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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

The proposed mechanism of the immunoenhancing effect caused by HOCl-mediated oxidation of protein antigens.Administration of adjuvants, infection or sterile injury trigger acute inflammation, characterized by recruitment and activation of neutrophils. Activated neutrophils produce HOCl which causes non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by DC through MR and SR-A, processed and presented as complexes with MHC-II on their surface. TCR-mediated cognate interactions of Th lymphocytes with peptide-MHC-II complexes on DC induce IL-2 production in Th lymphocytes (not shown) and up-regulate expression of CD40 on DC and of CD40L on lymphocytes. Upon ligation with CD40L, CD40 induces expression of CD86, a ligand for CD28 on Th lymphocytes. In the absence of PRR ligands, presentation of low density of peptide-MHC-II complexes on DC stimulates differentiation of naive Th lymphocytes towards Th2 cells. The Th2 polarization is reinforced by intracellular signalling triggered upon binding of HOCl-oxidised proteins to SR-A or MR, leading to the suppression of IL-12 and enhancement of IL-10 production by DC.
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pone.0123293.g012: The proposed mechanism of the immunoenhancing effect caused by HOCl-mediated oxidation of protein antigens.Administration of adjuvants, infection or sterile injury trigger acute inflammation, characterized by recruitment and activation of neutrophils. Activated neutrophils produce HOCl which causes non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by DC through MR and SR-A, processed and presented as complexes with MHC-II on their surface. TCR-mediated cognate interactions of Th lymphocytes with peptide-MHC-II complexes on DC induce IL-2 production in Th lymphocytes (not shown) and up-regulate expression of CD40 on DC and of CD40L on lymphocytes. Upon ligation with CD40L, CD40 induces expression of CD86, a ligand for CD28 on Th lymphocytes. In the absence of PRR ligands, presentation of low density of peptide-MHC-II complexes on DC stimulates differentiation of naive Th lymphocytes towards Th2 cells. The Th2 polarization is reinforced by intracellular signalling triggered upon binding of HOCl-oxidised proteins to SR-A or MR, leading to the suppression of IL-12 and enhancement of IL-10 production by DC.

Mentions: In summary, our results have revealed a mechanism of recognition of non-self proteins which is accomplished by co-operation between innate and adaptive immunity. We postulate that this mechanism may enable the immune system to detect infections caused by parasitic worms and possibly also by protozoan parasites, which, as being biochemically very similar, do not synthesize kinds of compounds absent in mammalian hosts, and therefore cannot be detected by innate immune cells through their PRR [68,69]. Main biochemical differences between mammalian hosts and these parasites seem to occur in amino acid sequences of proteins, but proteins of all eukaryotic organisms are built from the same set of amino acids, and as such are not good candidates for PRR ligands. However, in the detection of differences in amino acid sequences of proteins lymphocytes, the major cell type of adoptive immunity, are specialized, thanks to being furnished with recombined immunoglobulin receptors. Consequently, one may speculate that infections caused by protozoan and metazoan parasites might be an important part of selection pressure responsible for the evolving of adaptive immunity in jawed vertebrates. The first step in our proposed mechanism of recognition of non-self proteins is induction of acute inflammation, being a non-specific response triggered by essentially any disturbance of tissue homeostasis (Fig 12). One of the major goals of acute inflammation seems to be the recruitment of professional phagocytes. According to the model proposed, HOCl produced by these phagocytes would cause non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by APC through several endocytic receptors and presented on their surfaces as complexes with MHC molecules. In case of sterile injury, presentation of self proteins only by APC would not induce adaptive immune responses, whereas endocytic receptors, by scavenging apoptotic cells and tissue debris and by regulating production of growth factors and cytokines, would promote resolution of inflammation, immunosuppression and tissue repair [70,71]. In contrast, strongly increased affinity of non-self proteins to endocytic receptors, resulting from their HOCl-mediated oxidation, would allow the immune system to detect their even very small amounts. In the absence of PRR ligands, the major factor determining the type (polarisation) of adoptive immune response may be the dose of antigens. Presentation of high density of complexes of MHC-II with non-self peptides by APC, which either phagocytosed or became infected with intracellular protozoa, would result in the induction of Th1-polarised immune responses, characteristic of infections caused by this type of pathogens [72,73]. In turn, the presentation of only low density of MHC-II-peptide complexes would favour the Th2/Treg polarisation of adoptive immunity during infections with parasitic worms. The Th2/Treg polarisation under these conditions might be reinforced by intracellular signalling triggered upon ligation of endocytic receptors, leading to suppression of IL-12 and IL-6 and stimulation of IL-10 production and therefore creating a cytokine milieu favouring differentiation of Th2/Treg over Th1/Th17 lymphocytes. This possibility is consistent with the observed in this study inability of LPS to reverse the Th2 polarisation of immune responses to HOCl-oxidised proteins and supported by results of our previous studies on signalling abilities of endocytic receptors. We have demonstrated that upon ligation with specific Ab, DS or AcLDL SR-A mediates inhibition of LPS-stimulated IL-6 and IL-12, but enhancement of IL-10 production [18,31], whereas ligation of CD36 with Ab was sufficient alone to induce very high production of IL-10, capable of inhibiting IL-6 and IL-12 production in PEM [18]. Others reported that also MR mediates inhibition of IL-12 and enhancement of IL-10 release [74,75]. In this context, our observation that, unlike in the case of OVA-Cl, increasing the dose of HSA-Cl failed to produce the Th1-polarization of humoral response might be explained by binding of HSA-Cl to SR-A with a higher affinity than of OVA-Cl (Figs 3A and 7G) [18]. Thus, HOCl-oxidised proteins may share the mechanism of the Th2-polarising effect with products (proteins and glycoproteins) excreted/secreted by parasitic worms, which were found to be ligands of the same endocytic receptors as HOCl-modified proteins, including MR [76–78] and SR-A [79]. Acting on APC these products inhibit IL-12 and enhance IL-10 production and, consequently, induce strongly Th2-polarised immune responses in vivo [76–79].


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)

The proposed mechanism of the immunoenhancing effect caused by HOCl-mediated oxidation of protein antigens.Administration of adjuvants, infection or sterile injury trigger acute inflammation, characterized by recruitment and activation of neutrophils. Activated neutrophils produce HOCl which causes non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by DC through MR and SR-A, processed and presented as complexes with MHC-II on their surface. TCR-mediated cognate interactions of Th lymphocytes with peptide-MHC-II complexes on DC induce IL-2 production in Th lymphocytes (not shown) and up-regulate expression of CD40 on DC and of CD40L on lymphocytes. Upon ligation with CD40L, CD40 induces expression of CD86, a ligand for CD28 on Th lymphocytes. In the absence of PRR ligands, presentation of low density of peptide-MHC-II complexes on DC stimulates differentiation of naive Th lymphocytes towards Th2 cells. The Th2 polarization is reinforced by intracellular signalling triggered upon binding of HOCl-oxidised proteins to SR-A or MR, leading to the suppression of IL-12 and enhancement of IL-10 production by DC.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g012: The proposed mechanism of the immunoenhancing effect caused by HOCl-mediated oxidation of protein antigens.Administration of adjuvants, infection or sterile injury trigger acute inflammation, characterized by recruitment and activation of neutrophils. Activated neutrophils produce HOCl which causes non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by DC through MR and SR-A, processed and presented as complexes with MHC-II on their surface. TCR-mediated cognate interactions of Th lymphocytes with peptide-MHC-II complexes on DC induce IL-2 production in Th lymphocytes (not shown) and up-regulate expression of CD40 on DC and of CD40L on lymphocytes. Upon ligation with CD40L, CD40 induces expression of CD86, a ligand for CD28 on Th lymphocytes. In the absence of PRR ligands, presentation of low density of peptide-MHC-II complexes on DC stimulates differentiation of naive Th lymphocytes towards Th2 cells. The Th2 polarization is reinforced by intracellular signalling triggered upon binding of HOCl-oxidised proteins to SR-A or MR, leading to the suppression of IL-12 and enhancement of IL-10 production by DC.
Mentions: In summary, our results have revealed a mechanism of recognition of non-self proteins which is accomplished by co-operation between innate and adaptive immunity. We postulate that this mechanism may enable the immune system to detect infections caused by parasitic worms and possibly also by protozoan parasites, which, as being biochemically very similar, do not synthesize kinds of compounds absent in mammalian hosts, and therefore cannot be detected by innate immune cells through their PRR [68,69]. Main biochemical differences between mammalian hosts and these parasites seem to occur in amino acid sequences of proteins, but proteins of all eukaryotic organisms are built from the same set of amino acids, and as such are not good candidates for PRR ligands. However, in the detection of differences in amino acid sequences of proteins lymphocytes, the major cell type of adoptive immunity, are specialized, thanks to being furnished with recombined immunoglobulin receptors. Consequently, one may speculate that infections caused by protozoan and metazoan parasites might be an important part of selection pressure responsible for the evolving of adaptive immunity in jawed vertebrates. The first step in our proposed mechanism of recognition of non-self proteins is induction of acute inflammation, being a non-specific response triggered by essentially any disturbance of tissue homeostasis (Fig 12). One of the major goals of acute inflammation seems to be the recruitment of professional phagocytes. According to the model proposed, HOCl produced by these phagocytes would cause non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by APC through several endocytic receptors and presented on their surfaces as complexes with MHC molecules. In case of sterile injury, presentation of self proteins only by APC would not induce adaptive immune responses, whereas endocytic receptors, by scavenging apoptotic cells and tissue debris and by regulating production of growth factors and cytokines, would promote resolution of inflammation, immunosuppression and tissue repair [70,71]. In contrast, strongly increased affinity of non-self proteins to endocytic receptors, resulting from their HOCl-mediated oxidation, would allow the immune system to detect their even very small amounts. In the absence of PRR ligands, the major factor determining the type (polarisation) of adoptive immune response may be the dose of antigens. Presentation of high density of complexes of MHC-II with non-self peptides by APC, which either phagocytosed or became infected with intracellular protozoa, would result in the induction of Th1-polarised immune responses, characteristic of infections caused by this type of pathogens [72,73]. In turn, the presentation of only low density of MHC-II-peptide complexes would favour the Th2/Treg polarisation of adoptive immunity during infections with parasitic worms. The Th2/Treg polarisation under these conditions might be reinforced by intracellular signalling triggered upon ligation of endocytic receptors, leading to suppression of IL-12 and IL-6 and stimulation of IL-10 production and therefore creating a cytokine milieu favouring differentiation of Th2/Treg over Th1/Th17 lymphocytes. This possibility is consistent with the observed in this study inability of LPS to reverse the Th2 polarisation of immune responses to HOCl-oxidised proteins and supported by results of our previous studies on signalling abilities of endocytic receptors. We have demonstrated that upon ligation with specific Ab, DS or AcLDL SR-A mediates inhibition of LPS-stimulated IL-6 and IL-12, but enhancement of IL-10 production [18,31], whereas ligation of CD36 with Ab was sufficient alone to induce very high production of IL-10, capable of inhibiting IL-6 and IL-12 production in PEM [18]. Others reported that also MR mediates inhibition of IL-12 and enhancement of IL-10 release [74,75]. In this context, our observation that, unlike in the case of OVA-Cl, increasing the dose of HSA-Cl failed to produce the Th1-polarization of humoral response might be explained by binding of HSA-Cl to SR-A with a higher affinity than of OVA-Cl (Figs 3A and 7G) [18]. Thus, HOCl-oxidised proteins may share the mechanism of the Th2-polarising effect with products (proteins and glycoproteins) excreted/secreted by parasitic worms, which were found to be ligands of the same endocytic receptors as HOCl-modified proteins, including MR [76–78] and SR-A [79]. Acting on APC these products inhibit IL-12 and enhance IL-10 production and, consequently, induce strongly Th2-polarised immune responses in vivo [76–79].

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