Transient Activation of the Hedgehog-Gli Pathway Rescues Radiotherapy-Induced Dry Mouth via Recovering Salivary Gland Resident Macrophages

Abstract

Irreversible hypofunction of salivary glands is a common side effect of radiotherapy for head and neck cancer and is difficult to remedy. Recent studies indicate that transient activation of Hedgehog signaling rescues irradiation-impaired salivary function in animal models, but the underlying mechanisms are largely unclear. Here, we show in mice that activation of canonical Gli-dependent Hedgehog signaling by Gli1 gene transfer is sufficient to recover salivary function impaired by irradiation. Salivary gland cells responsive to Hedgehog/Gli signaling comprised small subsets of macrophages, epithelial cells, and endothelial cells, and their progeny remained relatively rare long after irradiation and transient Hedgehog activation. Quantities and activities of salivary gland resident macrophages were substantially and rapidly impaired by irradiation and restored by Hedgehog activation. Conversely, depletion of salivary gland macrophages by clodronate liposomes compromised the restoration of irradiation-impaired salivary function by transient Hedgehog activation. Single-cell RNA sequencing and qRT-PCR of sorted cells indicated that Hedgehog activation greatly enhances paracrine interactions between salivary gland resident macrophages, epithelial progenitors, and endothelial cells through Csf1, Hgf, and C1q signaling pathways. Consistently, expression of these paracrine factors and their receptors in salivary glands decreased following irradiation but were restored by transient Hedgehog activation. These findings reveal that resident macrophages and their prorepair paracrine factors are essential for the rescue of irradiation-impaired salivary function by transient Hedgehog activation and are promising therapeutic targets of radiotherapy-induced irreversible dry mouth. Significance: These findings illuminate a novel direction for developing effective treatment of irreversible dry mouth, which is common after radiotherapy for head and neck cancer and for which no effective treatments are available.

Differential Expression of CD11c Defines Two Types of Tissue-Resident Macrophages with Different Origins in Steady-State Salivary Glands

“…The present study showed that the phagocytic activity of SG macrophages was extremely weak; however, the gene expression pro le of SMG macrophages suggested that SG macrophages regulate gland development and functions in SGs. This view is partly supported by recent ndings indicating that murine SG macrophages allow the patrolling of tissue by tissue-resident memory CD8 + T cells for homeostatic organ surveillance 29 , and attenuate radiotherapy-induced dry mouth through the activation of the Hedgehog pathway 37 . The further characterization of SG macrophages is important for evaluating the contribution of SG macrophage subpopulations to SG homeostasis and to the pathology of SG dysfunctions and their therapeutic purposes.…”

A Novel Experimental Approach for In Vivo Analyses of the Salivary Gland Microvasculature

“…Although numerous pathological conditions (e.g., bacterial and viral infections, autoimmune diseases such as Sjögren’s syndrome, malignant tumors, or irradiation) may cause disorders of the head neck salivary glands, these diseases are all characterized by acute or chronic inflammatory changes arising from microvascular dysfunction ( 1 , 3 – 6 , 41 ). Immune cell accumulation in microvasculature and perivascular tissue as well as microvascular leakage are key components of inflammatory reactions ( 8 , 10 ) that are regulated by parenchymal sentinel cells such as tissue-resident macrophages ( 7 , 8 , 11 – 13 ). Targeting these events in the injured gland microvasculature might therefore represent a promising approach for the prevention and treatment of these pathologies.…”

“…The extravasation of leukocytes is associated with enhanced microvascular permeability, which leads to edema formation, reduced oxygenation, and remodeling of the underlying tissue, thus representing hallmarks of the inflammatory response ( 10 ). These microvascular processes are critically regulated by parenchymal sentinel cells such as tissue-resident macrophages (TRMs), which maintain microvascular homeostasis in intact and inflamed salivary glands ( 7 , 8 , 11 – 13 ).…”

Ophthalmologic Manifestations of Primary Sjögren’s Syndrome

“…Additionally, recent papers demonstrated the fundamental role of resident macrophages in salivary glands to repair and restore salivary function after damage through the secretion of pro-repair factors [ 56 , 57 ].…”