PPARγ activation modulates the balance of peritoneal macrophage populations to suppress ovarian tumor growth and tumor-induced immunosuppression.

BACKGROUND: Ovarian adenocarcinoma (OVAD) frequently metastasizes to the peritoneal cavity and manifests by the formation of ascites, which constitutes a tumor-promoting microenvironment. In the peritoneal cavity, two developmentally, phenotypically and functionally distinct macrophage subsets, immunocompetent large peritoneal macrophages (LPM) and immunosuppressive small peritoneal macrophages (SPM), coexist. Because peroxisome proliferator-activated receptor γ (PPARγ) is a critical factor participating in macrophage differentiation and cooperates with CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor essential for SPM-to-LPM differentiation, PPARγ could be also involved in the regulation of SPM/LPM balance and could be a promising therapeutic target. METHODS: To evaluate the 15(S)-hydroxyeicosatetraenoic acid (HETE), a PPARγ endogenous ligand, impact on ovarian tumor growth, we intraperitoneally injected 15(S)-HETE into a murine ovarian cancer model. This experimental model consists in the intraperitoneally injection of ID8 cells expressing luciferase into syngeneic C57BL/6 female mice. This ID8 orthotopic mouse model is a well-established experimental model of end-stage epithelial OVAD. Tumor progression was monitored using an in vivo imaging system. Peritoneal immune cells in ascites were analyzed by flow cytometry and cell sorting. To determine whether the impact of 15(S)-HETE in tumor development is mediated through the macrophages, these cells were depleted by injection of liposomal clodronate. To further dissect how 15(S)-HETE mediated its antitumor effect, we assessed the tumor burden in tumor-bearing mice in which the PPARγ gene was selectively disrupted in myeloid-derived cells and in mice deficient of the recombination-activating gene Rag2. Finally, to validate our data in humans, we isolated and treated macrophages from ascites of individuals with OVAD. RESULTS: Here we show, in the murine experimental model of OVAD, that 15(S)-HETE treatment significantly suppresses the tumor growth, which is associated with the differentiation of SPM into LPM and the LPM residency in the peritoneal cavity. We demonstrate that C/EBPß and GATA6 play a central role in SPM-to-LPM differentiation and in LPM peritoneal residence through PPARγ activation during OVAD. Moreover, this SPM-to-LPM switch is associated with the increase of the effector/regulatory T-cell ratio. Finally, we report that 15(S)-HETE attenuates immunosuppressive properties of human ovarian tumor-associated macrophages from ascites. CONCLUSION: Altogether, these results promote PPARγ as a potential therapeutic target to restrain OVAD development and strengthen the use of PPARγ agonists in anticancer therapy.

Differential immunomodulatory effects of six pesticides of different chemical classes on human monocyte-derived macrophage functions.

Exposure to pesticides through eyes, skin, ingestion and inhalation may affects human health by interfering with immune cells, such as macrophages. We evaluated, in vitro, the effect of six pesticides widely used in apple arboriculture on the functions of human monocyte-derived macrophages (hMDMs). hMDMs were cultured for 4 or 24 h with or without pesticides (0.01, 0.1, 1, 10 µmol.L-1). We showed that chlorpyrifos, thiacloprid, thiophanate, boscalid, and captan had little toxic effect at the tested concentrations, while dithianon had low-cytotoxicity at 10 µmol.L-1. While boscalid showed no effect on hMDMs function, thiophanate (0.01 µmol.L-1) stimulated with TPA and thiacloprid (1, 10 µmol.L-1) stimulated with zymosan activated ROS production. Chlorpyrifos, dithianon, and captan inhibited ROS production and TNF-α, IL-1ß pro-inflammatory cytokines. We established that dithianon (0.01-1 µmol.L-1) and captan (0.1, 1 µmol.L-1) induced mRNA expression of NQO1 and HMOX1 antioxidant enzymes. Dithianon also induced the mRNA expression of catalase, superoxide dismutase-2 at 10 µmol.L-1. Together, these results show that exposure to chlorpyrifos, dithianon, and captan induce immunomodulatory effects that may influence the disease fighting properties of monocytes/macrophages while pesticides such as thiacloprid, thiophanate and boscalid have little influence.

Bioactive fish collagen peptides weaken intestinal inflammation by orienting colonic macrophages phenotype through mannose receptor activation.

PURPOSE: Particular interest is now given to the potential of dietary supplements as alternative non-pharmacological approaches in intestinal inflammation handling. In this aim, this study evaluates the efficiency of fish collagen peptides, Naticol®Gut, on colonic inflammation. METHODS: Wild type and Mannose receptor-deficient in the myeloid lineage C57BL/6 mice were administered with Dextran Sodium Sulfate (DSS), Naticol®Gut, DSS, and Naticol®Gut or only water for 4 or 8 days. Inflammatory status was evaluated by establishing macroscopic and microscopic scores, by measuring cytokine and calprotectin production by ELISA and the myeloperoxidase activity by chemiluminescence. Colonic macrophages were phenotyped by measuring mRNA levels of specific markers of inflammation and oxidative status. Colonic immune populations and T-cell activation profiles were determined by flow cytometry. Mucosa-associated gut microbiota assessment was undertaken by qPCR. The phenotype of human blood monocytes from inflammatory bowel disease (IBD) subjects was characterized by RT-qPCR and flow cytometry and their oxidative activity by chemiluminescence. RESULTS: Naticol®Gut-treated DSS mice showed attenuated colonic inflammation compared to mice that were only exposed to DSS. Naticol®Gut activity was displayed through its ability to orient the polarization of colonic macrophage towards an anti-inflammatory and anti-oxidant phenotype after its recognition by the mannose receptor. Subsequently, Naticol®Gut delivery modulated CD4 T cells in favor of a Th2 response and dampened CD8 T-cell activation. This immunomodulation resulted in an intestinal eubiosis. In human monocytes from IBD subjects, the treatment with Naticol®Gut also restored an anti-inflammatory and anti-oxidant phenotype. CONCLUSION: Naticol®Gut acts as a protective agent against colitis appearing as a new functional food and an innovative and complementary approach in gut health.

Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation.

AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4+ Th1/Th2 balance towards a Th1 response and activates CD8+ T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

Comparative study of the effects of ziram and disulfiram on human monocyte-derived macrophage functions and polarization: involvement of zinc.

Ziram, a zinc dithiocarbamate is widely used worldwide as a fungicide in agriculture. In order to investigate ziram-induced changes in macrophage functions and polarization, human monocytes-derived macrophages in culture were treated with ziram at 0.01-10 µmol.L-1 for 4-24 h. To characterize zinc involvement in these changes, we also determined the effects of disulfiram alone (dithiocarbamate without zinc) or in co-incubation with ZnSO4. We have shown that ziram and disulfiram at 0.01 µmol.L-1 increased zymosan phagocytosis. In contrast, ziram at 10 µmol.L-1 completely inhibited this phagocytic process, the oxidative burst triggered by zymosan and the production of TNF-α, IL-1ß, IL-6, and CCL2 triggered by LPS. Disulfiram had the same effects on these macrophages functions only when combined with zinc (10 µmol.L-1). In contrast, at 10 µmol.L-1 ziram and zinc associated-disulfiram induced expression of several antioxidants genes HMOX1, SOD2, and catalase, which could suggest the induction of oxidative stress. This oxidative stress could be involved in the increase in late apoptosis induced by ziram (10 µmol.L-1) and zinc associated-disulfiram. Concerning gene expression profiles of membrane markers of macrophage polarization, ziram at 10 µmol.L-1 had two opposite effects. It inhibited the gene expression of M2 markers (CD36, CD163) in the same way as the disulfiram-zinc co-treatment. Conversely, ziram induced gene expression of other M2 markers CD209, CD11b, and CD16 in the same way as treatment with zinc alone. Disulfiram-zinc association had no significant effects on these markers. These results taken together show that ziram via zinc modulates macrophages to M2-like anti-inflammatory phenotype which is often associated with various diseases.

Circulating CD14high CD16low intermediate blood monocytes as a biomarker of ascites immune status and ovarian cancer progression.

BACKGROUND: Besides the interest of an early detection of ovarian cancer, there is an urgent need for new predictive and prognostic biomarkers of tumor development and cancer treatment. In healthy patients, circulating blood monocytes are typically subdivided into classical (85%), intermediate (5%) and non-classical (10%) populations. Although these circulating monocyte subsets have been suggested as biomarkers in several diseases, few studies have investigate their potential as a predictive signature for tumor immune status,tumor growth and treatment adaptation. METHODS: In this study, we used a homogeneous cohort of 28 chemotherapy-naïve patients with ovarian cancer to evaluate monocyte subsets as biomarkers of the ascites immunological status. We evaluated the correlations between circulating monocyte subsets and immune cells and tumor burden in peritoneal ascites. Moreover, to validate the use of circulating monocyte subsets tofollow tumor progression and treatment response, we characterized blood monocytes from ovarian cancer patients included in a phase 1 clinical trial at baseline and following murlentamab treatment. RESULTS: We demonstrate here a robust expansion of the intermediate blood monocytes (IBMs) in ovarian cancer patients. We establish a significant positive correlation between IBM percentage and tumor-associate macrophages with a CCR2high/CD163high/CD206high/CD86lowprofile. Moreover, IBM expansion is associated with a decreased effector/regulatory T-cell ratio in ascites and with the presence of soluble immunosuppressive mediators. We also establish that IBM proportion positively correlates with the peritoneum tumor burden. Finally, the study of IBMs in patients with ovarian cancer under immunotherapy during the phase clinical trial supports IBMs to follow the evolution of tumor development and the treatment adaptation. CONCLUSIONS: This study, which links IBM level with immunosuppression and tumor burden in peritoneum, identifies IBMs as apotential predictive signature of ascites immune status and as a biomarker ofovarian cancer development and treatment response. TRIAL REGISTRATION NUMBER: EudraCT: 2015-004252-22 NCT02978755.

Divergent Roles for Macrophage C-type Lectin Receptors, Dectin-1 and Mannose Receptors, in the Intestinal Inflammatory Response.

Colonic macrophages are considered to be major effectors of inflammatory bowel diseases (IBDs) and the control of gut inflammation through C-type lectin receptors is an emerging concept. We show that during colitis, the loss of dectin-1 on myeloid cells prevents intestinal inflammation, while the lack of mannose receptor (MR) exacerbates it. A marked increase in dectin-1 expression in dextran sulfate sodium (DSS)-exposed MR-deficient mice supports the critical contribution of dectin-1 to colitis outcome. Dectin-1 is crucial for Ly6ChighCCR2high monocyte population enrichment in the blood and their recruitment to inflamed colon as precursors of inflammatory macrophages. Dectin-1 also promotes inflammasome-dependent interleukin-1ß (IL-1ß) secretion through leukotriene B4 production. Interestingly, colonic inflammation is associated with a concomitant overexpression of dectin-1/CCL2/LTA4H and downregulation of MR on macrophages from IBD patients. Thus, MR and dectin-1 on macrophages are important mucosal inflammatory regulators that contribute to the intestinal inflammation.

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model.

The epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach.

Tumor cells educate mesenchymal stromal cells to release chemoprotective and immunomodulatory factors.

Factors released by surrounding cells such as cancer-associated mesenchymal stromal cells (CA-MSCs) are involved in tumor progression and chemoresistance. In this study, we characterize the mechanisms by which naïve mesenchymal stromal cells (MSCs) can acquire a CA-MSCs phenotype. Ovarian tumor cells trigger the transformation of MSCs to CA-MSCs by expressing pro-tumoral genes implicated in the chemoresistance of cancer cells, resulting in the secretion of high levels of CXC chemokine receptors 1 and 2 (CXCR1/2) ligands such as chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, and interleukin 8 (IL-8). CXCR1/2 ligands can also inhibit the immune response against ovarian tumor cells. Indeed, through their released factors, CA-MSCs promote the differentiation of monocytes towards M2 macrophages, which favors tumor progression. When CXCR1/2 receptors are inhibited, these CA-MSC-activated macrophages lose their M2 properties and acquire an anti-tumoral phenotype. Both ex vivo and in vivo, we used a CXCR1/2 inhibitor to sensitize ovarian tumor cells to carboplatin and circumvent the pro-tumoral effects of CA-MSCs. Since high concentrations of CXCR1/2 ligands in patients' blood are associated with chemoresistance, CXCR1/2 inhibition could be a potential therapeutic strategy to revert carboplatin resistance.

IL13-Mediated Dectin-1 and Mannose Receptor Overexpression Promotes Macrophage Antitumor Activities through Recognition of Sialylated Tumor Cells.

Macrophage-mediated cytotoxicity is controlled by surface receptor expression and activation. Despite the numerous studies documenting the role of macrophage C-type lectin receptors (CLR) in pathogen elimination, little is known about their contribution to antitumor responses. Here, we report that IL13 inhibits T-cell lymphoma and ovarian adenocarcinoma development in tumor-bearing mice through the conversion of tumor-supporting macrophages to cytotoxic effectors, characterized by a CLR signature composed of dectin-1 and mannose receptor (MR). We show that dectin-1 and MR are critical for the recognition of tumor cells through sialic acid-specific glycan structure on their surface and for the subsequent activation of macrophage tumoricidal response. Finally, we validated that IL13 antitumor effect mediated by dectin-1 and MR overexpression on macrophages can extend to various types of human tumors. Therefore, these results identify these CLRs as potential targets to promote macrophage antitumor response and represent an attractive approach to elicit tumor-associated macrophage tumoricidal properties.

P17, an Original Host Defense Peptide from Ant Venom, Promotes Antifungal Activities of Macrophages through the Induction of C-Type Lectin Receptors Dependent on LTB4-Mediated PPARγ Activation.

Despite the growing knowledge with regard to the immunomodulatory properties of host defense peptides, their impact on macrophage differentiation and on its associated microbicidal functions is still poorly understood. Here, we demonstrated that the P17, a new cationic antimicrobial peptide from ant venom, induces an alternative phenotype of human monocyte-derived macrophages (h-MDMs). This phenotype is characterized by a C-type lectin receptors (CLRs) signature composed of mannose receptor (MR) and Dectin-1 expression. Concomitantly, this activation is associated to an inflammatory profile characterized by reactive oxygen species (ROS), interleukin (IL)-1ß, and TNF-α release. P17-activated h-MDMs exhibit an improved capacity to recognize and to engulf Candida albicans through the overexpression both of MR and Dectin-1. This upregulation requires arachidonic acid (AA) mobilization and the activation of peroxisome proliferator-activated receptor gamma (PPARγ) nuclear receptor through the leukotriene B4 (LTB4) production. AA/LTB4/PPARγ/Dectin-1-MR signaling pathway is crucial for P17-mediated anti-fungal activity of h-MDMs, as indicated by the fact that the activation of this axis by P17 triggered ROS production and inflammasome-dependent IL-1ß release. Moreover, we showed that the increased anti-fungal immune response of h-MDMs by P17 was dependent on intracellular calcium mobilization triggered by the interaction of P17 with pertussis toxin-sensitive G-protein-coupled receptors on h-MDMs. Finally, we also demonstrated that P17-treated mice infected with C. albicans develop less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf Candida, to produce ROS and IL-1ß and to kill the yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts upstream PPARγ/CLRs axis and offer new immunomodulatory therapeutic perspectives in the field of infectious diseases.