Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Taurocholic Acid and Glycocholic Acid Inhibit Inflammation and Activate Farnesoid X Receptor Expression in LPS-Stimulated Zebrafish and Macrophages.

A hyperactive immune response can be observed in patients with bacterial or viral infection, which may lead to the overproduction of proinflammatory cytokines, or "cytokine storm", and a poor clinical outcome. Extensive research efforts have been devoted to the discovery of effective immune modulators, yet the therapeutic options are still very limited. Here, we focused on the clinically indicated anti-inflammatory natural product Calculus bovis and its related patent drug Babaodan to investigate the major active molecules in the medicinal mixture. Combined with high-resolution mass spectrometry, transgenic zebrafish-based phenotypic screening, and mouse macrophage models, taurochiolic acid (TCA) and glycoholic acid (GCA) were identified as two naturally derived anti-inflammatory agents with high efficacy and safety. Both bile acids significantly inhibited the lipopolysaccharide-induced macrophage recruitment and the secretion of proinflammatory cytokines/chemokines in in vivo and in vitro models. Further studies identified strongly increased expression of the farnesoid X receptor at both the mRNA and protein levels upon the administration of TCA or GCA, which may be essential for mediating the anti-inflammatory effects of the two bile acids. In conclusion, we identified TCA and GCA as two major anti-inflammatory compounds in Calculus bovis and Babaodan, which could be important quality markers for the future development of Calculus bovis, as well as promising lead compounds in the treatment of overactive immune responses.

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.

Anti-Inflammatory Activity of CIGB-258 against Acute Toxicity of Carboxymethyllysine in Paralyzed Zebrafish via Enhancement of High-Density Lipoproteins Stability and Functionality.

Background: Hyperinflammation is frequently associated with the chronic pain of autoimmune disease and the acute death of coronavirus disease (COVID-19) via a severe cytokine cascade. CIGB-258 (Jusvinza®), an altered peptide ligand with 3 kDa from heat shock protein 60 (HSP60), inhibits the systemic inflammation and cytokine storm, but the precise mechanism is still unknown. Objective: The protective effect of CIGB-258 against inflammatory stress of N-ε-carboxymethyllysine (CML) was tested to provide mechanistic insight. Methods: CIGB-258 was treated to high-density lipoproteins (HDL) and injected into zebrafish and its embryo to test a putative anti-inflammatory activity under presence of CML. Results: Treatment of CML (final 200 µM) caused remarkable glycation of HDL with severe aggregation of HDL particles to produce dysfunctional HDL, which is associated with a decrease in apolipoprotein A-I stability and lowered paraoxonase activity. Degradation of HDL3 by ferrous ions was attenuated by a co-treatment with CIGB-258 with a red-shift of the Trp fluorescence in HDL. A microinjection of CML (500 ng) into zebrafish embryos resulted in the highest embryo death rate, only 18% of survivability with developmental defects. However, co-injection of CIGB-258 (final 1 ng) caused the remarkable elevation of survivability around 58%, as well as normal developmental speed. An intraperitoneal injection of CML (final 250 µg) into adult zebrafish resulted acute paralysis, sudden death, and laying down on the bottom of the cage with no swimming ability via neurotoxicity and inflammation. However, a co-injection of CIGB-258 (1 µg) resulted in faster recovery of the swimming ability and higher survivability than CML alone injection. The CML alone group showed 49% survivability, while the CIGB-258 group showed 97% survivability (p < 0.001) with a remarkable decrease in hepatic inflammation up to 50%. A comparison of efficacy with CIGB-258, Infliximab (Remsima®), and Tocilizumab (Actemra®) showed that the CIGB-258 group exhibited faster recovery and swimming ability with higher survivability than those of the Infliximab group. The CIGB-258 group and Tocilizumab group showed the highest survivability, the lowest plasma total cholesterol and triglyceride level, and the infiltration of inflammatory cells, such as neutrophils in hepatic tissue. Conclusion: CIGB-258 ameliorated the acute neurotoxicity, paralysis, hyperinflammation, and death induced by CML, resulting in higher survivability in zebrafish and its embryos by enhancing the HDL structure and functionality.

Localization of BDNF and Calretinin in Olfactory Epithelium and Taste Buds of Zebrafish (Danio rerio).

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and it is involved in several fundamental functions in the central and peripheral nervous systems, and in sensory organs. BDNF regulates the chemosensory systems of mammals and is consistently expressed in those organs. In zebrafish, the key role of BDNF in the biology of the hair cells of the inner ear and lateral line system has recently been demonstrated. However, only some information is available about its occurrence in the olfactory epithelium, taste buds, and cutaneous isolated chemosensory cells. Therefore, this study was undertaken to analyze the involvement of BDNF in the chemosensory organs of zebrafish during the larval and adult stages. To identify cells displaying BDNF, we compared the cellular pattern of BDNF-displaying cells with those immunoreactive for calretinin and S100 protein. Our results demonstrate the localization of BDNF in the sensory part of the olfactory epithelium, mainly in the ciliated olfactory sensory neurons in larvae and adult zebrafish. Intense immunoreaction for BDNF was also observed in the chemosensory cells of oral and cutaneous taste buds. Moreover, a subpopulation of olfactory sensory neurons and chemosensory cells of olfactory rosette and taste bud, respectively, showed marked immunopositivity for calcium-binding protein S100 and calretinin. These results demonstrate the possible role of BDNF in the development and maintenance of olfactory sensory neurons and sensory cells in the olfactory epithelium and taste organs of zebrafish during all stages of development.

Alzheimer's Disease and Toxins Produced by Marine Dinoflagellates: An Issue to Explore.

This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in ß-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer's disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce ß-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.

Assessing Molecular Regulation of Vascular Permeability Using a VEGF-Inducible Zebrafish Model.

Vascular endothelial growth factor (VEGF) stimulates vascular permeability in a variety of human pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema, sepsis, acute lung injury, and acute respiratory distress syndrome. Comprehensive investigation of the molecular mechanisms of VEGF-induced vascular permeability has been hindered by the lack of in vivo models that easily facilitate genetic manipulation studies in real time. To address this need, we generated a heat-inducible VEGF transgenic zebrafish model of vascular permeability. Here, we describe how this zebrafish model can be used to monitor VEGF-induced vascular permeability through live in vivo imaging to identify genetic regulators that play key roles in vascular barrier integrity in physiological conditions and human disease processes.

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level.

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.

The effect of renin-angiotensin-aldosterone system inhibitors on organ-specific ace2 expression in zebrafish and its implications for COVID-19.

Among cases of SARS-CoV-2 infections that result in serious conditions or death, many have pre-existing conditions such as hypertension and are on renin-angiotensin-aldosterone system (RAAS) inhibitors. The angiotensin-converting-enzyme-2 (ACE2), a key protein of the RAAS pathway, also mediates cellular entry of SARS-CoV-2. RAAS inhibitors might affect the expression levels of ace2, which could impact patient susceptibility to SARS-CoV-2. However, multi-organ-specific information is currently lacking and no species other than rodents have been examined. To address this knowledge gap, we treated adult zebrafish with the RAAS inhibitors aliskiren, olmesartan, and captopril for 7 consecutive days and performed qRT-PCR analysis of major RAAS pathway genes in the brain, gill, heart, intestine, kidney, and liver. Both olmesartan and captopril significantly increased ace2 expression in the heart, gill, and kidney. Olmesartan also increased ace2 expression in the intestine. Conversely, aliskiren significantly decreased ace2 expression in the heart. Discontinuation of compound treatments for 7 days did not return ace2 expression to baseline levels. While potential risks or benefits of antihypertensive RAAS inhibitors to SARS-CoV-2 infections in humans remain uncertain, this study provides new insights regarding the impact of RAAS inhibitors on organ-specific ace2 expression in another vertebrate model, thereby providing comparative data and laying scientific groundwork for future clinical decisions of RAAS inhibitor use in the context of COVID-19.

The SARS-CoV-2 receptor and other key components of the Renin-Angiotensin-Aldosterone System related to COVID-19 are expressed in enterocytes in larval zebrafish.

People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with coronavirus SARS-CoV-2, which causes COVID-19. Hypertension and respiratory inflammation are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure via Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, counteracting its chronic effects, and serves as the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we do not yet understand how. To exploit zebrafish (Danio rerio) to help understand the relationship of the RAAS to COVID-19, we must identify zebrafish orthologs and co-orthologs of human RAAS genes and understand their expression patterns. To achieve these goals, we conducted genomic and phylogenetic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have one or more zebrafish orthologs or co-orthologs. Results identified a specific type of enterocyte as the specific site of expression of zebrafish orthologs of key RAAS components, including Ace, Ace2, Slc6a19 (SARS-CoV-2 co-receptor), and the Angiotensin-related peptide cleaving enzymes Anpep (receptor for the common cold coronavirus HCoV-229E), and Dpp4 (receptor for the Middle East Respiratory Syndrome virus, MERS-CoV). Results identified specific vascular cell subtypes expressing Ang II receptors, apelin, and apelin receptor genes. These results identify genes and cell types to exploit zebrafish as a disease model for understanding mechanisms of COVID-19.