Fig 2: CXCL1 is controlled by MyD88 in lung epithelial cells and prolongs survival in MyD88(−/−) mice following A. fumigatus challenge.(A) BALF (B) lung neutrophil recruitment in WT → WT (black bars), MyD88(−/−) → WT (dark grey bars), WT → MyD88(−/−) (light grey bars), and MyD88(−/−) → MyD88(−/−) (white bars) BM chimeric mice 10 h p.i. with 3 × 107 conidia. Data are expressed as the fold change when compared to the WT → WT group and were pooled from 3 experiments with 12–15 mice per group. (C) BALF and (D) lung neutrophil recruitment in IL1R(−/−) → WT (black circles), and WT → IL1R(−/−) (white circles) BM chimeric mice 10 h p.i. with 3 × 107 conidia. Data are expressed as mean (±SEM) and are from an experiment with 9 mice per group. (E-H) Mean (+SEM) BALF (E) neutrophil recruitment, (F) CXCL1, (G) CXCL2 and (H) CXCL5 levels, in MyD88(−/−) CC10-MyD88 (CC10-MyD88+; black bars) and in MyD88(−/−) transgene-negative littermate controls (CC10-MyD88−; grey bars) 10 h p.i. with 3 × 107 conidia. Data were pooled from 2 experiments and include 7–9 mice per genotype. (I) Kaplan-Meier survival plot of MyD88(−/−) mice challenged with 6–7 × 107 conidia and treated 4 h p.i. with 50 ng rCXCL1 (white circles, n = 11), or PBS vehicle (grey circles, n = 12). Data were pooled from 2 experiments (p = 0.026, Gehan-Breslow-Wilcoxon test).

Fig 3: CARD9-dependent induction of ELR+ chemokines in vitro and in vivo.(A) The plots show mean (+SEM) CXCL1 and CXCL2 secretion by WT (black bars) or CARD9(−/−) (light grey bars) BMMs following stimulation with A. fumigatus germlings (MOI = 1) as measured by ELISA. Data are from 4–5 replicates per condition from a representative experiment. (B) Strategy to generate CXCL2 reporter mouse. The graph shows CXCL2 (black lines) and mean GFP fluorescence (green lines) in transgene-positive (circle) and transgene-negative (non-Tg, square) littermates that were administered indicated amounts of Pam3Cys4 i.p. (C) The plots show neutrophils (left panel), inflammatory monocytes (middle panel) and CD11b+ DCs (right panel) that were isolated from CXCL2-GFP transgenic mice (upper panel) and non-transgenic littermates (lower panel) and analyzed for GFP expression. Representative data from 2 experiments is shown. Mice were administered 3 × 107 conidia and lung cell suspensions were analyzed 36 h p.i. (D) The graphs show mean number (+SEM) of GFP+ lung neutrophils, inflammatory monocytes, or CD11b+ DCs from Tg+ CARD9(+/+) (black bars), Tg+ CARD9(−/−) (grey bars), Tg− CARD9(+/+) (black crosshatched bars) and Tg− CARD9(−/−) mice (grey crosshatched bars).

Fig 4: TG2-dependent chemokine production in keratinocytes is critical for IL-17-producing CCR6+ γδT-cell and neutrophil dermal infiltration.a, b Effect of DMSO- or IMQ-treated WT- or TG2−/−-CM on CCR6+ γδT-cell (a) and CD11b+ Ly-6G+ cell (b) migration according to Transwell migration assays. Flow cytometric analysis of the percentage of cells migrating toward the CM in the presence or absence of anti-CXCL1 (a) and anti-CCL20 (b) antibodies (n = 3/group). c, d WT and TG2−/− mouse-derived CCR6+ γδT-cell (c) and CD11b+ Ly-6G+ cell (d) migration in response to recombinant mouse CCL20 and CXCL1, respectively. Data represent the mean ± SEM (n = 3/group). *p < 0.05; **p < 0.01. e, f Cells were isolated from the back skin of WT and TG2−/− mice treated with IMQ for four consecutive days and analyzed by flow cytometry. Representative flow cytometric profiles of CCR6+ γδTCRlow cells (e) and CD11bhigh Ly-6G+ cells (f). The percentage of cells is shown. Data represent the mean ± SEM (n = 9/group). g Skin sections were immunostained with CCR6 (green)- and IL-17 (red)-specific antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. h–j Back skin was separated into epidermis and dermis and analyzed by RT-PCR for IL-17-producing γδT-cell markers (h) (Ccr6 and Il23r), IL17 and related cytokines (i) (Il17a, Il17f, and Il22), and myeloid cell-derived cytokines (j) (Il12a and Il23a). Data represent the mean ± SEM (n = 11/group). *p < 0.05; **p < 0.01 vs. WT mice.

Fig 5: TG2 mediates IMQ-induced IL-6, CXCL8, and CCL20 expression in keratinocytes.a WT and TG2−/− mice (n = 11/group) were treated with IMQ for four consecutive days. Cytokine and chemokine mRNA levels in the back skin epidermis were measured by RT-PCR. b, c Primary keratinocytes prepared from WT and TG2−/− mice were treated with IMQ (200 μM) or Aldara cream (25 μM) for 3 h. Ccl20, Il6, and Cxcl1 mRNA (b) and protein levels in the media (c) were measured by RT-PCR and a multiplex cytometric bead array, respectively (n = 3/group). d, e HaCaT cells stably transfected with control and TG2 shRNA were treated with IMQ (200 μM) or Aldara cream (25 μM). IL6, CXCL8, and CCL20 mRNA (d) and protein levels in the media (e) were measured by RT-PCR after 3 h and a multiplex cytometric bead array after 6 h, respectively. Protein levels were normalized to total cellular soluble protein (n = 3/group). f Human primary keratinocytes stably transfected with control or TG2 shRNA were treated with IMQ (200 μM; n = 3). IL6, CXCL8, and CCL20 mRNA levels were measured by RT-PCR after 3 h. Data represent the mean ± SEM (n = 3/group). *p < 0.05; **p < 0.01.