Tumor Necrosis Factor and Interleukin-1β Upregulate NRP2 Expression and Promote SARS-CoV-2 Proliferation

Viruses. 2023 Jul 3;15(7):1498. doi: 10.3390/v15071498.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) and the auxiliary receptor Neuropilin-1 (NRP1) to enter host cells. NRP1 has another isoform, NRP2, whose function in COVID-19 has seldom been reported. In addition, although patients with severe cases of COVID-19 often exhibit increased levels of proinflammatory cytokines, the relationship between these cytokines and SARS-CoV-2 proliferation remains unknown. The aim of this study is to clarify the roles of proinflammatory cytokines in Neuropilin expressions and in SARS-CoV-2 infection. To identify the expression patterns of NRP under inflamed and noninflamed conditions, next-generation sequencing (RNA-seq), immunohistochemistry, quantitative real-time PCR, and Western blotting were performed using primary cultured fibroblast-like synoviocytes, MH7A (immortalized cell line of human rheumatoid fibroblast-like synoviocytes), immortalized MRC5 (human embryonic lung fibroblast), and synovial tissues. To measure viral proliferative capacity, SARS-CoV-2 infection experiments were also performed. NRP2 was upregulated in inflamed tissues. Cytokine-stimulated human fibroblast cell lines, such as MH7A and immortalized MRC5, revealed that NRP2 expression increased with co-stimulation of tumor necrosis factor α (TNFα) and interleukin-1 beta (IL-1β) and was suppressed with anti-TNFα antibody alone. TNFα and IL-1β promoted SARS-CoV-2 proliferation and Spike protein binding. The viral proliferation coincided with the expression of NRP2, which was modulated through plasmid transfections. Our results revealed that proinflammatory cytokines, including TNFα, contribute to NRP2 upregulation and SARS-CoV-2 proliferation in host human cells.

Keywords: Neuropilin-1 (NRP1); Neuropilin-2 (NRP2); coronavirus disease 2019 (COVID-19); severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); tumor necrosis factor alpha (TNFα).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19*
  • Cell Proliferation
  • Cytokines
  • Humans
  • Interleukin-1beta
  • SARS-CoV-2* / metabolism
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Cytokines
  • Interleukin-1beta
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • Tumor Necrosis Factor-alpha
  • neuropilin-2, human
  • IL1B protein, human
  • TNF protein, human

Grants and funding

This study was supported by funding from JSPS KAKENHI (grant numbers 17H04656 to J. Masumoto and 19K18499 and 22K08599 to S. Mokuda), AMED Research Grants for COVID-19 (grant numbers JP21fk0108558 and JP21fk0108550 to T. Sakaguchi), Mitsubishi Foundation to S. Mokuda, Takeda Science Foundation to S. Mokuda, Japanese Respiratory Foundation grant to S. Mokuda, Japan College of Rheumatology Grant for Promoting Research for Early RA to S. Mokuda, Nakatomi Foundation to S. Mokuda, Okinaka Memorial Institute for Medical Research to S. Mokuda, Tsuchiya Memorial Medical Foundation to S. Mokuda, and Rotary Club of Hiroshima to S. Mokuda.