IL-34- and M-CSF-induced macrophages switch memory T cells into Th17 cells via membrane IL-1α

International audienceMacrophages orchestrate the immune response via the polarization of CD4+ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M-CSF and IL-34 induce the differentiation of monocytes into IL-10high IL-12low immunoregulatory macrophages, which are similar to tumor-associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M-CSF (M-CSF macrophages) or IL-34 (IL-34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4+ T cells. Taken together, our results show that M-CSF-, IL-34 macrophages, and TAMs switch non-Th17 committed memory CD4+ T cells into conventional CCR4+ CCR6+ CD161+ Th17 cells, expressing or not IFN-gamma. Contrary, the pro-inflammatory GM-CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL-1α (mIL-1α), which is constitutively expressed by M-CSF-, IL-34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis

Pancreatic Ductal Adenocarcinoma: A Strong imbalance of Good and Bad immunological Cops in the Tumor Microenvironment

International audiencePancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers with very few available treatments. For many decades, gemcitabine was the only treatment for patients with PDAC. A recent attempt to improve patient survival by combining this chemotherapy with FOLFIRINOX and nab-paclitaxel failed and instead resulted in increased toxicity. Novel therapies are urgently required to improve PDAC patient survival. New treatments in other cancers such as melanoma, non-small-cell lung cancer, and renal cancer have emerged, based on immunotherapy targeting the immune checkpoints cytotoxic T-lymphocyte-associated antigen 4 or programmed death 1 ligand. However, the first clinical trials using such immune checkpoint inhibitors in PDAC have had limited success. Resistance to immunotherapy in PDAC remains unclear but could be due to tissue components (cancer-associated fibroblasts, desmoplasia, hypoxia) and to the imbalance between immunosuppressive and effector immune populations in the tumor microenvironment. In this review, we analyzed the presence of ``good and bad immunological cops'' in PDAC and discussed the significance of changes in their balance

Analysis of chemokine and chemokine receptor mRNA expression by macrophages.

<p><i>A Three-dimensional projection of PCA of IL-34-Mφ, M-CSF-Mφ and GM-CSF-Mφ</i>. Each dot represents one Mφ subset of one donor, stimulated or not for 6 h with LPS, based on values of all 18 studied chemokines/chemokine receptors. The percentage of variances is depicted on the three axes. <i>B,Two-dimensional unsupervised hierarchical clustering analysis of the chemokines/chemokine receptors expression in the Mφ subset</i>. The normalized values for each chemokine/chemokine receptors from four different donors is depicted according to the color scale, where red and green represent expression above and below the mean, respectively.</p

GM-CSF and IFNγ prevent the generation of IL-34-Mφ.

<p><i>A–C, GM-CSF and IFNγ prevent the differentiation of monocytes into immunosuppressive IL-34-Mφ.</i> IL-34-Mφ and GM-CSF-Mφ were generated in the absence or presence of GM-CSF or IFNγ before phenotype analysis. <i>D–F, IFNγ skews monocyte differentiation from IL-34-Mφ into immunostimulatory Mφ.</i> IL-34-Mφ and GM-CSF-Mφ were cultured for 3 days in the absence or presence of GM-CSF or IFNγ before phenotype analysis. The expression of CD14 and CD163 (<i>C</i>&<i>F</i>) was analyzed on non-stimulated cells. The production of IL-10 and IL-12 (<i>A</i>&<i>D</i>) and the expression of CD80 and CD86 (<i>B</i>&<i>E</i>) was analyzed after LPS stimulation. Results are expressed in MFI values, in pg/ml (IL-12) or ng/ml (IL-10), or as a percentage of inhibition (mean ± SD, n = 7), (<i>A</i>,<i>B</i>,<i>D</i>&<i>E</i>), * p<0.05, compared to IL-34-Mφ.</p

IL-34 induces the generation of immunosuppressive M2 independently of M-CSF.

<p><i>A</i>&<i>B, IL-34-Mφ are immunosuppressive.</i> LPS-stimulated IL-34-Mφ, M-CSF-Mφ and GM-CSF-Mφ were cultured with CFDA-SE labeled CD4⁺ T cells in MLR experiments (<i>A</i>) or with CFDA-SE labeled memory CD45RO⁺ T cells in T cell proliferation assays (<i>B</i>); results are expressed as a percentage of cells depending on the number of cycle division (mean ± SD, n = 4). * p<0.05, compared to GM-CSF-Mφ. Right panels, representative histograms of CFDA-SE labeling from one donor. <i>C</i>&<i>D, IL-34 switches monocytes into M2 in an M-CSF-independent manner.</i> Analysis of the expression of CD14 and CD163 (<i>C</i>), and of the production of IL-10 and IL-12 (quantified after a 24 h stimulation with LPS) (<i>D</i>) by IL-34-Mφ, M-CSF-Mφ and GM-CSF-Mφ generated in the absence or presence of a neutralizing anti-M-CSF mAb, an isotype control mAb, the kinase inhibitor GW2580, or the drug diluent. Results are expressed in MFI values or in ng/ml (mean ± SD, n = 4). <i>E, Analysis of cell viability.</i> The viability of monocytes cultured with IL-34, M-CSF or GM-CSF, in the absence of presence of a neutralizing anti-M-CSF mAb, an isotype control mAb, the kinase inhibitor GW2580, or the drug diluent, was determined at day 3 by annexinV labeling. Results are expressed as a percentage of living cells (mean ± SD, n = 4). † means >90% of died cells.</p

IL-34 induces M2 cell generation.

<p><i>A–E, IL-34 induces monocyte differentiation into M2</i>. Macrophage subsets were generated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056045#s2" target="_blank">Material & Methods</a> section. The expression of CD14 (<i>A</i>) and CD163 (<i>B</i>) was analyzed by flow cytometry on non stimulated cells. The expression of CD80 (<i>C</i>), CD86 (<i>D</i>) and ILT3 (<i>E</i>) was analyzed by flow cytometry after LPS stimulation. Results are expressed in MFI values (mean ± SD, n = 5). * p<0.05, compared to IL-34; # p<0.05, compared to IL-34+IL-6. <i>F, IL-6 favors IL-34 consumption</i>. IL-34 was quantified at different time-points in the supernatants of monocytes exposed to 50 ng/ml IL-34, without or with 50 ng/ml IL-6. Results are expressed in ng/ml (mean ± SD, n = 4). * p<0.05.</p

Analysis of cell surface markers on macrophage subsets.

<p>The expression of the indicated markers was analyzed by FACS on IL-34-Mφ, M-CSF-Mφ and GM-CSF-Mφ, stimulated or not for 48 h with 200 ng/ml LPS. Results are expressed as mean fluorescence intensity values (after subtraction of the MFI obtained with the isotype control mAb) and are representative of 4 separate experiments. +++++ indicates MFI greater than 1000; ++++, MFI greater than 400 and less than 1000; +++, MFI greater than 200 and less than 400; ++, MFI greater than 100 and less than 200; +, MFI greater than 20 and less than 100 and, − indicates MFI less than 20.</p