Transcriptional Profiling and Functional Analysis of N1/N2 Neutrophils Reveal an Immunomodulatory Effect of S100A9-Blockade on the Pro-Inflammatory N1 Subpopulation

Abstract

Neutrophils have been classically viewed as a homogenous population. Recently, neutrophils were phenotypically classified into pro-inflammatory N1 and anti-inflammatory N2 sub-populations, but the functional differences between the two subtypes are not completely understood. We aimed to investigate the phenotypic and functional differences between N1 and N2 neutrophils, and to identify the potential contribution of the S100A9 alarmin in neutrophil polarization. We describe distinct transcriptomic profiles and functional differences between N1 and N2 neutrophils. Compared to N2, the N1 neutrophils exhibited: i) higher levels of ROS and oxidative burst, ii) increased activity of MPO and MMP-9, and iii) enhanced chemotactic response. N1 neutrophils were also characterized by elevated expression of NADPH oxidase subunits, as well as activation of the signaling molecules ERK and the p65 subunit of NF-kB. Moreover, we found that the S100A9 alarmin promotes the chemotactic and enzymatic activity of N1 neutrophils. S100A9 inhibition with a specific small-molecule blocker, reduced CCL2, CCL3 and CCL5 chemokine expression and decreased MPO and MMP-9 activity, by interfering with the NF-kB signaling pathway. Together, these findings reveal that N1 neutrophils are pro-inflammatory effectors of the innate immune response. Pharmacological blockade of S100A9 dampens the function of the pro-inflammatory N1 phenotype, promoting the alarmin as a novel target for therapeutic intervention in inflammatory diseases.

Intratumoral pro-oxidants promote cancer immunotherapy by recruiting and reprogramming neutrophils to eliminate tumors

“…The N2 neutrophils produced significantly more IL-8 and were shown to be less effective at killing leishmania [ 51 ]. Similar findings were reported when transcriptomic profiles and functional differences between the N1 and N2 neutrophils were examined [ 73 ]. Compared to N2, the pro-inflammatory N1 neutrophils exhibited: i) higher levels of ROS and oxidative burst, ii) increased activity of MPO and MMP-9 and iii) enhanced chemotactic responses.…”

Cardiac repair after myocardial infarction: A two-sided role of inflammation-mediated

“…In contrast, recombinant S100A8/A9 treatment in the I/R mice model exacerbated myocardial injury and cardiac failure ( 158 ). In assessing whether S100A8/A9 can affect neutrophil phenotypic function, it was found that blocking S100A8/A9 reduced the migration rate of phosphorylation of ERK1/2 and p65 in N1 neutrophils, decreased the production of chemokines CCL2, CCL3, and CCL5, and attenuated the activity of NO, MPO, and MMP-9 ( 159 ). In addition, S100A8/A9 can induce NF-κB activation by promoting TNF-α secretion by macrophages and promoting MMP-9 expression by fibroblast cells, leading to cardiac rupture after MI ( 160 ).…”

Diverse Neutrophil Functions in Cancer and Promising Neutrophil-Based Cancer Therapies

“…The activation of A2AAR skewed N1 neutrophils to the N2 phenotype, while blocking A2AAR suppressed N2 polarization, which indicates the crucial role of the adenosine–A2AAR axis in N2 neutrophil polarization [ 66 ]. The discovery of the pro- and anti-inflammatory profiles of N1 and N2 neutrophils, respectively, has led to a wide investigation of these two phenotypes in several physiological and pathological conditions, including inflammatory diseases [ 67 , 68 ], bone regeneration [ 69 ], ischemia [ 70 ], myocardial infraction [ 71 ], and Alzheimer’s disease [ 72 ]. Of note, N1/N2 neutrophil classification in terms of infection could differ from N1/N2 TANs described in terms of tumor, which should be considered when moving from one research field to another.…”