Isabel Cabas Sánchez¹, Diana García Moreno², Victoria Gómez Abellán¹, Francisco Juan Martínez Navarro¹, Victoriano Mulero Méndez¹, Alfonsa García Ayala¹

(1) INMUNIDAD, INFLAMACIÓN Y CÁNCER, IMIB-Arrixaca, España
(2) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España

Estrogens are known to be involve in some chronic inflammatory diseases, such as psoriasis, as well as in infection. Despite their classical actions through nuclear estrogen receptors, additional estrogen-mediated effects on immune response could be mediated through G protein-coupled estrogen receptor 1 (GPER1), but almost unexplored. In zebrafish (Danio rerio), a recognized biomedical animal model, Gper1 has been identified showing high identity with human one. Thus, the aim of this study is to generate a gper1 deficient zebrafish line will allow to evaluate the impact and mechanism of Gper1 on key immune contexts such as infection and inflammation, in addition to analyze its relevance in a psoriasis dataset.

Zebrafish fertilized eggs/larvae/adults of wildtype, spint1a (also transgenic lyz:DsRED2nz50; red fluorescence neutrophils) and gper1 mutants. Single guide RNA (sgRNA) directed to a target sequence in the coding region of start of gper1-exon 2: ChopChop software and Veijnar et al. 2016 protocol. Microinjection of CRISPR/Cas9 system of eggs at one-cell stage. GPER1-selective agonist (G1), by bath immersion. Vibrio anguillarum R82 serogroup O1 intraperitoneal injection (50 bacteria/10 μl/adult fish). Whole kidney marrow (WKM) cell suspensions at 24 hours post infection for flow cytometry and gene expression analysis. FlowJo software to flow cytometry data analysis. RT-qPCR for il1b gene expression. GPER1 expression profile (ID 100692226) (180 samples, array) in the GEO dataset study “psoriasis lesional and non-lesional skin” (GDS4602). Neutrophil dispersion (towards the skin) using a Leica MZ16F fluorescence stereo microscope equipped with fluorescent filters and ImageJ. Statistical analysis by one or two-way ANOVA with Tukey post-test. p ≤ 0,05, using GraphPadPrism 7 software.

Using a specific sgRNA, disruption of gper1 was confirmed in F0 and F1-larvae by T7 endonuclease I mismatch cleavage assay, where a significant reduction of the wildtype product (943 bp) was observed. A deletion of 47 bp in mutant sequence was revealed in F1-adults by DNA sequencing. Offspring from heterozygotes were genotyped by PCR, the different genotypes where observed: wildtype (+/+; 943 bp), heterozygote (+/-; 943 and 896 bp) and homozygote (-/-; 896 bp). The disruption of gper1 was not able to significantly alter the percentage of any WKM cell populations neither in a control condition nor under infection. However, the infection-induced il1b mRNA levels were significantly higher in gper1 mutants. Moreover, significant lower GPER1-expression levels in affected epidermal sectors of psoriasis patients and a negative and significant correlation with IL1B expression were observed. The activation of Gper1 was not able to modulate the neutrophil dispersion observed in a zebrafish chronic skin inflammation model.

A gper1 mutant zebrafish line was successfully generated by CRISPR/Cas9 system. Although estrogens are known to regulate hematopoiesis, the absence of Gper1 did not modulate kidney marrow cell populations. In agreement with previous report, a negative correlation between Gper1 and inflammation are observed. The expression of GPER1 in psoriatic skin biopsies, Gper1 activation/overexpression/depletion in the spint1a model and/or evaluation of an induced-inflammatory state in gper1 mutants, will be carried out. The results could point to GPER1 as a potential therapeutic target in the immune disorders where estrogens taking part.