Sahil Gupta a,✉, Arpit Jain b, Shahzad Nawaz Syed a, Ryan G Snodgrass a, Beatrice Pflüger-Müller c,d, Matthias S Leisegang c,d, Andreas Weigert a, Ralf P Brandes c,d, Ingo Ebersberger b,e, Bernhard Brüne a,f, Dmitry Namgaladze
Posted: July 30, 2018
Abstract
Macrophages in the tumor microenvironment respond to complex cytokine signals. How these responses shape the phenotype of tumor-associated macrophages (TAMs) is incompletely understood. Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with pro-tumorigenic properties.
NeoBiotechnologies’ products were used in this study:
Differentiated macrophages were fixed in 1% paraformaldehyde, quenched with 0.125M glycine and washed in PBS. Cells were lysed in buffer I (20mM Tris-HCl pH 8.0, 85mM KCl, 0.5% NP-40) to release cytosolic proteins and debris and the nuclear pellet was lysed in 200µl nuclei lysis buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) and sonified with Branson Sonifier. Soluble chromatin was diluted with dilution buffer (0.01% SDS, 1.1% Triton X 100, 1.1mM EDTA, 20mM Tris-HCl pH 8.0, 167mM NaCl). The lysate was pre-cleared with sepharose CL-4B beads for 1h and 1% of input was stored at 4°C. The rest of soluble chromatin was pulled down overnight at 4°C using following primary antibodies: STAT6 (M-20, sc-981), STAT3 (C-20, sc-482) (both Santa-Cruz), BATF (m14-108, CDI/Neobiotechnologies), IgG (abcam, ab2410). Protein A/G beads were used to precipitate antibody-protein complexes for 2h at 4°C. The beads were washed once with low salt buffer (0.1% SDS, 1% Triton-X100, 2mM EDTA, 20mM Tris-HCl pH 7.4, 150mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton-X100, 2mM EDTA, 20mM Tris-HCl pH 7.4, 500mM NaCl) and twice with LiCl buffer (250mM LiCl, 10mM Tris-HCl, pH7.4, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA) at 4°C and twice with TE-buffer at room temperature. The beads were then eluted in 200µl of elution buffer (100mM NaHCO3, 1% SDS) at 55°C. The eluate was reverse crosslinked with RNAse and proteinase K at 65°C for 4h. The decrosslinked DNA was then purified using Qiagen Ampure purification kit and eluted in 80µl of elution buffer. The BATF ChIP was performed according to the company’s protocol using BATF-antibody coupled to Dynabeads and magnetic isolation (available upon request).
Keywords: primary human monocyte derived macrophages (hMDMs), interleukins, RNA sequencing, signal transducer and activator of transcription (STAT), basic leucine zipper ATF-like transcription factor (BATF), CRISPR interference (CRISPRi)
Publication History:
Oncoimmunology. 2018 Jul 30;7(10):e1494110. doi: 10.1080/2162402X.2018.1494110
