MACROPHAGE LRRK2 HYPERACTIVITY IMPAIRS AUTOPHAGY AND INDUCES PANETH CELL DYSFUNCTION AND INTESTINAL INFLAMMATION

Background: LRRK2 variants (G2019S/N2081D) that increase Parkinson’s disease (PD) and Crohn’s Disease (CD) susceptibility are associated with LRRK2 kinase hyperactivity and suppress autophagy, suggesting that LRRK2 kinase inhibition, a strategy being explored for PD treatment, may also benefit CD. The homeostasis of small intestinal Paneth cells, critical in innate immunity, is tightly regulated by autophagy, and Paneth cell dysfunction is a precursor to CD gut inflammation. Given the role of LRRK2 in autophagy regulation, we hypothesized that CD patients with LRRK2 kinase hyperactivity leads to Paneth cell defects.

Methods: Ileal biopsy or resection samples from 402 CD patients genotyped for the LRRK2 N2081D and G2019S variants were used for Paneth cell phenotype analysis by HD5 immunofluorescence. LRRK2 expression was defined by RNAscope, immunohistochemistry, and Western blot. A highly potent, selective, metabolically stable and peripherally restricted LRRK2 inhibitor was used in 2 mouse models to interrogate its role in modulating Paneth cell function: Lrrk2 G2019S mice, and Atg16l1 T300A mice with cigarette smoking. Bone marrow derived macrophages (BMDM) and ileal organoids were used for in vitro studies. BMDM secretome was defined by multiplex cytokine array and validated by ELISA.

Results: CD patients and mice carrying hyperactive LRRK2 variants possessed Paneth cell dysfunction. Surprisingly, myeloid-derived cells and not Paneth cells expressed detectable LRRK2. The LRRK2 inhibitor rescued Paneth cell defects in Lrrk2 G2019S mice and T300A-smoked mice. Culture media transfers from smoke condensate treated/T300A BMDM showed enhanced proinflammatory TNF production and reduced Paneth cells when transferred to T300A organoids. Enforced autophagy activation by BECN1 (pharmacologic or genetic approaches) also rescued Paneth cell defects.

Conclusion: LRRK2 kinase can be activated by LRRK2 variants, or in autophagy-deficient hosts exposed to smoking. LRRK2 kinase inhibitors maintain Paneth cell homeostasis by restoring autophagy, and may represent a novel therapeutic strategy for CD.