Data from: SiglecF-expressing neutrophils differentiate from mature neutrophils locally in the heart after myocardial infarction

Stephan, Jonah 1 ; Knerr, Taylor1; Isaacs, William1; Nong, Yibing1; Wysoczynski, Marcin 1

Published Jun 02, 2026 on Dryad. https://doi.org/10.5061/dryad.np5hqc090

Data files

Jun 02, 2026 version files 2.45 GB

Fig_1_2_3_SFig_1_2.zip

571.12 MB
Fig_4_6_SFig_5.zip

111.63 MB
Fig_5_SFig_3_4.zip

1.70 GB
Fig_6.zip

70.90 MB
README.md

8.34 KB

Abstract

Myocardial infarction (MI) triggers an immune response marked by rapid infiltration of neutrophils. Though once considered a uniform population, neutrophils display remarkable heterogeneity post-MI, including a subset expressing SiglecF, a marker typically associated with eosinophils. In this study, we investigated the origin of SiglecF^Pos neutrophils following MI. We found that these cells appear early in the heart after MI, coinciding with the influx of CD101^Neg immature neutrophils. However, SiglecF^Pos neutrophils were absent in hematopoietic organs, suggesting their local differentiation. Interestingly, SiglecF^Pos neutrophils co-expressed CD101, indicating their maturity. Neutrophil stimulation in vitro with TGF-β and GM-CSF induced co-expression of SiglecF and CD101. When sorted CD101^Neg and CD101^Pos neutrophils were exposed to the same stimuli, only the mature (CD101^Pos) subset could acquire SiglecF expression, indicating maturation as a prerequisite for SiglecF induction. Using EdU labeling prior to MI to track neutrophil origins in vivo, we found that most SiglecF^Pos neutrophils in infarcted hearts were derived from mature circulating neutrophils existing prior to MI. SiglecF^Pos neutrophils emerge during heightened inflammation, which drives emergency granulopoiesis in the bone marrow. Neutrophils from mice treated with G-CSF to stimulate emergency granulopoiesis exhibited enhanced capacity for SiglecF expression. Neutrophils exposed to G-CSF in vitro exhibited enhanced SiglecF induction by TGF-β, indicating that G-CSF primes neutrophils for SiglecF expression in vivo and ex vivo. These findings support a model in which SiglecF^Pos neutrophils originate from mature CD101^Pos precursors following MI, and emergency granulopoiesis enhancing their capacity to acquire this phenotype and contributing to neutrophil heterogeneity in cardiac injury. Flow cytometry was the primary endpoint for each of these experiments.

Dataset DOI: 10.5061/dryad.np5hqc090

Description of the data and file structure

Hearts, peripheral blood, bone marrow, and spleens were collected after myocardial infarction (MI), stained with fluorescently labeled antibodies, and assessed via flow cytometry. Flow cytometry (.fcs) files are deposited in compressed folders labeled with each corresponding figure in the manuscript. Each .fcs file is clearly labeled to group and timepoint or is included in a folder detailing group or timepoint.

Files and variables

File: Fig_4_6_SFig_5.zip

Description: Bone marrow neutrophils from mice treated with Vehicle (1X PBS) or G-CSF. Cells were stimulated with either TGF-β or GM-CSF, stained with fluorescent antibodies, then assessed via flow cytometry. Files are labeled as Vehicle (V1, V2, V3) or G-CSF (G1, G2, G3) followed by treatment.

Figure 4. GM-CSF, TGF-β stimulate SigecF expression in CD101^Pos neutrophils. Bone marrow neutrophils were cultured for 24 h in the presence of TGF-β, GM-CSF, or GM-CSF and TGF-β. After 24 h of culture, neutrophils were collected from the plates via washing and scraping. Collected cells were stained with fluorescently labeled antibodies to detect CD101 and SiglecF surface expression. SiglecF expression was assessed on Ly6G^PosCD101^Neg or Ly6G^PosCD101^Pos cells via flow cytometry and quantified as a percent of Ly6G^PosCD101^Neg or Ly6G^PosCD101^Pos and mean fluorescence intensity (MFI).

Figure 6. G-CSF enhances acquisition of SiglecF in response to GM-CSF and GM-CSF/TGF-β both in vivo and in vitro. Emergency granulopoiesis was stimulated in mice via injections of G-CSF (100 µg/kg/d) for 3d. At 24 h after the final injection of G-CSF neutrophils were sorted from the bone marrow via negative selection. These neutrophils were cultured for 24 h in the presence of TGF-β, GM-CSF, or GM-CSF and TGF-β. After 24 h of culture, neutrophils were collected from the plates via washing and scraping. Collected cells were stained with fluorescently labeled antibodies to detect Ly6G, CD101, and SiglecF surface expression.To determine G-CSF direct signaling on SiglecF expression, neutrophils sorted from the bone marrow were stimulated with in vitro G-CSF or vehicle in the presence of TGF-β, GM-CSF individually or combination of both. After 24 h, neutrophils were collected and surface expression of SiglecF was assessed via flow cytometry.

Supplemental Figure 5. Emergency granulopoiesis neutrophils stimulated to express SiglecF co-express CD101. Emergency granulopoiesis was stimulated in mice via injections of G-CSF (100 µg/kg/d) for 3d. At 24 h after the final injection of G-CSF neutrophils were sorted from the bone marrow and cultured for 24 h in the presence of TGF-β, GM-CSF, or GM-CSF and TGF-β. After 24 h of culture, neutrophils were collected and stained with fluorescently labeled antibodies to detect Ly6G, CD101, and SiglecF surface expression. Ly6G^PosCD101^Neg and Ly6G^PosCD101^Pos cells were analyzed for SiglecF expression by flow cytometry and quantified as a percent of Ly6GPos and mean fluorescence intensity (MFI).

File: Fig_1_2_3_SFig_1_2.zip

Description: Hearts, blood, bone marrow, and spleens were collected from mice at 0, 24, 48, 72, and 96 h after MI and stained with fluorescently labeled antibodies. Cells were assessed via flow cytometry. Files are organized by tissue, timepoint, and treatment.

Figure 1: The presence of SiglecF^Pos neutrophils in infarcted hearts after MI. At 24–96 h after MI, mouse hearts were harvested, perfused, and digested. Myocardial CD11b^Pos cells were gated as Ly6G^PosSiglecF^Neg or Ly6G^PosSiglecF^Pos neutrophils. Peripheral blood CD11b^Pos myeloid cells were gated as Ly6G^PosSiglecF^Neg or Ly6G^PosSiglecF^Pos neutrophils.

Figure 2. CD101^Neg immature neutrophils are recruited to the heart after MI. At 24–96 h after MI, mouse hearts were harvested, perfused, and digested. Myocardial myeloid cells were gated as either Ly6G^PosSiglecF^Neg or Ly6G^PosSiglecF^Pos neutrophils. CD11b^Pos cells were gated as Ly6G^PosCD101^Neg immature or Ly6G^PosCD101^Pos mature and represented as percentage of total cells in peripheral blood.

Figure 3. SiglecF^Pos neutrophils are primarily CD101^Pos mature neutrophils after MI. At 24–96 h after MI hearts were harvested, perfused, and digested. SiglecF surface expression was quantified in cells gated as either CD11b^PosLy6G^PosCD101^Neg or CD11b^PosLy6G^PosCD101^Pos and represented as a percent positive for SiglecF expression. Mean fluorescent intensity (MFI) was quantified in CD11b^PosLy6G^PosCD101^Neg and CD11b^PosLy6G^PosCD101^Pos populations.

Supplemental Figure 1. MI does not induce SiglecF surface expression in bone marrow or splenic neutrophils. At 24–96 h after the MI, cells were isolated from bone marrow and spleen and subjected to flow cytometry analysis. CD11b^Pos cells were gated as Ly6G^PosSiglecF^Neg or Ly6G^PosSiglecF^Pos and represented as percentage of total cells in bone marrow. CD11b^Pos cells were gated as Ly6G^PosSiglecF^Neg or Ly6G^PosSiglecF^Pos and represented as percentage of total cells in spleens.

Supplemental Figure 2. Mature and immature neutrophils accumulate in the bone marrow and spleen after MI. At 24–96 h after MI, single cell suspensions of bone marrow and spleens were subjected to flow cytometry analysis. CD11b^Pos cells were gated as Ly6G^PosCD101^Neg immature or Ly6G^PosCD101^Pos mature and represented as percentage of total cells in bone marrow. CD11bPos cells were gated as Ly6G^PosCD101^Neg immature or Ly6G^PosCD101^Pos mature and represented as percentage of total cells in spleens.

File: Fig_6.zip

Description: Bone marrow neutrophils were isolated and stimulated with either G-CSF, GM-CSF, or TGF-β. Cells were stained with fluorescently labeled antibodies then assessed via flow cytometry. Files are labeled as treatment.

See above.

File: Fig_5_SFig_3_4.zip

Description: Immature CD101- and mature CD101+ neutrophils were sorted from the bone marrow and stimulated with either GM-CSF or TGF-β. Cells were fluorescently labeled and assessed via flow cytometry. Files are labeled as which biological replicate, initial fraction (CD101- or CD101+), then the treatment and timepoint.

Figure 5. SiglecF^Pos neutrophils are derived from mature neutrophils. Immature CD101^Neg and mature CD101^Pos neutrophils were sorted from the mouse bone marrow via fluorescence activated cell sorting (FACS) based off surface expression of CD101. Immature and mature neutrophil fractions were cultured in the presence of GM-CSF and TGF-β. After 24 h of culture, collected cells were stained with fluorescently labeled antibodies to detect CD101 and SiglecF surface expression within the CD11b^PosLy6G^Pos population. Mature neutrophils in circulation were labeled via 3 injections of EdU every 6 h, then they were subjected to MI 4 d after the last EdU injection. At 3 d post-MI, hearts were harvested and dissociated to single cell suspensions. EdU^Pos cells were detected within the Ly6G^PosSiglecF^Pos population via flow cytometry.

Supplemental Figure 3. Gating strategy for sorting CD101Neg immature and CD101Pos mature neutrophils via fluorescence activated cells sorting (FACS). Mouse bone marrow cells were stained with fluorescently labeled antibodies and sorted for CD101^Neg and CD101^Pos neutrophil populations. Purity was assessed on post-sort fractions.

Supplemental Figure 4. Labeling of mature neutrophils with EdU. Mice were injected with EdU (200 µg/mouse) once every 6 h for 12 h (3x). At 4 d after the final injection, bone marrow and peripheral blood were harvested. EdUPos cells were quantified within CD101^Neg and CD101^Pos neutrophil populations.

Code/software

FlowJo v10 was used in analysis. Any software used to analyze flow cytometry may also be used.

Access information

Other publicly accessible locations of the data:

Data was derived from the following sources: