Double-walled carbon nanotubes trigger IL-1β release in human monocytes through Nlrp3 inflammasome activation

Because of their outstanding physical properties, carbon nanotubes (CNTs) are promising new materials in the field of nanotechnology. It is therefore imperative to assess their adverse effects on human health. Monocytes/macrophages that recognize and eliminate the inert particles constitute the main target of CNTs. In this article, we report our finding that double-walled CNTs (DWCNTs) synergize with Tolllike receptor agonists to enhance IL-1β release in human monocytes. We show that DWCNTs–induced IL-1β secretion is exclusively linked to caspase-1 and to Nlrp3 inflammasome activation in human monocytes. We also establish that this activation requires DWCNTs phagocytosis and potassium efflux, but not reactive oxygen specied (ROS) generation. Moreover, inhibition of lysosomal acidification or cathepsin-B activation reduces DWCNT-induced IL-1β secretion, suggesting that Nlrp3 inflammasome activation occurs via lysosomal destabilization. Thus, DWCNTs present a health hazard due to their capacity to activate Nlrp3 inflammasome, recalling the inflammation caused by asbestos and hence demonstrating that they should be used with caution. From the Clinical Editor: This is a very important biosafety/toxicity study regarding double walled carbon nanotubes. The investigators demonstrate that such nanotubes do represent a health hazard due to their capacity to activate Nlrp3 inflammasome, resembling the inflammation caused by asbestos. While further study of this phenomenon is definitely needed, the above findings clearly suggest that special precautions need to be taken when applying these nanoparticles in human disease research

PPARγ Controls Dectin-1 Expression Required for Host Antifungal Defense against Candida albicans

We recently showed that IL-13 or peroxisome proliferator activated receptor γ (PPARγ) ligands attenuate Candida albicans colonization of the gastrointestinal tract. Here, using a macrophage-specific Dectin-1 deficient mice model, we demonstrate that Dectin-1 is essential to control fungal gastrointestinal infection by PPARγ ligands. We also show that the phagocytosis of yeast and the release of reactive oxygen intermediates in response to Candida albicans challenge are impaired in macrophages from Dectin-1 deficient mice treated with PPARγ ligands or IL-13. Although the Mannose Receptor is not sufficient to trigger antifungal functions during the alternative activation of macrophages, our data establish the involvement of the Mannose Receptor in the initial recognition of non-opsonized Candida albicans by macrophages. We also demonstrate for the first time that the modulation of Dectin-1 expression by IL-13 involves the PPARγ signaling pathway. These findings are consistent with a crucial role for PPARγ in the alternative activation of macrophages by Th2 cytokines. Altogether these data suggest that PPARγ ligands may be of therapeutic value in esophageal and gastrointestinal candidiasis in patients severely immunocompromised or with metabolic diseases in whom the prevalence of candidiasis is considerable

PPARγ inhibition in M2 polarized macrophages abolishes the increase of Dectin-1.

<p>(A) The protein level of Dectin-1 on peritoneal macrophages was measured by flow cytometry after treatment with IL-13 (50 ng/mL) or rosiglitazone (RZ) (5 µM) in the presence of the PPARγ antagonists (GW9662 (5 µM) and T007 (2 µM)). Data are the means±SE of three separate experiments. (B) Dectin-1 mRNA level of peritoneal macrophages was quantified by quantitative real-time RT-PCR after treatment with IL-13 (50 ng/mL) or rosiglitazone (RZ) (5 µM) in the presence of the PPARγ antagonist (GW9662 (5 µM)). Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the untreated macrophages. (C) The surface protein level of Dectin-1 on peritoneal macrophages transfected with siRNA targeting PPARγ (PPARγ siRNA) or control siRNA (control siRNA) and stimulated by IL-13. Representative Dectin-1 FACS profiles of untreated (unfilled histograms) and treated (filled histograms) macrophages were obtained by flow cytometry. The changes in Dectin-1 receptor levels were normalized to the untreated macrophages transfected with the siRNA control. Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates significant difference compared with the untreated macrophages transfected with the siRNA control.</p

Dectin-1 and the Mannose Receptor are implicated in antifungal functions of macrophages treated with IL-13 or PPARγ ligand.

<p>Peritoneal macrophages were cultured with IL-13 (50 ng/mL) (A and B) or rosiglitazone (5 µM) (C and D). Mannan (mann) and/or soluble β-glucan (laminarin, lam) solutions were incubated at 4°C for 20 min until the phagocytosis and respiratory burst experiments. (A and C) The phagocytosis of non-opsonized <i>C.albicans</i> (ratio 1∶6) by macrophages was measured at 37°C after exposure to FITC-labeled <i>C.albicans</i> for 60 min. The amount of fluorescence was determined using a FACS based approach. The distinction between internalized yeast cells and those attached to macrophage surface was done <i>via</i> quenching the FITC-fluorescence with trypan blue. Data are expressed as percentage relative to untreated control macrophages and are means±SE of three separate experiments. (B and D) Non-opsonized <i>C.albicans</i>-induced respiratory burst of macrophages (ratio 1∶3) was measured by chimiluminescence. Total chemiluminescence emission (area under the curve expressed in counts x seconds) was observed continuously for 60 min in the presence or absence of non-opsonized <i>C. albicans</i>. The data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the untreated macrophages. ## (p<0.01) and # (p<0.05) indicates a significant difference compared with the treated control macrophages.</p

Dectin-1 and the Mannose Receptor are required in different stages of <i>C.albicans</i> clearance.

<p>Dectin-1 control (Cre 0) and Dectin-1 knockout (Cre Tg) peritoneal macrophages were cultured with IL-13 (50 ng/mL) (A–C) or with rosiglitazone (RZ) (5 µM) (D–F). Mannan (mann) solution was incubated at 4°C for 20 min until the binding, phagocytosis and respiratory burst experiments. (A and D) The binding experiment of non-opsonized <i>C.albicans</i> by macrophages was measured at 4°C after challenge with FITC-labeled <i>C.albicans</i> for 20 min (ratio 1∶6). The amount of fluorescence was determined using a FACS based approach. Data are expressed as the percentage relative to the untreated Dectin-1 control (Cre 0) macrophages and are the means±SE of three separate experiments. (B and E) The phagocytosis of non-opsonized <i>C.albicans</i> by macrophages was measured at 37°C after challenge with FITC-labeled <i>C.albicans</i> for 60 min (ratio 1∶6). The amount of fluorescence was determined using a FACS based approach. The distinction between internalized yeast cells and those attached to macrophage surface was done <i>via</i> quenching the FITC-fluorescence with trypan blue. Data are expressed as the percentage relative to the untreated Dectin-1 control (Cre 0) macrophages and are the means±SE of three separate experiments. (C and F) The respiratory burst of the Dectin-1 control (Cre 0) and Dectin-1 knockout (Cre Tg) macrophages induced by non-opsonized <i>C.albicans</i> was measured by chimiluminescence (ratio 1∶3). Total chemiluminescence emission (area under the curve expressed in counts x seconds) was observed continuously for 60 min. Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the Cre 0 untreated macrophages. ## (p<0.01) indicates a significant difference compared with the Cre Tg untreated macrophages. § (p<0.05) indicates a significant difference between Cre 0 and Cre Tg.</p

Dectin-1 expression depends on PPARγ activation by IL-13 or PPARγ-specific ligands.

<p>(A) The surface protein level of Dectin-1 on peritoneal macrophages was measured by flow cytometry after treatment with IL-13 (50 ng/mL), rosiglitazone (RZ) (5 µM), 15d-PGJ2 (1 µM), MCC555 (5 µM) or GW1929 (1 µM). The changes in Dectin-1 induction were normalized to the untreated control value. Data are the means±SE of three separate experiments. (B) Representative FACS profiles of Dectin-1 (filled histograms) and isotype control labeling (unfilled histograms) in treated macrophages. Representative Dectin-1 FACS profiles of untreated macrophages (unfilled histograms) and treated (filled histograms) macrophages. (C) The mRNA level of Dectin-1 on peritoneal macrophages was quantified by quantitative real-time RT-PCR after treatment with IL-13 (50 ng/mL), rosiglitazone (RZ) (5 µM) or 15d-PGJ2 (1 µM). Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the untreated macrophages. (D) The protein level of Dectin-1 on the murine cell line RAW264.7 transiently transfected with pCMV-luciferase (CMV-luc) or with pCMV-mPPARγ (CMV-PPARγ) and after treatment with IL-13 or rosiglitazone (RZ). Representative Dectin-1 FACS profiles of untreated (unfilled histograms) and treated (filled histograms) macrophages were obtained by flow cytometry. The changes in Dectin-1 induction were normalized to the untreated RAW 264.7 cells transfected with pCMV-luc. Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the untreated RAW 264.7 cells transfected with pCMV-luc.</p

cPLA2 is involved in Dectin-1 induction by IL-13.

<p>(A) The Dectin-1 mRNA level of peritoneal macrophages was measured by quantitative real-time RT-PCR after treatment of peritoneal macrophages by an irreversible cPLA2 antagonist (MAFP) and by IL-13. Data are the means±SE of three separate experiments. (B) The protein level of Dectin-1 was measured by flow cytometry on peritoneal macrophages after treatment with MAFP (10 µM and 20 µM) and with IL-13 (50 ng/mL). Data are the means±SE of three separate experiments. (C) The protein level of Dectin-1 was measured by flow cytometry on peritoneal macrophages after treatment with MAFP and IL-13 and/or 15d-PGJ2 (1 µM). Data are the means±SE of three separate experiments. ** (p<0.01) and * (p<0.05) indicates a significant difference compared with the IL-13-treated macrophages.</p

Dectin-1-knockout mice are more susceptible than Dectin-1-wildtype mice to <i>C. albicans</i> gastrointestinal infection.

<p>(A and B) Quantification of <i>C. albicans</i> fungal burden in the gastrointestinal tract (stomach and cecum) of Dectin-1-control mice Cre 0 (filled circles) and Dectin-1-knockout mice Cre Tg (open circles) at 5 day after oral infection with 5.10⁶ CFU (A, n = 6) or with 5.10⁷ CFU (B, n = 4) in standard conditions or after treatment with rosiglitazone (RZ) (2.8 µg/g of mouse). Each symbol represents an individual mouse. § (p<0.05) indicates a significant difference between group of mice. (C) Phagocytosis and ROS production were measured on peritoneal macrophages from Dectin-1 knockout (Cre Tg) mice at 5 day after oral infection with 5.10⁶ CFU in standard conditions or after treatment with rosiglitazone (RZ). The phagocytosis of non-opsonized <i>C.albicans</i> by macrophages was measured at 37°C after exposure to FITC-labeled <i>C.albicans</i> for 60 min (ratio 1∶6). The amount of fluorescence was determined using a FACS based approach. The distinction between internalized yeast cells and those attached to macrophage surface was done <i>via</i> quenching the FITC-fluorescence with trypan blue. Data are expressed as the percentage relative to untreated Dectin-1 control (Cre 0) macrophages and are the means±SE (n = 6). The respiratory burst of macrophages induced by non-opsonized zymosan (ZNO) (2 µg/mL) was measured by chimiluminescence. Total chemiluminescence emission (area under the curve expressed in counts x seconds) was observed continuously for 60 min. Data are the means±SE (n = 6). ** (p<0.01) indicates a significant difference compared with the Cre 0 untreated macrophages. § (p<0.05) indicates a significant difference between Cre 0 and Cre Tg. (D) The MR surface protein level was measured by flow cytometry on peritoneal macrophages from Dectin-1 control (Cre 0) or Dectin-1 knockout (Cre Tg) mice at day 5 after oral infection with 5.10⁷ CFU in standard conditions or after treatment with rosiglitazone (RZ). Data are the means±SE (n = 4). ** (p<0.01) indicates a significant difference compared with the Cre 0 control. § (p<0.05) indicates a significant difference between Cre 0 and Cre Tg.</p