Immunogenetics

, Volume 69, Issue 5, pp 341–349 | Cite as

CXCL12a/CXCR4b acts to retain neutrophils in caudal hematopoietic tissue and to antagonize recruitment to an injury site in the zebrafish larva

  • Susana Paredes-Zúñiga
  • Rodrigo A Morales
  • Salomé Muñoz-Sánchez
  • Carlos Muñoz-Montecinos
  • Margarita Parada
  • Karina Tapia
  • Carlos Rubilar
  • Miguel L Allende
  • Oscar A Peña
Original Article
First Online:
Received:
Accepted:

Abstract

Neutrophils are a major component of the innate immune response and the most abundant circulating cell type in humans and zebrafish. The CXCL12/CXCR4 ligand receptor pair plays a key role in neutrophil homeostasis, controlling definitive hematopoiesis and neutrophil release into circulation. Neutrophils overexpressing CXCR4 respond by migrating towards sources of CXCL12, which is abundant in hematopoietic tissues. However, the physiological role of CXCL12/CXCR4 signaling during inflammatory responses remains unknown. Here, we show that zebrafish mutants lacking functional CXCL12a or CXCR4b show disrupted granulopoiesis in the kidney and increased number of circulating neutrophils. Additionally, CXCL12a and CXCR4b mutants display exacerbated recruitment of neutrophils to wounds and not to infections, and migrating neutrophils to wounds show increased directionality. Our results show that CXCL12a/CXCR4b signaling antagonizes wound-induced inflammatory signals by retaining neutrophils in hematopoietic tissues as a part of a balance between both inflammatory and anti-inflammatory cues, whose dynamic levels control neutrophils complex migratory behavior.

Keywords

Neutrophil Inflammation CXCL12 CXCR4 Zebrafish 

Abbreviations

CHT

Caudal hematopoietic tissue

CXCL12

Chemokine (C-X-C motif) ligand 12

CXCR4

Chemokine (C-X-C motif) receptor 4

Dpf

Days post-fertilization

Hpf

Hours post-fertilization

Hpi

Hours post-injury

HSC

Hematopoietic stem cell

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Notes

Acknowledgements

We thank Pamela Vargas for expert fish care and Florencio Espinoza for technical help. Zebrafish strains were kindly provided by Darren Gilmour and Stephen Renshaw. This work was supported by grants to MA from FONDECYT (1140702) and FONDAP (15090007).

Compliance with ethical standards

All procedures complied with guidelines of the Animal Ethics Committee of the University of Chile.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

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Supplementary Figure 1

(a-d) DiOC6 stains of 3 dpf of cxcl12a t30516/t30516 (b) and cxcr4b t26035/t26035 (d) mutant larvae and their respective siblings (a and c) showing the location of posterior lateral line neuromasts (arrowheads). Both cxcl12a t30516/t30516 (b) and cxcr4b t26035/t26035 (d) mutant larvae lack or show decreased number of neuromasts in the trunk and the tail compared to their siblings (a and c, respectively). Note that neuromasts in the anterior lateral line (arrows) remain unaffected in mutant larvae (b and d). Scale bar, 500 μm. (GIF 241 kb)

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High resolution image (TIFF 14228 kb)
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Supplementary Figure 2 Table showing statistics of Fig. 1u. Sample size, mean, standard deviation (SD), standard error of the mean (SEM), and lower and upper 95% confidence interval (CI) of flow cytometry quantification of neutrophil percentage in cxcl12a t30516/t30516 mutant larvae and their siblings are shown. Each sample consisted of 30 larvae. (DOCX 10 kb)
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Supplementary Figure 3 Table showing statistics of Fig. 1v. Sample size, mean, standard deviation (SD), standard error of the mean (SEM), and lower and upper 95% confidence interval (CI) of flow cytometry quantification of neutrophil percentage in cxcr4b t26035/t26035 mutant larvae and their siblings are shown. Each sample consisted of 30 larvae. (DOCX 10 kb)
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Supplementary Figure 4 Table showing statistics of Fig. 1w. Mean, standard deviation (SD), and sample size of measurements of circulating neutrophils are displayed for both cxcl12a t30516/t30516 and their wild type siblings at 3, 5, 7, 9 and 13 dpf. (DOCX 11 kb)
251_2017_975_MOESM5_ESM.docx (11 kb)
Supplementary Figure 5 Table showing statistics of Fig. 1x. Mean, standard deviation (SD), and sample size of measurements of circulating neutrophils are displayed for both cxcr4b t26035/t26035 and their wild type siblings at 3, 5, 7, 9 and 13 dpf. (DOCX 11 kb)
251_2017_975_MOESM6_ESM.docx (12 kb)
Supplementary Figure 6 Table showing statistics of Fig. 2e. Mean, standard deviation (SD), and sample size of measurements of recruited neutrophils are displayed for both cxcl12a t30516/t30516 and their wild type siblings every 4 hours from 0 to 24 hours post injury. (DOCX 11 kb)
251_2017_975_MOESM7_ESM.docx (11 kb)
Supplementary Figure 7 Table showing statistics of Fig. 2f. Mean, standard deviation (SD), and sample size of measurements of recruited neutrophils are displayed for both cxcr4b t26035/t26035 and their wild type siblings every 4 hours from 0 to 24 hours post injury. (DOCX 11 kb)
251_2017_975_MOESM8_ESM.mov (14.8 mb)
Supplementary Figure 8 Neutrophil recruitment induced by tail transection in a wild type larva. At 3 dpf, a TgBAC (mpx: GFP) i114 transgenic larva was subjected to tail transection and immediately mounted for time-lapse imaging for 172 minutes under a fluorescence stereomicroscope. Imaging started 2 minutes after tail transection and images were captured every 30 seconds in the green channel. Interstitial migration of neutrophils from the caudal hematopoietic tissue (CHT) to the tail can be observed. Scale bar, 200 μm. Times are expressed as hh: mm: ss. (MOV 15116 kb)
251_2017_975_MOESM9_ESM.mov (17.7 mb)
Supplementary Figure 9 Recruitment of neutrophils after tail transection in a cxcl12a t30516/t30516 mutant larva. In vivo time-lapse imaging of a 3 dpf TgBAC (mpx: GFP) i114 ;TgBAC (neurod: EGFP)nl1;cxcl12a t30516/t30516 larva after tail transection. Image acquisition started 5 minutes after tail transection and images were captured every 30 seconds for 163 minutes under a fluorescence stereomicroscope. A large number of neutrophils migrate interstitially from the caudal hematopoietic tissue (CHT) to the wound, with a significant decrease in the amount of neutrophils remaining in the CHT by the end of the acquisition time. Scale bar, 200 μm. Times are expressed as hh:mm:ss. (MOV 18136 kb)
251_2017_975_MOESM10_ESM.docx (11 kb)
Supplementary Figure 10 Table showing statistics of Fig. 2g. Sample size, mean, standard deviation (SD), and standard error of the mean (SEM) of the speed of recruited neutrophils in mutant and wild type larvae are shown. (DOCX 10 kb)
251_2017_975_MOESM11_ESM.docx (11 kb)
Supplementary Figure 11 Table showing statistics of Fig. 2h. Sample size, mean, standard deviation (SD), and standard error of the mean (SEM) of the directionality of recruited neutrophils in mutant and wild type larvae are shown. (DOCX 10 kb)
251_2017_975_MOESM12_ESM.docx (12 kb)
Supplementary Figure 12 Table showing statistics of Fig. 2i. Mean, standard deviation (SD), and sample size (N) of quantifications of recruited neutrophils are displayed for both cxcr4b t26035/t26035 and their wild type siblings every 3 hours from 3 to 24 hours post infection. (DOCX 11 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Susana Paredes-Zúñiga
    • 1
  • Rodrigo A Morales
    • 1
  • Salomé Muñoz-Sánchez
    • 1
  • Carlos Muñoz-Montecinos
    • 1
  • Margarita Parada
    • 1
  • Karina Tapia
    • 1
  • Carlos Rubilar
    • 1
  • Miguel L Allende
    • 1
  • Oscar A Peña
    • 1
    • 2
  1. 1.FONDAP Center for Genome Regulation, Facultad de CienciasUniversidad de ChileSantiagoChile
  2. 2.University College LondonLondonUK

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