The presence of NLc liposomes in macrophage-like cells from the spleen was confirmed at 24, 48 and 72 h ( Fig. 2B). Fluorescent NLc liposomes were also found in macrophage-like cells isolated from head kidney ( Fig. 2C). The membrane-staining and the z-stack images enabled visualisation of the exact location of the liposomes, and the images demonstrated that the liposomes had been completely taken up by the cells; no fluorescent NLc liposomes attached to the plasma membrane were detected ( Fig. 2B and C(iii, iv)). In previous work, we showed that NLc liposomes induced the expression of immunologically

relevant genes in vitro [18]. Having determined, in the present work, that these liposomes target macrophage-like cells in vivo, we next studied the protective effect of the system against P. aeruginosa infection. Before the immunisation experiments, click here the PAO1 infection model in adult zebrafish was fully characterised by determining the LD50 = 5.3 × 107 cfu (supplementary Fig. 1), and then recovering Bortezomib and subsequently identifying the PAO1 strain by 16S rRNA sequencing (data not shown). The zebrafish were

immunised with the NLc liposomes, and then challenged with the PAO1 bacteria at 1 day, 1 week or 1 month post-immunisation. Their survival rates were assessed and the results were used to compare the different immunisation protocols ( Fig. 3 and supplementary Fig. 2 and Table 1). Neither the empty liposomes nor the mixture of free immunostimulants (poly(I:C) and LPS) protected the zebrafish against PAO1 infection when injected 1 day (supplementary Fig. 2) or 1 week ( Fig. 3A) before the challenge. In contrast, the fish that had received NLc liposomes exhibited significantly higher survival rates than the control group, regardless of the date of administration (RPS of 33.2% at 1 day; 47.1% at 1 week; and 36.3% at 1 month ( Fig. 3, supplementary Fig. 2 and Table 1). To determine the feasibility of using a storable version of the NLc liposomes Non-specific serine/threonine protein kinase (supplementary Fig. 3), we also evaluated the efficacy of lyophilised NLc liposomes against P. aeruginosa infection. Thus, adult zebrafish were treated with rehydrated

lyophilised NLc liposomes or with freshly prepared NLc liposomes, and then infected at 1 week post-injection ( Fig. 3A). Interestingly, the lyophilised liposomes were as effective as the freshly prepared ones (58.3% survival vs. 50% survival, respectively; Fig. 3A). This result confirmed that lyophilised liposomes are amenable to use after long-term storage. Supplementary Fig. 1.  Survival of adult zebrafish after challenge with P. aeruginosa (PAO1) by i.p. injection for LD50 determination. Fish were challenged with P. aeruginosa by i.p. injection of 20 μl of a bacterial suspension at concentrations ranging from 3.2 × 107 to 2.5 × 108 cfu/dose. Survival was recorded daily until 120 h post-injection. LD50 was determined to be 5.3 × 107 cfus.