A zebrafish model of Ifih1-driven Aicardi-Goutières syndrome reproduces the interferon signature and the exacerbated inflammation of patients.

Type I interferonopathies are a heterogenic group of rare diseases associated with an increase in type I interferon (IFN). The main challenge for the study of Type I interferonopathies is the lack of a well-founded animal model to better characterize the phenotype as well as to perform fast and large drug screenings to offer the best treatment options. In this study, we report the development of a transgenic zebrafish model of Type I interferonopathy overexpressing ifih1 carrying the mutation p.Arg742His (Tg(ifih1_mut)), corresponding to the human mutation p.Arg779His. RNA sequence analysis from Tg(ifih1_mut) larvae revealed a systemic inflammation and IFN signature upon a suboptimal poly I:C induction compared with wild-type larvae, confirming the phenotype observed in patients suffering from Type I interferonopathies. More interestingly, the phenotype was manifested in the zebrafish inflammation and Type I IFN reporters nfkb:eGFP and isg15:eGFP, respectively, making this zebrafish model suitable for future high-throughput chemical screening (HTS). Using the unique advantages of the zebrafish model for gene editing, we have generated Tg(ifih1_mut) knocked down for mavs and ikbke, which completely abrogated the Poly I:C induction and activation of the GFP of the reporters. Finally, we used an FDA-approved drug, Baricitinib (Jak1/Jak2 inhibitor), which was able to reduce the inflammation and the ISG expression. Our results demonstrate the potential of this model to further understand AGS pathological mechanisms and to identify novel therapeutic drugs by HTS.

Suramin, a drug for the treatment of trypanosomiasis, reduces the prothrombotic and metastatic phenotypes of colorectal cancer cells by inhibiting hepsin.

Recently, our group identified serine-protease hepsin from primary tumor as a biomarker of metastasis and thrombosis in patients with localized colorectal cancer. We described hepsin promotes invasion and thrombin generation of colorectal cancer cells in vitro and in vivo and identified venetoclax as a hepsin inhibitor that suppresses these effects. Now, we aspire to identify additional hepsin inhibitors, aiming to broaden the therapeutic choices for targeted intervention in colorectal cancer. METHODS: We developed a virtual screening based on molecular docking between the hepsin active site and 2000 compounds from DrugBank. The most promising drug was validated in a hepsin activity assay. Subsequently, we measured the hepsin inhibitor effect on colorectal cancer cells with basal or overexpression of hepsin via wound-healing, gelatin matrix invasion, and plasma thrombin generation assays. Finally, a zebrafish model determined whether hepsin inhibition reduced the invasion of colorectal cancer cells overexpressing hepsin. RESULTS: Suramin was the most potent hepsin inhibitor (docking score: -11.9691 Kcal/mol), with an IC50 of 0.66 µM. In Caco-2 cells with basal or overexpression of hepsin, suramin decreased migration and significantly reduced invasion and thrombin generation. Suramin did not reduce the thrombotic phenotype in the hepsin-negative colorectal cancer cells HCT-116 and DLD-1. Finally, suramin significantly reduced the in vivo invasion of Caco-2 cells overexpressing hepsin. CONCLUSION: Suramin is a novel hepsin inhibitor that reduces its protumorigenic and prothrombotic effects in colorectal cancer cells. This suggests the possibility of repurposing suramin and its derivatives to augment the repertoire of molecular targeted therapies against colorectal cancer.

Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes.

Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.

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.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

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.

Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells.

Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer. Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin. Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo. Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.

NAMPT and PARylation Are Involved in the Pathogenesis of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD+ and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD+, mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD+ and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.

Evolution of LPS recognition and signaling: The bony fish perspective.

Fish are the most diverse and successful group of vertebrate animals, with about 30,000 species. The study of fish immunity is of great importance for understanding the evolution of vertebrate immunity, as they are the first animals to show both innate and adaptive immune responses. Although fish immunity is similar to that of mammals, there are obvious differences, such as their dependence of ambient temperature, their poor antibody response, and lack of antibody switching and lymph nodes. In addition, several important differences have also been found between the innate immune responses of fish and mammals. Among these, we will discuss in this review the high resistance of fish to the toxic effects of lipopolysaccharide (LPS) which can be explained by the absence of a Toll-like receptor 4 (Tlr4) ortholog in most fish species or by the inability of the Tlr4/Md2 (Myeloid differentiation 2) complex to recognize LPS, together with the presence of a negative regulator of the LPS signaling complex formed by the TLR-like molecule Rp105 (Radioprotective 105) and Md1. Taken together, these data support the idea that, although TLR4 and RP105 arose from a common ancestor to fish and tetrapods, the TLR4/MD2 receptor complex for LPS recognition arose after their divergence about 450 million years ago.

Dual Role of DUOX1-Derived Reactive Oxygen Species in Melanoma.

Melanoma is the most serious type of skin cancer. Inflammation and oxidative stress play an essential role in the development of several types of cancer, including melanoma. Although oxidative stress promotes tumor growth, once cells escape from the primary tumor, they are subjected to a more hostile environment, with higher levels of oxidative stress typically killing most cancer cells. As Dual Oxidase 1 (DUOX1) is a major producer of reactive oxygen species (ROS) in epithelia, we used allotransplantation and autochthonous melanoma models in zebrafish together with in silico analysis of the occurrence and relevance of DUOX1 expression of the skin cutaneous melanoma (SKCM) cohort of The Cancer Genome Atlas (TCGA) to address the role of this enzyme in the aggressiveness of melanoma cells in vivo. It was found that high transcript levels of the gene encoding DUOX1 were associated with the poor prognosis of patients in the early-stage melanoma of TCGA cohort. However, DUOX1 transcript levels were not found to be associated to the prognosis of late-stage SKCM patients. In addition, the transcript level of DUOX1 in metastatic SKCM was lower than in primary SKCM. Using zebrafish primary melanoma and allotransplantation models, we interrogated the role of DUOX1 in vivo. Our results confirmed a dual role of DUOX1, which restrains melanoma proliferation but promotes metastasis. As this effect is only observed in immunocompromised individuals, the immune system appears to be able to counteract this elevated metastatic potential of DUOX1-deficient melanomas.

Analysis of 16S rRNA Gene Sequence of Nasopharyngeal Exudate Reveals Changes in Key Microbial Communities Associated with Aging.

Functional or compositional perturbations of the microbiome can occur at different sites, of the body and this dysbiosis has been linked to various diseases. Changes in the nasopharyngeal microbiome are associated to patient's susceptibility to multiple viral infections, supporting the idea that the nasopharynx may be playing an important role in health and disease. Most studies on the nasopharyngeal microbiome have focused on a specific period in the lifespan, such as infancy or the old age, or have other limitations such as low sample size. Therefore, detailed studies analyzing the age- and sex-associated changes in the nasopharyngeal microbiome of healthy people across their whole life are essential to understand the relevance of the nasopharynx in the pathogenesis of multiple diseases, particularly viral infections. One hundred twenty nasopharyngeal samples from healthy subjects of all ages and both sexes were analyzed by 16S rRNA sequencing. Nasopharyngeal bacterial alpha diversity did not vary in any case between age or sex groups. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant phyla in all the age groups, with several sex-associated. Acinetobacter, Brevundimonas, Dolosigranulum, Finegoldia, Haemophilus, Leptotrichia, Moraxella, Peptoniphilus, Pseudomonas, Rothia, and Staphylococcus were the only 11 bacterial genera that presented significant age-associated differences. Other bacterial genera such as Anaerococcus, Burkholderia, Campylobacter, Delftia, Prevotella, Neisseria, Propionibacterium, Streptococcus, Ralstonia, Sphingomonas, and Corynebacterium appeared in the population with a very high frequency, suggesting that their presence might be biologically relevant. Therefore, in contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated abundance changes were observed at phylum, family, and genus levels, as well as several sex-associated changes probably due to the different levels of sex hormones present in both sexes at certain ages. Our results provide a complete and valuable dataset that will be useful for future research aiming for studying the relationship between changes in the nasopharyngeal microbiome and susceptibility to or severity of multiple diseases.

The nasopharyngeal microbiome in COVID-19.

The development of novel culture-independent techniques of microbial identification has allowed a rapid progress in the knowledge of the nasopharyngeal microbiota and its role in health and disease. Thus, it has been demonstrated that the nasopharyngeal microbiota defends the host from invading pathogens that enter the body through the upper airways by participating in the modulation of innate and adaptive immune responses. The current COVID-19 pandemic has created an urgent need for fast-track research, especially to identify and characterize biomarkers to predict the disease severity and outcome. Since the nasopharyngeal microbiota diversity and composition could potentially be used as a prognosis biomarker for COVID-19 patients, which would pave the way for strategies aiming to reduce the disease severity by modifying such microbiota, dozens of research articles have already explored the possible associations between changes in the nasopharyngeal microbiota and the severity or outcome of COVID-19 patients. Unfortunately, results are controversial, as many studies with apparently similar experimental designs have reported contradictory data. Herein we put together, compare, and discuss all the relevant results on this issue reported to date. Even more interesting, we discuss in detail which are the limitations of these studies, that probably are the main sources of the high variability observed. Therefore, this work is useful not only for people interested in current knowledge about the relationship between the nasopharyngeal microbiota and COVID-19, but also for researchers who want to go further in this field while avoiding the limitations and variability of previous works.

The Spike protein of SARS-CoV-2 signals via Tlr2 in zebrafish.

One of the most studied defense mechanisms against invading pathogens, including viruses, are Toll-like receptors (TLRs). Among them, TLR3, TLR7, TLR8 and TLR9 detect different forms of viral nucleic acids in endosomal compartments, whereas TLR2 and TLR4 recognize viral structural and nonstructural proteins outside the cell. Although many different TLRs have been shown to be involved in SARS-CoV-2 infection and detection of different structural proteins, most studies have been performed in vitro and the results obtained are rather contradictory. In this study, we report using the unique advantages of the zebrafish model for in vivo imaging and gene editing that the S1 domain of the Spike protein from the Wuhan strain (S1WT) induced hyperinflammation in zebrafish larvae via a Tlr2/Myd88 signaling pathway and independently of interleukin-1ß production. In addition, S1WT also triggered emergency myelopoiesis, but in this case through a Tlr2/Myd88-independent signaling pathway. These results shed light on the mechanisms involved in the fish host responses to viral proteins.

Zebrafish models of COVID-19.

Although COVID-19 has only recently appeared, research studies have already developed and implemented many animal models for deciphering the secrets of the disease and provided insights into the biology of SARS-CoV-2. However, there are several major factors that complicate the study of this virus in model organisms, such as the poor infectivity of clinical isolates of SARS-CoV-2 in some model species, and the absence of persistent infection, immunopathology, severe acute respiratory distress syndrome, and, in general, all the systemic complications which characterize COVID-19 clinically. Another important limitation is that SARS-CoV-2 mainly causes severe COVID-19 in older people with comorbidities, which represents a serious problem when attempting to use young and immunologically naïve laboratory animals in COVID-19 testing. We review here the main animal models developed so far to study COVID-19 and the unique advantages of the zebrafish model that may help to contribute to understand this disease, in particular to the identification and repurposing of drugs to treat COVID-19, to reveal the mechanism of action and side-effects of Spike-based vaccines, and to decipher the high susceptibility of aged people to COVID-19.

Silica crystals activate toll-like receptors and inflammasomes to promote local and systemic immune responses in zebrafish.

Silica crystals are potent activators of the inflammasome that cause a fibrotic lung disease, called silicosis, with no effective treatment available. We report here that injection of silica crystals into the hindbrain ventricle of zebrafish embryos led to the initiation of local and systemic immune responses driven through both Toll-like receptors (TLR)- and inflammasome-dependent signaling pathways, followed by induction of pro-fibrotic markers. Genetic and pharmacological analysis revealed that the Nlrp3 inflammasome regulated silica crystal-induced inflammation and pyroptotic cell death, but not emergency myelopoiesis. In addition, Cxcl8a/Cxcr2-dependent recruitment of myeloid cells to silica crystals was required to promote emergency myelopoiesis and systemic inflammation. The zebrafish model of silicosis developed here shed light onto the molecular mechanisms involved in the activation of the immune system by silica crystals.

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.

Senescence-Independent Anti-Inflammatory Activity of the Senolytic Drugs Dasatinib, Navitoclax, and Venetoclax in Zebrafish Models of Chronic Inflammation.

Telomere shortening is the main molecular mechanism of aging, but not the only one. The adaptive immune system also ages, and older organisms tend to develop a chronic pro-inflammatory status with low-grade inflammation characterized by chronic activation of the innate immune system, called inflammaging. One of the main stimuli that fuels inflammaging is a high nutrient intake, triggering a metabolic inflammation process called metainflammation. In this study, we report the anti-inflammatory activity of several senolytic drugs in the context of chronic inflammation, by using two different zebrafish models: (i) a chronic skin inflammation model with a hypomorphic mutation in spint1a, the gene encoding the serine protease inhibitor, kunitz-type, 1a (also known as hai1a) and (ii) a non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) model with inflammation induced by a high-fat diet. Our results show that, although these models do not manifest premature aging, the senolytic drugs dasatinib, navitoclax, and venetoclax have an anti-inflammatory effect that results in the amelioration of chronic inflammation.

Differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern.

The coronavirus disease 2019 (COVID-19) pandemic turned the whole world upside down in a short time. One of the main challenges faced has been to understand COVID-19-associated life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). We report here the proinflammatory role of Spike (S) proteins from different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern in zebrafish. We found that wild-type/Wuhan variant S1 (S1WT) promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1γ was more proinflammatory S1δ was less proinflammatory than S1WT, and, notably, S1ß promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome alleviated S1-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and angiotensin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants.

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.