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-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

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