The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4⁺ and CD8⁺ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44^high/CD62L^low expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44^low/CD62L^high phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2-3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.

Preparation and histopathological analysis of tissues.

Mice were euthanized with increasing concentration of CO₂. The skin, tail, kidneys, liver, and lungs were removed and fixed for 48–72 h in 10% neutral buffered formalin. Tissues were processed and stained with haematoxylin and eosin (H&E) as described (16). For immunohistochemistry (IHC), antigen retrieval was performed using heat-induced epitope retrieval (HIER). Sections were treated at full pressure with Access Retrieval Unit (Menarini diagnostics, UK) in sodium citrate buffer (pH 6.0) for 90 sec at 125^oC and then rinsed in Tris/HCl Tween buffer (pH 7.5). The sections were treated for 5 min at 21^oC with 3% (v/v) hydrogen peroxide in phosphate buffered saline to quench endogenous peroxidase activity. After washing twice with TRIS/HCl Tween buffer (pH 7.5), the sections were incubated for 30 min at 21^oC with anti-CD3 (Dako) at 1:100, anti-PAX5 (Abcam) at 1:500, anti-IBA-1 (Alpha Lab) at 1:1500 and anti-p21 (Abcam) at 1:500 dilution. Sections stained with isotype control antibodies were used as negative controls. The sections were washed with Tris/HCl Tween buffer (pH 7.5) to remove the excess primary antibody, then incubated with EnVision+ system HRP Labelled Polymer anti-rabbit secondary antibody (Dako) for 30 min at 21^oC. The sections were washed with Tris/HCl Tween buffer pH 7.5 to remove excess labelled polymer from the sections, followed by two 5 min incubations with 3,3'-diaminobenzidine (DAB) substrate-chromogen (EnVision+ System, Dako) and two 5 min rinses with distilled water. Tissues were counterstained using Gill’s haematoxylin and mounted using DPX mounting media (Cellpath) and coverslips for long-term storage.

The tissue sections were assessed by a veterinary pathologist (C.S.) blinded to the genotype of the mice in the different cohorts. Pathological changes in H&E-stained sections of the skin, liver, lungs and thymus were scored using a non-linear semi-quantitative grading system from 0 to 5, where 0 = no significant change and 5 = whole organ or tissue affected. The frequency of IHC stained cells in the tissue were scored using a non-linear semi-quantitative grading system -, +, ++, +++) (17). Photomicrographs were captured using Nanozoomer software from whole slide image scans prepared using Hamamatsu Nanozoomer HT slide scanner. For preparation of the figures, images were obtained and processed using an Olympus CellSens Standard and resized using Adobe Photoshop.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed using the Click-iT™ Plus TUNEL Assay for In Situ detection of Apoptosis using Alexa Fluor™ 488 dye (ThermoFisher) according to the manufacturer’s instructions. 15 images per mouse were acquired using a Zeiss 710 Xenon microscope and analyzed using Volocity 3D Image Analysis Software. TUNEL positive cells were quantified as the percentage of the total number of cells (DAPI-positive).