Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

Abstract

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.

A high-sensitivity bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real time

“…6B, bottom panel). This is in agreement with previous studies showing that microinjection of bacteria in these tissues results in an inflammatory response that includes the recruitment of leukocytes (Levraud et al, 2008;Nguyen-Chi et al, 2014). We also observed an accumulation of GFP-expressing immune cells (Fig.…”

“…Morpholino at a concentration of 0.3 mM or 1 mM together with 100 mM KCl was injected into the blastomere of zebrafish embryos at the one-cell stage. Nonpathogenic Gram-negative Escherichia coli BL21 expressing HcRed (Aghaallaei et al, 2010) were microinjected into the transgenic pSGNluc embryos at 3 dpf as described previously (Nguyen-Chi et al, 2014).…”

TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity

“…Neutrophils, and to a less extent macrophages, have been described as the main source of IL-1b and TNF-a (19,43,57), but at the Prim-5 stage macrophages are hardly detected and neutrophils are absent (30,31,56,58). However, we demonstrate that if protective chorion is removed in advance, zebrafish has already developed yet various types of epidermal cells at the surface and inner layer (59) that cope with common stressors (60).…”

Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

“…All morpholinos were injected into single cell stage larvae in total volume of 2 nl. Knockdown of il-1β was carried out using the antisense morpholino against il-1β (5 -CCCACAAACTGCAAAATATCAGCTT-3 ) targeting the splice site between intron 2 and exon 3 according to 24 . In order to block neutrophil development, we used the previously described MO combination 15 For live confocal imaging, zebrafish larvae were anesthetized in PBS containing 0.02% MS222 and mounted in 1.5% low melting point agarose (Ultra-PureTM, Invitrogen).…”

“…For this, we used the pharmacological inhibitor YVAD that is functional in zebrafish 23 ( Fig.7A-E). Secondly, we used an established morpholino to disrupt il-1β RNA splicing 24 . Finally, we targeted il-1β with gRNA in a CRISPR approach (Fig.…”