Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Insulin-like growth factor 1 promotes an anti-inflammatory phenotype of neutrophils in myocardial infarction
R. Nederlof1, S. Reidel1, T. Lautwein2, A. Spychala1, K. Köhrer2, A. Gödecke1
1Institut für Herz- und Kreislaufphysiologie, Universitätsklinikum Düsseldorf, Düsseldorf; 2Biologisch-Medizinisches Forschungszentrum (BMFZ), Genomics & Transcriptomics Labor (GTL), Heinrich-Heine-Universität Düsseldorf, Düsseldorf;
Insulin-like growth factor 1 (IGF1) controls growth and metabolism of many cell types and has been shown to provide cardioprotective effects after acute myocardial infarction (AMI). Previously, we have shown that short-term treatment with IGF1 after AMI significantly reduces scar size, increases angiogenesis and improves cardiac function. This cardioprotective effect of IGF1 was dependent on the IGF1 receptor on myeloid cells, indicating that these cells are responsible for the protective effect. In addition, we could show that IGF1 treatment created an anti-inflammatory phenotype in macrophages both in vitro and in vivo. The effect of IGF1 on neutrophils is unknown.
For in vitro experiments, neutrophils were derived from bone marrow from mice using Percoll gradient centrifugation. Neutrophils were left untreated, or treated 4 hours with (ng/ml) IL4 (20), LPS/IFNγ (10/2), IGF1 (10).  LPS/IFNγ treatment upregulated pro-inflammatory markers (Tnf, Il12a and Nos2), whereas IL4 treatment upregulated anti-inflammatory markers (Arg1, Retnla and Chil3). IGF1 treatment also upregulated these anti-inflammatory genes, showing that IGF1 treatment created an anti-inflammatory phenotype in vitro.
To study whether IGF1 treatment also creates an anti-inflammatory phenotype in neutrophils in vivo, mice (n=3 per treatment) were exposed to 45 min ischemia by LAD occlusion, followed by 3 days reperfusion. At the end of ischemia, mice were injected with a bolus IGF1 (40 ng/kg i.p.) or vehicle (0.1% BSA), followed by continuous treatment for 3 days via osmotic mini pumps (1 µg/g/d s.c.). After 3 days, cardiac myeloid cells (CD45+CD11b+) were obtained using FACS sorting. Single-cell RNA sequencing was performed with 10X Genomics Chromium Platform. Cells were first clustered with low resolution. A clear distinction between neutrophils and other myeloid cells (monocytes, macrophages, dendritic cells) could be observed. Both subsets were reclustered with a higher resolution. The neutrophil subset contained in total 6635 cells, 3533 from control and 3102 from IGF1 treated mice. Unsupervised clustering created 8 neutrophil clusters. The percentage of neutrophils in each cluster was comparable for each replicate. No difference could be observed between control and IGF1 treated neutrophils in percentage of neutrophils in each cluster, indicating that IGF1 does not create a specific type of neutrophil, or increases a certain population of neutrophils. Analysis of differential gene expression of all neutrophils combined showed that IGF1 treatment upregulated, amongst others, Retnlg and downregulated Tnf, Il23a, Nfkbia and Icam1, showing a less inflammatory phenotype in IGF1 treated neutrophils. The same was observed when data was analyzed for each cluster separately. Genes involved in NFκB signaling were reduced after IGF1 treatment in most neutrophil clusters. For example, Tnf was downregulated in 7 of 8 clusters and Nfkbia and Icam1 in 6 of 8 clusters. IPA pathway analysis of differently regulated genes showed a downregulation of pro-inflammatory upstream regulators (TNF, NFκB, Il1β) in most clusters, whereas more anti-inflammatory upstream regulators (CISH, SOCS1) were upregulated after IGF1 treatment.  
In conclusion, IGF1 creates an anti-inflammatory phenotype in neutrophils, both in vitro and in vivo after myocardial infarction. These anti-inflammatory neutrophils might be responsible for the cardioprotective effects of IGF1 after AMI.

Funded by SFB1116/A06

https://dgk.org/kongress_programme/jt2022/aV1047.html