Polarization dictates the macrophage iron phenotype.

<p>(A) mRNA expression of iron regulated genes FPN, IRP2, TfR, HAMP, CP, and FTL was quantified in primary human macrophages using qRT-PCR. Results were normalized to the unstimulated control. Protein expression of FPN (B) and FTL (C) was determined in macrophages by FACS analysis. (D) Iron content in macrophage supernatants was quantified by AAS measurement. (E) MCF-7 breast cancer cells were stimulated with macrophage conditioned media (CM) and proliferation was analyzed by qRT-PCR of (E) PCNA, Ki-67, (F) Western analysis of PCNA, and (G) xCELLigence. Data are means ± S.D.M, n>6, *p<0.05, **p<0.01, ***p<0.001 vs. control/CM_control.</p

Chelator treatment induces a macrophage phenotype shift towards iron-sequestration.

<p>(A) mRNA expression of iron regulated genes FPN, IRP2, TfR, HAMP, CP, and FTL was quantified using qRT-PCR. Protein expression of FPN (B) and FTL (C) was determined by FACS analysis. (D) Iron content in macrophage supernatants was quantified by AAS measurement. Data are shown as means ± S.D.M, n>6, *p<0.05, **p<0.01, ***p<0.001 vs. DMSO.</p

GM-CSF deficiency does not protect mice from IMQPD.

<p>(a) Time course of erythema, scaling, stiffness and total PASI scores in IMQ-treated wild-type (Wt n = 12) and GM-CSF^-/- (n = 13) mice. (b) <i>Csf2</i> mRNA expression relative to actin mRNA in skin of wild-type mice (days 1–3 n = 5; day 6 n = 9) following IMQ treatment. (c) Neutrophil distribution in skin at day 6 after IMQ application by immunofluorescence staining Ly6B.2 (Wt n = 6; GM-CSF^-/- n = 5). Graphs show mean values ± SEM. *p-value<0.05.</p

Hypothesis for the alternative pathogenic pathway of IMQ-induced skin disease in GM-CSF deficient mice.

<p>GM-CSF has a role as a differentiation factor in DC development; GM-CSF inhibits FLT3L-driven pDC production from lineage-negative DC precursor subsets, while promoting conventional DC growth via modulation of a complex network of transcriptional factors. Thus, the upregulation of the transcription factor E2-2, which is induced by FLT3L, is essential for the development and maintenance of a mature pDC phenotype. However, the E2-2-dependent pDC promoting-transcriptional program can be antagonized by GM-CSF via upregulation of the transcription factor ID2 that directs conventional DC development.</p

Cytokine levels in supernatants of GM-CSF-deficient T cells activated by costimulation.

<p>Isolated splenocytes of wild-type (Wt n = 5) and GM-CSF-deficient (GM-CSF^-/- n = 5) mice were activated by CD3 and CD28 stimulation, and concentrations of cytokines in supernatant were measured. IL-22 levels were determined by ELISA and IL-17A levels were measured by cytometric bead array. Bars show mean values ± SEM. *p-value<0.05, compared with control group as determined by the Mann-Whitney-U-Test.</p

GM-CSF deficiency enhances the pDC compartment.

<p>(a) Quantification of pDC in skin draining lymph nodes (LN) and spleen (CD45⁺Siglec-H⁺, CD11cint) in healthy wild-type (Wt n = 4) and GM-CSF^-/- (n = 4) mice without prior IMQ application. Cells were analyzed by flow cytometry and normalized to counting beads. (b) Representative Siglec-H staining (red) in PFA-fixated spleens of Wt and GM-CSF^-/- mice (right). The graph shows mean values ± SEM. *p-value<0.05, **p-value<0.01 compared with control group as determined by the Mann-Whitney-U-Test. Scale bars equal 400 μm.</p

Iron chelator functionality in primary human macrophages.

<p>(A) Time dependent activation of HIF-1α in primary human macrophages was determined by Western blot analysis. DMOG- as well as (TC3-SH)₂-treated cells were compared to DMSO-stimulated control macrophages. (B) Cell viability was measured by AnnexinV/PI staining using FACS measurement. The percentage of living, apoptotic, and necrotic cells is represented. Staurosporine (Sts) was used as a positive control. Data are shown as mean ± S.D.M, n>4, *p<0.05, **p<0.01, ***p<0.001 vs. DMSO.</p

Functional consequences of chelator-dependent reprogramming of the macrophage iron phenotype in breast cancer cells.

<p>As a functional readout, proliferation of macrophage conditioned media (CM)-stimulated MCF-7 cells was measured by qRT-PCR of (A) PCNA and Ki-67, (B) Western analysis of PCNA, as well as using (C) the xCELLigence real-time analysis system. (D) Migration and (E) adhesion to either collagen I or fibronectin matrix of MCF-7 tumor cells upon MΦ-conditioned media treatment. (F) Data are shown as means ± S.D.M, n>3, *p<0.05, **p<0.01, ***p<0.001 vs. CM_DMSO.</p

GM-CSF deficiency does not alter epidermal thickening during IMQPD.

<p>Mice were sacrificed at day 6 after IMQ application. (a) Representative hematoxylin and eosin (HE) staining of healthy (upper panel) and inflamed skin following IMQ application (bottom panel) in wild-type (Wt) and GM-CSF^-/- mice. (b) Quantification of epidermal thickness of HE-stained histological samples (left: Wt n = 5; GM-CSF^-/- -n = 4) and <i>Ki67</i> mRNA expression in skin by qPCR (right: Wt n = 12; GM-CSF^-/- n = 13; each symbol represents the quantified measured mRNA of one mouse). Scale bar as indicated.</p

Prochelation approach and stability in cell culture medium.

<p>(A) Scheme of the redox directed chelation system: The disulfide-based prochelator (TC3-S)₂ undergoes intracellular reduction to produce the active chelator TC3-SH, which readily binds iron forming a stable complex. (B) Stability of prochelator (TC3-S)₂ at 12.0 μM in phenol red-free EMEM: relative concentrations over the course of 24 hours were determined by UV-Visible absorption spectroscopy (λ_max = 318 nm, 37°C). No measureable loss of the prochelator is observed over the course of 2 hours, and less than 10% loss is observed over 24 hours.</p