Fig 2: Validation and characterization of the two clones exhibiting large deletions in the TNFAIP6 gene.TSG-6+/+ and TSG-6–/– (a and b) RHE stimulated or not stimulated for 48 hours with IL-4 and IL-13. (a) HE staining and immunofluorescence (in green) of K10, IVL, LOR, and FLG. Bar = 20 µm. (b) TSG-6-encoding mRNA expression of TSG-6+/+ and TSG-6–/– RHE assessed by RT-qPCR. The reference gene is RPLP0 (mean ± SD; n = 3; two-way ANOVA; **P < 0.01; ***P < 0.001 vs. TSG-6+/+). (c) TSG-6-encoding mRNA detection using in situ hybridization and hemalun counterstaining of TSG-6+/+ and TSG-6–/– RHE. Bar = 20 µm. (d) TSG-6 protein concentration of TSG-6+/+ and TSG-6–/– RHE assayed using ELISA (mean ± SD; n = 3; two-way ANOVA; ***P < 0.001 vs. TSG-6+/+). CTL, control; HE, hemalun/eosin; IVL, involucrin; K, keratin; LOR, loricrin; RHE, reconstructed human epidermis.

Fig 3: Lack of TSG-6 slows down KCs migration. Monolayers cultures of TSG-6+/+ or TSG-6–/– (a and b) N/TERT KCs at confluence stage treated with IL-4 and IL-13 for 24 h. KCs were growth arrested by mitomycin C before performing a scratch. KCs migration was monitored for 24 h in the presence of IL-4 and IL-13. (a) Scratch closure was analyzed every 6 h by Cytonote and was quantified by ImageJ software (mean ± SD; n = 5; two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001 vs. TSG-6+/+). (b) Recombinant human TSG-6 protein (50 ng/ml) was added in the culture medium just after scratching monolayers cultures of TSG-6–/– (a) and (b) cells (mean ± SD; n = 3 for TSG-6–/– (a) and n = 4 for TSG-6–/– (b); two-way ANOVA; *P < 0.05; **P < 0.01 vs. 0 ng/ml rhTSG-6). Scratch closure was monitored by Cytonote and quantified by ImageJ software. h, hour; KC, keratinocyte; rhTSG-6, recombinant human TSG-6.

Fig 4: Analytes in the synovial fluid (SF) and blood of participants with knee injury in the Knee Injury Cohort at the Kennedy (KICK) and healthy controls. A, Flow chart indicating the schedule of study visits in the KICK cohort over 3 months and timing of clinically indicated surgical treatment. (Surgical interventions are detailed in Supplementary Table 1, http://onlinelibrary.wiley.com/doi/10.1002/art.39677/abstract.) The numbers of individuals for whom Knee Injury and Osteoarthritis Outcome Score (KOOS) data were available (at baseline and 3 months) and from whom blood and SF samples were obtained are shown. To be eligible for the study, participants had to have sustained 1 or more of the following injuries within 8 weeks of the baseline visit: meniscal tear, cruciate ligament rupture, colateral ligament tear, posterolateral corner injury, traumatic chondral defects, articular or periarticular fracture, or patellofemoral or tibiofemoral dislocation. B, Baseline levels of markers of interest in SF and matched blood samples, either plasma (for interleukin-6 [IL-6], monocyte chemotactic protein 1 [MCP-1], and activin A) or serum (all other markers), from KICK participants with knee injury and healthy, age- and sex-matched controls. All samples were centrifuged to remove cells. Supernatants were measured in duplicate by electrochemiluminescence or enzyme-linked immunosorbent assay (activin A only). Values were obtained in all participants for all SF analytes except for IL-6 and tumor necrosis factor–stimulated gene 6 (TSG-6). IL-6 levels were below the lower limit of quantitation in 12 of 136 samples (8.8%), and TSG-6 levels were below the lower limit of quantitation in 8 of 135 samples (6%). Measurements for each of 6 analytes are shown, plotted on a log10 y-axis. Symbols represent individual samples; horizontal and vertical lines show the median and interquartile range. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001, by Mann-Whitney U test. MMP-3 = matrix metalloproteinase 3; TIMP-1 = tissue inhibitor of metalloproteinases 1. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39677/abstract.

Fig 5: Increase of TSG-6-encoding mRNA in lesional AD skin. (a) TSG-6 and PPIB (CTL+) mRNA detection using in situ hybridization and hemalun counterstaining of healthy and lesional AD skin. Bar = 20 µm. (b) TSG-6-encoding mRNA expression in normal and lesional AD skin assessed by RT-qPCR. The reference gene is RPLP0 (mean ± SD; n = 5 for normal skin and n = 10 for AD skin; unpaired t-test with Welch’s correction). (c) TSG-6-encoding mRNA expression in normal, nonlesional AD, and lesional AD skins analyzed from GEO Profiles database (accession GSE5667; GPL96; 206026_s_at) (mean ± SD; n = 5 or 6; one-way ANOVA). (d) UMAP visualization of undifferentiated and differentiated KC in healthy and lesional AD skins found in single-cell RNA-seq dataset (https://developmentcellatlas.ncl.ac.uk/datasets/ hca_skin_portal) (n = 5). Normalized gene expressions of K14, K10, CA2, NELL2, and TNFAIP6 are represented in green for a low expression and in red for a strong expression. AD, atopic dermatitis; CTL, control; GEO, Gene Expression Omnibus; K, keratin; KC, keratinocyte; RNA-seq, RNA sequencing; UMAP, Uniform Manifold Approximation and Projection.