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Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

Wang M, Mukherjee PK, Chandra J, Lattif AA, McCormick TS, Ghannoum MA - BMC Microbiol. (2008)

Bottom Line: The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml.CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio, USA. mingyuewang@gmail.com

ABSTRACT

Background: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.

Results: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.

Conclusion: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

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CA-SIIF inhibits macrophage IL-12 production both (A) in vitro and (B) in vivo in a murine model. (A) murine peritoneal Macrophages (MØ) were elicited by TG treatment and collected from the peritoneum of C57Bl/6 mice 5 days after elicitation. 2 × 106/ml macrophages were cultured in the presence or absence of LPS (50 ng) and IFN-γ (0.5 ng) stimulation for 16 h with or without 1 mg CA-SIIF. Next, IL-12 levels were measured in the supernatant by ELISA using antibodies specific for murine IL-12 p70. (B) LPS (100 ng) was administered intraperitoneally immediately followed with or without 1 mg CA-SIIF in mice 1 h prior to intravenous injection of LPS (1 μg) to stimulate IL-12 production. Serum IL-12 levels were then measured. Macrophages alone served as controls for base line IL-12 production.
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Figure 3: CA-SIIF inhibits macrophage IL-12 production both (A) in vitro and (B) in vivo in a murine model. (A) murine peritoneal Macrophages (MØ) were elicited by TG treatment and collected from the peritoneum of C57Bl/6 mice 5 days after elicitation. 2 × 106/ml macrophages were cultured in the presence or absence of LPS (50 ng) and IFN-γ (0.5 ng) stimulation for 16 h with or without 1 mg CA-SIIF. Next, IL-12 levels were measured in the supernatant by ELISA using antibodies specific for murine IL-12 p70. (B) LPS (100 ng) was administered intraperitoneally immediately followed with or without 1 mg CA-SIIF in mice 1 h prior to intravenous injection of LPS (1 μg) to stimulate IL-12 production. Serum IL-12 levels were then measured. Macrophages alone served as controls for base line IL-12 production.

Mentions: Monocytes differentiate into macrophages and are the first line of defense against Candida infection [12]. Therefore, we determined whether CA-SIIF affects IL-12 production by murine macrophages. Macrophages were isolated from the peritoneum of C57BL/6 mice following TG elicitation, and then exposed to CA-SIIF in combination with IFN-γ/LPS and the levels of IL-12 were determined. We found that macrophages co-incubated with CA-SIIF produced 32% less IL-12 than those grown in its absence (IL-12 level = 571 ± 24 pg/ml vs. 387 ± 87 pg/ml, respectively; P = 0.05, Figure 3A). Next, we established a murine model to investigate the influence of CA-SIIF on IL-12 production in vivo. In this regard, CA-SIIF toxicity assays in mice showed no signs of toxicity after injection of up to 1 mg CA-SIIF, over a 2 week period. RBC lysis induction tests at various CA-SIIF concentrations (100–1200 μg) were also negative (data not shown). IL-12 production in vivo in the presence or absence of CA-SIIF was examined using C57BL/6 mice pretreated for 5 days with TG peritoneally in combination with a priming ip injection of LPS (100 ng) and another iv LPS (1 μg) injection 1 h later. Prior to the first LPS treatment, CA-SIIF was injected ip, and six hours later, serum was collected (Figure 1). Our data showed that the level of IL-12 detected in the sera of mice treated with CA-SIIF was significantly decreased from 262 ± 6 pg/ml to 144 ± 30 pg/ml (P < 0.05) (Figure 3B). These results demonstrated that CA-SIIF can decrease IL-12 production by murine macrophages both in vitro and in vivo.


Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

Wang M, Mukherjee PK, Chandra J, Lattif AA, McCormick TS, Ghannoum MA - BMC Microbiol. (2008)

CA-SIIF inhibits macrophage IL-12 production both (A) in vitro and (B) in vivo in a murine model. (A) murine peritoneal Macrophages (MØ) were elicited by TG treatment and collected from the peritoneum of C57Bl/6 mice 5 days after elicitation. 2 × 106/ml macrophages were cultured in the presence or absence of LPS (50 ng) and IFN-γ (0.5 ng) stimulation for 16 h with or without 1 mg CA-SIIF. Next, IL-12 levels were measured in the supernatant by ELISA using antibodies specific for murine IL-12 p70. (B) LPS (100 ng) was administered intraperitoneally immediately followed with or without 1 mg CA-SIIF in mice 1 h prior to intravenous injection of LPS (1 μg) to stimulate IL-12 production. Serum IL-12 levels were then measured. Macrophages alone served as controls for base line IL-12 production.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2289826&req=5

Figure 3: CA-SIIF inhibits macrophage IL-12 production both (A) in vitro and (B) in vivo in a murine model. (A) murine peritoneal Macrophages (MØ) were elicited by TG treatment and collected from the peritoneum of C57Bl/6 mice 5 days after elicitation. 2 × 106/ml macrophages were cultured in the presence or absence of LPS (50 ng) and IFN-γ (0.5 ng) stimulation for 16 h with or without 1 mg CA-SIIF. Next, IL-12 levels were measured in the supernatant by ELISA using antibodies specific for murine IL-12 p70. (B) LPS (100 ng) was administered intraperitoneally immediately followed with or without 1 mg CA-SIIF in mice 1 h prior to intravenous injection of LPS (1 μg) to stimulate IL-12 production. Serum IL-12 levels were then measured. Macrophages alone served as controls for base line IL-12 production.
Mentions: Monocytes differentiate into macrophages and are the first line of defense against Candida infection [12]. Therefore, we determined whether CA-SIIF affects IL-12 production by murine macrophages. Macrophages were isolated from the peritoneum of C57BL/6 mice following TG elicitation, and then exposed to CA-SIIF in combination with IFN-γ/LPS and the levels of IL-12 were determined. We found that macrophages co-incubated with CA-SIIF produced 32% less IL-12 than those grown in its absence (IL-12 level = 571 ± 24 pg/ml vs. 387 ± 87 pg/ml, respectively; P = 0.05, Figure 3A). Next, we established a murine model to investigate the influence of CA-SIIF on IL-12 production in vivo. In this regard, CA-SIIF toxicity assays in mice showed no signs of toxicity after injection of up to 1 mg CA-SIIF, over a 2 week period. RBC lysis induction tests at various CA-SIIF concentrations (100–1200 μg) were also negative (data not shown). IL-12 production in vivo in the presence or absence of CA-SIIF was examined using C57BL/6 mice pretreated for 5 days with TG peritoneally in combination with a priming ip injection of LPS (100 ng) and another iv LPS (1 μg) injection 1 h later. Prior to the first LPS treatment, CA-SIIF was injected ip, and six hours later, serum was collected (Figure 1). Our data showed that the level of IL-12 detected in the sera of mice treated with CA-SIIF was significantly decreased from 262 ± 6 pg/ml to 144 ± 30 pg/ml (P < 0.05) (Figure 3B). These results demonstrated that CA-SIIF can decrease IL-12 production by murine macrophages both in vitro and in vivo.

Bottom Line: The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml.CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio, USA. mingyuewang@gmail.com

ABSTRACT

Background: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.

Results: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.

Conclusion: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

Show MeSH
Related in: MedlinePlus