Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells

BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer

Correction: Orsi et al. Carbon Nanotubes under Scrutiny: Their Toxicity and Utility in Mesothelioma Research. Appl. Sci. 2020, 10, 4513

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Characterization of a new intermediate macrophage subpopulation : SDC-1 positive SPM-like macrophages possess immunosuppressive functions in early mesotheliomagenic responses to carbon nanotubes

Malignant mesothelioma is a disease caused by inhalation of needle-like shaped particles and whose pathogenesis is not yet fully elucidated. A long-lasting general inflammation caused by persistent mesotheliomagenic particles is not sufficient to explain mesothelioma onset. Indeed, challenging data have shown that mesothelioma occurs without chronic inflammation. Our research team previously helped to discover that, beside inflammation, particles such as silica also induce a selective, rapid and sustained accumulation of immunosuppressive cells (regulatory T lymphocytes, myeloid derived suppressive cells and M2/regulatory macrophages) participating to fibrogenesis. To determine whether carcinogenic particles such as asbestos and carbon nanotubes (CNT) also elicit immunosuppressive responses, we investigated the impact of these particles on macrophage turnover, phenotype and immunosuppressive activity. With that purpose, we intraperitoneally injected mesotheliomagenic CNT-7 (needle-like, Mitsui & Co) and non-mesotheliomagenic CNT-T (tangled, Nagoya University) particles in Wistar rats and compared the effects on peritoneal macrophage subpopulations. We showed that macrophages die very rapidly in the attempt to phagocyte mesotheliomagenic CNT. They are later replenished by monocytic-derived small peritoneal macrophages (SPM-like macrophages) possessing comparable immunosuppressive functions and signatures to Tumor-Associated Macrophages (TAM), which infiltrate mesothelioma and block T cell antitumor activities. Early immunosuppressive SPM-like macrophages express and release the shed form of the immunoregulatory syndecan-1 glycoprotein, which could explain the immunosuppression that they exert on T cells. Non-mesotheliomagenic peritoneal responses induced by CNT-T are, in contrast, characterized by a recruitment of self-proliferating large peritoneal macrophages (LPM-like macrophages) that correspond to homeostatic macrophages without immunosuppressive activity. Our observations indicate that mesotheliomagenic particles elicit a prompt immunosuppressive response involved in an early environment favouring tumoral cell evasion from T cell surveillance. Since our RNA sequencing data indicated that immunosuppressive SPM-like macrophages have an intermediate polarization state between M1/inflammatory and M2/regulatory macrophages, we suggest that they may represent the gap between the short-term (inflammation) and long-term (tumor immunosuppression) responses to mesotheliomagenic particles. These observations offer new therapeutic targets (SPM or syndecan-1) and in vivo bioassays (macrophage replenishment) to anticipate particle hazard and carcinogenicity.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

Early and sustained immunosupressive macrophages in mesothelioma.

Malignant mesothelioma (MM) is a rare cancer caused by carcinogenic fibers such as asbestos or carbon nanotubes (CNT) exposure and affecting serous membrane of pleural and peritoneal cavities. It is accepted that fiber-induced chronic inflammation, M1-macrophage and neutrophil infiltration are crucial in tumor establishment. However, there is growing evidence that immunosuppressive leukocytes preventing efficient anti-tumor immune responses are also implicated in development of MM. Our experimental study aims to determine if the carcinogenic responses to asbestos and Mitsui-7 CNT are associated with the accumulation of immunosuppressive macrophages. We used a Wistar rat peritoneum model which allows directly exposing mesothelial cells to asbestos or CNT, and easily sampling the mesothelial cavity for monitoring macrophage responses during the carcinogenic process. We show that FACS-sorted eosinophilic macrophages present in CNT-7-induced mesothelioma microenvironment suppress polyclonal activation of T lymphocytes in vitro, during the early response to carcinogenic CNT or asbestos (day 1 to 30), well before the cancer establishment. They also express CD163, a marker of M2 phenotype, known to inhibit human T lymphocytes activation and proliferation in vitro. Peritoneal macrophages purified from CNT-treated rats (day 1) highly express the immunosuppressive mediators IL-10 and Arginase-1. The levels of TGF-beta in peritoneal fluids were also increased by CNT-7 or asbestos injection. Altogether, our data demonstrate that carcinogenic CNT possess the intrinsic capacity to induce a rapid and sustained accumulation of immunosuppressive macrophages and their related mediators before mesothelioma is established

Carbon nanotubes under scrutiny: Their toxicity and utility in mesothelioma research

Research on the toxicity of engineered carbon nanotubes (CNT) was initiated by Belgian academic chemists and toxicologists more than 15 years ago. It is now undisputed that some of these attractive nanomaterials induce serious illness such as fibrosis and cancer. The physico-chemical determinants of CNT-induced adverse effects are now elucidated and include shape, nanoscale diameter, structural defects and scavenger capacity. Generated in vitro and in vivo data on their inflammogenic and fibrogenic activities were combined and translated in worldwide accepted AOP (Adverse Outcome Pathways) available for risk assessment and regulatory policies. The asbestos-like carcinogenic effects of CNT, notably their capacity to induce malignant mesothelioma (MM), remain a cause of concern for public health and strongly curb the craze for CNT in industries. MM still represents a real challenge for clinicians and a highly refractory cancer to existing therapeutic strategies. By comparing mesotheliomagenic CNT (needle-like CNT-N) to non mesotheliomagenic CNT (tangled-like CNT-T), our group generated a relevant animal model that highlights immune pathogenic pathways specifically associated to the carcinogenic process. Evidence indicate that only CNT-N possess the intrinsic capacity to induce a preferential, rapid and sustained accumulation of host immunosuppressive cells that subvert immune surveillance and suppress T lymphocyte anti-mesothelioma immunity. This new concept offers novel horizons for clinical management of mesothelioma and represents an additional tool for predicting mesotheliomagenic activity of newly elaborated CNT or nanoparticles

Early and sustained immunosuppressive macrophage in rat mesothelioma

Malignant mesothelioma (MM) is a rare cancer caused by carcinogenic fibers such as asbestos or carbon nanotubes (CNT) exposure and affecting serous membrane of pleural and peritoneal cavities. It is accepted that fiber-induced chronic inflammation, M1-macrophage and neutrophil infiltration are crucial in tumor establishment. However, there is growing evidence that immunosuppressive leukocytes preventing efficient anti-tumor immune responses are also implicated in development of MM. Our experimental study aims to determine if the carcinogenic responses to asbestos and Mitsui-7 CNT are associated with the accumulation of immunosuppressive macrophages. We used a Wistar rat peritoneum model which allows directly exposing mesothelial cells to asbestos or CNT, and easily sampling the mesothelial cavity for monitoring macrophage responses during the carcinogenic process. We show that FACS-sorted eosinophilic macrophages present in CNT-7-induced mesothelioma microenvironment suppress polyclonal activation of T lymphocytes in vitro, during the early response to carcinogenic CNT or asbestos (day 1 to 30), well before the cancer establishment. They also express CD163, a marker of M2 phenotype, known to inhibit human T lymphocytes activation and proliferation in vitro. Peritoneal macrophages purified from CNT-treated rats (day 1) highly express the immunosuppressive mediators IL-10 and Arginase-1. The levels of TGF-beta in peritoneal fluids were also increased by CNT-7 or asbestos injection. Altogether, our data demonstrate that carcinogenic CNT possess the intrinsic capacity to induce a rapid and sustained accumulation of immunosuppressive macrophages and their related mediators before mesothelioma is established

Monocytic Ontogeny of Regenerated Macrophages Characterizes the Mesotheliomagenic Responses to Carbon Nanotubes

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres