DOI: 10.5061/dryad.zkh1893k1
title: Mitochondrial respiratory complex III sustains IL-10 production in activated macrophages and promotes tumor-mediated immune evasion

description: The cytokine interleukin (IL)-10 limits the immune response and promotes
 resolution of acute and chronic inflammation. Because of its
 immunosuppressive effects, IL-10 upregulation is a common feature of tumor
 progression and metastasis. Most recently, IL-10 regulation has been shown
 to depend on mitochondria and redox-sensitive signals. We have found that
 Suppressor of site IIIQo Electron Leak (S3QEL) 1.2, a specific inhibitor
 of reactive oxygen species (ROS) production from mitochondrial Complex
 III, and Myxothiazol, a Complex III inhibitor, decrease IL-10 production
 from Lipopolysaccharide (LPS) activated macrophages. IL-10 downregulation
 is likely to be mediated by the suppression of c-Fos, which is a subunit
 of Activator Protein (AP)-1, a transcription factor required for IL-10
 gene expression. S3QEL 1.2 impairs IL-10 production in vivo after LPS
 challenge and promotes the survival of mice bearing B16F10 melanoma, by
 lowering tumor growth. Our data identify a link between Complex
 III-dependent ROS generation and IL-10 production in macrophages, the
 specific targeting of which could have potential in the effort to boost
 anti-tumor immunity.
description: This dataset was collected after performing experiments on
 primary cultures of murine bone marrow-derived macrophages (BMDMs), murine
 B16F10 melanoma cells, human macrophages differentiated from peripheral
 blood mononuclear cells (PBMCs) and from <em>in
 vivo </em>experiments on mice.
description: # Mitochondrial respiratory complex III sustains IL-10 production in
 activated macrophages and promotes tumor-mediated immune evasion
 [https://doi.org/10.5061/dryad.zkh1893k1](https://doi.org/10.5061/dryad.zkh1893k1) ## Description of the data and file structure Fig 1: Analysis of ROS production by Amplex Red assay (described in M&M) in Bone marrow-derived macrophages (BMDMs) pre-treated with DMSO, Myxothiazol, S3QEL 1.2 and MitoTEMPO and stimulated with LPS. Analysis of Maximal Respiration, Spare Respiratory Capacity and Complex III activity assessed by Seahorse technology in BMDMs pre-treated with DMSO, Myxothiazol, S3QEL 1.2, and stimulated with LPS. All values are from 3 independent experiments (biological replicate, indicated as 'replicate' in the Excel file on Dryad). Fig 2: p values and counts of genes differentially expressed (downregulated) after S3QEL 1.2 and MYX pre-treated and LPS stimulated BMDMs, as obtained from bulk RNA-seq analysis. Data are from 3 mice from 1 independent experiment. Fig 3: RT-qPCR (Fold change) and ELISA (pg/mL) experiments, analysing IL-10 expression in BMDMs pre-treated with DMSO, S3QEL 1.2 or Myxothiazol and primed with LPS. 2-3 mice were tested on 3 different weeks (3 independent times). Fig 4: Quantitative data regarding phospho-c-Fos signal intensity in the nucleus, performed by confocal microscopy (and analysed and obtained with Imaris Software). The experiment shown in Fig.4 is representative of different independent experiments. Fig 5: Geometric mean of fluorescent intensity (MFI) for IL-10 positive immune cells as performed by flow cytometry staining and analysis (see M&M and supplementary figure S8).  Cell Lines: BMDMs. Other samples: individual mice from in vivo experiments. **Description of the data and file structure** Data in this upload are organized based on the main figures they appear in, in the publication: Mitochondrial respiratory complex III sustains IL-10 production in activated macrophages and promotes tumor-mediated immune evasion. ### Files and variables #### File: S3QL\_heatmap.R **Description:** RNA-seq analysis of genes downregulated by S3QEL 1.2 and MYX in LPS-activated macrophages. #### File: Figure\_4\_raw\_data.xlsx **Description:** Analysis of AP-1 presence in the nucleus of macrophages pre-treated with S3QEL 1.2 and MYX and stimulated with LPS. ##### Variables * phospho-c-fos signal intensity #### File: geo\_mean\_IL-10\_calculations\_Alessia\_S3QEL\_raw\_data\_figure\_5.xls **Description:** Analysis of IL-10 MFI in immune cells isolated from tumors of mice treated with PBS or S3QEL 1.2. ##### Variables * IL-10 MFI #### File: Figure\_3\_raw\_data.xlsx **Description:**  Analysis of IL-10 expression after Complex III inhibition by S3QEL 1.2 and MYX and LPS priming. ##### Variables * IL-10 mRNA and concentration of released protein. #### File: Figure\_1\_raw\_data.xlsx **Description:** Analysis of ROS production, respiration and Complex III activity in macrophages after S3QEL 1.2 and MYX treatment and LPS stimulation. ##### Variables * Fluorescence intensity and Oxygen Consumption Rate (OCR) ## Code/software To visualize and analyse the data contained in this data repository include GraphPad Prism v10.2.2, FlowJo v10.10 and Imaris v10.2. ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A