TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals

José M. Jiménez, Michelle Salazar, Sergio Arancibia, Javiera Villar, Fabian Salazar, Gordon D. Brown, Ed C. Lavelle, Luisa Martínez-Pomares, Jafet Ortiz-Quintero, Sergio Lavandero, Augusto Manubens, María Inés Becker (Corresponding Author)

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Abstract

Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.
Original languageEnglish
Article number1136
Number of pages20
JournalFrontiers in Immunology
Volume10
DOIs
Publication statusPublished - 31 May 2019

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Hemocyanin
Toll-Like Receptor 4
Mollusca
Antigen-Presenting Cells
Mammals
Dendritic Cells
dectin 1
mouse dectin-2
Interleukin-6
Glycoproteins
Macrophages
Phosphorylation
Interleukin-12 Subunit p40
C-Type Lectins
Antigens
Immunologic Adjuvants
Blocking Antibodies
Inbred C3H Mouse
Endocytosis
Protein C

Keywords

  • mollusk hemocyanins
  • antigen presenting cells
  • inflammation
  • Dectin-1
  • Dectin-2
  • Toll-like receptor 4
  • antigen-presenting cells
  • Antigen-presenting cells
  • Inflammation
  • Mollusk hemocyanins

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals. / Jiménez, José M.; Salazar, Michelle; Arancibia, Sergio; Villar, Javiera; Salazar, Fabian; Brown, Gordon D.; Lavelle, Ed C.; Martínez-Pomares, Luisa; Ortiz-Quintero, Jafet; Lavandero, Sergio; Manubens, Augusto; Becker, María Inés (Corresponding Author).

In: Frontiers in Immunology, Vol. 10, 1136, 31.05.2019.

Research output: Contribution to journalArticle

Jiménez, JM, Salazar, M, Arancibia, S, Villar, J, Salazar, F, Brown, GD, Lavelle, EC, Martínez-Pomares, L, Ortiz-Quintero, J, Lavandero, S, Manubens, A & Becker, MI 2019, 'TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals', Frontiers in Immunology, vol. 10, 1136. https://doi.org/10.3389/fimmu.2019.01136
Jiménez, José M. ; Salazar, Michelle ; Arancibia, Sergio ; Villar, Javiera ; Salazar, Fabian ; Brown, Gordon D. ; Lavelle, Ed C. ; Martínez-Pomares, Luisa ; Ortiz-Quintero, Jafet ; Lavandero, Sergio ; Manubens, Augusto ; Becker, María Inés. / TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals. In: Frontiers in Immunology. 2019 ; Vol. 10.
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abstract = "Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.",
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note = "Funding This study was supported by CONICYT-CHILE FONDECYT Regular Grant 1151337 to MB. FONDAP 15130011 to SL. CONICYT-CHILE National Ph.D. Fellowships were awarded to JJ (CONICYT-PCHA/Doctorado Nacional/2013-21130683) and to JO-Q (CONICYT-PFCHA/Doctorado Nacional/2017-21171588). FS holds a postdoctoral fellowship from the National Commission for Scientific and Technological Research (CONICYT), Chile. Funding was provided by the Wellcome Trust (102705, 097377), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1) to GB. Acknowledgments We thank Dr. Mar{\'i}a Rosa Bono, Dr. Sergio Vargas, and Dr. Juan Carlos Aguill{\'o}n from Universidad de Chile and Dr. M{\'o}nica Imarai from Universidad de Santiago, Chile for helpful comments.",
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TY - JOUR

T1 - TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals

AU - Jiménez, José M.

AU - Salazar, Michelle

AU - Arancibia, Sergio

AU - Villar, Javiera

AU - Salazar, Fabian

AU - Brown, Gordon D.

AU - Lavelle, Ed C.

AU - Martínez-Pomares, Luisa

AU - Ortiz-Quintero, Jafet

AU - Lavandero, Sergio

AU - Manubens, Augusto

AU - Becker, María Inés

N1 - Funding This study was supported by CONICYT-CHILE FONDECYT Regular Grant 1151337 to MB. FONDAP 15130011 to SL. CONICYT-CHILE National Ph.D. Fellowships were awarded to JJ (CONICYT-PCHA/Doctorado Nacional/2013-21130683) and to JO-Q (CONICYT-PFCHA/Doctorado Nacional/2017-21171588). FS holds a postdoctoral fellowship from the National Commission for Scientific and Technological Research (CONICYT), Chile. Funding was provided by the Wellcome Trust (102705, 097377), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1) to GB. Acknowledgments We thank Dr. María Rosa Bono, Dr. Sergio Vargas, and Dr. Juan Carlos Aguillón from Universidad de Chile and Dr. Mónica Imarai from Universidad de Santiago, Chile for helpful comments.

PY - 2019/5/31

Y1 - 2019/5/31

N2 - Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

AB - Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

KW - mollusk hemocyanins

KW - antigen presenting cells

KW - inflammation

KW - Dectin-1

KW - Dectin-2

KW - Toll-like receptor 4

KW - antigen-presenting cells

KW - Antigen-presenting cells

KW - Inflammation

KW - Mollusk hemocyanins

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