Disruption of Trim9 function abrogates macrophage motility in vivo

Abstract

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as "brain specific," its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.

Keywords: chemotaxis; leukocyte; ubiquitin; zebrafish.

Figure 1

Immune agonist exposure and summary of microarray results. (A) Zebrafish larvae (72 hpf) were exposed to 5 μg/ml Pam3CSK4, 10 μg/ml PolyIC or no agonist for 4, 8, 12, 24, or 36 h. RNA was isolated from larvae at each time point and used for microarray analyses. (B and C) Exposure to Pam3CSK4 or PolyIC led to the identification of 574 and 1035 genes, respectively, that had significantly increased or decreased transcript levels at 1 or more time points (as compared to control larvae) (dataset 1). Venn diagrams indicate the number of genes with altered transcript levels at each time point. (D) Hierarchal clustering and heat map of 1110 zebrafish genes with significantly different transcript levels (P < 0.05) after exposure to either Pam3CSK4 or PolyIC at any time point. Gene clusters with similar expression patterns are annotated on the right. (E) Hierarchal clustering and heat map of 121 zebrafish genes with significantly different transcript levels after exposure to Pam3CSK4 at any time point and significantly different transcript levels after exposure to PolyIC at any time point. The position of trim9, which displayed increased transcript levels at 8 and 12 hpe to both Pam3CSK4 and PolyIC is indicated by red text.

Figure 2

Immune agonist exposure induces increased TRIM9 transcript levels in Mϕs. (A) Relative trim9 transcript levels were determined by qPCR from zebrafish larvae after 8 h of exposure to 5 μg/ml Pam3CSK4 or 10 μg/ml PolyIC. Larval exposure was initiated at 72 hpf. The means ± sem are shown (n = 3). *P < 0.05. (B) Relative trim9 transcript levels were determined by qPCR from larval Mϕs after 8 and 12 h exposures to 5 μg/ml Pam3CSK4 or 10 μg/ml PolyIC. Zebrafish larvae (120 hpf) of the Tg (mpeg1.1:EGFP) transgenic line were exposed to immune agonists and EGFP⁺ Mϕs isolated by cell sorting. The means ± sem are shown (n = 3). *P < 0.05; **P < 0.10. Relative increases for each biologic replicate are PolyIC at 8 hpe = 2.034, 2.39, and 1.99; PolyIC at 12 hpe = 10.9, 3.92, and 2.30; Pam3CSK4 at 8 hpe = 1.33, 1.48, and 1.34; Pam3CSK4 at 12 hpe = 1.52, 2.42, and 3.82. (C) The human promonocytic cell line U937 was differentiated to a Mϕ‐like phenotype and exposed to 10 μg/ml PolyIC, 0.1 μg/ml Pam3CSK4 or 0.1 μg/ml LPS for 4, 8 or 12 h. Relative transcript levels were determined by qPCR. The means ± sem are shown (n = 3). *P < 0.05.

Figure 3

Mϕ‐specific disruption of Trim9 function results in reduced cellular chemotaxis in vivo. (A) Three transgenes were constructed that employed the Mϕ‐specific promoter of the zebrafish mpeg1.1 gene [13]. The Tg (mpeg1.1:Trim9,EGFP) transgene expresses full‐length zebrafish Trim9 and EGFP from the same transcript but produces both proteins via a viral 2A peptide cleavage site [43]. The Tg (mpeg1.1:∆RINGTrim9,EGFP) transgene expresses both zebrafish Trim9 that lacks the RING domain and EGFP. The Tg (mpeg1.1:EGFP) transgene expresses EGFP. When these transgenes are injected into 1‐cell zebrafish embryos of the stable transgenic line Tg (mpeg1.1:mCherry) the resultant larvae are mosaic with all Mϕs expressing mCherry [13] but only a subpopulation of Mϕs expressing Tg (mpeg1.1:Trim9,EGFP), Tg (mpeg1.1:∆RINGTrim9,EGFP) or Tg (mpeg1.1:EGFP). Trim9 includes RING, B‐box (BB), coiled‐coil (CC), COS, fibronectin type‐III (FN3) and SPla and RYanodine receptor (SPRY) domains [51]. (B and C) The in vivo cellular migration index for chemotaxis toward PolyIC or Pam3CSK4 is shown for zebrafish Mϕs expressing Tg (mpeg1.1:Trim9,EGFP), Tg (mpeg1.1:∆RINGTrim9,EGFP) or Tg (mpeg1.1:EGFP) on the Tg (mpeg1.1:mCherry) genetic background as compared to Mϕs within the same individual expressing only mCherry. Each data point (dot) represents the migration index for a single larva. Raw data are provided in dataset 3. Data are presented as box‐and‐whisker plots (n = 20–39 larvae). *P < 0.05.

Figure 4

In vivo disruption of Trim9 function in Mϕs significantly alters cell shape. (A) Zebrafish Mϕs expressing Tg (mpeg1.1:EGFP), Tg (mpeg1.1:Trim9,EGFP), or Tg (mpeg1.1:∆RINGTrim9,EGFP) on the Tg (mpeg1.1:mCherry) background were photographed to assess cell shape. Cells from each transgenic background were photographed for quantifying circularity. Scale bar, 20 μm. (B) Mϕs from Tg (mpeg1.1:mCherry) larvae injected with Tg (mpeg1.1:∆RINGTrim9,EGFP), but not expressing ∆RINGTrim9 (e.g., EGFP⁻) were photographed to assess cell shape. Scale bar, 20 μm. (C) Circularity scores are shown for individual cells. Mϕs that are mCherry‐positive, but EGFP‐negative were analyzed from Tg (mpeg1.1:∆RINGTrim9,EGFP) mosaic larvae including those displayed in (B). Mϕs that are mCherry‐positive and EGFP‐positive were analyzed from Tg (mpeg1.1:EGFP), Tg (mpeg1.1:Trim9,EGFP) and Tg (mpeg1.1:∆RINGTrim9,EGFP) mosaic larvae including those displayed in (A). Scale: 0–1, where 1 indicates a perfect circle. Data are presented as box‐and‐whisker plots (n = 17). *P < 0.05.

Figure 5

In vivo disruption of Trim9 function in Mϕs significantly disrupts Mϕ velocity. Time‐lapse video recordings were collected documenting the in vivo movement of zebrafish Mϕs expressing Tg (mpeg1.1:EGFP), Tg (mpeg1.1:Trim9,EGFP) or Tg (mpeg1.1:∆RINGTrim9,EGFP) (see Supplemental Videos 1, 2, and 3, respectively). (A and B) The mean and maximum velocities of individual Mϕs are displayed (n = 9–16). *P < 0.05. (C) Plots of 2 h migration tracks for individual Mϕs (n = 6–8).