TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control T_H2-Mediated Immunopathology

<div><p>Persistent T_H2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of T_H2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (<i>Map3k8</i>; Cot) regulated T_H2-mediated intestinal, hepatic and pulmonary immunopathology following <i>Schistosoma mansoni</i> infection or <i>S</i>. <i>mansoni</i> egg injection. Elevated inflammation, T_H2 cell responses and exacerbated fibrosis in <i>Map3k8</i>^–/–mice was observed in mice with myeloid cell-specific (LysM) deletion of <i>Map3k8</i>, but not CD4 cell-specific deletion of <i>Map3k8</i>, indicating that TPL-2 regulated myeloid cell function to limit T_H2-mediated immunopathology. Transcriptional and metabolic assays of <i>Map3k8</i>^–/–M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated T_H2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing T_H2 cell expansion and downstream immunopathology and fibrosis.</p></div

TPL-2 regulates lipolysis in M2 macrophages and regulation of T_H2 cell differentiation and proliferation.

<p>Bone marrow-derived macrophages (BMDM) were stimulated with IL-4 and IL-13 for 24 hours. A) Analysis of genes involved in lipid metabolism was performed by Ingenuity pathways analysis (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005783#ppat.1005783.s007" target="_blank">S1 Table</a>) with <i>Map3k8</i>-dependent genes depicted in a heat map. B) After 24hrs of stimulation with IL-4 and IL-13 oxygen consumption rates (OCR) were determined in M2 macrophages using an XF-96 Extracellular Flux Analyzer (EFA) during sequential treatments with oligomycin, FCCP, and rotenone/antimycin. Spare respiratory capacity (SRC), the quantitative difference between maximal uncontrolled OCR, and the initial basal OCR, is depicted in the plot. C) Basal oxygen consumption rates (OCR) and spare respiratory capacity (SRC) in WT and <i>Map3k8</i>^{<i>–/–</i>}M2 macrophages. D) WT or Map3k8-deficent bone marrow-derived macrophages (BMDM) were generated from 3 individual mice and co-cultured with a pool of cell trace Violet (CTV)-labelled naïve OTII CD4⁺CD44⁻<i>Il4</i>^{<i>gfp</i>–}T cells and stimulated with IL-4 and IL-13 for 3 days. Th2 cell differentiation (<i>Il4</i>^<i>gfp</i> expression) and proliferation (CTV dilution) was determined by FACS after 3 days. In some wells BMDM were pre-treated with Orlistat for 6 hours and washed, prior to co-culture. Data is representative of 2–3 independent experiments with a minimum of 3 biological replicates per experiment. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

TPL-2 is required for M2 activation of Macrophages, <i>in vitro</i>.

<p>A-D) Bone marrow-derived macrophages (BMDM) were stimulated with IL-4 and IL-13 for 6 or 24 hours, as indicated. Cells were harvested, RNA extracted and gene expression was determined by qRT—PCR and expressed relative to un-stimulated genotype control cells. E) BMDM’s were stimulated for 1.5, 3 and 6 hours, as indicated. Total Protein was extracted with phosphorylated and total protein levels of STAT6, ERK, p38a, JNK and α-tubulin determined by western blot. All experiments are representative of 2–3 independent experiments with 3–5 biological replicates and 3 technical replicates in each experiment. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

TPL-2 regulates pro-fibrotic and immuno-regulatory pathways in M2 macrophages.

<p>Bone marrow-derived macrophages (BMDM) were stimulated with IL-4 and IL-13 for 24 hours. Cells were harvested, RNA extracted and genome-wide transcriptional expression was determined by microarray analysis using 3 biological replicates. A) Heat map of differentially regulated genes in un-stimulated and IL-4+IL-13 stimulated cells. B) Ingenuity pathways analysis of transcriptional profiles of differentially regulated genes. C-F) Venn diagram and bar graphs of TPL-2 dependent (1), common (2) and TPL-2-regulated genes (3). G and H) Ratio of Ratios graph (top) and bar graphs (H) showing increased pro-fibrotic (red) and decreased Immunoregulatory (blue) genes in <i>Map3k8</i>^{<i>–/–</i>}macrophages, relative to WT macrophages (x-axis) and un-stimulated macrophages (y-axis).</p

T cell-intrinsic <i>Map3k8</i> does not contribute to exacerbated inflammation and pathology following <i>S</i>. <i>mansoni</i> infection.

<p><i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>+/+</i> and <i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/fl</i> mice were infected percutenously with 50 <i>S</i>. <i>mansoni</i> cercariae and analysed at 8 weeks post-infection. A–C) Perfused tissue was fixed and embedded in paraffin before sectioning and staining with Masson’s trichrome. B) Granuloma size was determined from 10–20 individual granulomas per sample measured using Image J. D) Expression of <i>Col3</i> and <i>Col6</i> was determined from RNA extracted from liver or small intestinal tissue. Data is expressed relative to HPRT and shown as a fold-change relative to uninfected mice. E) Mesenteric lymph node cells were re-stimulated with anti-CD3 for 3 days. Cytokines were measured in supernatants, by ELISA. F) Naive T cells (CD4⁺CD44⁻CD25⁻CD62L⁺) were FACS purified from WT and <i>Map3k8</i>^{<i>–/–</i>}mice and cultured under T_H1 and T_H2 conditions. Frequencies of CD44⁺IFNγ⁺ and CD44⁺IL-4⁺ cells were determined by intracellular FACS analysis on day 7. All experiments are representative of 2–3 independent experiments with 5–10 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

TPL-2 is required for M2 activation of Macrophages, <i>in vivo</i>.

<p>WT C57BL/6 mice were lethally irradiated (900rad) and reconstituted with 50% CD45.1+ WT bone marrow and 50% CD45.2+ <i>Map3k8</i>^–/–bone marrow and left for 6–8 weeks, prior to infection with 50 <i>S</i>. <i>mansoni</i> cercariae. A) After 8 weeks of infection, mice were sacrificed and CD3⁻CD19⁻CD11b⁺F4/80⁺ Macrophages were FACS-sorted. B-C) Expression of <i>Arg1</i>, <i>Relma</i>, <i>Chi3l3</i>, <i>Col1a1</i>, <i>Col3a1</i> and <i>Ctgf</i> was determined from RNA extracted from purified macrophages. Data is expressed relative to HPRT and presented as a fold-change relative in genotype-controlled naïve bone marrow derived macrophages. Experiments are representative of 2 independent experiments with 5 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

<i>Map3k8</i>^–/–mice develop increased hepatic and intestinal inflammation and fibrosis following <i>S</i>. <i>mansoni</i> infection.

<p>WT and <i>Map3k8</i>^{<i>–/–</i>}mice were infected percutenously with 50 <i>S</i>. <i>mansoni</i> cercariae and analysed at 8 weeks post-infection. A & C) Perfused tissue was fixed and embedded in paraffin before sectioning and staining with Masson’s trichrome. B) Granuloma size was determined from 10–20 individual granulomas per sample measured using Image J. Scale bars are 1000μm (top), 200μm (middle) and 100μm (bottom). D) Intestinal pathology score, as described in methods. E) Expression of <i>Col3</i> and <i>Col6</i> was determined from RNA extracted from liver or small intestinal tissue. Data is expressed relative to HPRT. F) Hydroxyproline was quantified in liver tissue from naïve and infected animals. G) Frequency of T_REG (CD4⁺CD25⁺<i>Foxp3</i>^<i>RFP</i>+) and T_H2 (CD4⁺CD44⁺<i>Il4</i>^<i>GFP</i>+) cells in the spleen, mesenteric lymph nodes (MLN) and liver were determined by FACS. All experiments are representative of 2–3 independent experiments with 5–10 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p