease symptoms and eliminated progression to overt diabetes. The effect involves several GH-mediated mechanisms; GH altered the cytokine environment, triggered anti-inflammatory macrophage (M2) polarization, maintained activity of the suppressor T-cell population, and limited Th17 cell plasticity. In addition, GH reduced apoptosis and/or increased the proliferative rate of β-cells. These results support a role for GH in immune response regulation and identify a unique target for therapeutic intervention in type 1 diabetes.

Remodeling our concept of chemokine receptor function: From monomers to oligomers

The chemokines direct leukocyte recruitment in both homeostatic and inflammatory conditions, and are therefore critical for immune reactions. By binding to members of the class A G protein‐coupled receptors, the chemokines play an essential role in numerous physiological and pathological processes. In the last quarter century, the field has accumulated much information regarding the implications of these molecules in different immune processes, as well as mechanistic insight into the signaling events activated through their binding to their receptors. Here, we will focus on chemokine receptors and how new methodological approaches have underscored the role of their conformations in chemokine functions. Advances in biophysical‐based techniques show that chemokines and their receptors act in very complex networks and therefore should not be considered isolated entities. In this regard, the chemokine receptors can form homo‐ and heterodimers as well as oligomers at the cell surface. These findings are changing our view as to how chemokines influence cell biology, identify partners that regulate chemokine function, and open new avenues for therapeutic intervention.

Chemokine Detection Using Receptors Immobilized on an SPR Sensor Surface

Chemokines and their receptors take part in many physiological and pathological processes, and their dysregulated expression is linked to chronic inflammatory and autoimmune diseases, immunodeficiencies, and cancer. The chemokine receptors, members of the G protein-coupled receptor family, are integral membrane proteins, with seven-transmembrane domains that bind the chemokines and transmit signals through GTP-binding proteins. Many assays used to study the structure, conformation, or activation mechanism of these receptors are based on ligand-binding measurement, as are techniques to detect new agonists and antagonists that modulate chemokine function. Such methods require labeling of the chemokine and/or its receptor, which can alter their binding characteristics. Surface plasmon resonance (SPR) is a powerful technique for analysis of the interaction between immobilized receptors and ligands in solution, in real time, and without labeling. SPR measurements nonetheless require expression and purification steps that can alter the conformation, stability, and function of the chemokine and/or the chemokine receptor. In this review, we focus on distinct methods to immobilize chemokine receptors on the surface of an optical biosensor. We expose the advantages and disadvantages of different protocols used and describe in detail the method to retain viral particles as receptor carriers that can be used for SPR determinations.

Dido mutations trigger perinatal death and generate brain abnormalities and behavioral alterations in surviving adult mice.

Significance The primary cilium is an organelle protruding from most postmitotic vertebrate cells. A growing body of data supports the crucial role of primary cilia in developmental signaling pathways. Recent studies describe the main stages in ciliogenesis at the morphological level and components of some of the mechanisms involved, including the selective acetylation of tubulin. How this acetylation is modulated in cilia nonetheless remains poorly understood. Here we show that the death inducer-obliterator (dido) gene product, which regulates histone deacetylase 6 deacetylase activity, is necessary for orofacial development in the mouse embryo and influences brain patterning and neuromuscular activity. Mice deficient in dido function present neonatal mortality and various ciliopathies including cleft palate and hydrocephalus, as well as hippocampal and commissural dysplasia. Nearly all vertebrate cells have a single cilium protruding from their surface. This threadlike organelle, once considered vestigial, is now seen as a pivotal element for detection of extracellular signals that trigger crucial morphogenetic pathways. We recently proposed a role for Dido3, the main product of the death inducer-obliterator (dido) gene, in histone deacetylase 6 delivery to the primary cilium [Sánchez de Diego A, et al. (2014) Nat Commun 5:3500]. Here we used mice that express truncated forms of Dido proteins to determine the link with cilium-associated disorders. We describe dido mutant mice with high incidence of perinatal lethality and distinct neurodevelopmental, morphogenetic, and metabolic alterations. The anatomical abnormalities were related to brain and orofacial development, consistent with the known roles of primary cilia in brain patterning, hydrocephalus incidence, and cleft palate. Mutant mice that reached adulthood showed reduced life expectancy, brain malformations including hippocampus hypoplasia and agenesis of corpus callosum, as well as neuromuscular and behavioral alterations. These mice can be considered a model for the study of ciliopathies and provide information for assessing diagnosis and therapy of genetic disorders linked to the deregulation of primary cilia.

Agent-based model of macrophage action on endocrine pancreas

This paper proposes an agent-based model of the action of macrophages on the beta cells of the endocrine pancreas. The aim of this model is to simulate the processes of beta cell proliferation and apoptosis and also the process of phagocytosis of cell debris by macrophages, all of which are related to the onset of the autoimmune response in type 1 diabetes. We have used data from the scientific literature to design the model. The results show that the model obtains good approximations to real processes and could be used to shed light on some open questions concerning such processes.