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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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Cytokine profiles of supernatants obtained from IFN-γ and LPS stimulated monocytes cultured in presence or absence of CA-SIIF. 1 × 106/ml human monocytes were grown in the absence or presence of CA-SIIF with IFN-γ and LPS for 40 h, their supernatants were collected, and the cytokines present in these supernatants were measured using the preprinted human cytokine antibody arrays 5 (Ray Biotech, Inc.). (A) differentially expressed cytokines/chemokines with P value less than 0.05 (n = 3). (B) representative images of cytokine array membranes. (C) cytokine map of the membrane used, showing location of cytokines on the membrane.
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Figure 4: Cytokine profiles of supernatants obtained from IFN-γ and LPS stimulated monocytes cultured in presence or absence of CA-SIIF. 1 × 106/ml human monocytes were grown in the absence or presence of CA-SIIF with IFN-γ and LPS for 40 h, their supernatants were collected, and the cytokines present in these supernatants were measured using the preprinted human cytokine antibody arrays 5 (Ray Biotech, Inc.). (A) differentially expressed cytokines/chemokines with P value less than 0.05 (n = 3). (B) representative images of cytokine array membranes. (C) cytokine map of the membrane used, showing location of cytokines on the membrane.

Mentions: Although CA-SIIF was shown to inhibit monocyte IL-12 production, its effect on production of other cytokines/chemokines was unknown. Since microbial pathogens are known to overcome host immune responses by modulating levels of pro- and anti-inflammatory cytokines, we used membrane arrays to evaluate cytokine/chemokine profile of monocytes exposed to CA-SIIF. We found that in addition to decreased IL-12, CA-SIIF also inhibited production of other cytokines/chemokines including GRO, IL-8, IL-10, IL-13, MCP-1, MCP-2, MIP-1Δ, RANTES, Leptin, Eotaxin-2, LIF and TIMP-2 by monocytes. In contrast, other chemokines like MCP-4, MIF and MIP-3α were increased significantly (P < 0.05, Figure 4). These studies suggested that inhibition of pro-inflammatory cytokines/chemokines and increase of anti-inflammatory ones (e.g. MIF) may be one of the mechanisms by which the inhibitory activity of CA-SIIF is mediated. Alternatively, the balance of pro- and anti- inflammatory cytokines may be changing dynamically following CA-SIIF treatment. For example, IL-10, an anti-inflammatory cytokine was decreased following CA-SIIF treatment, perhaps following an increase in levels of IL-10 prior to our sampling point.


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)

Cytokine profiles of supernatants obtained from IFN-γ and LPS stimulated monocytes cultured in presence or absence of CA-SIIF. 1 × 106/ml human monocytes were grown in the absence or presence of CA-SIIF with IFN-γ and LPS for 40 h, their supernatants were collected, and the cytokines present in these supernatants were measured using the preprinted human cytokine antibody arrays 5 (Ray Biotech, Inc.). (A) differentially expressed cytokines/chemokines with P value less than 0.05 (n = 3). (B) representative images of cytokine array membranes. (C) cytokine map of the membrane used, showing location of cytokines on the membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Cytokine profiles of supernatants obtained from IFN-γ and LPS stimulated monocytes cultured in presence or absence of CA-SIIF. 1 × 106/ml human monocytes were grown in the absence or presence of CA-SIIF with IFN-γ and LPS for 40 h, their supernatants were collected, and the cytokines present in these supernatants were measured using the preprinted human cytokine antibody arrays 5 (Ray Biotech, Inc.). (A) differentially expressed cytokines/chemokines with P value less than 0.05 (n = 3). (B) representative images of cytokine array membranes. (C) cytokine map of the membrane used, showing location of cytokines on the membrane.
Mentions: Although CA-SIIF was shown to inhibit monocyte IL-12 production, its effect on production of other cytokines/chemokines was unknown. Since microbial pathogens are known to overcome host immune responses by modulating levels of pro- and anti-inflammatory cytokines, we used membrane arrays to evaluate cytokine/chemokine profile of monocytes exposed to CA-SIIF. We found that in addition to decreased IL-12, CA-SIIF also inhibited production of other cytokines/chemokines including GRO, IL-8, IL-10, IL-13, MCP-1, MCP-2, MIP-1Δ, RANTES, Leptin, Eotaxin-2, LIF and TIMP-2 by monocytes. In contrast, other chemokines like MCP-4, MIF and MIP-3α were increased significantly (P < 0.05, Figure 4). These studies suggested that inhibition of pro-inflammatory cytokines/chemokines and increase of anti-inflammatory ones (e.g. MIF) may be one of the mechanisms by which the inhibitory activity of CA-SIIF is mediated. Alternatively, the balance of pro- and anti- inflammatory cytokines may be changing dynamically following CA-SIIF treatment. For example, IL-10, an anti-inflammatory cytokine was decreased following CA-SIIF treatment, perhaps following an increase in levels of IL-10 prior to our sampling point.

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