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. 2021 Jun 10:12:700429.
doi: 10.3389/fimmu.2021.700429. eCollection 2021.

Immunomodulation: Immunoglobulin Preparations Suppress Hyperinflammation in a COVID-19 Model via FcγRIIA and FcαRI

Fabian Bohländer  1   2 , Dennis Riehl  1   2 , Sabrina Weißmüller  2   3 , Marcus Gutscher  1   2 , Jörg Schüttrumpf  2 , Stefanie Faust  1   2
Affiliations

Immunomodulation: Immunoglobulin Preparations Suppress Hyperinflammation in a COVID-19 Model via FcγRIIA and FcαRI

Fabian Bohländer et al. Front Immunol. .

Abstract

The rapid spread of SARS-CoV-2 has induced a global pandemic. Severe forms of COVID-19 are characterized by dysregulated immune response and "cytokine storm". The role of IgG and IgM antibodies in COVID-19 pathology is reasonably well studied, whereas IgA is neglected. To improve clinical outcome of patients, immune modulatory drugs appear to be beneficial. Such drugs include intravenous immunoglobulin preparations, which were successfully tested in severe COVID-19 patients. Here we established a versatile in vitro model to study inflammatory as well as anti-inflammatory processes by therapeutic human immunoglobulins. We dissect the inflammatory activation on neutrophil-like HL60 cells, using an immune complex consisting of latex beads coated with spike protein of SARS-CoV-2 and opsonized with specific immunoglobulins from convalescent plasma. Our data clarifies the role of Fc-receptor-dependent phagocytosis via IgA-FcαRI and IgG-FcγR for COVID-19 disease followed by cytokine release. We show that COVID-19 associated inflammation could be reduced by addition of human immunoglobulin preparations (IVIG and trimodulin), while trimodulin elicits stronger immune modulation by more powerful ITAMi signaling. Besides IgG, the IgA component of trimodulin in particular, is of functional relevance for immune modulation in this assay setup, highlighting the need to study IgA mediated immune response.

Keywords: COVID-19; Fc-receptors; ITAMi; IVIG; SARS-CoV-2; immune modulation; neutrophils; trimodulin.

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Conflict of interest statement

All authors are employees of Biotest AG, Dreieich, Germany. The authors declare that this study received funding from Biotest. The funder had the following involvement in the study: study design, interpretation of data, the writing of this article and the decision to submit it for publication.

Figures

Figure 1
Figure 1
Characterization of SARS-CoV-2-like particles. (A) Schematic overview of fluorescent latex beads coated with recombinant (re-) SARS-CoV-2 spike protein; specific immunoglobulins of IgG (red), IgA (blue), and IgM (green) classes bound to the spike protein are shown. (B) IgG, IgA, and IgM bound on surface of SARS-CoV-2-like latex beads opsonized with different convalescent COVID-19 plasma donations (donor numbers 1–6). Coated beads were incubated with plasma from indicated donors for 45 min at 37°C. After washing, beads were stained with anti-IgG, anti-IgA, and anti-IgM detection antibodies and analyzed by flow cytometry. Percentage of positive beads for IgG, IgA, and IgM is shown; data represents mean of four independent experiments. (C) Control experiments show specific binding of anti-SARS-CoV-2 antibodies to spike protein. Coated or non-coated beads were incubated with indicated plasma, immunoglobulins, or buffer for 45 min at 37°C and were stained as described in (B). Data represents mean of six independent experiments.
Figure 2
Figure 2
Establishment of COVID-19 like inflammation model (A) Heat map of chemokines and cytokines secreted by neutrophil-like HL60 cells after stimulation with anti-SARS-CoV-2 IgG-IC. HL60 cells were stimulated for 18 h at 37°C with the indicated immune complex. Qualitative cytokine secretion was measured by using human cytokine arrays. Relative signal intensities for every measured cytokine of untreated cells were set as 1 (yellow). The x-fold induction (red) or reduction (green) in comparison to untreated cells was calculated. Not detected cytokines are shown in white. Results represent mean of three independent experiments. (B) Evaluation of COVID-19-like inflammation model. Cells were incubated for 1 h with SARS-CoV-2 spike protein coated or not coated beads opsonized with different immunoglobulins, BSA, or PBS controls. Phagocytic index (gray bars, left y-axis) for bead uptake and corresponding IL-8 release (red dots, right y-axis) in cell culture supernatant were measured. Data represents mean of eight independent experiments. (C) Analysis of phagocytic activity and IL-8 release with different COVID-19 plasma donors [experimental procedure as in (B)]. (D) Overview of human Fc-receptors (FcR), immunoglobulin binding specificity, cellular function, and their expression on neutrophil-like HL60 cells. Fc-receptor expression on HL60 cells, 4 days differentiated with 1.3% DMSO, was measured as percentage of positive cells; results represent mean of three independent experiments. (E) FcR blocking experiments with COVID-19 plasma opsonized SARS-CoV-2-like particles. Cells were pre-incubated with 5 µg/ml of indicated blocking antibodies or combinations of those 20 min before addition of immune complex. Phagocytic index of not blocked cells was referred as 100% phagocytic activity, and the remaining phagocytic activity is shown as mean of six independent experiments. Statistics: One way ANOVA; Dunetts multiple comparisons test, 95% confidence interval. ns, not significant, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 3
Figure 3
Immune modulation in COVID-19-like model by IVIG and trimodulin preparation. (A) HL60 cells were incubated for 1 h with SARS-CoV-2 spike protein coated beads opsonized with COVID-19 plasma. IVIG (IgG Next Generation, Biotest AG) or trimodulin (Biotest AG) was added in the indicated concentrations to the cell immune complex mixture. Phagocytosis of SARS-CoV-2-like immune complex was measured with trimodulin (light gray bars) or IVIG (dark gray bars) addition. (B–F) Same as (A) instead phagocytosis cytokine release into cell culture supernatant was measured with trimodulin (dots, blue line) or IVIG (square, red line) addition. IL1-ra (B), IL-10 (C), MCP-1 (D), MIP-1α (E), and IL-8 (F) were measured. Values represent mean of six independent experiments. Statistics: Two way ANOVA; Tukey’s multiple comparisons test, 95% confidence interval. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
Figure 4
Figure 4
Stronger immune modulation by trimodulin due to more potent ITAMi signaling. (A) Phosphorylation of SHP-1 at tyrosine 536 (pY536). Cells were treated for 1 h with COVID-19 plasma-IC and indicated trimodulin or IVIG concentrations for 90 min. Cells were fixed, permeabilized, and stained with anti-phospho SHP-1 pY536 antibody. Fluorescence signal was measured by flow cytometry and normalized to buffer treated cells. (B) Blocking of SHP-1 phosphatase reduces immune modulatory effects. Cells were treated as in (A), but additional pre-incubation with 200 µM SHP-1 inhibitor NSC-87877. IL-8 release was normalized and compared between NSC-87877 (dotted lines) or buffer treatment (solid lines). Incubation of cells with trimodulin (blue lines) or IVIG (red lines) is shown with or without SHP-1 inhibitor. Values represent mean of six independent experiments. Statistics: Two way ANOVA; Tukey’s multiple comparisons test, 95% confidence interval. ns, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
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References

    1. Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (Covid-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA (2020) 323(13):1239. 10.1001/jama.2020.2648 - DOI - PubMed
    1. Henss L, Scholz T, von Rhein C, Wieters I, Borgans F, Eberhardt FJ, et al. . Analysis of Humoral Immune Responses in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection. J Infect Dis (2021) 223(1):56–61. 10.1093/infdis/jiaa680 - DOI - PMC - PubMed
    1. Hoepel W, Chen H-J, Allahverdiyeva S, Manz X, Aman J, Amsterdam UMC COVID-19 Biobank et al. . Anti-SARS-Cov-2 IgG From Severely Ill COVID-19 Patients Promotes Macrophage Hyper-Inflammatory Responses. Immunology (2020) 11:eabf8654. 10.1101/2020.07.13.190140 - DOI
    1. McGonagle D, Sharif K, O’Regan A, Bridgewood C. The Role of Cytokines Including Interleukin-6 in COVID-19 Induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Autoimmun Rev (2020) 19(6):102537. 10.1016/j.autrev.2020.102537 - DOI - PMC - PubMed
    1. Gong J, Dong H, Xia SQ, Huang YZ, Wang D, Zhao Y, et al. . Correlation Analysis Between Disease Severity and Inflammation-related Parameters in Patients With COVID-19 Pneumonia. Infect Dis (Except HIV/AIDS) (2020) 20(1):963. 10.1101/2020.02.25.20025643 - DOI - PMC - PubMed

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