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Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

Biedroń R, Konopiński MK, Marcinkiewicz J, Józefowski S - PLoS ONE (2015)

Bottom Line: Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses.Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins.Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Jagiellonian University Medical College, Cracow, Poland.

ABSTRACT
The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

No MeSH data available.


Related in: MedlinePlus

OVA and OVA-Cl bind to the same receptors, which are inhibited by mannan (Man), DS and CS and also shared with other HOCl-modified proteins and glycoproteins.(A, B, C) BM-DC were pre-incubated for 20 min with 1 mg/ml of indicated unlabelled proteins (A), 6 mg/ml Man, 0.2 mg/ml DS or CS (B, C) before the same volume of double-concentrated solution of AF-OVA or AF-OVA-Cl was added to give the final concentration of 5 μg/ml and the incubation was continued for 1 h (A, B) or 2 h (C) in a cell culture incubator. Following washing, cell-associated fluorescence was quantified by flow cytometry. (D) BM-DC were incubated for 1 h with 5 μg/ml AF-OVA-Cl, washed and either directly assessed for antigen uptake or incubated for another 1 h in medium alone before the cell-associated fluorescence was measured. (E) Following pre-incubation with DS or Man, BM-DC were incubated for 1 h on ice with 20 μg/ml DQ-OVA. Unbound DQ-OVA was washed out and the cells were either directly assessed for DQ-OVA binding (“4°C”) or transfer to 37°C for 2 h before the measurement. Results shown are averages ± SEM of triplicates obtained in single experiments, each repeated at least 3 times with similar results. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test (A-C, E) or with the Student’s t-test (D). *, p < 0.05; NS, non-significant.
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pone.0123293.g005: OVA and OVA-Cl bind to the same receptors, which are inhibited by mannan (Man), DS and CS and also shared with other HOCl-modified proteins and glycoproteins.(A, B, C) BM-DC were pre-incubated for 20 min with 1 mg/ml of indicated unlabelled proteins (A), 6 mg/ml Man, 0.2 mg/ml DS or CS (B, C) before the same volume of double-concentrated solution of AF-OVA or AF-OVA-Cl was added to give the final concentration of 5 μg/ml and the incubation was continued for 1 h (A, B) or 2 h (C) in a cell culture incubator. Following washing, cell-associated fluorescence was quantified by flow cytometry. (D) BM-DC were incubated for 1 h with 5 μg/ml AF-OVA-Cl, washed and either directly assessed for antigen uptake or incubated for another 1 h in medium alone before the cell-associated fluorescence was measured. (E) Following pre-incubation with DS or Man, BM-DC were incubated for 1 h on ice with 20 μg/ml DQ-OVA. Unbound DQ-OVA was washed out and the cells were either directly assessed for DQ-OVA binding (“4°C”) or transfer to 37°C for 2 h before the measurement. Results shown are averages ± SEM of triplicates obtained in single experiments, each repeated at least 3 times with similar results. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test (A-C, E) or with the Student’s t-test (D). *, p < 0.05; NS, non-significant.

Mentions: Uptake of both AF-OVA and AF-OVA-Cl was inhibited more strongly by unlabelled OVA-Cl than by unlabelled OVA (Fig 5A, left panel). This mutual cross-competition indicates that OVA and OVA-Cl bind to shared receptors, exhibiting higher affinity for OVA-Cl as compared to OVA.


Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

Biedroń R, Konopiński MK, Marcinkiewicz J, Józefowski S - PLoS ONE (2015)

OVA and OVA-Cl bind to the same receptors, which are inhibited by mannan (Man), DS and CS and also shared with other HOCl-modified proteins and glycoproteins.(A, B, C) BM-DC were pre-incubated for 20 min with 1 mg/ml of indicated unlabelled proteins (A), 6 mg/ml Man, 0.2 mg/ml DS or CS (B, C) before the same volume of double-concentrated solution of AF-OVA or AF-OVA-Cl was added to give the final concentration of 5 μg/ml and the incubation was continued for 1 h (A, B) or 2 h (C) in a cell culture incubator. Following washing, cell-associated fluorescence was quantified by flow cytometry. (D) BM-DC were incubated for 1 h with 5 μg/ml AF-OVA-Cl, washed and either directly assessed for antigen uptake or incubated for another 1 h in medium alone before the cell-associated fluorescence was measured. (E) Following pre-incubation with DS or Man, BM-DC were incubated for 1 h on ice with 20 μg/ml DQ-OVA. Unbound DQ-OVA was washed out and the cells were either directly assessed for DQ-OVA binding (“4°C”) or transfer to 37°C for 2 h before the measurement. Results shown are averages ± SEM of triplicates obtained in single experiments, each repeated at least 3 times with similar results. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test (A-C, E) or with the Student’s t-test (D). *, p < 0.05; NS, non-significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g005: OVA and OVA-Cl bind to the same receptors, which are inhibited by mannan (Man), DS and CS and also shared with other HOCl-modified proteins and glycoproteins.(A, B, C) BM-DC were pre-incubated for 20 min with 1 mg/ml of indicated unlabelled proteins (A), 6 mg/ml Man, 0.2 mg/ml DS or CS (B, C) before the same volume of double-concentrated solution of AF-OVA or AF-OVA-Cl was added to give the final concentration of 5 μg/ml and the incubation was continued for 1 h (A, B) or 2 h (C) in a cell culture incubator. Following washing, cell-associated fluorescence was quantified by flow cytometry. (D) BM-DC were incubated for 1 h with 5 μg/ml AF-OVA-Cl, washed and either directly assessed for antigen uptake or incubated for another 1 h in medium alone before the cell-associated fluorescence was measured. (E) Following pre-incubation with DS or Man, BM-DC were incubated for 1 h on ice with 20 μg/ml DQ-OVA. Unbound DQ-OVA was washed out and the cells were either directly assessed for DQ-OVA binding (“4°C”) or transfer to 37°C for 2 h before the measurement. Results shown are averages ± SEM of triplicates obtained in single experiments, each repeated at least 3 times with similar results. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test (A-C, E) or with the Student’s t-test (D). *, p < 0.05; NS, non-significant.
Mentions: Uptake of both AF-OVA and AF-OVA-Cl was inhibited more strongly by unlabelled OVA-Cl than by unlabelled OVA (Fig 5A, left panel). This mutual cross-competition indicates that OVA and OVA-Cl bind to shared receptors, exhibiting higher affinity for OVA-Cl as compared to OVA.

Bottom Line: Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses.Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins.Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Jagiellonian University Medical College, Cracow, Poland.

ABSTRACT
The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

No MeSH data available.


Related in: MedlinePlus