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Expression of O-glycosylated oncofetal fibronectin in alternatively activated human macrophages

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A Correction to this article was published on 10 November 2022

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Abstract

Macrophage (Mϕ) polarization is an essential phenomenon for the maintenance of homeostasis and tissue repair, and represents the event by which Mϕ reach divergent functional phenotypes as a result to specific stimuli and/or microenvironmental signals. Mϕ can be polarized into two main phenotypes, M1 or classically activated and M2 or alternatively activated. These two categories diverge in many aspects, such as secreted cytokines, markers of cell surface, and biological functions. Over the last 10 years, many potential markers have been proposed for both M1 and M2 human Mϕ. However, there is scarce information regarding the glycophenotype adopted by these cells. Here, we show that M2- but not M1-polarized Mϕ expresses high levels of an unusual glycoform of fibronectin (FN), named O-glycosylated oncofetal FN (onf-FN), found in fetal/cancer cells, but not in healthy tissues. The onf-FN expression was confirmed in vitro by Western blot and real-time RT-qPCR in primary and cell line monocyte-derived Mϕ. onf-FN was induced by IL-4 and IL-13, but not by pro-inflammatory stimuli (LPS and INF-γ). RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization. In conclusion, we show by the first time that O-glycosylated onf-FN is expressed by M2-polarized Mϕ.

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Acknowledgements

Manuscript dedicated to the memory of Dr. Sen-Itiroh Hakomori, a researcher who was always been ahead of his time. To Professor Hakomori, our eternal gratitude and respect.

Funding

This work was supported by the FAPERJ, CNPq, CAPES, and Brazilian Cancer Foundation.

Author information

Author notes

  1. Marcos Andre Rodrigues da Costa Santos, Jhenifer Santos dos Reis, and Carlos Antonio do Nascimento Santos contributed equally to this work.

Authors and Affiliations

  1. Instituto de Biofisica Carlos Chagas Filho, Laboratório de Biologia Celular de Glicoconjugados, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil

    Marcos Andre Rodrigues da Costa Santos, Jhenifer Santos dos Reis, Carlos Antonio do Nascimento Santos, Kelli Monteiro da Costa, Pedro Marçal Barcelos, Karen Queiroz de Oliveira Francisco, Pedro Antônio Guimarães Notaroberto Barbosa, Emanuelle Damasceno Souza da Silva, Celio Geraldo Freire-de-Lima, Israel Diniz-Lima, Jose Osvaldo Previato, Lucia Mendonca-Previato, Leonardo Marques da Fonseca & Leonardo Freire-de-Lima

  2. Fiocruz, Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, 21040-360, Brazil

    Alexandre Morrot

  3. Faculdade de Medicina, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil

    Alexandre Morrot

  4. Departamento de Microbiologia E Imunologia Veterinária, Instituto de Veterinária, Universidade Federal Rural Do Rio de Janeiro, Rio de Janeiro, RJ, 23890-000, Brazil

    Debora Decote-Ricardo

Authors
  1. Marcos Andre Rodrigues da Costa Santos
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  2. Jhenifer Santos dos Reis
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Contributions

Conceptualization: Marcos André Rodrigues da Costa Santos, Jhenifer Santos dos Reis, Kelli Monteiro da Costa, Israel Diniz-Lima, Carlos Antônio do Nascimento Santos, Debora Decote-Ricardo, Alexandre Morrot, Leonardo Marques da Fonseca, Leonardo Freire-de-Lima. Methodology: Marcos André Rodrigues da Costa Santos, Jhenifer Santos dos Reis, Kelli Monteiro da Costa. Formal analysis and investigation: Marcos André Rodrigues da Costa Santos, Jhenifer Santos dos Reis, Kelli Monteiro da Costa, Israel Diniz-Lima, Pedro Marçal Barcelos, Karen Queiroz de Oliveira Francisco, Pedro Antônio Guimarães Notaroberto Barbosa, Emanuelle Damasceno Souza da Silva. Writing—original draft preparation: Marcos André Rodrigues da Costa Santos, Leonardo Freire-de-Lima, Leonardo Marques da Fonseca, Celio Geraldo Freire- de-Lima, Lucia Mendonça Previato, José Osvaldo Previato. Writing—review and editing: Marcos André Rodrigues da Costa Santos, Leonardo Freire-de-Lima, Leonardo Marques da Fonseca, Celio Geraldo Freire- de-Lima, Lucia Mendonça Previato, José Osvaldo Previato. Funding acquisition: Leonardo Freire-de-Lima, Leonardo Marques da Fonseca, Celio Geraldo Freire- de-Lima, Lucia Mendonça Previato, José Osvaldo Previato. Resources: Leonardo Freire-de-Lima, Celio Geraldo Freire- de-Lima, Lucia Mendonça Previato, José Osvaldo Previato, Leonardo Marques da Fonseca. Supervision: Leonardo Freire-de-Lima.

Corresponding authors

Correspondence to Leonardo Marques da Fonseca or Leonardo Freire-de-Lima.

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Ethics approval

The study was approved by Institutional Ethics Committee and was carried out in accordance with the relevant guidelines and regulations (CAAE 48917418.2.0000.5257).

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The original online version of this article was revised: The authors have noticed mistakes during figure production, where supplementary figure 3 was incomplete, missing Western blot data.

Supplementary Information

Supplementary Figure 1.

Analysis of CD68 expression in THP-1 derived macrophages. THP-1 derived macrophages were generated after treatment with 320 nM PMA for 48 hours as described in the materials and methods section. The expression of the marker CD68 was monitored by flow cytometry, which the mean fluorescent intensity (MIF) was determined between cells incubated or not with the specific monoclonal antibody. *p ≤ 0.05. (PNG 14 kb)

High resolution image (TIF 72 kb)

Supplementary Figure 2.

Analysis of the profile of cytokines expressed/secreted by THP-1 derived macrophages after polarization for M1 and M2 phenotypes. After differentiation, Mɸ were polarized to the M1 and M2 phenotypes following the treatment with INF-γ + LPS (A and B) or IL-4 + IL-3 (C and D), respectively, as described in the materials and methods section. To confirm the polarization, the levels of transcripts (mRNA) (A and C) and cytokines (B and D) were quantified by qPCR and ELISA, respectively. * p ≤ 0.05 vs. Mϕ and # p ≤ 0.05 vs. M1- Mϕ. n.s. not significant in relation to Mɸ. (PNG 47 kb)

High resolution image (TIF 112 kb)

Supplementary Figure 3.

Analysis of GalNAc-T6 expression in THP-1 derived macrophages by qPCR and Western blot. After differentiation from Monɸ to Mɸ with PMA, cells were classically or alternatively activated as detailed in the material and methods section. mRNA and protein levels for pp-GalNAc-T6 in both M1- and M2-Mɸ were monitored by real time PCR (A) and Western blot (B and C), respectively. Graphs representative of three individual experiments. n.s. not significant in relation to Mɸ. (PNG 658 kb)

High resolution image (TIF 134 kb)

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da Costa Santos, M.A.R., dos Reis, J.S., do Nascimento Santos, C.A. et al. Expression of O-glycosylated oncofetal fibronectin in alternatively activated human macrophages. Immunol Res 71, 92–104 (2023). https://doi.org/10.1007/s12026-022-09321-9

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