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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

Humoral immune response stimulated by native and HOCl-oxidised proteins in vivo.(A, C, D) CBA mice were immunized with the indicated antigens. Two weeks later all mice received boost immunization with 20 μg of native proteins and 8 days later sera were collected for the determination of titers of specific Ab by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. (B) Uptake of fluorescently-labelled ligands by PEM was determined by flow cytometry. The data were analysed by ANOVA and the Benferroni post-test was applied to compare the indicated pairs of columns. *, p < 0.05; NS, non-significant.
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pone.0123293.g002: Humoral immune response stimulated by native and HOCl-oxidised proteins in vivo.(A, C, D) CBA mice were immunized with the indicated antigens. Two weeks later all mice received boost immunization with 20 μg of native proteins and 8 days later sera were collected for the determination of titers of specific Ab by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. (B) Uptake of fluorescently-labelled ligands by PEM was determined by flow cytometry. The data were analysed by ANOVA and the Benferroni post-test was applied to compare the indicated pairs of columns. *, p < 0.05; NS, non-significant.

Mentions: Another potential cause of increased immunogenicity of OVA-Cl may be enhanced uptake and intracellular processing by APC. In order to test this possibility, we labelled OVA and OVA-Cl with the Alexa Fluor 647 fluorescent dye (AF) and studied their uptake by APC. We found that both PEM (Fig 2B) and BM-DC (Fig 1B) endocytosed much more AF-OVA-Cl than AF-OVA. Of note, stimulation of lymphocyte proliferation in the presence of LPS correlated with the level of antigen uptake (compare Fig 1A, left panel, and 1B). Thus, it seems that in the presence of LPS the major limiting factors in antigen presentation are amounts of endocytosed antigens and the ability of BM-DC to process and present these antigens. Consistent with this interpretation, increasing the duration of co-culture to 3 days resulted in the shift of OVA-Cl concentrations producing the maximal effect to higher concentrations (Fig 1A, right panel). In contrast, there was no correlation between antigen uptake and lymphocyte proliferation in the absence of LPS (Fig 1A and 1B), which indicates that also factors other than enhanced uptake contribute to increased immunogenicity of HOCl-modified 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)

Humoral immune response stimulated by native and HOCl-oxidised proteins in vivo.(A, C, D) CBA mice were immunized with the indicated antigens. Two weeks later all mice received boost immunization with 20 μg of native proteins and 8 days later sera were collected for the determination of titers of specific Ab by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. (B) Uptake of fluorescently-labelled ligands by PEM was determined by flow cytometry. The data were analysed by ANOVA and the Benferroni post-test was applied to compare the indicated pairs of columns. *, p < 0.05; NS, non-significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g002: Humoral immune response stimulated by native and HOCl-oxidised proteins in vivo.(A, C, D) CBA mice were immunized with the indicated antigens. Two weeks later all mice received boost immunization with 20 μg of native proteins and 8 days later sera were collected for the determination of titers of specific Ab by ELISA. Points represent titer values in individual mice and horizontal lines geometric means. (B) Uptake of fluorescently-labelled ligands by PEM was determined by flow cytometry. The data were analysed by ANOVA and the Benferroni post-test was applied to compare the indicated pairs of columns. *, p < 0.05; NS, non-significant.
Mentions: Another potential cause of increased immunogenicity of OVA-Cl may be enhanced uptake and intracellular processing by APC. In order to test this possibility, we labelled OVA and OVA-Cl with the Alexa Fluor 647 fluorescent dye (AF) and studied their uptake by APC. We found that both PEM (Fig 2B) and BM-DC (Fig 1B) endocytosed much more AF-OVA-Cl than AF-OVA. Of note, stimulation of lymphocyte proliferation in the presence of LPS correlated with the level of antigen uptake (compare Fig 1A, left panel, and 1B). Thus, it seems that in the presence of LPS the major limiting factors in antigen presentation are amounts of endocytosed antigens and the ability of BM-DC to process and present these antigens. Consistent with this interpretation, increasing the duration of co-culture to 3 days resulted in the shift of OVA-Cl concentrations producing the maximal effect to higher concentrations (Fig 1A, right panel). In contrast, there was no correlation between antigen uptake and lymphocyte proliferation in the absence of LPS (Fig 1A and 1B), which indicates that also factors other than enhanced uptake contribute to increased immunogenicity of HOCl-modified 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