Background/Purpose: NEMO-deleted 5 autoinflammatory syndrome (NEMO-NDAS) is an inflammatory disease caused by mosaic splice-site variants that lead to exon 5 skipping in IKBKG, encoding NEMO. Patients (pts) present with systemic inflammation, gdT-cells expansion, high serum IFNg, portal hypertension (PH) and porto-sinusoidal vascular disorders (PSVD). Here, we characterize the effect of exon 5-skipped NEMO on NFkB signaling in U937 cells, and assess intestinal barrier and gut microbiome dysregulation in the pathogenesis of PH and PSVD.

Methods: U937 cells with exon 5-skipped NEMO were created by CRISPR (MUT) and compared to wildtype U937 cells (WT). NFkB signaling, cell viability, p65 nuclear translocation, NEMO/IKKb colocalization, and mRNA transcription profiling were assessed in TNF-stimulated MUT and WT U937 cells. We assessed immune activation and target killing in pts’ PBMCs cocultured with WT and/or MUT U937s and quantified live U937 cells by flow cytometry. To characterize the gut-liver-axis, we analyzed intestinal fatty-acid-binding protein (I-FABP) concentrations in serum, and the stool microbiome (including virome), in NEMO-NDAS pts, inflammatory controls, and healthy household controls (HCs).

Results: Following TNF stimulation, nuclear translocation of p65 was significantly attenuated in MUT U937 cells consistent with impaired NFkB signaling. Additionally, increased susceptibility to TNF-induced cell death of MUT U937 cells was associated with a lack of upregulation of the anti-apoptotic genes BIRC3/cIAP2 and CFLAR/c-FLIP.To test the killing potential of expanded gd T cells in pts’ PBMCs, we cocultured pts’ PBMCs with WT or MUT U937 cells. While viability of WT U937 cells is not affected by the pts’ PBMC, mutant U937 cells are killed. When mixed WT and MUT U937s were cocultured with PBMCs, both WT and MUT U937s had decreased survival compared to PBMC-free conditions. Exploration of contributing cell types and pathomechanisms is ongoing.Liver-gut-axis evaluation suggested that NEMO-NDAS pts have increased IFABP serum levels compared to CANDLE pts and HCs, suggestive of increased intestinal permeability and impaired gut barrier function. Microbiome studies showed increased abundance of Faecalibacterium phages and a decrease in metabolic protective markers, which is consistent with previously reported data in liver disease.

Conclusion: Exon 5-skipping in NEMO impairs NFkB signaling, sensitizes U937 cells to TNF-induced cell death, and leads to U937 cell death in cocultures with PBMCs from NEMO-NDAS pts. We hypothesize that exon 5-skipped NEMO impairs gut epithelial cell function and leads to dysbiosis and increased microbial translocation that contribute to PH and PSVD in NEMO-NDAS. Whether impaired gut barrier and dysbiosis contribute to activation and expansion of gd T cells in NEMO-NDAS is under investigation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-characterization-of-nemo-ndas-causing-variants-in-patients-pbmcs-and-in-wildtype-and-mutant-u937-cells/