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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

Immunogenicity of proteins is enhanced as the result of oxidation by endogenous, neutrophils-derived HOCl.(A) Luminol- or lucigenin-enhanced chemiluminescence stimulated by zymosan (Zym) or heat-killed S. aureus (Sa) in WT and MPO-/- neutrophils. (B) Presentation of OVA, as assessed by IL-2 production, by PEM pre-incubated with OVA, S. aureus and WT or MPO-/- neutrophils (PMN) to subsequently added CD4+ OT-II lymphocytes. (C) Uptake of pHrodo-labelled native or HOCl-oxidised YAD or HSA by BM-DC, assessed by flow cytometry. Results of single experiments shown were repeated 2 more times with similar results. (D) Titers of YAD-specific IgG in sera of mice primed with 20 μg YAD and hot alkali-treated zymosan or TNF-α plus WKYMVm peptide and boosted 2 weeks later with 20 μg of YAD alone. Sera were collected 8 days after the boost immunization and titers of YAD-specific IgG were determined by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test, to compare all pairs of groups (B, D) or by the Dunnett’s test, to make comparisons with the control group (“Autofluor.”) (C). *, p < 0.05; NS, non-significant.
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pone.0123293.g006: Immunogenicity of proteins is enhanced as the result of oxidation by endogenous, neutrophils-derived HOCl.(A) Luminol- or lucigenin-enhanced chemiluminescence stimulated by zymosan (Zym) or heat-killed S. aureus (Sa) in WT and MPO-/- neutrophils. (B) Presentation of OVA, as assessed by IL-2 production, by PEM pre-incubated with OVA, S. aureus and WT or MPO-/- neutrophils (PMN) to subsequently added CD4+ OT-II lymphocytes. (C) Uptake of pHrodo-labelled native or HOCl-oxidised YAD or HSA by BM-DC, assessed by flow cytometry. Results of single experiments shown were repeated 2 more times with similar results. (D) Titers of YAD-specific IgG in sera of mice primed with 20 μg YAD and hot alkali-treated zymosan or TNF-α plus WKYMVm peptide and boosted 2 weeks later with 20 μg of YAD alone. Sera were collected 8 days after the boost immunization and titers of YAD-specific IgG were determined by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test, to compare all pairs of groups (B, D) or by the Dunnett’s test, to make comparisons with the control group (“Autofluor.”) (C). *, p < 0.05; NS, non-significant.

Mentions: The results presented above confirm that treatment with HOCl added as a reagent increases immunogenicity of OVA. The purpose of the next series of experiments was to verify the physiological significance of this process, i.e. evaluating the possibility that oxidation by endogenous HOCl, produced by activated neutrophils, leads to increased immunogenicity of proteins. For this purpose, adherent PEM were co-cultured with neutrophils in the presence of native OVA. Production of HOCl in neutrophils was induced by heat-killed S. aureus. As shown in Fig 6A, both the bacteria and zymosan stimulated ~10-times stronger luminol chemiluminescence in WT than in MPO-/- neutrophils (as estimated by calculating the areas under curves), confirming that chemiluminescence under these conditions depends on MPO activity. Consistently, the zymosan-stimulated luminol chemiluminescence in WT neutrophils was inhibited in ~63% by 0.5 mM ABAH, an inhibitor of MPO. In contrast, both zymosan (Fig 6A, right panel) and S. aureus (S2 Fig) stimulated ~2-fold stronger lucigenin chemiluminescence in MPO-/- than in WT neutrophils. Zymosan-stimulated lucigenin chemiluminescence was quenched completely by 5 kU/ml SOD (Fig 6A, right panel), but unaffected by ABAH (S2 Fig), confirming the major role of extracellular O2- in generating lucigenin chemiluminescence.


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)

Immunogenicity of proteins is enhanced as the result of oxidation by endogenous, neutrophils-derived HOCl.(A) Luminol- or lucigenin-enhanced chemiluminescence stimulated by zymosan (Zym) or heat-killed S. aureus (Sa) in WT and MPO-/- neutrophils. (B) Presentation of OVA, as assessed by IL-2 production, by PEM pre-incubated with OVA, S. aureus and WT or MPO-/- neutrophils (PMN) to subsequently added CD4+ OT-II lymphocytes. (C) Uptake of pHrodo-labelled native or HOCl-oxidised YAD or HSA by BM-DC, assessed by flow cytometry. Results of single experiments shown were repeated 2 more times with similar results. (D) Titers of YAD-specific IgG in sera of mice primed with 20 μg YAD and hot alkali-treated zymosan or TNF-α plus WKYMVm peptide and boosted 2 weeks later with 20 μg of YAD alone. Sera were collected 8 days after the boost immunization and titers of YAD-specific IgG were determined by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test, to compare all pairs of groups (B, D) or by the Dunnett’s test, to make comparisons with the control group (“Autofluor.”) (C). *, p < 0.05; NS, non-significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g006: Immunogenicity of proteins is enhanced as the result of oxidation by endogenous, neutrophils-derived HOCl.(A) Luminol- or lucigenin-enhanced chemiluminescence stimulated by zymosan (Zym) or heat-killed S. aureus (Sa) in WT and MPO-/- neutrophils. (B) Presentation of OVA, as assessed by IL-2 production, by PEM pre-incubated with OVA, S. aureus and WT or MPO-/- neutrophils (PMN) to subsequently added CD4+ OT-II lymphocytes. (C) Uptake of pHrodo-labelled native or HOCl-oxidised YAD or HSA by BM-DC, assessed by flow cytometry. Results of single experiments shown were repeated 2 more times with similar results. (D) Titers of YAD-specific IgG in sera of mice primed with 20 μg YAD and hot alkali-treated zymosan or TNF-α plus WKYMVm peptide and boosted 2 weeks later with 20 μg of YAD alone. Sera were collected 8 days after the boost immunization and titers of YAD-specific IgG were determined by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test, to compare all pairs of groups (B, D) or by the Dunnett’s test, to make comparisons with the control group (“Autofluor.”) (C). *, p < 0.05; NS, non-significant.
Mentions: The results presented above confirm that treatment with HOCl added as a reagent increases immunogenicity of OVA. The purpose of the next series of experiments was to verify the physiological significance of this process, i.e. evaluating the possibility that oxidation by endogenous HOCl, produced by activated neutrophils, leads to increased immunogenicity of proteins. For this purpose, adherent PEM were co-cultured with neutrophils in the presence of native OVA. Production of HOCl in neutrophils was induced by heat-killed S. aureus. As shown in Fig 6A, both the bacteria and zymosan stimulated ~10-times stronger luminol chemiluminescence in WT than in MPO-/- neutrophils (as estimated by calculating the areas under curves), confirming that chemiluminescence under these conditions depends on MPO activity. Consistently, the zymosan-stimulated luminol chemiluminescence in WT neutrophils was inhibited in ~63% by 0.5 mM ABAH, an inhibitor of MPO. In contrast, both zymosan (Fig 6A, right panel) and S. aureus (S2 Fig) stimulated ~2-fold stronger lucigenin chemiluminescence in MPO-/- than in WT neutrophils. Zymosan-stimulated lucigenin chemiluminescence was quenched completely by 5 kU/ml SOD (Fig 6A, right panel), but unaffected by ABAH (S2 Fig), confirming the major role of extracellular O2- in generating lucigenin chemiluminescence.

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