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. 2023 May 12:14:1193977.
doi: 10.3389/fimmu.2023.1193977. eCollection 2023.

The manipulation of cell suspensions from zebrafish intestinal mucosa contributes to understanding enteritis

Xuyang Zhao  1   2 , Yuhang Liu  1   2 , Jiayuan Xie  1   3 , Lei Zhang  1   3 , Qingsong Zhu  1   2 , Lian Su  1   3 , Cheng Guo  1   3 , Heng Li  1   3 , Guangxin Wang  1 , Wanting Zhang  1 , Yingyin Cheng  1 , Nan Wu  1   3 , Xiao-Qin Xia  1   3
Affiliations

The manipulation of cell suspensions from zebrafish intestinal mucosa contributes to understanding enteritis

Xuyang Zhao et al. Front Immunol. .

Abstract

Background: Although zebrafish are commonly used to study intestinal mucosal immunity, no dedicated procedure for isolating immune cells from zebrafish intestines is currently available. A speedy and simple operating approach for preparing cell suspension from mucosa has been devised to better understanding of intestinal cellular immunity in zebrafish.

Methods and results: The mucosal villi were separated away from the muscle layer by repeated blows. The complete deprivation of mucosa was done and evidenced by HE and qPCR results. Higher expression of both innate (mpeg1, mpx, and lck) and adaptive immune genes (zap70, blnk, foxp3a, and foxp3b) was revealed compared to cells obtained by typical mesh rubbing. The cytometric results also revealed that the tested operation group had a higher concentration and viability. Further, fluorescent-labelled immune cells from 3mo Tg(lyz:DsRED2), Tg(mpeg1:EGFP), Tg(Rag2:DsRED), and Tg(lck:EGFP), were isolated and evaluated for the proportion, and immune cells' type could be inferred from the expression of marker genes. The transcriptomic data demonstrated that the intestinal immune cell suspension made using the new technique was enriched in immune-related genes and pathways, including il17a/f, il22, cd59, and zap70, as well as pattern recognition receptor signaling and cytokine-cytokine receptor interaction. In addition, the low expression of DEG for the adherent and close junctions indicated less muscular contamination. Also, lower expression of gel-forming mucus-associated genes in the mucosal cell suspension was consistent with the current less viscous cell suspension. To apply and validate the developed manipulation, enteritis was induced by soybean meal diet, and immune cell suspensions were analyzed by flow cytometry and qPCR. The finding that in enteritis samples, there was inflammatory increase of neutrophils and macrophages, was in line with upregulated cytokines (il8 and il10) and cell markers (mpeg1 and mpx).

Conclusion: As a result, the current work created a realistic technique for studying intestinal immune cells in zebrafish. The immune cells acquired may aid in further research and knowledge of intestinal illness at the cellular level.

Keywords: SBMIE; cell suspension; flow cytometry; intestine; transcriptome; zebrafish.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Operating processes for preparing cell suspension from zebrafish intestinal mucosa. (A) Schematic diagram of operating processes. (B) Photographs of the detail operation, including dissection, intestinal separation, blowing and filtering.
Figure 2
Figure 2
Quality control of prepared intestinal single cell suspension from Tg(lyz:DsRED2);Tg(mpeg1:EGFP). (A) Imaging by automated cell counter, cell under brightfield and fluorescence channels (FL1: 488nm; FL2:558nm). Scale bar: 100μm. The cellular diameter of the prepared intestinal mucosal cell suspension was calculated besides. (B) Cell viability reflected AO/PI staining during cytometric analysis. The stained cell number was counted by automatic cell counter. After AO/PI staining, 75% cell obtained from robbing method (RM) was alive, meanwhile for the blowing method, 80% of all cells (AC) and 90% of genetically fluorescence labeled immune cells, which were the target cells (TC), were alive for flow cytometric analysis. (C) Images of intestinal mucosal cells from Tg(lyz:DsRED2);Tg(mpeg1:EGFP). In single cell suspensions, DsRed labeled lyz+ cell and EGFP labeled mpeg1+ cells could be clearly observed, together with other unstained cells (shown in the brightfield). ** represented p < 0.01, **** represented p < 0.0001.
Figure 3
Figure 3
Enriching intestinal immune cells via stripping the muscular layer. (A) Morphological analysis of HE staining in comparison between intestinal tissue after blowing treatment and normal intestinal tissue. (B) qPCR analysis of genes involved in immune cell differentiation. All values are means ± SEM; statistical significance was determined by independent-samples T-test. For the comparison of IMC (intestinal mucosa cells) vs IT (intestinal tissue), *** represented p < 0.01.
Figure 4
Figure 4
Enriched KEGG pathways and GO terms as well as heatmaps of mucosal immune related DEGs. Intestinal pathways (A) and terms (B) for the comparison between IMC and IT. (C) The heat map of immune cells maker genes. (D) The heat map of mucus related genes. (E) The heat map of mast cell related genes.
Figure 5
Figure 5
Visualization of involved genes in innate immune related KEGG pathways by cnetplot analysis. The pattern recognition related “C-type lectin receptor signaling pathway”, “NOD like receptor signaling pathway”, and “Toll like receptor signaling pathway”, as well as cytokine signaling related “cytokine-cytokine receptor interaction” were shown in details.
Figure 6
Figure 6
Flow cytometric analysis of transgenic zebrafish, including Tg(lyz:DsRED2), Tg(rag2:DsRed), Tg(lck:EGFP), and Tg(mpeg1:EGFP). (A) The gating strategies in cytometric analysis for intestinal neutrophils (with red fluorescence labeled Lyz), immature lymphocytes (with red fluorescence labeled Rag2), mature T lymphocytes (with green fluorescence labeled Lck), and macrophages (with green fluorescence labeled Mpeg1) in zebrafish. Compare with wild type samples, all types of fluorescence labeled immune cells could be accurately sorted. (B) Comparing samples made by mechanical dissociation of whole intestine and blowing off mucosal cells from muscularis, the fluorescence labeled cells were significantly enriched. * represented p < 0.05, **** represented p < 0.0001.
Figure 7
Figure 7
Proportion of fluorescent labeled immune cells, including neutrophils, macrophages, lymphocytes, and activated T cells, in immune organs (the periphery blood, intestine, liver, kidney and spleen). * represented p < 0.05, ** represented 0.01< p < 0.05. and *** represented p < 0.01.
Figure 8
Figure 8
The SBMIE induced mucosa pathology was related to the altered composition of immune cells. The single cell suspension, obtained using current optimized method, accurately reflects altered composition of intestinal immune cells upon soybean induced inflammation. (A) Validation of inflammation by pathological analysis of intestinal mucosa. The length of intestinal villi was reduced at the condition of SBM (soybean meal) feeding. (B) qPCR analysis of genes involved in intestinal inflammation. (C) the increased proportion of intestinal mpeg+ and lyz+ cells upon SBMIE. *** represented p < 0.01. ns, no significance.
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