Fig 2: CRAE induced to the M1-type macrophage polarization and the M1-type polarization markers were inhibited by the TLR4 protein inhibitors TAK-242 and NF-?B inhibitor Bay 11–7085 (Bay). (A) Protein expression levels of M1 macrophages markers and M2 macrophages markers were analyzed and semi-quantified through WB. (B) The corresponding expression of CD163, iNOS, CD86 and IL-1ß levels are shown as histograms (one-way ANOVA or two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (C) The expression level and localization of p65 protein in macrophages were detected by immunofluorescence assay. Magnification, ×400.

Fig 3: Phenotypic characterization (A) and effect on the levels of the co-stimulatory proteins CD54 and CD86 (B) of HRW and EO from E. globulus leaves in THP-1 monocytes. The cells were treated in the absence or presence of 0.16 mg/mL EO or 0.8 µg/mL HRW for 24 h. In addition, skin sensitization was induced with 8 µM 1-fluoro-2,4-dinitrobenzene (DNFB) for 24 h. The cells treated with the medium alone were used as the CTRL. The blue line represents unstained cells, and the red line represents the specific levels of the tested cells. The levels of CD54 and CD86 were measured by flow cytometry. The results—expressed as a percentage (%) of the fluorescence intensity (RFI) relative to the CTRL—represent the mean ± SEM of at least three independent experiments. The statistical analysis was performed by one-way ANOVA, followed by Dunnett’s and Sidak’s multiple comparison tests. * p < 0.05: significantly different compared to the CTRL.

Fig 4: Uptake of CNPs promotes a pro-inflammatory phenotype of DCs.Human and murine DCs (DC2.4) were either left untreated or incubated with 100 µg/ml empty (90/10) or SIINFEKL-loaded 90/10-CNPs for 5, 24 and 48 hours. A) Flow cytometric analysis of CD80, CD86, HLA-DR and PD-L1 cell surface levels in human DCs. Median fluorescence intensity (MFI) ratio was calculated by normalizing MFI of each surface marker to the MFI of its respective isotype control. Then, MFI ratio of CNP treated cells was normalized to untreated cells and expressed as n-fold MFI of untreated cells. B+C) Relative mRNA levels of TGF-ß1, IL-10, IL-1ß, IL-6 and TNF-a in B) primary human DCs and C) murine DC2.4 cells was determined by RT-qPCR. Expression levels were normalized to expression of the housekeeping gene TBP/GAPDH and normalized to values determined for untreated cells. Data are presented as mean + SEM or median with 75th and 25th percentile of three independent experiments. *p < 0.05.

Fig 5: 341G2 h2 Potently Activates Dendritic Cells(A) hCD40Tg and hCD40Tg/Fc?Rnull mice received 30 µg anti-CD40 mAbs intravenously and spleens were harvested on day 2. Expression levels of CD80 and CD86 on splenic CD11c+CD8+DEC205+ DCs were analyzed by flow cytometry. Histograms representative of six to seven mice from two experiments.(B) Experiments same as in (A). MFI values for CD80 and CD86 are quantified, and the frequency of CD11c+CD8+DEC205+ DC expressed as the percentage of CD11c+ cells. Means ± SEM, n = 6–7, data pooled from two experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.(C) Human monocyte-derived DCs were stimulated with various anti-CD40 mAbs, CD40L or LPS as indicated for 48 h and then stained for CD86, CD70, and CD80. The MFI values for each marker in response to treatments were normalized against the MFI values corresponding to the unstimulated control. Means ± SEM, data pooled from six donors. Two-tailed, paired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001.(D) Human monocyte-derived DCs pre-treated with anti-CD40 mAbs or LPS for 24 h were washed and then co-cultured with allogeneic CD4+ T cells at different ratios as indicated for 5 days. CD4+ T cell proliferation was measured by 3H-thymidine incorporation. Means ± SEM, n = 3, data representative of three experiments involving multiple donors.See also Figure S3.