{
  "abstract": "Background Current evidence indicates that a monocyte/macrophage-driven cytokine storm is a major pathogenic event in severe COVID-19. Excessive chemokine release promotes monocyte recruitment to the lungs, leading to hyper-inflammation, tissue damage, acute respiratory distress syndrome and multi-organ failure. Among the key pathways involved are CCL2/CCR2 and CCR5 ligands, which regulate monocyte/macrophage migration and infiltration. This study assessed the impact of CCR2 and CCR5 blockade during SARS-CoV-2 infection using a 3D co-culture model of fully differentiated primary human airway epithelium and primary monocytes.Materials and Methods Primary bronchial conditionally reprogrammed cells (CRC) were seeded onto transwell inserts and cultured at air–liquid interface (ALI) for 5 weeks to generate a differentiated, polarized epithelium (CRC-ALI). Cultures were apically infected with SARS-CoV-2 (BetaCov/Italy/CDG1/2020). Twenty-four hours post-infection, cells were treated with the antibodies plozalizumab (anti-CCR2) or PRO-140 (anti-CCR5). In selected experiments, freshly isolated primary monocytes (3×10 5/100 µL/well) were added to the apical surface of infected CRC-ALI cultures in the presence or absence of the antibodies. After 4 hours at 37°C, the apical medium was removed and cultures were maintained under ALI culture conditions. Every 24 hours up to 96 hours post-infection, apical washes and cell lysates were collected for RT-qPCR quantification of the SARS-CoV-2 N gene. Surface phenotypic and activation markers were analyzed by flow cytometry.Results Basal expression of CCR2 and CCR5 in CRC-ALI cultures was low; infection induced a slight increase in CCR5 expression. Treatment of infected cultures with plozalizumab or PRO-140 inhibited viral replication with distinct kinetics and a more pronounced and sustained effect observed with CCR2 blockade. Both antibodies modulated epithelial activation, as indicated by changes in HLA-DR expression. Notably, addition of primary monocytes to infected CRC-ALI cultures determined a reduction of viral replication, suggesting an active role of recruited monocytes in controlling infection within this model. Experiments are in progress to assess the effect of CCR2 and CCR5 blockade in infected co-cultures of CRC-ALI and monocytes.Conclusions CCR2 and CCR5 blockade modulates SARS-CoV-2 replication and epithelial activation in a 3D airway model. These findings support a dual role for chemokine receptor pathways in regulating viral replication and inflammatory responses and provide experimental evidence for targeting CCR2/CCR5 axes to modulate host responses in severe COVID-19.This work was supported by the Italian Ministry of Health (R.I.Pr.E.I. 2023_88a81b92ca9f to LF).",
  "authors": [
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "C Antonucci"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "M Andreotti"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "Z Michelini"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "CM Galluzzo"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "R Amici"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "A Eramo"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "G Castelli"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "S Lo Cicero"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "AM Cerio"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "S Cecchetti"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "F Spadaro"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "R Bona"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "MC Gauzzi"
    },
    {
      "affiliations": [
        "Istituto Superiore di Sanità, Rome, Italy"
      ],
      "name": "L Fantuzzi"
    }
  ],
  "title": "OC39 A 3D airway epithelium-monocyte co-culture model to assess the impact of CCR2 and CCR5 inhibition on SARS-CoV-2 replication and inflammatory responses",
  "uid": "d833dc15-cced-56c2-9dee-415ba3b58d3b"
}