DOI: 10.5061/dryad.ns1rn8pv2
title: Why are the phenotypes of TRAF6 knock-in and TRAF6 knock-out mice so different?

description: 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 CD44high/CD62Llow expression on the cell surface. In
 contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh 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.
description: <strong>Preparation
 and histopathological analysis of tissues.</strong> Mice were euthanized with
 increasing concentration of CO<sub>2</sub>. 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<sup>o</sup>C and
 then rinsed in Tris/HCl Tween buffer (pH 7.5). The sections were treated
 for 5 min at 21<sup>o</sup>C 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<sup>o</sup>C 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<sup>o</sup>C. 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).