Microbial colonization induces dynamic temporal and spatial patterns of NF-κB activation in the zebrafish digestive tract

Abstract

Background & aims: The nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) transcription factor pathway is activated in response to diverse microbial stimuli to regulate expression of genes involved in immune responses and tissue homeostasis. However, the temporal and spatial activation of NF-κB in response to microbial signals have not been determined in whole living organisms, and the molecular and cellular details of these responses are not well understood. We used in vivo imaging and molecular approaches to analyze NF-κB activation in response to the commensal microbiota in transparent gnotobiotic zebrafish.

Methods: We used DNA microarrays, in situ hybridization, and quantitative reverse transcription polymerase chain reaction analyses to study the effects of the commensal microbiota on gene expression in gnotobiotic zebrafish. Zebrafish PAC2 and ZFL cells were used to study the NF-κB signaling pathway in response to bacterial stimuli. We generated transgenic zebrafish that express enhanced green fluorescent protein under transcriptional control of NF-κB, and used them to study patterns of NF-κB activation during development and microbial colonization.

Results: Bacterial stimulation induced canonical activation of the NF-κB pathway in zebrafish cells. Colonization of germ-free transgenic zebrafish with a commensal microbiota activated NF-κB and led to up-regulation of its target genes in intestinal and extraintestinal tissues of the digestive tract. Colonization with the bacterium Pseudomonas aeruginosa was sufficient to activate NF-κB, and this activation required a functional flagellar apparatus.

Conclusions: In zebrafish, transcriptional activity of NF-κB is spatially and temporally regulated by specific microbial factors. The observed patterns of NF-κB-dependent responses to microbial colonization indicate that cells in the gastrointestinal tract respond robustly to the microbial environment.

Fig. 1. Bacterial stimulation of zebrafish cell lines results in activation of the canonical NF-κB pathway and induces expression of NF-κB target genes

(A) Western blot of zebrafish PAC2 cells shows rapid phosphorylation of IκBα proteins following LPS (10 μg/ml) stimulation. (B) Immunofluorescence of PAC2 cells using anti-RelA antibody reveals rapid nuclear localization following LPS stimulation. Original magnification set at 400×. (C) qRT-PCR using primers for ikbaa, a predicted NF-κB target gene, demonstrating time-dependent accumulation upon stimulation with LPS (10 μg/ml) or P. aeruginosa PAK lysate (43.5 μg/ml; normalized to 18S rRNA). (D) Chromatin immunoprecipitation of LPS-stimulated PAC2 cells reveals increased binding of RelA to the zebrafish ikbaa promoter. (E) Zebrafish ZFL cells transfected with ikbaa-luciferase gene reporter (pikbaa:Luc) show increased luciferase activity upon stimulation with LPS (10 μg/ml) or P. aeruginosa PAK lysates (43.5 μg/ml). (F) qRT-PCR of ikbaa shows that induction upon LPS stimulation of PAC2 cells is attenuated after treatment with NAI (200 nM; normalized to 18S rRNA). Data in panels C–F are expressed as mean ± SD. See also Figure S3.

Fig. 2. Tg(NFkB:EGFP) zebrafish express EGFP in diverse tissues during development

(A) Schematic depiction of pNFkB:EGFP transgene. Brightfield and EGFP fluorescence images of CONV-R Tg(NFkB:EGFP) zebrafish at 24hpf (B), 50hpf (C), 74hpf (D), and 6dpf (E). (F) Higher magnification images at 50hpf and 6dpf illustrate diverse tissues expressing EGFP including neuromasts (nm), dorsal root ganglia (drg), pharyngeal teeth (pt), cloaca (cl), liver (lv), and intestine (in). Scale Bars: 500μm. See also Figures S4, S5, and S6.

Fig. 3. Colonization of GF Tg(NFkB:EGFP) zebrafish with a microbiota stimulates binding of RelA to reporter transgene and ikbaa promoter

Measurement of RelA binding in whole 6dpf GF and CONVD zebrafish (10 fish/condition) was performed using anti-RelA antibody and primers targeting the synthetic NF-κB binding site in the NFkB:EGFP transgene (A) and the promoter of predicted NF-κB target gene ikbaa (B). Results are normalized to internal input controls and expressed as mean fold-difference ± SD.

Fig. 4. Conventionalization of GF Tg(NFkB:EGFP) zebrafish stimulates EGFP expression in diverse cell types

Brightfield (A) and EGFP fluorescence images shown as heatmaps (A–E) of GF 3dpf (A) and GF and CONVD 4dpf (B), 5dpf (C), 6dpf (D), and 8dpf Tg(NFkB:EGFP) zebrafish (E). (F) Quantification of relative EGFP fluorescence reveals a significant increase in CONVD compared to GF controls by 5dpf which continues through 8dpf. Treatment with 200nM NAI from 3–6dpf results in a significant decrease in relative EGFP fluorescence of 6dpf CONVD zebrafish compared to vehicle-treated CONVD controls (6–10 fish/condition). Scale bars: 200μm.

Fig. 5. Microbiota activates NF-κB in specific tissues in gnotobiotic zebrafish

Quantification of relative EGFP fluorescence in specific tissues of 6dpf GF and CONVD Tg(NFkB:EGFP) zebrafish. (A) Areas quantified (outlined in green) include intestinal segment 1 (seg 1), segment 2 (seg 2), segment 3 including cloaca (seg 3), liver (lv), swim bladder (sb), neuromasts (nm), dorsal root ganglia (drg), and muscle (m). (B) Quantification of EGFP fluorescence shows significant induction in intestine and other digestive tract tissues of CONVD zebrafish compared to GF controls (10 fish/condition). (C) FACS histogram showing Log EGFP fluorescence in DsRed-positive intestinal epithelial cells (IECs) from 6dpf Tg(-4.5ifabp:DsRed)(NFKB:EGFP) GF and CONVD zebrafish. Data is representative of two biological replicates. (D) Wholemount confocal microscopy of EGFP fluorescence in swim bladder and liver in 6dpf GF and CONVD Tg(NFkB:EGFP) zebrafish. Scale bars: 10μm.

Fig. 6. Conventionalization of GF zebrafish stimulates NF-κB dependent immune responses in diverse cell types

(A–C) Semi-quantitative wholemount in situ hybridization of 6dpf GF and CONVD zebrafish using RNA probes for complement factor b (cfb), serum amyloid a (saa), and myeloperoxidase (mpo). (A) CONVD zebrafish show increased cfb expression in the liver (black arrowhead) compared to GF controls. (B) CONVD zebrafish also display increased saa expression in liver (black arrowhead), swim bladder (white asterisk) and segment 3 of the intestine (black arrow) compared to GF controls. (C) CONVD larvae robustly express mpo in neutrophils in the caudal hematopoietic tissue (black arrowhead) and other locations (black arrow) compared to GF zebrafish. (D) qRT-PCR assays of whole 6dpf GF and CONVD zebrafish treated with DMSO vehicle from 3–6dpf shows that cfb, saa, and mpo are all significantly induced by the microbiota, and that treatment with 200nM NAI from 3–6dpf attenuates microbial induction of cfb and saa, but not mpo. (E) qRT-PCR assays of whole 6dpf GF and CONVD larvae injected at the 1-cell stage with either standard control morpholino (Std Ctrl MO) or a morpholino targeting myd88 (myd88 MO). qRT-PCR data in panels D–G are from biological duplicate pools (5–20 fish/pool) normalized to 18S rRNA levels and expressed as mean mRNA fold-difference ± SEM. Scale bars: 200μm (A–C).

Fig. 7. P. aeruginosa flagellar function is required for NF-κB activation in gnotobiotic Tg(NFkB:EGFP) zebrafish

Representative EGFP fluorescence heatmap images of 6dpf Tg(NFkB:EGFP) larvae raised GF (A), or mono-associated since 3dpf with wild type P. aeruginosa strain PAK (B) or deletion mutants PAK ΔfliC (C) and ΔmotABCD (D). (E) Densitometric quantification of relative EGFP fluorescence for whole animal, liver, swim bladder, intestinal segments 1, 2, and 3, neuromasts, dorsal root ganglia (DRG), and muscle (10 fish/condition). One-way ANOVA P-values are shown in each panel, with significant differences (Tukey’s post-test P<0.05) compared to GF (a) and PAK WT (b) indicated. Scale bars: 500μm (A–D).