Fig 1: α-IgM caused strong cytokine production in monocytes. (A) t-SNE plots from mixed PBMCs (monocytes highlighted in red) that were stimulated with α-IgM for 6 h (top) and 24 h (bottom) as well as t-SNE plots displayed as a heatmap for IL1β, TNF, MCP1, IL6, IL8, IL10 (n = 6). (B) t-SNE plots from samples stained for intracellular cytokines that were unstimulated (black) or stimulated with α-IgM (blue) for 6 and 24 h after gating on CD14+ monocytes (n = 6). (C) Staining of intracellular IL1β, TNF, MCP1, IL6, IL8 and IL10 in monocytes after ODN2006, α-IgM or ODN2006 + α-IgM stimulation for 6 h (top) and 24 h (bottom) via flow cytometry (n = 6). (D) Heat map diagram of secreted IL1β, IFNα2, IFNγ, TNF, IL10, IL12p70, IL17A, IL18, IL23, IL33 (measured via Legendplex, n = 6) and MCP1, IL6, IL8 (measured via ELISA, n = 6–8) in supernatant of mixed PBMCs (top) or monocyte-depleted fraction (bottom) after 24 h of stimulation with α-IgM. (E) Secreted IL1β, TNF, MCP1, IL6, IL8 and IL10 in supernatant of mixed PBMCs and monocyte depleted fraction after α-IgM stimulation for 24 h measured with Legendplex for IL1β, TNF and IL10 (n = 6; statistical asterisks above bars refer to the unstimulated sample of the same cells) and ELISA for MCP1, IL6 and IL8 (n = 7–8; statistical asterisks above bars refer to the unstimulated sample of the same cells). n.d.: not detectable. No statistical test was performed for cytokines whose level was not detectable in unstimulated sample. One-Way ANOVA or mixed-effects analysis with Geisser–Greenhouse correction and Tukey’s multiple comparisons test was used for analyzing data including more than two stimulation conditions (C). Paired t test was used for the comparison of unstimulated vs. stimulated conditions within the same cell type as well as for comparing data from PBMCs with data from monocytes depleted fraction (D,E); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Fig 2: Monoclonal α-IgM also led to an upregulated cytokine production in monocytes. (A) Intracellular cytokine staining for IL1β, TNF, MCP1, IL6, IL8 and IL10 in monocytes after stimulation with monoclonal and polyclonal α-IgM for 6 h via flow cytometry staining (n = 6). (B) Secreted IL1β, TNF, MCP1, IL6, IL8 and IL10 (measured via Legendplex, n = 6) in supernatant of mixed PBMCs after stimulation with monoclonal and polyclonal α-IgM for 24 h. One-Way ANOVA with Geisser–Greenhouse correction and Tukey’s multiple comparisons test was used for analyzing data including more than two conditions (A,B); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Fig 3: α-IgM acted directly on monocytes. (A,B) Heat map (A) and bar diagrams (B) of secreted IL1β, IFNα2, IFNγ, TNF, IL10, IL12p70, IL17A, IL18, IL23, IL33 (measured via Legendplex) and MCP1, IL6, IL8 (measured via ELISA) in supernatant of mixed PBMCs (top or left) or B-cell-depleted fraction (bottom or right) after 24 h of stimulation without or with α-IgM (n = 6–8, statistical asterisks in heat map refer to the unstimulated sample of the same cells). (C) Representative histograms of unstimulated B cells and monocytes using α-IgM-FITC (n = 8). Empty histograms: negative controls. (D) Quantification of MFI of unstimulated B cells, monocytes using α-IgM-FITC (n = 8). (E,F) Representative histograms showing unstimulated monocytes stained with commercially available IgM-FITC (E) and α-IgM-FITC without (blue) or with (green) FCR blocking (F). Empty histograms: negative controls. n.d.: not detectable. No statistical test was performed for cytokines whose level was not detectable in unstimulated sample. Paired t test was used for the comparison of unstimulated vs. stimulated conditions within the same cell type as well as for comparing data from PBMCs with data from B-cell-depleted fraction (A,B,D); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Fig 4: Strong cytokine release after α-IgM exposure in mixed PBMCs mainly not attributable to B cells. (A,B) Heat map diagram of secreted IL1β, IFNα2, IFNγ, TNF, IL10, IL12p70, IL17A, IL18, IL23, IL33 (measured via Legendplex, n = 7) and MCP1, IL6, IL8 (measured via ELISA, n = 9–11) in supernatant of mixed PBMCs 24 h after treatment with ODN2006 + α-IgM (A) or both individually (B). (C) Intracellular cytokine staining for IL1β, TNF, MCP1, IL6, IL8 and IL10 in B cells after ODN2006, α-IgM or ODN2006 + α-IgM stimulation for 6 h (top) and 24 h (bottom) via flow cytometry staining (n = 6). (D) Heat map diagram of cytokines and chemokines released in supernatant of isolated B cells after 24 h of stimulation with ODN2006, α-IgM or their combination obtained by Legendplex Human Inflammatory Panel 1 (n = 5). N.d.: not detectable. No statistical test was performed for cytokines whose level was not detectable in unstimulated sample. Paired t test was used for the comparison of unstimulated vs. stimulated conditions (A). One-Way ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparisons test was used for analyzing data including more than two stimulation conditions (A–D); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n.d. = not detectable.
Fig 5: Sex-specific cytokine secretion of monocytes after DR stimulation is dependent of B cells. A Representative flow cytometry plots of mixed PBMCs and CD19+ B cell-depleted PBMCs. B, C IL8 levels in supernatant from PBMCs and B cell-depleted PBMCs from women (B) and men (C) after 24 h in culture, with and without stimulation by A68930 (A, 10–7 M) or Ropinirole (R, 10–6 M) measured via ELISA; normalized to unstimulated control; n = 8–9 per group. D, E MCP1 levels in supernatant from PBMCs and B cell-depleted PBMCs from women (D) and men (E) after 24 h in culture, with and without stimulation by A68930 (A, 10–7 M) or Ropinirole (R, 10–6 M) measured via ELISA; normalized to unstimulated control; n = 8 per group. F, G Diagrams illustrating the interplay between B cells and monocytes after DR stimulation, showing effects on activation markers and cytokine secretion for cells from women (F) and men (G); created in BioRender.com. Wilcoxon test was used for paired data comparisons including PBMCs vs. B cells depleted as well as unstimulated vs. stimulated samples. Mann–Whitney test was used for testing statistical significance between unpaired data of women and men; *p ≤ 0.05
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