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

Expression of MHC-II and co-stimulatory molecules on BM-DC (A) and cytokine production (B) in the co-culture of BM-DC with OT-II Th lymphocytes.Purified BM-DC (2.5 × 105) were co-cultured with CD4+ OT-II splenocytes (7.5 × 105) for 2 days in 1 ml of medium, with or without 10 μg/ml OVA or OVA-Cl. When indicated, 5 μg/ml of blocking anti-MHC-II mAb or 25 μg/ml anti-CD40L mAb was additionally included. Expression of proteins on BM-DC surfaces was assessed by flow cytometry and cytokine concentration in culture supernatants by ELISA. The results shown are averages +SEM from 4 independent experiments (A) or means +SEM of 4 replicates obtained in a single, representative experiment (B). The data were analysed by ANOVA, combined with the Tukey-Kramer post-test. *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123293.g004: Expression of MHC-II and co-stimulatory molecules on BM-DC (A) and cytokine production (B) in the co-culture of BM-DC with OT-II Th lymphocytes.Purified BM-DC (2.5 × 105) were co-cultured with CD4+ OT-II splenocytes (7.5 × 105) for 2 days in 1 ml of medium, with or without 10 μg/ml OVA or OVA-Cl. When indicated, 5 μg/ml of blocking anti-MHC-II mAb or 25 μg/ml anti-CD40L mAb was additionally included. Expression of proteins on BM-DC surfaces was assessed by flow cytometry and cytokine concentration in culture supernatants by ELISA. The results shown are averages +SEM from 4 independent experiments (A) or means +SEM of 4 replicates obtained in a single, representative experiment (B). The data were analysed by ANOVA, combined with the Tukey-Kramer post-test. *, p < 0.05.

Mentions: In order to gain insight into mechanisms of this efficient Ag presentation in the absence of PRR ligands, we assessed effects of co-culturing BM-DC and OT-II CD4+ T cells, with or without OVA antigens, on the expression of MHC-II and co-stimulatory molecules on BM-DC as well as on cytokine release. Even in the absence of specific antigens, 2-days co-culture of BM-DC with CD4+ T lymphocytes resulted in slight up-regulation of CD40 and CD86 expression on BM-DC (Fig 4A). BM-DC also spontaneously released interleukin (IL)-12p40 subunit, but no production of IL-2 and IFN-γ was observed in the absence of antigen (Fig 4B). Inclusion of OVA in the co-culture led to strong up-regulation of MHC-II and co-stimulatory molecules expression on BM-DC, enhanced IL-12p40 and induced IL-2 and IFN-γ secretion. In comparison to OVA, OVA-Cl induced much higher CD40 expression as well as IL-2 and IFN-γ secretion, but similar MHC-II and CD86 expression. Despite stimulating much higher IFN-γ production, OVA-Cl stimulated similar IL-12p40 production as OVA. Moreover, no detectable production of bioactive IL-12p70 was observed in the co-culture, which, together, indicate that in this system IFN-γ production is IL-12-independent. The role of recognition of peptide-MHC-II complexes on BM-DC by Th lymphocytes as well as of CD40-CD40L interactions in the observed phenomena was assessed with the use of blocking mAb. Blocking MHC-II reversed completely OVA-Cl-stimulated increases of MHC-II and CD86 expression (Fig 4A) as well as of IL-2, IFN-γ and IL-12p40 secretion (Fig 4B). Surprisingly, while anti-MHC-II mAb blocked recognition of peptide-MHC-II complexes by Th lymphocytes, binding of this mAb to BM-DC gave rise to selective activation of these cells, reflected by very strong up-regulation of CD40, but not MHC-II or CD86 expression, and IL-2 production by BM-DC themselves. Acting on BM-DC anti-MHC-II mAb also induced production of IL-12p70, likely related to the induction of IFN-γ production in these cells. In turn, blocking interactions between CD40 and CD40L with the use of anti-CD40L mAb, largely reversed the OVA-Cl-stimulated up-regulation of CD86, but not CD40 expression, and OVA-Cl-stimulated IL-12p40 and IFN-γ secretion, but had no such effect on IL-2 production.


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)

Expression of MHC-II and co-stimulatory molecules on BM-DC (A) and cytokine production (B) in the co-culture of BM-DC with OT-II Th lymphocytes.Purified BM-DC (2.5 × 105) were co-cultured with CD4+ OT-II splenocytes (7.5 × 105) for 2 days in 1 ml of medium, with or without 10 μg/ml OVA or OVA-Cl. When indicated, 5 μg/ml of blocking anti-MHC-II mAb or 25 μg/ml anti-CD40L mAb was additionally included. Expression of proteins on BM-DC surfaces was assessed by flow cytometry and cytokine concentration in culture supernatants by ELISA. The results shown are averages +SEM from 4 independent experiments (A) or means +SEM of 4 replicates obtained in a single, representative experiment (B). The data were analysed by ANOVA, combined with the Tukey-Kramer post-test. *, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123293.g004: Expression of MHC-II and co-stimulatory molecules on BM-DC (A) and cytokine production (B) in the co-culture of BM-DC with OT-II Th lymphocytes.Purified BM-DC (2.5 × 105) were co-cultured with CD4+ OT-II splenocytes (7.5 × 105) for 2 days in 1 ml of medium, with or without 10 μg/ml OVA or OVA-Cl. When indicated, 5 μg/ml of blocking anti-MHC-II mAb or 25 μg/ml anti-CD40L mAb was additionally included. Expression of proteins on BM-DC surfaces was assessed by flow cytometry and cytokine concentration in culture supernatants by ELISA. The results shown are averages +SEM from 4 independent experiments (A) or means +SEM of 4 replicates obtained in a single, representative experiment (B). The data were analysed by ANOVA, combined with the Tukey-Kramer post-test. *, p < 0.05.
Mentions: In order to gain insight into mechanisms of this efficient Ag presentation in the absence of PRR ligands, we assessed effects of co-culturing BM-DC and OT-II CD4+ T cells, with or without OVA antigens, on the expression of MHC-II and co-stimulatory molecules on BM-DC as well as on cytokine release. Even in the absence of specific antigens, 2-days co-culture of BM-DC with CD4+ T lymphocytes resulted in slight up-regulation of CD40 and CD86 expression on BM-DC (Fig 4A). BM-DC also spontaneously released interleukin (IL)-12p40 subunit, but no production of IL-2 and IFN-γ was observed in the absence of antigen (Fig 4B). Inclusion of OVA in the co-culture led to strong up-regulation of MHC-II and co-stimulatory molecules expression on BM-DC, enhanced IL-12p40 and induced IL-2 and IFN-γ secretion. In comparison to OVA, OVA-Cl induced much higher CD40 expression as well as IL-2 and IFN-γ secretion, but similar MHC-II and CD86 expression. Despite stimulating much higher IFN-γ production, OVA-Cl stimulated similar IL-12p40 production as OVA. Moreover, no detectable production of bioactive IL-12p70 was observed in the co-culture, which, together, indicate that in this system IFN-γ production is IL-12-independent. The role of recognition of peptide-MHC-II complexes on BM-DC by Th lymphocytes as well as of CD40-CD40L interactions in the observed phenomena was assessed with the use of blocking mAb. Blocking MHC-II reversed completely OVA-Cl-stimulated increases of MHC-II and CD86 expression (Fig 4A) as well as of IL-2, IFN-γ and IL-12p40 secretion (Fig 4B). Surprisingly, while anti-MHC-II mAb blocked recognition of peptide-MHC-II complexes by Th lymphocytes, binding of this mAb to BM-DC gave rise to selective activation of these cells, reflected by very strong up-regulation of CD40, but not MHC-II or CD86 expression, and IL-2 production by BM-DC themselves. Acting on BM-DC anti-MHC-II mAb also induced production of IL-12p70, likely related to the induction of IFN-γ production in these cells. In turn, blocking interactions between CD40 and CD40L with the use of anti-CD40L mAb, largely reversed the OVA-Cl-stimulated up-regulation of CD86, but not CD40 expression, and OVA-Cl-stimulated IL-12p40 and IFN-γ secretion, but had no such effect on IL-2 production.

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