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• W4377695520 abstract "A20 protein, encoded by TNFAIP3, is involved in NF-kB pathway regulation. Mutations in TNFAIP3 can lead to A20 haploinsufficiency (A20HA), an important monogenic etiology of Behçet’s disease (BD). Understanding the effect of A20 in immune cells can help to comprehend BD pathophysiology, and pave the road for functional assays for patients with monogenic NF-κB disorders. THP-1 (monocytic), HL-60 (neutrophilic) and Daudi (B-lymphoid) cells were transfected with a GFP-tagged plasmid containing either TNFAIP3 or mutated TNFAIP3-L227X genes. Cells were assessed for A20 protein expression, proliferation rates, phagocytic activity and reactive oxygen species (ROS) production. Flow cytometry was used for all assays. A20 expression (figure 1) was increased in Daudi-TNFAIP3 cells, compared to Daudi [mean of fluorescence intensity (MFI) 241 vs 295, p < 0.01]; THP-1-TNFAIP3 had higher A20 expression (MFI 1396 vs 1169), not statistically significant. A20 expression was reduced in THP-1-TNFAIP3-L227X (MFI 1169 vs 581, p < 0.05) and HL-60-TNFAIP3-L227X (MFI 572 vs 450, p < 0.05), compared to non-transfected cells. THP-1-TNFAIP3-L227X showed higher phagocytic activity than THP-1 and THP-1-TNFAIP3 (MFI 94748 vs 34039.33 vs 18476; p < 0.001) at baseline and after stimulus (MFI 76265 vs 35835 vs 25379; p < 0.05). Similarly, HL-60-TNFAIP3-L227X cells showed higher phagocytic activity than HL-60 (17480 vs 5862; p < 0.01) at baseline and after stimulus (16869 vs 5482; p < 0.01) (figure 2). Constitutional ROS production by THP-1-TNFAIP3 was higher than THP-1 and TNFAIP3-L227X (MFI 291919 vs 98226 vs 230711; p < 0.001). PMA-induced Ki-67 expression of THP-1-TNFAIP3-L227X was increased compared with THP-1 and THP-1-TNFAIP3 (19184 vs 2434 vs 4463; p < 0.01). Ki-67 expression was lower at baseline for Daudi-TNFAIP3-WT, compared to Daudi (MFI 10250 vs 12310; p < 0.01) (figure 3).Download : Download high-res image (87KB)Download : Download full-size imageFigure 1. A20 protein assessed by anti-TNFAIP3 C-terminal (500–600 aa) rabbit IgG antibody, analyzed by flow cytometry. (A) THP-1 cells, showing reduced A20 expression in THP-1-TNFAIP3-L227X cells, compared to non-transfected cells. (B) HL-60 cells, showing reduced A20 expression in HL-60-TNFAIP3-L227X cells, compared to non-transfected cells. (C) Daudi cells, showing increased A20 expression in Daudi-TNFAIP3 cells, compared to non-transfected cells. * p < 0.05; p < 0.01.Download : Download high-res image (111KB)Download : Download full-size imageFigure 2. Phagocytosis of zymosan fluorescent (Alexa Fluor 594) particles by THP-1 and HL-60 cells. HL-60 were differentiated with all-trans-retinoic acid for 5 days prior to this experiment (dHL-60). (A) THP-1-TNFAIP3-L227X cells show higher phagocytic activity than THP-1 and THP-1-TNFAIP3, at baseline and upon stimulation with soluble CD40 ligand. (B) dHL-60-TNFAIP3-L227X cells showed higher phagocytic activity than dHL-60 at baseline and after stimulation; dHL-60-TNFAIP3 also had increased phagocytosis compared to dHL-60. * p < 0.05; p < 0.01; p < 0.001Download : Download high-res image (111KB)Download : Download full-size imageFigure 3. Expression of Ki-67 in THP-1 and Daudi cells, showing reduced expression in cells transfected with TNFAIP3, and increased expression in THP-1-TNFAIP3-L227X upon PMA stimulation. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure 1. A20 protein assessed by anti-TNFAIP3 C-terminal (500–600 aa) rabbit IgG antibody, analyzed by flow cytometry. (A) THP-1 cells, showing reduced A20 expression in THP-1-TNFAIP3-L227X cells, compared to non-transfected cells. (B) HL-60 cells, showing reduced A20 expression in HL-60-TNFAIP3-L227X cells, compared to non-transfected cells. (C) Daudi cells, showing increased A20 expression in Daudi-TNFAIP3 cells, compared to non-transfected cells. * p < 0.05; p < 0.01. Figure 2. Phagocytosis of zymosan fluorescent (Alexa Fluor 594) particles by THP-1 and HL-60 cells. HL-60 were differentiated with all-trans-retinoic acid for 5 days prior to this experiment (dHL-60). (A) THP-1-TNFAIP3-L227X cells show higher phagocytic activity than THP-1 and THP-1-TNFAIP3, at baseline and upon stimulation with soluble CD40 ligand. (B) dHL-60-TNFAIP3-L227X cells showed higher phagocytic activity than dHL-60 at baseline and after stimulation; dHL-60-TNFAIP3 also had increased phagocytosis compared to dHL-60. * p < 0.05; p < 0.01; p < 0.001 Figure 3. Expression of Ki-67 in THP-1 and Daudi cells, showing reduced expression in cells transfected with TNFAIP3, and increased expression in THP-1-TNFAIP3-L227X upon PMA stimulation. * p < 0.05; ** p < 0.01; *** p < 0.001. Reduced expression of A20 in TNFAIP3-L227X cells is relevant, considering previous reports of A20HA did not show a dominant negative effect. THP-1-TNFAIP3-L227X presented higher phagocytic activity, a pattern also observed by neutrophilic cells (HL-60-TNFAIP3-L227X), suggesting a specific mutation-induced phenotype. Decreased THP-1-TNFAIP3-L227X oxidative burst suggests a state of cell exhaustion. Lower Ki-67 expression in THP-1-TNFAIP3 and Daudi-TNFAIP3 cells at baseline reinforces the role of A20 as an NF-κB pathway inhibitor." @default.
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• W4377695520 date "2023-05-01" @default.
• W4377695520 modified "2023-10-01" @default.
• W4377695520 title "The search for an in vitro model for monogenic Behçet’s disease: the influence of A20 protein on functional cell assays" @default.
• W4377695520 doi "https://doi.org/10.1016/j.clim.2023.109552" @default.
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