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.
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