Semaphorin 3F signaling actively retains neutrophils at sites of inflammation
Tracie Plant, … , Moira K.B. Whyte, Sarah R. Walmsley
Tracie Plant, … , Moira K.B. Whyte, Sarah R. Walmsley
Published March 19, 2020
Citation Information: J Clin Invest. 2020;130(6):3221-3237. https://doi.org/10.1172/JCI130834.
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Research Article Inflammation Pulmonology

Semaphorin 3F signaling actively retains neutrophils at sites of inflammation

Abstract

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Authors

Tracie Plant, Suttida Eamsamarng, Manuel A. Sanchez-Garcia, Leila Reyes, Stephen A. Renshaw, Patricia Coelho, Ananda S. Mirchandani, Jessie-May Morgan, Felix E. Ellett, Tyler Morrison, Duncan Humphries, Emily R. Watts, Fiona Murphy, Ximena L. Raffo-Iraolagoitia, Ailiang Zhang, Jenna L. Cash, Catherine Loynes, Philip M. Elks, Freek Van Eeden, Leo M. Carlin, Andrew J.W. Furley, Moira K.B. Whyte, Sarah R. Walmsley

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Figure 5

Overexpression of sema3f in a zebrafish model of inflammation delays neutrophil recruitment and resolution of the inflammatory response.

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Overexpression of sema3f in a zebrafish model of inflammation delays neu...
(A–D) sema3fa or sema3fb RNA (50 ng/μL) was injected into 1-cell-stage zebrafish mpx:GFP embryos, with 50 ng/μL mCherry RNA used as a negative control. Tail fin transection was performed at 2 dpf, and neutrophils counted at 6 and 24 hpi. (A) Neutrophil counts at the 6 hpi time point with (B) overlaid fluorescence and bright-field photomicrographs (recruitment). (C) Neutrophil counts at the 24 hpi time point with (D) overlaid fluorescence and bright-field photomicrographs (resolution). Scale bars: 60 μm (B and D). Data are mean ± SEM with individual data points from 3 independent experiments (n = 30). (E) sema3fa and/or sema3fb RNA (50 ng/μL) were injected into 1-cell-stage zebrafish mpx:kaede embryos, and tail fin transection was performed at 2 dpf. Neutrophils recruited to the wound at 6 hpi were photoconverted, and red fluorescent neutrophils were tracked for 3.5 hours. Data are mean ± SEM from 3 independent experiments (n = 9). (F and G) sema3fa or sema3fb RNA (50 ng/μL) was injected into 1-cell-stage zebrafish mpx:GFP embryos, with 50 ng/μL mCherry RNA used for control. Tail fin transection was performed at 2 dpf. Neutrophil movement was tracked over 1 hour by time-lapse microscopy during the recruitment phase of inflammation (1–2 hpi) and speed of neutrophil migration (F) and meandering index (displacement/path length) (G) were determined. Data are mean ± SEM from 3 independent experiments (each point represents a single neutrophil) (n = 15). (H) sema3fa or sema3fb RNA (50 ng/μL) was injected into 1-cell-stage Tg(lyz:PHAkt-EGFP) embryos with noninjected controls, tail fin transection was performed at 2 dpf, and polarity indices were calculated for neutrophils recruited to the tail region. Data are mean ± SEM from a single experiment (n = 8). Statistical analysis was by 1-way ANOVA and Bonferroni’s post hoc test. ***P < 0.001.