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. 2021 Dec 3;9(12):1828.
doi: 10.3390/biomedicines9121828.

The Functional Role of IgA in the IgM/IgA-Enriched Immunoglobulin Preparation Trimodulin

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

The Functional Role of IgA in the IgM/IgA-Enriched Immunoglobulin Preparation Trimodulin

Fabian Bohländer et al. Biomedicines. .

Abstract

In comparison to human immunoglobulin (Ig) G, antibodies of IgA class are not well investigated. In line with this, the functional role of the IgA component in IgM/IgA-enriched immunoglobulin preparations is also largely unknown. In recent years, powerful anti-pathogenic and immunomodulatory properties of human serum IgA especially on neutrophil function were unraveled. Therefore, the aim of our work is to investigate functional aspects of the trimodulin IgA component, a new plasma-derived polyvalent immunoglobulin preparation containing ~56% IgG, ~23% IgM and ~21% IgA. The functional role of IgA was investigated by analyzing the interaction of IgA with FcαRI, comparing trimodulin with standard intravenous IgG (IVIG) preparation and investigating Fc receptor (FcR)-dependent functions by excluding IgM-mediated effects. Trimodulin demonstrated potent immunomodulatory, as well as anti-pathogenic effects in our neutrophil model (neutrophil-like HL-60 cells). The IgA component of trimodulin was shown to induce a strong FcαRI-dependent inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) signaling, counteract lipopolysaccharide-induced inflammation and mediate phagocytosis of Staphylococcus aureus. The fine-tuned balance between immunomodulatory and anti-pathogenic effects of trimodulin were shown to be dose-dependent. Summarized, our data demonstrate the functional role of IgA in trimodulin, highlighting the importance of this immunoglobulin class in immunoglobulin therapy.

Keywords: Fc receptors; FcαRI; ITAMi; IVIG; IgA; cytokines; neutrophils; phagocytosis; trimodulin.

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Conflict of interest statement

Authors are employees of Biotest AG, Dreieich, Germany. The authors declare that the research was conducted in the absence of any other commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Trimodulin facilitates immune homeostasis by three modes of action. (a) Incubation of neutrophil-like HL-60 cells with trimodulin or IVIG reduce IL-8 secretion. Indicated concentrations of trimodulin (blue line) or IVIG (red line) were added to cells for 4 h; after centrifugation, supernatant was analyzed for IL-8 concentration via ELISA. (b) Trimodulin or IVIG are able to bind free IL-8 in PBS. Recombinant IL-8 was dissolved at 300 ng/mL in PBS and depicted concentrations of trimodulin (blue line) or IVIG (red line) were added; the mixture was incubated for 1 h and the remaining IL-8 was measured. (c) Activation of IL-8 release in correlation with blocking of ITIM signaling. 10 µM SHIP-1 inhibitor 3AC were pre-incubated with cells (30 min) and different concentrations of trimodulin or IVIG were added for 4 h. Cells were treated with depicted concentrations of trimodulin (blue lines) or IVIG (red lines). IL-8 release of cells without inhibitor 3AC (solid lines) and with inhibitor 3AC (dotted lines) was compared. (d) Inhibitory ITAMi signaling is activated by incubating HL-60 cells with trimodulin or IVIG. Same as in (c), except cells were pre-incubated with 200 µM NSC-87877 (SHP-1 Inhibitor) or not. The data are shown as mean values of six independent experiments. Statistics: Two-way ANOVA, Sidak multiple comparison test between trimodulin and IVIG group (a,b) or within trimodulin/IVIG group +/− inhibitor treatment (c,d), 95% confidence interval. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.
Figure 2
Figure 2
LPS-induced inflammatory phenotype of neutrophil-like HL-60 cells. (a) Heat map of released cytokines after treatment with LPS. HL-60 cells were incubated with 500 ng/mL LPS (18 h); supernatant of cell culture was analyzed by semi-quantitative cytokine arrays. Signal intensities of the measured cytokines of untreated cells were referenced as 1 (yellow) and the x-fold increase (red) or decrease (green) calculated. Cytokines below the detection limit are shown in white (n.d. = not detected). Data show the mean of 3 independent experiments. (b) IL-8 release is dose-dependently induced by LPS. Indicated concentrations of LPS were added to HL-60 cells for 24 h. IL-8 release was measured by ELISA. (c) LPS treatment modulates FcR expression. Neutrophil-like HL-60 cells were incubated with 500 ng/mL LPS for 24 h (light gray bars) or 48 h (dark gray bars). By immunological staining using flow cytometry, FcR expression was analyzed. The x-fold change in fluorescence value of untreated cells to treated cells was evaluated. Signal of untreated cells was set to 1 and change was calculated. Six independent experiments were performed for evaluation. Statistics: Two-way ANOVA, Sidak multiple comparison test calculated between untreated cells (baseline) and 24/48 h LPS-treated cells, 95% confidence interval. **** p ≤ 0.0001.
Figure 3
Figure 3
Modulation of LPS-induced cytokine release and FcR expression by trimodulin and IVIG. (a) Addition of trimodulin or IVIG decrease LPS-induced IL-8 secretion. Neutrophil-like HL-60 cells were treated with 500 ng/mL LPS for 48 h and in the following 24 h, with 15 g/L trimodulin (blue points), IVIG (red points) or equal volume buffer (black points). Via ELISA, the IL-8 level in the cell culture supernatant was analyzed. (b–f) Modulation of LPS-induced FcR expression. Cells were incubated with LPS (48 h; 500 ng/mL) and afterwards, with 15 g/L trimodulin, IVIG or equal volume buffer (for 24 h). FcR expression was examined by flow cytometry. Measured fluorescence value of LPS-treated cells was referenced as 1 (baseline) and x-fold change after immunoglobulin treatment was determined. The mean of 6 independent experiments is depicted. Statistics: One way ANOVA; Dunnett’s multiple comparisons test calculated between buffer control and trimodulin or IVIG, 95% confidence interval. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ns = not significant.
Figure 4
Figure 4
Antibody-dependent phagocytosis of S. aureus. (a) Schematic overview of the S. aureus–trimodulin immune complex (IC). (b) S. aureus particles were bound by IgM, IgA and IgG species. Binding of immunoglobulins to S. aureus bioparticles after 30 min of incubation were detected using specific detection antibodies. Percentage of IgM (green triangle), IgA (blue dots) and IgG (red square) on positive S. aureus particles was determined by flow cytometry. * Purified IgA and IgM from human serum. (c) Cytokine heat map after stimulation of HL-60 cells with S. aureus–IVIG IC. Neutrophil-like HL-60 cells were stimulated with S. aureus–IVIG IC (18 h); supernatant of cell culture was analyzed by semi-quantitative cytokine arrays. Signal intensities of cytokines measured by untreated cells were referenced as 1 (yellow) and the x-fold increase (red) or decrease (green) was calculated. Cytokines below the detection limit are shown in white (n.d. = not detected). Data show the mean of 3 independent experiments. (d) Phagocytosis of S. aureus bioparticles with different immunoglobulin preparations; 50 µg/mL immunoglobulin preparations or buffer were added for opsonization to S. aureus particles for 1 h. Phagocytosis was measured as percentage fluorescence positive cells (gray bars). Corresponding IL-8 release was analyzed by ELISA (red dots). * As controls, purified IgA and IgM from human serum was used. (e) FcR blocking experiments with S. aureus–trimodulin IC. Blocking antibodies (5 µg/mL) were added 20 min before S. aureus–trimodulin IC to cells. Phagocytic index (percentage positive cells multiplied with median fluorescence intensity) of non-blocked cells was referenced as 100% and remaining phagocytosis was calculated. Values represent mean of 6 independent experiments. Statistics: One way ANOVA; Dunnett’s multiple comparisons test between non-blocked cells (100% control) and treatment with indicated blocking antibodies, 95% confidence interval. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.
Figure 5
Figure 5
Dose-dependent effects of trimodulin on S. aureus phagocytosis. (a) Dose dependency of trimodulin on phagocytosis of S. aureus. Indicated concentrations of trimodulin were added with S. aureus bioparticles for 1 h to neutrophil-like HL-60 cells. Phagocytosis (gray bars) and corresponding IL-8 release (red dots) are shown. Low doses of trimodulin mediate anti-pathogenic effects, as shown by increased phagocytosis and cytokine release. Higher doses mediate immunomodulatory effects with reduced phagocytosis and inflammation. (b) ITAMi signaling induced by high concentrations of trimodulin. Tyrosine 536 (pY536) phosphorylation of phosphatase SHP-1 was measured. Therefore, S. aureus–trimodulin IC and depicted trimodulin concentrations were added to cells for 90 min. Intracellular staining with anti-phospho-SHP-1 pY536 antibody was performed. Fluorescence was normalized to buffer control. (c) SHP-1 important for immunomodulation. Cell treatment as in (b), with additional 30 min of pre-incubation with 200 µM NSC-87877. IL-8 release between NSC-87877 (dotted lines) or buffer treatment (solid lines) is shown. Values represent mean of 6 independent experiments. Statistics: Two-way ANOVA; Tukey’s multiple comparisons test between S. aureus–trimodulin IC control (100%) and trimodulin addition (b) or within trimodulin + buffer and trimodulin + NSC-87877 treatment (c). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.
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