A Newborn Screening Program for Sickle Cell Disease in Murcia (Spain).

Sickle cell disease (SCD) is an inherited autosomal recessive hemoglobin disorder caused by the presence of hemoglobin S, a mutant abnormal hemoglobin caused by a nucleotide change in codon 6 of the ß-globin chain gene. SCD involves a chronic inflammatory state, exacerbated during vaso-occlusive crises, which leads to end-organ damage that occurs throughout the lifespan. SCD is associated with premature mortality in the first years of life. The process of sickling provokes asplenia in the first years of life with an increased risk of infection by encapsulated germs. These complications can be life-threatening and require early diagnosis and management. The most important interventions recommend an early diagnosis of SCD to ensure that affected newborns receive immediate care to reduce mortality and morbidity. The newborn screening program in the region of Murcia for SCD began in March 2016. We aimed to determine the incidence of sickle cell anemia and other structural hemoglobinopathies in the neonatal population of the region of Murcia, an area of high migratory stress, and to systematically assess the benefit of newborn screening for SCD, leading to earlier treatment, as well as to offer genetic counseling to all carriers. The prevalence of SCD in our region is similar to others in Spain, except for Catalonia and Madrid. The newborns with confirmed diagnoses of SCD received early attention, and all the carriers received genetic counseling.

ZAKα/P38 kinase signaling pathway regulates hematopoiesis by activating the NLRP1 inflammasome.

Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress-induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.

Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment.

BACKGROUND: Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. METHODS: Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. RESULTS: We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1ß cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. CONCLUSIONS: Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.

Impact of Comorbidities of Patients with Psoriasis on Phototherapy Responses.

A retrospective study of 200 psoriasis patients and 100 healthy donors in a Spanish cohort was carried out to study the comorbidities associated with psoriasis and their association with the response to phototherapy. The results showed a higher incidence of psychiatric disease, liver disease, kidney disease, hypertension, heart disease, vascular disease, diabetes, gastrointestinal disease, autoimmune and infectious diseases, dyslipidemia, and psoriatic arthritis in patients with psoriasis than in the control group. The incidence of comorbidities was higher in psoriasis patients over 40 years old than in the control individuals of the same age, which could be indicative of premature aging. Phototherapy was seen to be an effective treatment in cases of moderate-severe psoriasis, total whitening being achieved in more than 30% of patients, with women showing a better response than men. Narrow-band ultraviolet B was found to be the most effective type of phototherapy, although achievement of PASI100 was lower in patients with liver disease, hypertension, heart disease, vascular disease, or diabetes. Strikingly, liver disease and anemia comorbidities favored therapeutic failure. Finally, zebrafish and human 3D organotypic models of psoriasis point to the therapeutic benefit of inhibiting the glucose transporter GLUT1 and the major regulator of blood glucose dipeptidyl peptidase 4. Our study reveals that specific comorbidities of psoriasis patients are associated to failure of phototherapy and, therefore, need to be considered when planning treatment for these patients.

Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 in fish granulocytes.

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.

Posttranslational Regulation of Inflammasomes, Its Potential as Biomarkers and in the Identification of Novel Drugs Targets.

In this review, we have summarized classical post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, and SUMOylation of the different components of one of the most studied NLRP3, and other emerging inflammasomes. We will highlight how the discovery of these modifications have provided mechanistic insight into the biology, function, and regulation of these multiprotein complexes not only in the context of the innate immune system but also in adaptive immunity, hematopoiesis, bone marrow transplantation, as well and their role in human diseases. We have also collected available information concerning less-studied modifications such as acetylation, ADP-ribosylation, nitrosylation, prenylation, citrullination, and emphasized their relevance in the regulation of inflammasome complex formation. We have described disease-associated mutations affecting PTMs of inflammasome components. Finally, we have discussed how a deeper understanding of different PTMs can help the development of biomarkers and identification of novel drug targets to treat diseases caused by the malfunctioning of inflammasomes.

New Insights Into Pathophysiology of ß-Thalassemia.

ß-thalassemia is a disease caused by genetic mutations including a nucleotide change, small insertions or deletions in the ß-globin gene, or in rare cases, gross deletions into the ß-globin gene. These mutations affect globin-chain subunits within the hemoglobin tetramer what induces an imbalance in the α/ß-globin chain ratio, with an excess of free α-globin chains that triggers the most important pathogenic events of the disease: ineffective erythropoiesis, chronic anemia/chronic hypoxia, compensatory hemopoietic expansion and iron overload. Based on advances in our knowledge of the pathophysiology of ß-thalassemia, in recent years, emerging therapies and clinical trials are being conducted and are classified into three major categories based on the different approach features of the underlying pathophysiology: correction of the α/ß-globin disregulation; improving iron overload and reverse ineffective erythropoiesis. However, pathways such as the dysregulation of transcriptional factors, activation of the inflammasome, or approach to mechanisms of bone mineral loss, remain unexplored for future therapeutic targets. In this review, we update the main pathophysiological pathways involved in ß-thalassemia, focusing on the development of new therapies directed at new therapeutic targets.

Gasdermin E mediates pyroptotic cell death of neutrophils and macrophages in a zebrafish model of chronic skin inflammation.

Chronic diseases and hematopoietic disorders are associated with dysregulation of the inflammasome. Our group has recently reported the relevance of the inflammasome in the differentiation of hematopoietic stem and progenitor cells. However, the impact of the inflammasome of myeloid cells in the regulation of hematopoiesis is largely unknown. In this study, we used the unique advantages of the zebrafish model to demonstrate that genetic inhibition of macrophage inflammasome resulted in increased number of macrophages in larvae with skin inflammation without affecting erythrocyte and neutrophil counts. Similarly, the inhibition of the neutrophil inflammasome by the same strategy resulted in increased number of neutrophils in larvae with skin inflammation but did not affect erythrocytes and macrophages. Consistently, hyperactivation of the inflammasome in neutrophils in this model promoted neutrophil death, which was recovered by pharmacological inhibition of Gasdermin E. We conclude that the myeloid inflammasome autonomously regulates pyroptotic cell death in chronic inflammation through a Gasdermin E-dependent pathway in zebrafish.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA (terc) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.

Bcl-xL: A Focus on Melanoma Pathobiology.

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors.

The molecular, functional and phylogenetic characterization of PGE2 receptors reveals their different roles in the immune response of the teleost fish gilthead seabream (Sparus aurata L.).

Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.

Non-canonical roles of NAMPT and PARP in inflammation.

Nicotinamide adenine dinucleotide (NAD+) is the most important hydrogen carrier in cell redox reactions. It is involved in mitochondrial function and metabolism, circadian rhythm, the immune response and inflammation, DNA repair, cell division, protein-protein signaling, chromatin remodeling and epigenetics. Recently, NAD+ has been recognized as the molecule of life, since, by increasing NAD+ levels in old or sick animals, it is possible to improve their health and lengthen their lifespan. In this review, we summarize the contribution of NAD+ metabolism to inflammation, with special emphasis in the major NAD+ biosynthetic enzyme, nicotinamide phosphoribosyl transferase (NAMPT), and the NAD+-consuming enzyme, poly(ADP-ribose) polymerase (PARP). The extracurricular roles of these enzymes, i.e. the proinflammatory role of NAMPT after its release, and the ability of PARP to promote a novel form of cell death, known as parthanatos, upon hyperactivation are revised and discussed in the context of several chronic inflammatory diseases.

Zebrafish Models to Study Inflammasome-Mediated Regulation of Hematopoiesis.

Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

The vitamin B6-regulated enzymes PYGL and G6PD fuel NADPH oxidases to promote skin inflammation.

Psoriasis is a skin inflammatory disorder that affects 3% of the human population. Although several therapies based on the neutralization of proinflammatory cytokines have been used with relative success, additional treatments are required. The in silico analysis of gene expression data of psoriasis lesional skin and an analysis of vitamin B6 metabolites in the sera of psoriasis patients point to altered vitamin B6 metabolism at both local and systemic levels. Functional studies showed that vitamin B6 vitamers reduced skin neutrophil infiltration, oxidative stress and Nfkb activity in two zebrafish models of skin inflammation. Strikingly, inhibition of glycogen phosphorylase L (Pygl) and glucose-6-phosphate dehydrogenase (G6pd), two vitamin B6-regulated enzymes, alleviated oxidative-stress induced inflammation in zebrafish skin inflammation models. Despite the central role of G6pd in antioxidant defenses, the results of the study demonstrate that glycogen stores and G6pd fuel NADPH oxidase to promote skin inflammation, revealing novel targets for the treatment of skin inflammatory disorders.

Hydrogen peroxide in neutrophil inflammation: Lesson from the zebrafish.

The zebrafish has become an excellent model for the study of inflammation and immunity. Its unique advantages for in vivo imaging and gene and drug screening have allowed the visualization of dual oxidase 1 (Duox1)-derived hydrogen peroxide (H2O2) tissue gradients and its crosstalk with neutrophil infiltration to inflamed tissue. Thus, it has been shown that H2O2 directly recruits neutrophils via the Src-family tyrosine kinase Lyn and indirectly by the activation of several signaling pathways involved in inflammation, such as nuclear factor κB (NF-κB), mitogen activated kinases and the transcription factor AP1. In addition, this model has also unmasked the unexpected ability of H2O2 to induce the expression of the gene encoding the key neutrophil chemoattractant CXC chemokine ligand 8 by facilitating the accessibility of transcription factors to its promoter through histone covalent modifications. Finally, zebrafish models of psoriasis have shown that a H2O2/NF-κB/Duox1 positive feedback inflammatory loop operates in this chronic inflammatory disorder and that pharmacological inhibition of Duox1, but not of downstream mediators, inhibits inflammation and restores epithelial homeostasis. Therefore, these results have pointed out DUOX1 and H2O2 as therapeutic targets for the treatment of skin inflammatory disorders, such as psoriasis.

WDR90 is a new component of the NLRC4 inflammasome involved in Salmonella Typhimurium resistance.

Inflammasomes are pivotal cytosolic molecular platforms involved in infection resistance. As multiprotein complexes, they consist of NOD-like receptors (NLRs), the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and the effector molecules caspase-1 and caspase-11, whose assembly and activation depends on homotypic interactions. Here we describe WD repeat containing protein 90 (WDR90) as a new inflammasome component. We found that zebrafish wdr90 is highly induced by guanylate binding protein 4 (Gbp4) independently of inflammasome activation and caspase-1 activity. This gene encodes an evolutionarily conserved protein with unknown functions that contains several WD40 domains, which are involved in coordinating multiprotein complex assembly. Functional studies in zebrafish larvae showed that forced expression of wdr90 increased caspase-1 activity and inflammasome-dependent resistance to Salmonella enterica serovar Typhimurium infection. Wdr90 acted upstream of zebrafish caspase a (Caspa), the functional homolog of mammalian caspase-1, and Asc. Reconstitution experiments of the human inflammasome in HEK293 cells demonstrated that WDR90 was able to physically interact with and to alter the cellular distribution of NLRC4, but not of NLRP3 and AIM2. These results highlight the complexity of the inflammasome and the interest of studying fish immunity to understand not only the evolution of the immune system but also human immunity.

Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1.

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

The zebrafish: A research model to understand the evolution of vertebrate immunity.

The zebrafish has unique advantages for understanding the evolution of vertebrate immunity and to model human diseases. In this review, we will firstly give an overview of the current knowledge on vertebrate innate immune receptors with special emphasis on the inflammasome and then summarize the main contribution of the zebrafish model to this field, including to the identification of novel inflammasome components and to the mechanisms involved in its activation, assembly and clearance of intracellular bacteria.