Neutrophils are necessary for cardiac healing after MI. By producing pro-inflammatory cytokines, chemokines and high amounts of ROS, they clear the infarcted area of cell debris, but also increase injury. Therefore, they are generally thought to have a negative effect on cardiac remodeling. However, recent studies show that neutrophils might also have a positive effect. Neutrophil depletion after MI leads to increased fibrosis, reduced cardiac function and finally heart failure. Also, by interacting with macrophages, they might have a direct, or indirect, anti-inflammatory effect. Moreover, it has been shown that neutrophils, like macrophages, can be divided in at least pro-inflammatory N1, and reparative N2 neutrophils, which have both been found after MI. Influencing neutrophil phenotype might improve cardiac remodeling after MI. To study this, it is important to have good markers for the different neutrophils phenotypes. Markers used until now, are markers that are also used for macrophage. The aim of this study was to find neutrophil specific markers.

Neutrophils were isolated from mouse bone marrow and left untreated, or treated 4 hours with the polarizers IL4 (20 ng/mL) or LPS+ IFNγ (10+2 ng/mL). qPCR analysis showed that IL4 treatment upregulated the classic markers Arg1, Retnlα and Chi3l3 and LPS+ IFNγ upregulated TNFα, IL12a, and NOS2. RNA sequencing of polarized neutrophils was performed to find more neutrophil specific N1 and N2 markers. Car4 was the highest upregulated marker after IL4 treatment, with a fold change (FC) of 228.8 (p=7.4E-19) when compared to untreated. Other significantly high upregulated markers, that were not affected by LPS treatment were the classic marker Retnlα (FC 68.9 p=2.97E-32), Lct4s (FC 40.8 p=2.97E-09) and Slc28a3 (FC 26.6 p=6.97E-37). Interestingly, RNA sequencing showed that these genes were not upregulated in IL4 treated macrophages. qPCR analysis confirmed that these marker were significantly upregulated after IL4 treatment. To be able to differentiate between neutrophils with another method, FACS analysis was performed with the markers arginase 1, Car4 and CD206 (mannose receptor), which is a common marker for reparative M2-macrophages. Unfortunately, these markers were not observed in FACS analysis of neutrophils. Looking at the gene expression of these genes, we observed, that they are little expressed in neutrophils (RPKM 0.2, 0.65 and 0.7 respectively). Therefore, we searched for genes that were significantly upregulated after IL4 treatment with a RPKM>40 reads. Fcgr2b (FC 3.0 p=1.9E-45) and Itgb3 (FC 2.7 p=3.0E-8) were one of the highest upregulated markers, and qPCR confirmed their increase after IL4 treatment. Interestingly, Itgb3 was significantly reduced after LPS+IFNγ treatment. These markers might be of interest to use in FACS analysis.

The same was done for ILPS+ IFNγ treated neutrophils. The highest upregulated markers in the RNAseq analysis were Cxcl9 (FC 5293 p=9.3E-13), Cxcl11 (FC 4754 p=4.1E-73) and Batf2 (FC 4013 p=1.8E-102). Again, qPCR analysis confirmed the RNAseq results. FACS analysis of TNFα showed an upregulation in LPS+ IFNγ treated cells. CD274 (FC 110 p=1.5E-60) might be another protein of interest for FACS analysis, since this is expressed on the membrane.

Cxcl9 and CD274 and Fcgr2b and Itgb3 are promising markers to study neutrophil phenotype, which now have to be proven in vivo.