FATE MAPPING HUMAN MONOCYTES DURING INFLAMMATION

INTRODUCTION: The mononuclear phagocyte system consists of three populations: macrophages, dendritic cells and monocytes. Human monocytes can be divided into three principle subsets based on the relative expression of CD14 and CD16: classical monocytes (CD14++CD16-), intermediate monocytes (CD14+CD16+) and non-classical monocytes (CD14lowCD16++). Previously, we demonstrated that a developmental relationship exists between each monocyte subset in both mouse and humans and their functional phenotype appears distinct between these populations. To assess the role of these populations in a human in vivo inflammatory setting, we examined the temporal contribution of each monocyte subset during a bacterial insult.

MATERIALS AND METHODS: The human experimental skin blister examines the function and kinetics of leucocytes in response to an inflammatory insult. Briefly, a blister was elicited between 6 to 48-hours following an intradermal UV-killed E. coli. Blister fluids were collected and leucocyte populations analysed using spectral flow cytometry or single cell RNA-Seq and cytokine secretion was measured in the exudate via multiplex ELISA.

RESULTS AND DISCUSSION: At 6 hours neutrophils dominated the inflammatory site, the mononuclear phagocytes started to emerge by 12 hours, classical monocytes appeared peaking at 24 hours while the intermediate-like monocytes were present only at 24 hours, at later time points these monocytes differentiate into a distinct monocytes-derived population. Cytokine analysis revealed an elevated production of cytokine CSF2 and the chemokines CX3CL1 and CCL2, peaking at 12 and 24 hours, respectively. Next, we examined monocytes chemotaxis towards these chemokines and observed classical but not non-classical monocytes migrate towards CCL2.

CONCLUSION: Taken together, these data highlight the sequential leucocyte infiltration kinetics during dermal inflammation. Specifically, how infiltrating human classical human monocytes migrate towards CCL2 and their ability to efficiently clear bacteria. In addition, our transcriptomic data reveal the development of two distinct monocyte-derived populations at the site of inflammation. Further studies will resolve how each of these monocyte-derived populations aid in the inflammatory process. A greater understanding of the chemokine-leucocyte axis may aid in the rational development of therapeutic agents.