Eveline Verheugen

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.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

Proof of concept: enthesitis and new bone formation in spondyloarthritis are driven by mechanical strain and stromal cells

Objectives Spondyloarthritides (SpA) are characterised by both peripheral and axial arthritis. The hallmarks of peripheral SpA are the development of enthesitis, most typically of the Achilles tendon and plantar fascia, and new bone formation. This study was undertaken to unravel the mechanisms leading towards enthesitis and new bone formation in preclinical models of SpA. Results First, we demonstrated that TNFΔARE mice show typical inflammatory features highly reminiscent of SpA. The first signs of inflammation were found at the entheses. Importantly, enthesitis occurred equally in the presence or absence of mature T and B cells, underscoring the importance of stromal cells. Hind limb unloading in TNFΔARE mice significantly suppressed inflammation of the Achilles tendon compared with weight bearing controls. Erk1/2 signalling plays a crucial role in mechanotransduction-associated inflammation. Furthermore, new bone formation is strongly promoted at entheseal sites by biomechanical stress and correlates with the degree of inflammation. Conclusions These findings provide a formal proof of the concept that mechanical strain drives both entheseal inflammation and new bone formation in SpA.

A20 inhibition of STAT1 expression in myeloid cells: a novel endogenous regulatory mechanism preventing development of enthesitis.

Objectives A20 is an important endogenous regulator of inflammation. Single nucleotide polymorphisms in A20 have been associated with various immune-mediated inflammatory diseases, and cell-specific deletion of A20 results in diverse inflammatory phenotypes. Our goal was to delineate the underlying mechanisms of joint inflammation in myeloid-specific A20-deficient mice (A20myelKO mice). Methods Inflammation in A20myelKO mice was assessed in a time-dependent manner. Western blot analysis and quantitative PCR analysis were performed on bone marrow-derived macrophages from A20myelKO and littermate control mice to study the effect of A20 on STAT1/STAT3 expression and STAT1/STAT3-dependent gene transcription in myeloid cells. The in vivo role of Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling in the development of enthesitis in A20myelKO mice was assessed following administration of a JAK inhibitor versus placebo control. Results Enthesitis was found to be an early inflammatory lesion in A20myelKO mice. A20 negatively modulated STAT1-dependent, but generally not STAT3-dependent gene transcription in myeloid cells by suppressing STAT1 but not STAT3 expression, both in unstimulated conditions and after interferon-γ or interleukin-6 stimulation. The increase in STAT1 gene transcription in the absence of A20 was shown to be JAK-STAT-dependent. Moreover, JAK inhibition in vivo resulted in significant reduction of enthesitis, both clinically and histopathologically. Conclusions Our data reveal an important and novel interplay between myeloid cells and tissue resident cells at entheseal sites that is regulated by A20. In the absence of A20, STAT1 but not STAT3 expression is enhanced leading to STAT1-dependent inflammation. Therefore, A20 acts as a novel endogenous regulator of STAT1 that prevents onset of enthesitis.

A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis

BACKGROUND & AIMS Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. METHODS The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. RESULTS The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. CONCLUSIONS Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells.

NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20

Drennan et al. use a new mouse to show that A20-deficient NKT cells are hyperresponsive to TCR-dependent stimuli and have severely impaired NKT cell development.

A1.11 A20 controls activation of STAT1, but has no effect on STAT3: Implications for development of enthesitis

Background and objectives A20 is a negative regulator of NF-kB with potent anti-inflammatory properties. SNPs in A20 have been associated with a variety of IMIDs, including RA, psoriasis and PsA. Cell specific deletion of A20 results in a myriad of inflammatory disorders: for example, myeloid deletion results in polyarthritis but epidermal loss rather leads to psoriatic like lesions. Our goal was to delineate the underlying mechanisms and earliest phases leading to joint inflammation in A20 myeloid deficient mice. Materials and methods We used mice with a myeloid specific deficiency of A20 (A20myelKO mice using LysM-cre). This model is IL-6 dependent, but TNF independent. We isolated BMDMs from A20myelKO and wildtype mice and stimulated them with IL-6 or IFN-γ. qPCR and western blot analysis were performed to study whether A20 has an effect on the activation of the JAK-STAT pathway. Transfection experiments were conducted on HEK293T cells to examine which part of A20 is responsible for this effect. In vivo experiments with a JAK-STAT inhibitor (tofacitinib) were performed: A20myelKO mice were divided into 2 groups: a tofacitinib and a placebo group. Mice were treated bidaily with respectively 50mg/kg tofacitinib orplacebo. Results In the earliest phase of disease, inflammation was markedly confined to enthesial sites. We also found that A20 has a marked suppressive effect in myeloid cells on the expression of STAT1, but not STAT3 induced genes. Western blot showed that A20 has an inhibitory effect on STAT1, but not STAT3 phosphorylation. Reporter assays confirmed these findings and highlighted that this effect is probably due to ZnF motif 4 and 7, rather than to the OTU domain. We therefore assessed the role of JAK-STAT inhibition in vivo and found significantly lower clinical scores and significantly less inflammation of the synovio-entheseal complex in tofacitinib compared to placebo treated mice. Conclusions Our data reveal a novel interplay between myeloid cells and tissue resident cells at entheseal sites that relies on A20. In the absence of A20, STAT1 but not STAT3 signalling is engaged leading to activation of a marked STAT1 dependent inflammation. Interestingly, the effects of JAK-STAT are operating independently from TNF.

A20 (TNFAIP3) deficiency in myeloid cells triggers rheumatoid arthritis

Background and objectives Rheumatoid arthritis is an inflammatory autoimmune disease characterised by chronic inflammation of the joints associated with progressive destruction of cartilage and bone. Deregulated cytokine production is known to contribute to the aetiology of rheumatoid arthritis but the underlying molecular mechanism is still unclear. A20 (also known as TNFAIP3) is a protein that is involved in the negative feedback regulation of nuclear factor-κB (NF-κB) signalling in response to specific proinflammatory stimuli in different cell types. To define the contribution of A20 to rheumatoid arthritis pathology, the authors generated mice deficient in A20 in myeloid cells, B cells or T cells. Materials and methods Conditional A20/tnfaip3 knockout mice were generated. A20FL/FL mice were crossed with LysM-Cre transgenic mice to generate a myeloid-specific A20 knockout mouse. T and B cell-specific A20 knockout mice were obtained by crossing with CD4-Cre and CD19-Cre transgenic mice, respectively. Mice were scored twice a week for development of peripheral arthritis, until they were killed. Histological analysis was performed on paraffin embedded mouse paws. Immunofluorescence stainings for B220, CD3, F4/80 were performed and analysed by confocal microscopy. In separate experiments, in vivo monitoring of inflammation by positron emission tomography-CT was conducted using fluorodeoxyglucose. Peritoneal and bone marrow-derived macrophages were isolated and stimulated with various toll-like receptor (TLR) ligands. Cell lysates were subject to Western blot analysis for IκBα, phospho-IκBα, phospho-JNK, phospho-Erk and phospho-p38, A20 and actin. Quantitative real-time PCR for interleukin 1β (IL-1β), IL-6, IL-23 was conducted on joint tissue. In vitro induction of osteoclast formation was done by macrophage colony-stimulating factor and receptor activator of NF-κB. Tartrate resistant acid phosphatase staining and Pit-forming assays were conducted. Results Specific ablation of A20 in myeloid cells, but not in B or T cells, resulted in spontaneous development of a severe destructive polyarthritis with many features of rheumatoid arthritis. Myeloid A20 deficient mice have high levels of tumour necrosis factor (TNF) and other inflammatory cytokines in their serum, consistent with a sustained NF-κB activation and higher TNF production by cultured macrophages. Remarkably, the authors observed no histological evidence of inflammation or damage in other tissues of myeloid A20 deficient mice. Secondary lymphoid organs were markedly enlarged and the number of myeloid cells in the spleen was increased. Destructive polyarthritis in myeloid A20 knockout mice was TLR4/MyD88 dependent. Myeloid A20 deficiency also promoted osteoclastogenesis in mice. Conclusions These observations indicate a critical and cell-specific function for A20 in the aetiology of rheumatoid arthritis, supporting the concept of developing A20 modulatory drugs as cell targeted therapies.