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. 2021 Jul 30;22(15):8234. doi: 10.3390/ijms22158234

Figure 4.

Figure 4

Inflammation potentiates migration of unstimulated T cells in response to CXCL12. (A) Barrier integrity of HMEC-1 vessels is affected by TNFα pretreatment. Apparent permeability (Papp) values are normalized against Papp values of mono-cultured HMEC-1 vessels. Shown are mean ± SD, and data points represent individual chips (N = 6–13, n = 2–7). Statistical analysis was performed on non-normalized data. Data were log transformed and analyzed using mixed-effects models indicating significant differences between chips containing HMEC-1 vessels and empty chips (**** p < 0.0001) and no significant difference between mono-cultured HMEC-1 vessels and vessels co-cultured with either unstimulated T cells (U) or stimulated T cells (S). Effect of inflammation on barrier function was assessed by analyzing transformed data using Kruskall–Wallis tests showing a significant effect of TNFα pretreatment at 0 h (bold grey, **** p < 0.0001) and no (S) or a reduced effect (U) at 48 h after co-culture with respectively stimulated and unstimulated T cells (bold black, ns = p > 0.05 and * p = 0.0360 respectively). (B) Expression of Intercellular Adhesion Molecule 1 (ICAM-1) per HMEC-1 nucleus of mono-cultured vessels and vessels co-cultured with either unstimulated or stimulated T cells in response to TNFα pretreatment. Shown are mean ± SD, and data points represent individual chips (N = 3, n = 2–6). Data were normalized against mono-cultured, non-treated HMEC-1 vessels, and dose-responses were analyzed after log transformation using Brown–Forsythe and Welch ANOVA tests, showing a significant concentration-dependent effect of TNFα pretreatment on ICAM-1 expression for mono-cultured vessels (*** p = 0.0001) and vessels co-cultured with unstimulated T cells (** p = 0.0019). Differences between mono-culture and co-cultures were analyzed using Two-way ANOVA tests after log transformation, showing a significantly different response to TNFα pretreatment between the mono-cultured vessels and vessels co-cultured with unstimulated T cells (* p = 0.0120) and stimulated T cells (**** p < 0.0001) as well as between both co-cultures (**** p < 0.0001). (C) Quantification of T cell numbers in the ECM compartment after 48 h of co-culture in response to TNFα pretreatment in the presence or absence of CXCL12. Shown are mean ± SD, and data points represent individual chips (N = 2–7, n = 3–5). Data were analyzed using One-way and Two-way ANOVA tests after log transformation, showing a significant effect of CXCL12 on the migration of T cells after TNFα pretreatment for both unstimulated (blue) and stimulated (red) T cells (**** p < 0.0001) as well as a TNFα effect on migration of unstimulated T cells in the presence of CXCL12 (*** p = 0.0008). ns = not significant.