Short-term treatment with insulin-like growth factor (IGF1) preserves cardiac function after myocardial infarction (MI) targeting myeloid cells. Until recently neutrophils were thought to play a detrimental role in ischaemic heart disease. However, recent data show, that neutrophils also play an important role in cardiac remodeling and repair post-MI. Comparable to macrophages, it was reported, that neutrophils can exhibit an anti-inflammatory N2-phenotype or a pro-inflammatory N1 phenotype. Based on the finding that myeloid cells (neutrophils, macrophages) mediate the cardioprotective effect of IGF1, it was the aim to analyse to what extent IGF1 modulates neutrophil function.

Murine bone marrow neutrophils were isolated by density gradient centrifugation and were polarized for 4 hours in vitro with IL-4 (20 ng/ml), IGF1 (10 ng/ml), insulin (10 and 100 ng/ml) or LPS+IFN-γ (10/2 ng/ml). RNAseq and qPCR analysis showed that IL-4 induces a reparative N2-like neutrophil phenotype, characterised by the induction of Arg1, Retnlα and Chi3l3. In contrast, LPS+IFN-γ treatment induced a pro-inflammatory N1 phenotype, by inducing upregulation of N1 marker genes TNFα, IL12a and NOS2. Interestingly, IGF1 also skewed neutrophils towards a N2-like neutrophil phenotype, which is comparable to that obtained by the classical N2 polarizer IL-4. As expected, RNAseq analysis revealed, that IL-4 treatment upregulated genes involved in the IL-4 pathway, and LPS+IFN-γ treatment upregulated genes involved in inflammatory pathways. Surprisingly, IGF1 treatment induced almost the same pathways in neutrophils as IL-4, and when comparing IL-4 to IGF1 treated neutrophils there were no genes that were differentially expressed.

Unexpectedly, insulin, despite the structural and functional similarities to IGF1, showed hardly any effects on neutrophil polarization. When comparing RNAseq results of IGF1 treated neutrophils to insulin treated neutrophils around 280 genes were altered, mainly downregulated, which is in line with the almost unaltered gene expression profile in insulin treated in comparison to untreated neutrophils.

It was also of interest to retrace signaling pathways responsible for IGF1 mediated neutrophil polarization. Therefore, the canonical IL-4 signaling molecule Jak was examined. Jak1/2/3/Tyk2 was inhibited with pharmacological inhibitors (InSolution Jak Inhibitor I and Ruxolitinib). Inhibition of all Jaks prevented IL-4 and LPS+IFN-γ induced polarization. Also IGF1 induced polarization was prevented by Jak inhibition. Specific inhibition of Jak-isoforms showed that IL-4- and IGF1-induced polarization is dependent on Jak2 activation. In contrast, N1 polarization by LPS+IFN-γ involves phosphorylation and activation of Jak1 and STAT1.