Fig. 7: IL-10 loop controls DC communication with keratinocytes, neutrophils, and pDCs.

a RT-PCR analysis of the expression of TNF and IL-1b mRNA in HaCat cells incubated with medium, LPS or with supernatant (diluted 1:10) of the indicated DCs for 4 h. Blocking antibodies for the cytokines IL-19, IL-36g, OSM and TNF were added to LPS + aIL-10R-DC supernatant for 1-h incubation before culturing with HaCat cells. Data are represented as mean values ± SEM, n = 4 or n = 8 biologically independent samples depending on the conditions, *p < 0.05 (two-sided paired t-test without correction). b, c Expression of maturation markers CD86, HLA-DR, and ICOSL (b) or CD11b and CD62L (c) analyzed by flow cytometry with surface staining on pDCs (n = 18 or n = 6 biologically independent samples depending on the conditions) cultured with supernatant (diluted 1:10) of the indicated DC for 24 h (b) and neutrophils (n = 9 or n = 6 biologically independent samples depending on the conditions) (c) cultured with supernatant (diluted 1:100) of the indicated DC for 1 h. Blocking antibodies for the cytokines GCSF, GM-CSF, TNF, and IL-12 (for pDC) or IL-6 (neutrophils) were added to LPS + aIL-10R-DC supernatant for 1 h incubation before culture. Each biological replicate comprised independent DC donor paired to independent pDCs/neutrophils donor. Data are represented as mean values ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided paired t-test without correction). d For each target cell, we reduced the different activation markers to a single parameter normalized between 0 (Ø) and 1 (max) in the rectangles. The value 0 corresponds to the activation level induced by supernatants from untreated DC, while 1 corresponds to the maximum activation level from all the observed conditions. These experimentally validated activation scores were in qualitative agreement with the ICELLNET communication scores between DC and the target cells, represented by the width of the edges. Some schematic art pieces were used and modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License. http://smart.servier.com/ (a, b, c).