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

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant immune cell in the salivary glands and are attractive therapeutic targets due to their unrivaled capacity to drive tissue repair. Yet, the nature and role of macrophages in salivary gland homeostasis and how they may contribute to tissue repair after injury are not well understood. Here, we show that at least two phenotypically and transcriptionally distinct CX3CR1+ macrophage populations are present in the adult salivary gland, which occupy anatomically distinct niches. CD11c+CD206-CD163- macrophages typically associate with gland epithelium, whereas CD11c-CD206+CD163+ macrophages associate with blood vessels and nerves. Using a suite of complementary fate mapping systems, we show that there are highly dynamic changes in the ontogeny and composition of salivary gland macrophages with age. Using an in vivo model of radiation-induced salivary gland injury combined with genetic or antibody-mediated depletion of macrophages, we demonstrate an essential role for macrophages in clearance of cells with DNA damage. Furthermore, we show that epithelial-associated macrophages are indispensable for effective tissue repair and gland function after radiation-induced injury, with their depletion resulting in reduced saliva production. Our data, therefore, provide a strong case for exploring the therapeutic potential of manipulating macrophages to promote tissue repair and thus minimize salivary gland dysfunction after radiotherapy.

The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury.

Salivary glands are damaged by radiotherapy for head and neck cancers, which often culminates in radiation-induced hyposalivation and xerostomia that may be permanent. Here, we identified a central role for YAP in the regenerative response of the salivary gland. Activation of the Hippo signaling pathway inhibits the phosphorylation of YAP, leading to its nuclear translocation and transcriptional activity. Using mice with salivary gland injury induced by surgical ligation and salivary gland­derived organoids, we found that YAP nuclear localization in the salivary gland epithelium changed dynamically between homeostasis and regeneration. Whereas local injury had no effect on nuclear YAP localization in saliva-producing acinar cells, it triggered nuclear accumulation of YAP in saliva-transporting ductal cells. Injury also stimulated the proliferation of ductal cells, which were mainly quiescent under homeostatic conditions and in nonregenerating areas distal to the injury site, thus enabling salivary gland regeneration. Overexpressing YAP or driving YAP nuclear translocation by inhibiting upstream Hippo pathway kinases increased the capacity of mouse and human salivary gland cells, including human cells that had been irradiated, to form lobed organoids in vitro. Our results identify a YAP-driven regeneration program in salivary gland ductal cells that could be used to promote salivary gland regeneration after irradiation-induced damage.

The evolving definition of salivary gland stem cells.

Dysfunction of the salivary gland and irreversible hyposalivation are the main side effects of radiotherapy treatment for head and neck cancer leading to a drastic decrease of the quality of life of the patients. Approaches aimed at regenerating damaged salivary glands have been proposed as means to provide long-term restoration of tissue function in the affected patients. In studies to elucidate salivary gland regenerative mechanisms, more and more evidence suggests that salivary gland stem/progenitor cell behavior, like many other adult tissues, does not follow that of the hard-wired professional stem cells of the hematopoietic system. In this review, we provide evidence showing that several cell types within the salivary gland epithelium can serve as stem/progenitor-like cells. While these cell populations seem to function mostly as lineage-restricted progenitors during homeostasis, we indicate that upon damage specific plasticity mechanisms might be activated to take part in regeneration of the tissue. In light of these insights, we provide an overview of how recent developments in the adult stem cell research field are changing our thinking of the definition of salivary gland stem cells and their potential plasticity upon damage. These new perspectives may have important implications on the development of new therapeutic approaches to rescue radiation-induced hyposalivation.

Mouth-Watering Results: Clinical Need, Current Approaches, and Future Directions for Salivary Gland Regeneration.

Permanent damage to the salivary glands and resulting hyposalivation and xerostomia have a substantial impact on patient health, quality of life, and healthcare costs. Currently, patients rely on lifelong treatments that alleviate the symptoms, but no long-term restorative solutions exist. Recent advances in adult stem cell enrichment and transplantation, bioengineering, and gene transfer have proved successful in rescuing salivary gland function in a number of animal models that reflect human diseases and that result in hyposalivation and xerostomia. By overcoming the limitations of stem cell transplants and better understanding the mechanisms of cellular plasticity in the adult salivary gland, such studies provide encouraging evidence that a regenerative strategy for patients will be available in the near future.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.

Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals.

Adult stem cells are the ultimate source for replenishment of salivary gland (SG) tissue. Self-renewal ability of stem cells is dependent on extrinsic niche signals that have not been unraveled for the SG. The ductal compartment in SG has been identified as the location harboring stem cells. Here, we report that rare SG ductal EpCAM(+) cells express nuclear ß-catenin, indicating active Wnt signaling. In cell culture experiments, EpCAM(high) cells respond potently to Wnt signals stimulating self-renewal and long-term expansion of SG organoids, containing all differentiated SG cell types. Conversely, Wnt inhibition ablated long-term organoid cultures. Finally, transplantation of cells pre-treated with Wnt agonists into submandibular glands of irradiated mice successfully and robustly restored saliva secretion and increased the number of functional acini in vivo. Collectively, these results identify Wnt signaling as a key driver of adult SG stem cells, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation.

Purification and ex vivo expansion of fully functional salivary gland stem cells.

Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24(hi)/CD29(hi)). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.