Characterization of Zymosan-Modulated Neutrophils With Neuroregenerative Properties

Abstract

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

“…S3 ). In addition, five clusters (clusters 3, 4, 8, 9, and 10) were transcriptionally similar to developing PMNs found in the bone marrow, suggesting a more intermediate phenotype ( 33 ), while cluster 6 was transcriptionally similar to immature, Ly6G lo neutrophils found in the peritoneal cavity ( 32 ) characterized by expression of several immunomodulatory chemokines ( Supplementary Fig. S3 ).…”

“…Four clusters were transcriptionally similar to mature PMNs found in the peritoneal cavity (clusters 1, 5, 11, and 13) expressing Cfs3r , Mmp9 , Cd9 , and Il1rn (ref. 32 ; Supplementary Fig. S3 ).…”

Human neutrophil‐like cells demonstrate antimicrobial responses to the chronic cyst form of Toxoplasma gondii

“…Neutrophils can differentiate into multiple subsets, some of which do not possess the canonical antimicrobial qualities of ‘classical’ neutrophils 27 . There is a new appreciation for these ‘non‐classical’ neutrophils as they can be beneficial and even protective 50,51 . Aged neutrophils demonstrate decreased effector functions like phagocytosis and instead adopt a more regulatory phenotype as reviewed by Peiseler and Kubes 52 .…”

“…27 There is a new appreciation for these 'non-classical' neutrophils as they can be beneficial and even protective. 50,51 Aged neutrophils demonstrate decreased effector functions like phagocytosis and instead adopt a more regulatory phenotype as reviewed by Peiseler and Kubes. 52 Our results reveal a decrease in the ability of aged HNLCs to consume cyst material, supporting these previous studies.…”

The double‐edged role of neutrophil heterogeneity in inflammatory diseases and cancers

“…Neutrophil subpopulations also have fundamental effects on periodontal health regulation, as CD177 + neutrophils were recruited to gingival crevicular fluid in periodontitis preferentially 230–232 . Some researchers have identified specific neutrophil subsets, such as CD14 + Ly6G low neutrophils, that could secrete growth factors NGF and IGF‐1, thereby promoting neuronal survival 233,234 . Ly‐6G + SiglecF + neutrophils specifically exist in the allergic mouse nasal mucosa, exhibiting an activated phenotype 235 .…”