Limits...
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

Effects of SR-A or CD36 deficiency on antigen uptake, expression of endocytic receptors and antigen presentation to CD4+ OT-II splenocytes.(A, D) Uptake of indicated, fluorescently labelled proteins, present at 5 μg/ml by BM-DC (A) and PEM (D) was assessed by flow cytometry. (B, E) Specific binding of Ab to receptors (geometric mean fluorescence intensity) on BM-DC (B) and PEM (E) which was obtained by subtracting binding of control Ab from the total binding of receptor-specific Ab. (C, F) IL-2 production in 1- or 2-days co-cultures of BM-DC (C) or PEM (F) with CD4+ OT-II lymphocytes. Directly before the co-incubation with lymphocytes, APC were pulsed for 3.5 h with 20 μg/ml OVA or 7 μg/ml OVA-Cl. The data shown on graphs A-G are means +SEM from 6–8 independent experiments. (H) Titers of OVA- or HSA-specific IgM in sera of mice immunized 8 days earlier with 20 μg OVA-Cl or HSA-Cl. Points represent titer values in individual mice and horizontal lines geometric means. The data were analysed with the regular (H) or repeated measures (A-G) ANOVA and the Dunnett’s post-test was used to make comparisons with the control groups (WT). *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g003: Effects of SR-A or CD36 deficiency on antigen uptake, expression of endocytic receptors and antigen presentation to CD4+ OT-II splenocytes.(A, D) Uptake of indicated, fluorescently labelled proteins, present at 5 μg/ml by BM-DC (A) and PEM (D) was assessed by flow cytometry. (B, E) Specific binding of Ab to receptors (geometric mean fluorescence intensity) on BM-DC (B) and PEM (E) which was obtained by subtracting binding of control Ab from the total binding of receptor-specific Ab. (C, F) IL-2 production in 1- or 2-days co-cultures of BM-DC (C) or PEM (F) with CD4+ OT-II lymphocytes. Directly before the co-incubation with lymphocytes, APC were pulsed for 3.5 h with 20 μg/ml OVA or 7 μg/ml OVA-Cl. The data shown on graphs A-G are means +SEM from 6–8 independent experiments. (H) Titers of OVA- or HSA-specific IgM in sera of mice immunized 8 days earlier with 20 μg OVA-Cl or HSA-Cl. Points represent titer values in individual mice and horizontal lines geometric means. The data were analysed with the regular (H) or repeated measures (A-G) ANOVA and the Dunnett’s post-test was used to make comparisons with the control groups (WT). *, p < 0.05.

Mentions: Efficient presentation to naïve CD4+ lymphocytes by DC of protein antigens alone, not contaminated with microbial products, is inconsistent with the currently dominating paradigm, according to which such presentation requires activation of pattern-recognition receptors (PRR) on DC [21]. As DC activation might be caused by GM-CSF used in the culture of BM-DC, we decided to assess whether OVA-Cl alone would also be able to initiate immune response in the culture of splenocytes isolated from naïve OT-II mice. Also in the culture of unfractionated splenocytes OVA-Cl stimulated proliferation of lymphocytes which was higher than that stimulated by OVA (Fig 1C, left panel). There were, however, two major differences with previous experiments employing BM-DC. First, in the case of unfractionated splenocytes proliferation was also stimulated by native OVA. This difference might be caused by higher expression of endocytic receptors on splenic DC than on BM-DC or the presence among splenocytes of other cell types. Consistent with the former possibility, also BM-DC effectively presented OVA when they were allowed to endocytose it for a longer period (Fig 3C). Second, unlike in the co-culture with BM-DC, in unfractionated splenocytes lymphocyte proliferation was not enhanced by LPS. However, under these conditions LPS did promote differentiation of naïve T lymphocytes towards Th1 cells, as indicated by strongly increased interferon-γ (IFN-γ) production. Interestingly, very high IFN-γ production was also stimulated by OVA-Cl alone, which was not only higher than that stimulated by OVA alone but also by OVA plus LPS (Fig 1C, right panel).


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)

Effects of SR-A or CD36 deficiency on antigen uptake, expression of endocytic receptors and antigen presentation to CD4+ OT-II splenocytes.(A, D) Uptake of indicated, fluorescently labelled proteins, present at 5 μg/ml by BM-DC (A) and PEM (D) was assessed by flow cytometry. (B, E) Specific binding of Ab to receptors (geometric mean fluorescence intensity) on BM-DC (B) and PEM (E) which was obtained by subtracting binding of control Ab from the total binding of receptor-specific Ab. (C, F) IL-2 production in 1- or 2-days co-cultures of BM-DC (C) or PEM (F) with CD4+ OT-II lymphocytes. Directly before the co-incubation with lymphocytes, APC were pulsed for 3.5 h with 20 μg/ml OVA or 7 μg/ml OVA-Cl. The data shown on graphs A-G are means +SEM from 6–8 independent experiments. (H) Titers of OVA- or HSA-specific IgM in sera of mice immunized 8 days earlier with 20 μg OVA-Cl or HSA-Cl. Points represent titer values in individual mice and horizontal lines geometric means. The data were analysed with the regular (H) or repeated measures (A-G) ANOVA and the Dunnett’s post-test was used to make comparisons with the control groups (WT). *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123293.g003: Effects of SR-A or CD36 deficiency on antigen uptake, expression of endocytic receptors and antigen presentation to CD4+ OT-II splenocytes.(A, D) Uptake of indicated, fluorescently labelled proteins, present at 5 μg/ml by BM-DC (A) and PEM (D) was assessed by flow cytometry. (B, E) Specific binding of Ab to receptors (geometric mean fluorescence intensity) on BM-DC (B) and PEM (E) which was obtained by subtracting binding of control Ab from the total binding of receptor-specific Ab. (C, F) IL-2 production in 1- or 2-days co-cultures of BM-DC (C) or PEM (F) with CD4+ OT-II lymphocytes. Directly before the co-incubation with lymphocytes, APC were pulsed for 3.5 h with 20 μg/ml OVA or 7 μg/ml OVA-Cl. The data shown on graphs A-G are means +SEM from 6–8 independent experiments. (H) Titers of OVA- or HSA-specific IgM in sera of mice immunized 8 days earlier with 20 μg OVA-Cl or HSA-Cl. Points represent titer values in individual mice and horizontal lines geometric means. The data were analysed with the regular (H) or repeated measures (A-G) ANOVA and the Dunnett’s post-test was used to make comparisons with the control groups (WT). *, p < 0.05.
Mentions: Efficient presentation to naïve CD4+ lymphocytes by DC of protein antigens alone, not contaminated with microbial products, is inconsistent with the currently dominating paradigm, according to which such presentation requires activation of pattern-recognition receptors (PRR) on DC [21]. As DC activation might be caused by GM-CSF used in the culture of BM-DC, we decided to assess whether OVA-Cl alone would also be able to initiate immune response in the culture of splenocytes isolated from naïve OT-II mice. Also in the culture of unfractionated splenocytes OVA-Cl stimulated proliferation of lymphocytes which was higher than that stimulated by OVA (Fig 1C, left panel). There were, however, two major differences with previous experiments employing BM-DC. First, in the case of unfractionated splenocytes proliferation was also stimulated by native OVA. This difference might be caused by higher expression of endocytic receptors on splenic DC than on BM-DC or the presence among splenocytes of other cell types. Consistent with the former possibility, also BM-DC effectively presented OVA when they were allowed to endocytose it for a longer period (Fig 3C). Second, unlike in the co-culture with BM-DC, in unfractionated splenocytes lymphocyte proliferation was not enhanced by LPS. However, under these conditions LPS did promote differentiation of naïve T lymphocytes towards Th1 cells, as indicated by strongly increased interferon-γ (IFN-γ) production. Interestingly, very high IFN-γ production was also stimulated by OVA-Cl alone, which was not only higher than that stimulated by OVA alone but also by OVA plus LPS (Fig 1C, right panel).

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