A Salivary Gland Resident Macrophage Subset Regulating Radiation Responses

Abstract

Radiotherapy of head and neck cancers frequently leads to irreversible hypofunction of salivary glands, which severely compromises the quality of life and is extremely difficult to treat. We found recently that salivary gland resident macrophages are sensitive to radiation and interact with epithelial progenitors and endothelial cells through homeostatic paracrine factors. Heterogeneous subpopulations of resident macrophages are present in other organs with distinct functions, whereas subpopulations of salivary gland resident macrophages with distinct functions or transcriptional profiles have not been reported yet. Using single-cell RNA sequencing, we found that mouse submandibular glands (SMGs) contain 2 distinct self-renewing resident macrophage subsets, an MHC-IIhi subset present in many other organs and an uncommon Csf2r+ subset. The main source of Csf2 in SMGs are innate lymphoid cells (ILCs) that rely on IL15 for maintenance, while the main source of IL15 protein is Csf2r+ resident macrophages, indicating a homeostatic paracrine interaction between these cells. Csf2r+ resident macrophages are the major source of hepatocyte growth factor (Hgf) that regulates homeostasis of SMG epithelial progenitors. Meanwhile, Csf2r+ resident macrophages are responsive to Hedgehog signaling that can rescue salivary function impaired by radiation. Consistently, irradiation persistently decreased numbers of ILCs and levels of IL15 and Csf2 in SMGs, which were all recovered by transient activation of Hedgehog signaling after radiation. Csf2r+ resident macrophages and MHC-IIhi resident macrophages share transcriptome profiles of perivascular macrophages and macrophages associated with nerves and/or epithelial cells in other organs, respectively, and such niche preferences were supported by lineage tracing and immunofluorescent staining. These findings reveal an uncommon resident macrophage subset that regulates the homeostasis of the salivary gland and is promising as the target to restore salivary gland function impaired by radiation.

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration after radiation-induced injury

“…In our study, CD206 + CD163 + macrophages expressed Tim4 and LYVE-1, albeit at low and variable levels, and FRβ, aligning them with so-called "TLF" (Tim4 and/or LYVE-1 and/or FRβ)-expressing macrophages found across many different tissues by the Epelman group (6,27,28). This is in disagreement with Zhao et al (29), where the authors conclude that Folr2 (FRβ), Lyve1, and Timd4 are not expressed in any macrophage subset. Clusters 0 and 1 appeared to represent discrete transcriptional states of the CD11c + CD206 − CD163 − subset.…”

“…Similarly, we did not find Csf2rb -defined subsets of macrophages as recently suggested (fig. S2B) ( 29 ). We subsequently confirmed the presence of CD163 + macrophages in the SMG by immunofluorescence.…”

“…Previous work has leveraged scRNA-seq technologies to chart the cellular composition of the salivary gland ( 29 , 32 , 69 ). However, most of these studies have sought to create a cellular atlas of the salivary gland and thus have lacked sufficient resolution to comprehensively and specifically profile the macrophage compartment.…”

“…Our targeted scRNA-seq provided sufficient resolution to identify four clusters of macrophages, which fell into three phenotypically discrete subsets identifiable by flow cytometry: CD163 + CD206 + , CD163 − CD206 − , and proliferating macrophages (Ki67 + ). During revision of our manuscript, another study describing SMG macrophage heterogeneity was published ( 29 ). Whereas both studies describe macrophage heterogeneity, the approaches used to define this heterogeneity are quite different.…”

“…Zhao et al. ( 29 ) define heterogeneity on the basis of Csf2rb , which encodes the β subunit of the receptor for GM-CSF, and suggest dependence of this Csf2rb + subset on innate lymphoid cell–derived GM-CSF ( 29 ). In contrast, we found no heterogeneity on the basis of expression or reliance on GM-CSF receptors.…”