Figure 1

Natural IgG collaborates with ficolin to recognize bacteria and form immune complexes on monocytes.

(a) FACS analysis to detect nIgG bound to 10⁶ cfu P. aeruginosa opsonized with proteins (see key below the figure) and incubated with primary anti-mouse IgG3, followed by staining with Alexa488-conjugated secondary antibody. nIgG purified from undepleted pooled sera of uninfected mice (n = 8) does not bind to P. aeruginosa (red). It binds to the bacteria only when aided by dose-dependent increase in IgG⁻ serum (1:20; blue and 1:10; orange). Further depletion of ficolin from the IgG⁻ serum (1:10 ficolin⁻ IgG⁻ serum; green) shows reduction in binding of nIgG to P. aeruginosa. nIgG binding decreases when bacteria were incubated with 1:10 ficolin⁻ serum (light grey) as compared to 1:10 undepleted serum (dark grey), indicated by the shift (arrow). The right panel shows quantitative comparison of the FACS plots of different samples indicated by mean fluorescence intensity (MFI) of nIgG binding. (b–d) U937 monocytes were incubated with GlcNac-beads opsonized with H-ficolin, nIgG or the nIgG:ficolin complex. (b) Confocal microscopy to detect co-localization of nIgG (green) and ficolin (red) on the monocytes, which were pre-incubated for 30 min at room temperature with GlcNAc-beads opsonized with H-ficolin, nIgG or the nIgG:H-ficolin complex. The cell nuclei were stained with DAPI (blue). 63× objective; scale bar, 5 μm. (c) In situ proximity ligation assay (PLA) to identify nIgG:ficolin interaction under normal and infection-inflammation conditions on the monocytes, using anti-human IgG3 and anti-H-ficolin antibodies. Protein-protein interactions are seen as PLA signals – each red dot represents an interaction. 63× objective; scale bar, 5 μm. (d) Quantification of the number of PLA signals of nIgG:H-ficolin complex. The interaction complexes per cell were scored using Image J software. Duplicates of 50 cells each were enumerated for each condition tested. Three replicates per condition were tested and three independent experiments were performed. *p < 0.05; **p < 0.01; n.s., not significant.