Cardiovascular diseases are the leading cause of death in western societies and neutrophils were long thought to play a detrimental role in ischaemic heart disease. But recent data showed that neutrophils also play an important role in cardiac remodelling and can exhibit an anti-inflammatory N2-phenotype or a pro-inflammatory N1 phenotype. Previous experiments showed that short-term treatment with insulin-like growth factor (IGF1) preserves cardiac function after myocardial infarction (MI). This effect was mediated by myeloid cells. Therefore, it was the aim to analyse to what extent and how IGF1 modulates neutrophil function.
Murine bone marrow neutrophils were isolated by density gradient centrifugation and were left untreated or polarized for 4 hours in vitro with IL-4 (20 ng/ml) or IGF1 (10 ng/ml), Insulin (10 ng/ml) or LPS/IFN-γ (10 ng/ml / 2 ng/ml).
Transcript expression analysis and qRT-PCR were performed to analyse to what extent transcriptional changes were triggered by growth factor and cytokine induced polarization after 4 hours. These analyses revealed that IL-4, and interestingly also IGF1, induces a reparative N2-like neutrophil phenotype, characterised by the induction of Arg1, Retnlα and Chi3l3. Surprisingly, Insulin, despite the high homology to IGF1, did not induce an antiinflammatory phenotype. LPS/IFN-y treatment induced upregulation of proinflammatory genes (TNFα, IL12a, iNOS). As expected, RNAseq analysis showed, that IL-4 treatment upregulated genes involved in the IL-4 pathway. Surprisingly, IGF1 treatment induced almost the same pathways as IL-4. Therefore, the canonical IL-4 signaling pathway Jak-Stat was analysed. This revealed that IL-4, and also IGF1, induced phosphorylation of Stat6. Additionally, Jak1/2/3/Tyk2 was inhibited with pharmacological inhibitors (InSolution Jak Inhibitor I, Ruxolitinib, BMS-911543). Inhibition of all Jaks prevented IL-4, and also IGF1, induced upregulation of N2 marker genes. Specific inhibition of Jak2 showed that IL-4- and IGF1-induced polarization is most likely dependent on Jak2 activation, as N2 marker gene expression was reduced. Western blot analysis as well revealed that Stat6 phosphorylation was reduced after Jak2 inhibition and Proximity ligation assay proved an interaction of the IGF1R and Jak2. Regarding the missing effect of insulin also the canonical insulin/IGF1 pathway, as for example the Akt and Ras-Raf-Erk pathway, were analysed. By using Tie2 Akt1 and Tie2 Akt2 KO mice, we showed the independency of IL-4 and IGF1 induced polarization on those Akt isoforms, as upregulation of antiinflammatory genes was not reduced.
Additionally, the effect of different polarizers on the formation of neutrophil extracellular traps, an inflammatory mechanism often found in heart failure, was analysed. This revealed that both, IL-4 and IGF1, are able to reduce the formation of NETs, whereas LPS/IFN-y treatment induces the formation of NETs.
In conclusion, these findings indicate that IGF1 is able to polarize neutrophils to a reparative N2-like phenotype most likely via the non-canonical Jak2-Stat6 axis, comparable to that induced by the classical M2/N2 polarizer IL-4. Additionally, IGF1 is able to reverse proinflammatory mechanisms.
