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Autophagy is required for the development and functionality of lacrimal gland-like organoids

ABSTRACT: Lacrimal glands (LGs) serve as pivotal exocrine glands crucial for protecting the ocular surface. Dysfunction in LG cell composition or secretion is implicated in dry eye disease (DED). While autophagy plays a vital role in tissue. homeostasis in many organs, how it affects LG development and secretory function is not known. Here we have undertaken a genetic study by utilizing autophagy-deficient human embryonic stem cells and differentiating them into LG-like organoids. Autophagy-deficient LG-like organoids exhibited improper development and secretion, along with increased protein aggregation, proliferation, and cell death. These phenotypes were associated with an accumulation of PAX6, a transcription factor crucial for brain and eye development, which we identified as an autophagy substrate. Pharmacological interventions with nicotinamide mononucleotide and melatonin were able to rescue the cellular dysfunction in autophagy-deficient LG-like organoids. Together, our study highlights the role of autophagy in LG along with potential therapeutic interventions for DED.

ORGANISM(S): Homo sapiens

PROVIDER: GSE280811 | GEO | 2025/12/18

REPOSITORIES: GEO

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Project description:Purpose: CD25KO mice have severe autoimmune disease and are used as a model of Sjögren disease, a prototype autoimmune dry eye. CD25KO mice have severe inflammation and infiltrating lymphocytes to multiple self-tissues, including the exocrine glands. We investigated the immunological profile of CD25KO mice and compared it to wild-type (WT) mice. Methods: 8-week-old WT and CD25KO mice were used. LGs (n=5) were submitted for RNA bulk sequencing. Tears washes were collected from mice and analyzed for protein expression using a luminex assay. A time course of adoptive transfer of CD25KO CD4+T cells was performed and LGs were evaluated by histology and qPCR. Co-adoptive transfer of CD25KO CD4+T cells with either 2M or 20M B6 CD25+GITR+T regulatory cells (Tregs) was performed in RAG1KO mice. LGs were evaluated in H&E-stained sections and by qPCR. Results: Investigation by RNA bulk sequencing found 3481 genes differentially expressed in CD25KO mice compared to B6, including upregulation of genes involved in T cell activation and Type I interferon signaling. CD25KO mice produced the same volume of tears as wild-type littermate controls, but had elevated protein expression of TNF, IFN-γ, and CCL5 and decreased protein expression of IL-12p40 and VEGF-A. Adoptive transfer recipients begin to develop inflammation and disease three weeks after adoptive transfer. In mice that received adoptive transfer of CD25KO CD4+ T cells, LGs had inflammation scores significantly higher than naive mice. They had increased expression of transcripts for inflammatory cytokines and chemokines. However, in recipient mice that received CD25KO CD4+T cells + 2M B6 Tregs, LGs had significantly reduced inflammatory scores and reduced expression of some inflammatory cytokines. Recipients of CD25KO CD4+T cells + 20M B6 Tregs had more inflamed lacrimal glands by both histology and qPCR, suggesting aged Tregs have less suppressive capacity.Conclusions: CD25KO mice have phenotypic and genetic changes resulting in increased inflammation, systemic autoimmunity and severe lymphocytic infiltration to the exocrine glands. These results are prominent in the tear film and LG. However, this autoimmunity can be controlled by addition of Tregs from healthy young mice, suggesting a possible avenue for therapeutic research.

Project description:Crohn’s disease (CD) is associated with multiple Paneth cell dysfunctions such as altered antimicrobial secretion that relies on autophagy - an essential process controlling the turnover of cellular components. Genome-wide association studies have linked CD to mutations in the ATG16l1 gene, encoding an important component of the autophagy machinery. Patients carrying the ATG16l1 CD risk allele have Paneth cell abnormalities, as reproduced in Atg16l1-deficient mouse models. However, the direct effect of the ATG16L1-deficiency and autophagy-impairment in Paneth cells has not been analysed in detail. To investigate this, we generated a mouse model lacking ATG16L1 specifically in intestinal epithelial cells (Atg16l1△IEC) making these cells impaired in autophagy. Using a 3D small intestinal organoid culture model, which we enriched for Paneth cells and compared the proteomic profiles of organoids derived from the wild-type (WT) and the Atg16l1△IEC mice. We used an integrated computational approach combining protein-protein interaction networks, list of proteins potentially targeted by autophagy and functional information on the proteins with altered protein levels to identify the mechanistic link between autophagy-impairment and disrupted cellular process. 198 (70%) of the 284 altered proteins were more abundant in autophagy-impaired organoids than WT organoids that could be due to a reduced protein turnover. Combining the proteomic dataset with the potential target proteins of selective autophagy proteins revealed that 116 (41%) of the differentially abundant proteins could rely on autophagy-mediated turnover. Taken together, our results suggest that proteins with increased levels in autophagy-impaired background require an autophagy-mediated turnover mechanism, and that their altered levels in CD could affect key cellular processes. Consequently, we pointed out the importance of autophagy in controlling the turnover of proteins relevant in key Paneth cell functions such as exocytosis, apoptosis and DNA damage repair. Our study unravels autophagy-dependent cellular processes of Paneth cells that not directly relies on the autophagy process, rather than a selective protein turnover mechanism. The identified proteins and processes open new avenues for targeted, cell type-specific therapeutic interventions in CD.

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Autophagy is required for the development and functionality of lacrimal gland-like organoids

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