Lars Vereecke

The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of immunopathology

Nuclear factor (NF)-kappaB has an important role in immunity and inappropriate NF-kappaB activity has been linked with many autoimmune and inflammatory diseases. Multiple mechanisms normally ensure the proper termination of NF-kappaB activation. In this context, the intracellular ubiquitin-editing protein A20 (also known as Tumor Necrosis Factor Alpha-Induced Protein 3 or TNFAIP3) is a key player in the negative feedback regulation of NF-kappaB signaling in response to multiple stimuli. Moreover, A20 also regulates tumor necrosis factor (TNF)-induced apoptosis. Recent genetic studies demonstrate a clear association between several mutations in the human A20 locus and immunopathologies such as Crohns disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis and type 1 diabetes. These findings further illustrate the importance of A20 in the resolution of inflammation and the prevention of human disease.

A20 in inflammation and autoimmunity

Although known for many years as a nuclear factor (NF)-κB inhibitory and antiapoptotic signaling protein, A20 has recently attracted much attention because of its ubiquitin-regulatory activities and qualification by genome-wide association studies (GWASs) as a susceptibility gene for inflammatory disease. Here, we review new findings that have shed light on the molecular and biochemical mechanisms by which A20 regulates inflammatory signaling cascades, and discuss recent experimental evidence characterizing A20 as a crucial gatekeeper preserving tissue homeostasis.

A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis

A20 (TNFAIP3) is a protein that is involved in the negative feedback regulation of NF-κB signaling in response to specific proinflammatory stimuli in different cell types and has been suggested as a susceptibility gene for rheumatoid arthritis. To define the contribution of A20 to rheumatoid arthritis pathology, we generated myeloid-specific A20-deficient mice and show that specific ablation of Tnfaip3 in myeloid cells results in spontaneous development of a severe destructive polyarthritis with many features of rheumatoid arthritis. Myeloid-A20–deficient mice have high levels of inflammatory cytokines in their serum, consistent with a sustained NF-κB activation and higher TNF production by macrophages. Destructive polyarthritis in myeloid A20 knockout mice was TLR4-MyD88 and IL-6 dependent but was TNF independent. Myeloid A20 deficiency also promoted osteoclastogenesis in mice. Together, these observations indicate a critical and cell-specific function for A20 in the etiology of rheumatoid arthritis, supporting the idea of developing A20 modulatory drugs as cell-targeted therapies.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL‐4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL‐4‐dependent E‐cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL‐4‐induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1‐deficient macrophages display rather enhanced IL‐4‐induced polyamine production, suggesting that an Arg1‐independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS‐induced expression of several proinflammatory genes was increased significantly in polyamine‐depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL‐4‐induced expression of several AAM mediators, they inhibit the LPS‐mediated expression of proinflammatory genes in CAMs.

Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease.

A20 [also known as TNFAIP3 (tumour necrosis factor α-induced protein 3)] restricts and terminates inflammatory responses through modulation of the ubiquitination status of central components in NF-κB (nuclear factor κB), IRF3 (interferon regulatory factor 3) and apoptosis signalling cascades. The phenotype of mice with full or conditional A20 deletion illustrates that A20 expression is essential to prevent chronic inflammation and autoimmune pathology. In addition, polymorphisms within the A20 genomic locus have been associated with multiple inflammatory and autoimmune disorders, including SLE (systemic lupus erythaematosis), RA (rheumatoid arthritis), Crohns disease and psoriasis. A20 has also been implicated as a tumour suppressor in several subsets of B-cell lymphomas. The present review outlines recent findings that illustrate the effect of A20 defects in disease pathogenesis and summarizes the identified A20 polymorphisms associated with different immunopathologies.

Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20EKO) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20EKO mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

A20 controls intestinal homeostasis through cell-specific activities

The transcription factor NF-κB is indispensable for intestinal immune homeostasis, but contributes to chronic inflammation and inflammatory bowel disease (IBD). A20, an inhibitor of both NF-κB and apoptotic signalling, was identified as a susceptibility gene for multiple inflammatory diseases, including IBD. Despite absence of spontaneous intestinal inflammation in intestinal epithelial cell (IEC) specific A20 knockout mice, we found additional myeloid-specific A20 deletion to synergistically drive intestinal pathology through cell-specific mechanisms. A20 ensures intestinal barrier stability by preventing cytokine-induced IEC apoptosis, while A20 prevents excessive cytokine production in myeloid cells. Combining IEC and myeloid A20 deletion induces ileitis and severe colitis, characterized by IEC apoptosis, Paneth and goblet cell loss, epithelial hyperproliferation and intestinal microbiota dysbiosis. Continuous epithelial cell death and regeneration in an inflammatory environment sensitizes cells for neoplastic transformation and the development of colorectal tumours in aged mice.

Optineurin deficiency in mice is associated with increased sensitivity to Salmonella but does not affect proinflammatory NF-κB signaling.

Optineurin (OPTN) is an evolutionary conserved and ubiquitously expressed ubiquitin‐binding protein that has been implicated in glaucoma, Paget bone disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. From in vitro studies, OPTN was shown to suppress TNF‐induced NF‐κB signaling and virus‐induced IRF signaling, and was identified as an autophagy receptor required for the clearance of cytosolic Salmonella upon infection. To assess the in vivo functions of OPTN in inflammation and infection, we generated OPTN‐deficient mice. OPTN knockout mice are born with normal Mendelian distribution and develop normally without any signs of spontaneous organ abnormality or inflammation. However, no differences in NF‐κB activation could be observed in OPTN knockout mice or fibroblasts derived from these mice upon TNF or LPS treatment. Primary bone marrow‐derived macrophages from OPTN‐deficient mice had slightly impaired IRF signaling and reduced IFN type I production in response to LPS or poly(I,C). Finally, OPTN‐deficient mice were more susceptible to infection with Salmonella, confirming in vivo the importance of OPTN in bacterial clearance.