Citation

  • Authors: Cavounidis A. et al.
  • Year: 2022
  • Journal: Mucosal Immunol 15 1431-1446
  • Applications: in vitro / siRNA / INTERFERin
  • Cell type: HAP1

Method

For siRNA transfections, we used the Rab32 siGENOME (Horizon Discovery, SMARTPool, M-009920-02-0005) and the non-targeting siRNA pool #1 (Horizon Discovery, D-001206-13-05). Cells were transfected with 40 nM siRNA and INTERFERin (Polyplus transfection, 409-10) according to the manufacturer’s protocol. Experiments were performed 72 h following transfection.

Abstract

Hermansky-Pudlak syndrome (HPS) types 1 and 4 are caused by defective vesicle trafficking. The mechanism for Crohn's disease-like inflammation, lung fibrosis, and macrophage lipid accumulation in these patients remains enigmatic. The aim of this study is to understand the cellular basis of inflammation in HPS-1. We performed mass cytometry, proteomic and transcriptomic analyses to investigate peripheral blood cells and serum of HPS-1 patients. Using spatial transcriptomics, granuloma-associated signatures in the tissue of an HPS-1 patient with granulomatous colitis were dissected. In vitro studies were conducted to investigate anti-microbial responses of HPS-1 patient macrophages and cell lines. Monocytes of HPS-1 patients exhibit an inflammatory phenotype associated with dysregulated TNF, IL-1α, OSM in serum, and monocyte-derived macrophages. Inflammatory macrophages accumulate in the intestine and granuloma-associated macrophages in HPS-1 show transcriptional signatures suggestive of a lipid storage and metabolic defect. We show that HPS1 deficiency leads to an altered metabolic program and Rab32-dependent amplified mTOR signaling, facilitated by the accumulation of mTOR on lysosomes. This pathogenic mechanism translates into aberrant bacterial clearance, which can be rescued with mTORC1 inhibition. Rab32-mediated mTOR signaling acts as an immuno-metabolic checkpoint, adding to the evidence that defective bioenergetics can drive hampered anti-microbial activity and contribute to inflammation.

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