P0039 Deletion of the autophagy-related gene 16 like 1 (Atg16l1) in the myeloid lineage induces systemic inflammation
Authors: Wobbe M , Falk-Paulsen M , Luzius A , Pimenta Bernardes J , Kimmig F , Rosenstiel P 
Published: January 2025

Abstract

Background
The autophagy-related gene 16 like 1 (ATG16L1) is essential for the formation of the autophagosome and deletion of this gene leads, among other things, to the disruption of autophagy.1,2 In 2007, a Genome Wide Association Study identified ATG16L1 as a risk gene for Crohn’s Disease.3 This study aims to understand the mechanisms causing the hyperinflammatory systemic phenotype observed in our mouse model carrying a conditional myeloid deletion of Atg16l1 (Atg16l1LysM).

Methods
The immune phenotype of Atg16l1LysM and Atg16l1fl/fl mice was characterized using histology staining and flow cytometry of lymphatic organs. In addition, single cell RNA sequencing of splenocytes and determination of cytokine levels in the serum were performed to investigate the inflammatory status of the animals. LPS-stimulated bone marrow-derived macrophages (BMDMs) of Atg16l1LysM and Atg16l1fl/fl mice were used as an in-vitro model to study signaling pathways that might be responsible for the inflammatory phenotype.

Results
Atg16l1LysM mice display a spontaneous hyperinflammatory phenotype that is accompanied by hepatosplenomegaly and increased cytokine levels in serum. Histology staining revealed a disrupted spleen architecture with an increase of red pulp tissue compared to floxed littermates. Flow cytometry and single cell transcriptome data of splenocytes revealed alterations in the cellular composition of Atg16l1LysM spleens confirming an increase of activated myeloid cells and a simultaneous reduction of lymphoid cells, especially T cells. Additionally, the proportion of myeloid-derived suppressor cells (MDSC) in Atg16l1LysM spleen was found to be increased. Moreover, a significant overexpression of type-I interferon signaling genes was discovered in monocytic cells derived from the Atg16l1LysM spleen. Bulk RNA sequencing data of LPS-stimulated Atg16l1 knockout BMDMs confirmed the overactivation of type-I IFN-related genes compared to wild type BMDMs, pointing towards a role for the Stimulator of IFN genes (STING) and Rig-I-like (RLR) pathways in the Atg16l1-deficient scenario. However, neither inhibiting the STING pathway in vitro nor creating Atg16l1STINGLysM double knockout mice lead to a significant reduction of IFN-β production.

Conclusion
The increased type-I IFN response observed in Atg16l1LysM mice does not seem to be influenced by STING signaling. Future studies are needed to uncover whether RIG-I-like receptors are major contributors of the increased IFN-β response in absence of Atg16l1 and to determine the internal triggers of this reaction. The results also prompt further investigations on aberrant cell activation in autophagy-deficient myeloid cells.

References
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2.Fujita N, Itoh T, Omori H, Fukuda M, Noda T, Yoshimori T. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol Biol Cell. 2008;19(5):2092-2100. doi:10.1091/mbc.e07-12-1257
3.Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet. 2007;39(2):207-211. doi:10.1038/ng1954