Ontogenetically distinct neutrophils differ in function and transcriptional profile in zebrafish.

The current view of hematopoiesis considers leukocytes on a continuum with distinct developmental origins, and which exert non-overlapping functions. However, there is less known about the function and phenotype of ontogenetically distinct neutrophil populations. In this work, using a photoconvertible transgenic zebrafish line; Tg(mpx:Dendra2), we selectively label rostral blood island-derived and caudal hematopoietic tissue-derived neutrophils in vivo during steady state or upon injury. By comparing the migratory properties and single-cell expression profiles of both neutrophil populations at steady state we show that rostral neutrophils show higher csf3b expression and migration capacity than caudal neutrophils. Upon injury, both populations share a core transcriptional profile as well as subset-specific transcriptional signatures. Accordingly, both rostral and caudal neutrophils are recruited to the wound independently of their distance to the injury. While rostral neutrophils respond uniformly, caudal neutrophils respond heterogeneously. Collectively, our results reveal that co-existing neutrophils populations with ontogenically distinct origin display functional differences.

Articaine in functional NLC show improved anesthesia and anti-inflammatory activity in zebrafish.

Anesthetic failure is common in dental inflammation processes, even when modern agents, such as articaine, are used. Nanostructured lipid carriers (NLC) are systems with the potential to improve anesthetic efficacy, in which active excipients can provide desirable properties, such as anti-inflammatory. Coupling factorial design (FD) for in vitro formulation development with in vivo zebrafish tests, six different NLC formulations, composed of synthetic (cetyl palmitate/triglycerides) or natural (avocado butter/olive oil/copaiba oil) lipids were evaluated for loading articaine. The formulations selected by FD were physicochemically characterized, tested for shelf stability and in vitro release kinetics and had their in vivo effect (anti-inflammatory and anesthetic effect) screened in zebrafish. The optimized NLC formulation composed of avocado butter, copaiba oil, Tween 80 and 2% articaine showed adequate physicochemical properties (size = 217.7 ± 0.8 nm, PDI = 0.174 ± 0.004, zeta potential = - 40.2 ± 1.1 mV, %EE = 70.6 ± 1.8) and exhibited anti-inflammatory activity. The anesthetic effect on touch reaction and heart rate of zebrafish was improved to 100 and 60%, respectively, in comparison to free articaine. The combined FD/zebrafish approach was very effective to reveal the best articaine-in-NLC formulation, aiming the control of pain at inflamed tissues.

Intestinal Inflammation Induced by Soybean Meal Ingestion Increases Intestinal Permeability and Neutrophil Turnover Independently of Microbiota in Zebrafish.

Intestinal inflammation is a condition shared by several intestinal chronic diseases, such as Crohn's disease and ulcerative colitis, with severely detrimental consequences in the long run. Current mammalian models have considerably increased understanding of this pathological condition, highlighting the fact that, in most of the cases, it is a highly complex and multifactorial problem and difficult to deal with. Thus, there is an increasingly evident need for alternative animal models that could offer complementary approaches that have not been exploited in rodents, thereby contributing to a different view on the disease. Here, we report the effects of a soybean meal-induced intestinal inflammation model on intestinal integrity and function as well as on neutrophil recruitment and microbiota composition in zebrafish. We find that the induced intestinal inflammation process is accompanied by an increase in epithelial permeability in addition to changes in the mRNA levels of different tight junction proteins. Conversely, there was no evidence of damage of epithelial cells nor an increase in their proliferation. Of note, our results show that this intestinal inflammatory model is induced independently of the presence of microbiota. On the other hand, this inflammatory process affects intestinal physiology by decreasing protein absorption, increasing neutrophil replacement, and altering microbiota composition with a decrease in the diversity of cultivable bacteria.

Zebrafish, a model to develop nanotherapeutics that control neutrophils response during inflammation.

Neutrophils are crucial modulators of the inflammation process, and their uncontrolled response worsens several chronic pathologies. The p38 mitogen-activated protein kinases (MAPKs) activity is critical for normal immune and inflammatory response through the regulation of pro-inflammatory cytokines synthesis. In this work, we study the effect of hybrid lipid-polymer nanoparticles loaded with the p38 MAPK inhibitor SB203580 in an acute and chronic inflammatory model in zebrafish containing a transgenic neutrophil cell line that constitutively expresses a green fluorescent protein. We identify the existence of at least two neutrophils subpopulation involved in the response during the acute inflammation triggered; a first-responder p38α-independent subset and a second-responder p38α-dependent subset. In the case of chronic inflammation, neutrophils recruited in the intestine only during the inflammation process, migrate in a p38α-dependent manner. Likewise, we establish that SB203580-loaded in NPs exerts their action during at least a double period than the inhibitor administers directly in both types of inflammation. Our results demonstrate the exceptional potential of the zebrafish as an inflammatory model for studying novel nanotherapeutics that selectively inhibit the neutrophils response, and to identify functional neutrophils subpopulations involved in the inflammation process.