Detection and Localization of IL-8 and CXCR1 in Rainbow Trout Larvae in Response to Pseudomonas aeruginosa Lipopolysaccharide

Abstract

The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

Keywords: CXCR1; IL-8; LPS; immunological capacity; trout larvae.

Figure 1

Structure and identification of epitope peptide from omCXCR1. (A) The predicted topology and sequence of omCXCR1 is shown with putative seven transmembrane segments positioned in the cell membrane. The amino acid sequence of the chosen epitope peptide is indicated in red. Furthermore, two conserved sites for cathepsin G and elastase in extracellular regions are indicated. (B) Three-dimensional structure of omCXCR1, where the chosen epitope peptide is shown in red.

Figure 2

Characterization of synthetic peptide epitope antisera to detect omCXCR1. (A) Three-dimensional structure of epitope peptide of omCXCR1. (B) Spectra of reverse-phase high-performance chromatography (RP-HPLC) revealed a purity of the epitope peptide higher than 90%. (C) ESI-MS showing the molecular ion [M+1H-H₂O] with m/z of 1672.99 Da. (D) Mouse antiserum anti-omCXCR1sp was characterized by dot blot and indirect ELISA, performing serial dilutions of synthetic peptides from 4 to 0.125 µg/µL and from 3 to 0.09 ng/µL, respectively.

Figure 3

Effect of LPS challenge on the protein expression of omIL-8 and omCXCR1 in rainbow trout larvae at 10, 13, 16, 19, and 33 days post-hatching (dph). Indirect ELISA showing omCXCR1 (A) and omIL-8 (B) bioavailability in trout larvae 2 h and 8 h after LPS challenge. Statistically significant differences are indicated with **** p < 0.01.

Figure 4

Location of omIL-8 and omCXCR1 in rainbow trout fry at 19 days post-hatching after LPS challenge. Immunolocalization of omCXCR1/IL-8 in tissues of rainbow trout larvae 8 h after LPS challenged. Above: Representative photos of trout larvae at ×40 magnification illustrating the localization by immunohistochemistry (A–F) of omCXCR1 in fish skin, gill, and gut in control (A,C,E) and LPS-challenged larvae (B,D,F). Red arrows denote corresponding cells which are omCXCR1 positive. The letters in black are defined as follows: mc: mucous cells; ep: epidermis; ec: epithelial cells; p: pillar cell; sl: secondary lamellae; pl: primary lamellae; cc: chloride cells; RBC: red blood cells; e: enterocyte; mv: microvilli, and lp: lamina propia. Bar: 50 μm. Below: Co-localization of omCXCR1 with omIL-8 is shown in fish skin (G–I), gill (J–L), and gut (M–O). omCXCR1 was detected using mouse antiserum anti-omCXCR1sp (1:500 dilution) and Alexa Fluor 568 (G,J,M), and omIL-8 was detected using rabbit antiserum anti-omIL-8 (1:500 dilution) and Alexa Fluor 635 (H,K,N). Nuclear staining was performed using SYTO 9. Bar: 20 μm.