DNA damage triggers a cascade of cellular responses leading to inflammation and immune activation. These processes have been studied primarily in tumors, leaving their significance for normal tissues less well understood.
In the treatment of head and neck cancers, irradiation inevitably affects healthy salivary glands. As a result, normal tissues can be damaged, salivary gland function may decline, and xerostomia (dry mouth) can develop, severely impairing patients’ quality of life.
Tissue repair largely depends on stem cells. Previous studies have shown that preserving regions of salivary glands enriched in stem and progenitor cells during radiotherapy markedly reduces the risk of xerostomia. Stress-induced changes influence the ability of these cells to proliferate and self-renew in the microenvironment. One important factor is type I interferons (IFN-I), whose production can be enhanced after radiation-induced DNA damage and can trigger immune responses. Therefore, it is crucial to understand how irradiation induces IFN responses and how these responses affect stem cells in normal tissues.
Studying these mechanisms directly in vivo is challenging, as salivary gland stem cells are rare and usually quiescent. For this reason, salivary gland organoids – three-dimensional structures derived from stem cells – are widely used. They faithfully recapitulate key properties of normal tissue and enable detailed analysis of self-renewal, differentiation, and responses to DNA damage.
Responses of normal tissues depend on the extent of DNA damage induced by different irradiation modalities, particularly when comparing photon radiotherapy, which remains the standard treatment for head and neck tumors, with proton therapy. Proton therapy allows more precise dose delivery to tumors while sparing surrounding tissues. In addition, it can elicit distinct cellular responses by activating specific signaling pathways and, in some cases, more effectively killing cancer cells.
Dutch researchers studied mouse salivary gland organoids enriched in stem and progenitor cells and showed that proton irradiation induces a stronger IFN-I response following activation of transposable elements (TEs) and formation of cytoplasmic dsRNA. As a result, stem and progenitor cell activity and self-renewal capacity were enhanced, both in organoids and in mouse experiments.
Proton Irradiation Induces Stronger Interferon Responses and Stem Cell Activity Than Photon Irradiation
Irradiation of salivary gland organoids with photons or protons at a dose of 7 Gy caused a similar reduction in survival. However, proton irradiation increased self-renewal capacity, as evidenced by higher organoid-forming efficiency, suggesting differences in stem and progenitor cell responses.
Both irradiation modalities induced marked changes in the expression of genes related to immune and inflammatory responses, but the IFN-I response, particularly interferon-β, was significantly stronger after proton irradiation, accompanied by increased expression of interferon-stimulated genes (ISGs) and stronger activation of the STAT1 signaling pathway.
The IFN-I signaling pathway is a key regulator of stem cell activity and tissue regeneration. Single-cell RNA analysis revealed enhanced IFN-I responses across all cell populations in irradiated organoids, especially after proton irradiation. The strongest IFN-I response was observed in Sox9+ stem and progenitor cell populations, including a subset of actively proliferating cells. In these cells, proton irradiation induced more pronounced activation of mitotic cell cycle–related processes.
Together, these findings indicate that proton irradiation more strongly activates IFN-I responses, which may positively influence the activity and proliferation of salivary gland stem and progenitor cells, potentially explaining their enhanced self-renewal capacity compared with photon irradiation.
The cGAS–STING Pathway Mediates Radiation-Induced IFN-I Activation
Analysis of salivary gland organoids showed that photon and proton irradiation induced similar early DNA damage, accompanied by activation of γ-H2AX and p-P53, and resulted in comparable mitotic activity. Both irradiation types led to a transient increase in the number of cGAS-positive micronucleated cells.
Both modalities activated immune and inflammatory processes and increased expression of Ifnb, ISGs, and STAT1 activation at early time points after irradiation. Depletion of cGAS or inhibition of STING significantly reduced ISG expression.
These results demonstrate that the cGAS–STING pathway is a key mediator of early IFN-I activation in response to both photon and proton irradiation.
Mitochondrial Damage Contributes to Radiation-Induced IFN-I Signaling
At later stages after irradiation, when micronuclei numbers declined, a sustained IFN-I response persisted in salivary gland organoids. Photon and proton irradiation led to the accumulation of cytoplasmic double-stranded DNA, increased levels of the cytosolic nucleic acid sensor ZBP1, increased mitochondrial mass, and reduced mitochondrial membrane potential, indicating accumulation of dysfunctional mitochondria, accompanied by a marked increase in mitochondrial DNA in the cytoplasm.
Experimental mitochondrial DNA depletion significantly reduced ISG expression following both irradiation modalities. These findings indicate that radiation-induced release of mitochondrial DNA can amplify inflammatory responses and IFN-I signaling in salivary gland cells, but does not fully explain the stronger inflammatory effects observed after proton irradiation.
Proton Irradiation Enhances IFN-I Responses Through Activation of Transposable Elements and Accumulation of Cytoplasmic dsRNA
Six days after irradiation, salivary gland organoids exhibited increased expression of dsRNA-binding components, with higher levels after proton irradiation than after photon irradiation. In particular, expression of the dsRNA sensors RIG-I and MDA5 was markedly increased following proton irradiation. Functional blockade of RIG-I reduced ISG expression, confirming its key role in activating IFN-I responses after irradiation.
Microscopic analysis revealed accumulation of intracellular dsRNA, more pronounced after proton irradiation, accompanied by late activation of transposable elements, including endogenous retroviruses and LINE-1 elements, which was also stronger after proton irradiation. This effect was associated with reduced levels of histone H3.3 and the repressive histone mark H3K9Me, both of which are key regulators that suppress transposable element activity.
Pharmacological inhibition of histone methylation in non-irradiated organoids reproduced these effects, inducing transposon activation, dsRNA accumulation, and enhanced IFN-I responses.
These results indicate that proton irradiation disrupts repression of transposable elements more strongly, leading to dsRNA accumulation and a more pronounced IFN-I response.
IFN-β Enhances the Activity of Salivary Gland Stem and Progenitor Cells After Irradiation
Addition of IFN-β after photon irradiation significantly enhanced the proliferation of stem and progenitor cells and increased organoid-forming efficiency. In contrast, neutralization of IFN-β or inhibition of the dsRNA sensor RIG-I reduced the self-renewal capacity of stem and progenitor cells, confirming the central role of IFN-β and RIG-I in regulating these cells after irradiation.
These findings were validated in mice: administration of IFN-β after local irradiation of salivary glands restored the organoid-forming capacity of cells. IFN-β treatment increased the number of Sox9+ stem and progenitor cells and enhanced their proliferation in both control and irradiated tissues.
Collectively, these data support a beneficial role of IFN-β in maintaining salivary gland stem and progenitor cells and enhancing their proliferative capacity.
Conclusion
Photon and proton irradiation of salivary gland organoids activate IFN-I signaling, but proton irradiation induces a stronger and more sustained response. This effect is associated with activation of transposable elements and accumulation of cytoplasmic dsRNA, leading to enhanced ISG expression.
At early stages after irradiation, IFN-I responses are initiated via the cGAS–STING pathway and are linked to micronuclei formation. At later stages, increased expression of the dsRNA sensor RIG-I plays a central role, particularly after proton irradiation. Proton irradiation more strongly promotes transposable element expression and amplification of IFN-I responses.
These changes result in higher activity and self-renewal capacity of Sox9+ stem and progenitor cells in proton-irradiated organoids. Administration of IFN-β enhanced self-renewal after photon irradiation, whereas blockade of IFN-β or inhibition of RIG-I reduced organoid-forming efficiency.
Thus, proton irradiation is associated with activation of the TE–dsRNA–RIG-I–IFN-I axis and increased regenerative activity of stem cells, which may be relevant for optimizing radiotherapy strategies that consider normal tissue protection and regeneration.
Reference
IFN-I signaling enhances salivary gland stem and progenitor cell activity after irradiation
