Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

Abstract

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

Figure 1. TNFAIP3 mutations cause a dominantly inherited systemic inflammatory disease

(a) Pedigrees of 5 families with heterozygous mutations in the TNFAIP3 gene. WT indicates wild-type TNFAIP3 alleles; M1, M2, M3, M4 and M5 indicate mutant alleles. The individuals selected for exome sequencing are marked with asterisk. NA: an affected sister of P6 had similar symptoms but was not available for this study. (b) Clinical manifestations in patients: chorioretinal scarring and macular fibrosis secondary to retinal vasculitis causing a significant visual impairment (upper panel), recurrent oral ulcers (middle panel), axillary dermal abscesses and a pathergy-positive response (bottom panel). (c) Schematic of TNFAIP3/A20 protein domains. Deubiquitinating (DUB) activity of A20 is mediated by the ovarian tumor (OTU) domain. ZFs mediate A20 ubiquitin ligase activity and binding to K63-linked ubiquitin chains. The locations of the mutations are indicated with red arrows. These mutations are predicted to affect A20 protein interactions, dimerization and its enzymatic activities. (d-e) Reduced of TNFAIP3/A20 expression in patients’ PBMCs and fibroblasts. (d) Whole cell lysates were prepared from PBMCs isolated from three patients from Family 1, a healthy donor (C1), and purified control lymphocytes (Lym). Immunoblotting was performed against the N-terminus of A20. (e) Whole cell lysates from a healthy donor (C2) and patient P6 cultured fibroblast cells were either blotted with A20 N-terminal specific antibody (lane 3 and 4) or immunoprecipitated with one A20 N-terminal antibody and subsequently blotted with another A20 N-terminal antibody (lane 5 and 6). 293T lysates transfected with either FLAG-tagged wild-type (WT) A20 (lane 1) or A20 mutant L227* (lane 2) served as controls.

Figure 2. Enhanced NFκB signaling in transiently transfected HEK293T cells and patients’ cells

(a) The TNFAIP3/A20 mutants do not antagonize the inhibitory effects of wild type TNFAIP3/A20 on TNF-induced NFκB activation. HEK293T cells were transiently transfected with a NFκB reporter plasmid, Renilla luciferase control vector, and expression plasmids for either GFP-tagged wild-type or mutant A20. Plasmid expressing IκBα served as a positive control. Results are plotted as luc/ren to compensate for differences in transfection efficiency. One representative result of three independent experiments is shown. Values are expressed as mean of duplicates ± S.E.M. Protein expression of the transfected plasmids was confirmed by western blotting of cell lysates. (b) Stimulated cells from A20-deficient patients show increased IκBα degradation and increased IKKα/β and MAPK activities. PBMCs from patient P6 were stimulated with TNF for the time period indicated in the graph. Whole cell lysates were immunoblotted against respective target proteins. Healthy individuals and an unrelated patient with SLE served as controls. (c) Fibroblasts derived from A20-deficient patients show increased levels of phospho-IκBα, phospho-IKKα/β and MAPK activity. Whole cell lysates from TNF-stimulated fibroblasts were immunoblotted against respective target proteins. (d) Cytosolic and nuclear fractions were prepared from patient P9 and immunoblotted with antibodies against p65, A20, and β-actin, respectively. (e) Quantified nuclear NFκB p65 immunofluorescence staining in patients’ and controls’ fibroblasts under no-stimulation (left panel) and stimulation (right panel). Patient fibroblasts are significantly more activated than control fibroblasts (P<0.0001, Mann Whitney test). Data points represent mean ± S.E.M from analysis of quadruplicates of each individual. Data from patients (n = 4) and controls (n = 2) were pooled respectively.

Figure 3. Impaired TNFR signaling and deubiquitinase function of mutant TNFAIP3/A20

(a) Reduced recruitment of mutant A20 to the TNFR signaling complex. PBMCs from patient P4 and a healthy control were stimulated with TNF for the indicated times. Whole cell lysates were immunoprecipitated with antibodies against TNFR1, TRAF2, or RIP1, blotted with antibodies against N-terminal A20 and re-blotted with antibodies against TNFR1, TRAF2, RIP1 and Ubc13. (b-d) The A20 mutants lose their ability to deubiquitinate K63-ubiquitin chains but do not antagonize the deubiquitinase function of WT A20. 293T cells were transiently transfected with expression plasmids for A20 target proteins RIP1 with Myc tag (b), NEMO (c) and TRAF6 (d), together with plasmids for HA-K63Ub and either GFP-tagged WT or mutant A20. Cells were harvested 48 hrs later and an equal amount of whole cell lysates was immunoprecipitated with antibodies against respective target proteins. High-molecular weight (MW) Ub-aggregates (top panel) were indicated by immunoblotting of precipitates with a HA-specific antibody. As controls for transfection efficiency, cell lysates were also blotted with antibodies against each target protein (middle panel) or antibodies against the N-terminus of A20 or GFP tag (bottom panel). (e-f) TNF-stimulated patient PBMCs or fibroblasts showed increased levels and increased molecular weight of the K63-ubiquitinated NEMO protein as a result of insufficient A20 deubiquitinase activity. Primary cells from patients and healthy controls were stimulated for the indicated times. Whole cell lysates were subjected to immunoprecipitation with antibodies against K63-linked ubiquitin and subsequently blotted with antibodies against NEMO and Ub. Cell lysates were also blotted with antibodies against the target protein and β-actin as internal controls.

Figure 4. Patients’ immune cells have a strong inflammatory signature

Cytokine profiles comparing A20-deficient patients with healthy controls. (a) Serum cytokine levels from 6 patients and 8 controls. (b) Cytokine levels from patients (n = 5) or controls (n = 6) PBMCs stimulated either with bacterial lipopolysaccharide (LPS) or Staphylococcal enterotoxin B (SEB). Each sample was assessed in duplicates or triplicates. Values are represented as mean ± S.E.M. (c) Representative flow plots of CD3+ CD4+ CD45RO+ single live cells: Upper panels show IL-9 expression by intra-cellular cytokine (ICC) staining. The A20-deficient patients (n = 4) expressed significantly more IL-9 compared to paired controls (n = 5), and ex vivo PU.1 transcript levels were likewise significantly increased in patient PBMCs. Lower panels show representative flow plots of IL-17A and IL-4 expression in an A20-deficient patient compared to a paired control. A significant increase in intracellular IL-17A expression was observed in patients (n = 5) compared to controls (n = 8). Consistent with an absence of clinical allergic disease, no significant difference in intracellular IL-4 was seen. * P<0.05; Mann-Whitney test. Values are represented as mean ± S.E.M. (d) Increased inflammatory monocyte subsets in patients. Top panel: Representative flow-cytometry analysis of monocyte subsets in one control (left) and one A20-deficient patient (right). Three monocyte subsets are identified as CD14^highCD16^neg (classical, P1), CD14^highCD16^pos (inflammatory, P2), CD14^lowCD16^pos (non-classical, P3). The population (%) of different subsets is shown in different gates. Bottom panel: Quantification of CD14^highCD16^pos (inflammatory) monocyte subsets in 4 A20-deficient patients and in 4 controls. Data are shown as mean ± SD (p = 0.00013, n = 4).

Figure 5. Spontaneous NLRP3 inflammasome activity in PBMCs of patients with TNFAIP3 mutations

(a) Immunoblots of total cell lysates from LPS-stimulated patient cells and control show increased expression of pro IL-1β, NLRP3 and activated Caspase-1 (p10) and increased production of mature IL-1β. (b) IL-1β secretion in supernatants of LPS-stimulated patient cells is regulated by the activity of the NLRP3 inflammasome. Activation of adenylate cyclase (NKH477: adenylate cyclase activator), a PLC inhibitor (U73122), and a small molecule MCC950 attenuate the NLRP3 inflammasome activation. U73343: inactive analog of U73122. (c) Treatment with an IL-1 inhibitor, anakinra, normalized markers of systemic inflammation in Patient 6. Anakinra was initially given at a dose of 200mg daily (2014 May), later increased to 400mg daily (2014 Jun-2014 Sep), and then increased to 500 mg daily (2014 Oct-2015 Aug). The patient was also on prednisone 10mg and azathioprine 200 mg daily. Besides anakinra, she had no other medication changes from 2012-2014.