Semaphorin 3F signaling actively retains neutrophils at sites of inflammation
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
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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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 3

Neutrophil-specific loss of Sema3f results in more rapid neutrophil recruitment to and clearance from the lungs in a murine acute lung injury model.

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Neutrophil-specific loss of Sema3f results in more rapid neutrophil recr...
(A and B) Fold change in Sema3f and Nrp2 gene expression following acute lung injury with LPS. Mice were sacrificed at 6, 24, and 48 hours after instillation. BAL neutrophils were collected and cDNA was extracted. TaqMan analysis of cDNA was performed with data normalized to murine Actb gene expression. Data are mean ± SEM of fold change compared with peripheral blood neutrophils (T0 PB) from 2 individual experiments (n = 4–6). An acute lung injury was induced by intratracheal LPS instillation, mice were sacrificed at 24 hours, and lung sections were stained for expression of the Ly6G neutrophil marker and SEMA3F (C), NRP1, and NRP2 (D). Scale bars: 50 μm. (E and F) Sema3ffl/flMrp8Cre–/– (WT) and Sema3ffl/flMrp8Cre+/– (KO) mice were challenged with LPS, sacrificed at 2, 6, 24, and 48 hours, and BAL fluid was obtained. Cell counts were performed by hemocytometer and the differential cell count was established by cytospins. Time to 50% reduction in peak neutrophil number was calculated individually for each genotype (T50) (E). BAL fluid IgM content was measured by ELISA. Data are shown as log-transformed fold change from WT (F). Apoptosis was assessed by morphology, with data as mean ± SEM (G) from 3 individual experiments (n = 6–12). Statistical analysis was by 1-way ANOVA and Bonferroni’s post hoc test (A and B) and 2-way ANOVA with Sidak’s post hoc test (E–G). *P < 0.05; **P < 0.01; ***P < 0.001.