Direct Binding of Bovine IgG-Containing Immune Complexes to Human Monocytes and Their Putative Role in Innate Immune Training

Abstract

Bovine milk IgG (bIgG) was shown to bind to and neutralize the human respiratory synovial virus (RSV). In animal models, adding bIgG prevented experimental RSV infection and increased the number of activated T cells. This enhanced activation of RSV-specific T cells may be explained by receptor-mediated uptake and antigen presentation after binding of bIgG-RSV immune complexes (ICs) with FcγRs (primarily CD32) on human immune cells. This indirect effect of bIgG ICs on activation of RSV-specific T cells was confirmed previously in human T cell cultures. However, the direct binding of ICs to antigen-presenting cells has not been addressed. As bovine IgG can induce innate immune training, we hypothesized that this effect could be caused more efficiently by ICs. Therefore, we characterized the expression of CD16, CD32, and CD64 on (peripheral blood mononuclear cells (PBMCs), determined the optimal conditions to form ICs of bIgG with the RSV preF protein, and demonstrated the direct binding of these ICs to human CD14⁺ monocytes. Similarly, bIgG complexed with a murine anti-bIgG mAb also bound efficiently to the monocytes. To evaluate whether the ICs could induce innate immune training more efficiently than bIgG itself, the resulted ICs, as well as bIgG, were used in an in vitro innate immune training model. Training with the ICs containing bIgG and RSV preF protein-but not the bIgG alone-induced significantly higher TNF-α production upon LPS and R848 stimulation. However, the preF protein itself nonsignificantly increased cytokine production as well. This may be explained by its tropism to the insulin-like growth factor receptor 1 (IGFR1), as IGF has been reported to induce innate immune training. Even so, these data suggest a role for IgG-containing ICs in inducing innate immune training after re-exposure to pathogens. However, as ICs of bIgG with a mouse anti-bIgG mAb did not induce this effect, further research is needed to confirm the putative role of bIgG ICs in enhancing innate immune responses in vivo.

Keywords: RSV; bovine IgG; immune complex; preF protein; trained immunity.

Figure 1

Histogram comparing the mean fluorescence intensity (MFI) of the bovine IgG (bIgG) signal on CD14⁺ monocytes: Peripheral blood mononuclear cells (PBMCs) were incubated with bIgG (500, 50, 5, or 0 µg/mL) for 20 min (A). A dose-dependent increase in the bIgG signal on the CD14⁺ monocytes was detected with an increase in the concentration of bIgG used (B).

Figure 2

The curves drawn from MFI of the bIgG detection signal: CD14⁺ monocytes were exposed to bIgG only (0–100 µg/mL), preF protein only (50 µg/mL), and the bIgG: preF ICs in one representative donor (donor A); the MFI was used to generate the detection curve (A). The delta MFI (ΔMFI) was produced when the MFI of the bIgG only signal was deducted from the MFI of the bIgG: preF ICs signal (B).

Figure 3

The curves drawn from MFI of the bIgG detection signal: the monocytes were exposed to bIgG only (0–100 µg/mL), α-bIgG (5 µg/mL) only, α-bIgG only (1 µg/mL), and the bIgG: α-bIgG ICs in one representative donor (donor A) (A). The delta MFI (ΔMFI) was produced when the MFI of the bIgG only signal was deducted from the MFI of the bIgG: α-bIgG ICs signal (B).

Figure 4

The fold changes in TNF-α (A) and IL-6 (B) production of the cells trained with WGP and stimulated with LPS in comparison to the untrained control. The TNF-α (C) and IL-6 (D) production fold changes for WGP-trained monocytes after stimulation with R848. The average (range) of cytokines in the RPMI controls of all 8 donors tested were 1805 (719–4825 pg/mL), 3181 (1044–7232 pg/mL), 3592 (1839–7434 pg/mL), and 9047 (3732–14230 pg/mL) for conditions A to D, respectively. The boxes represent 50% of the data, and the line is the median value where upper and lower whiskers present the upper and lower 25% of the data, respectively. The significance of differences is shown as p-value < 0.05 (*) and < 0.01 (**).

Figure 5

The fold changes in the TNF-α (A) and IL-6 (B) production in the cells trained with preF only (50 µg/mL) bIgG alone (10 µg/mL), or bIgG: preF ICs compared to untrained monocytes (RPMI) upon stimulation with LPS. Also, the TNF-α (C) and IL-6 (D) production fold changes compared to the RPMI control in the group of monocytes trained with single components or the bIgG: preF ICs after stimulation with and R848. The average (range) of cytokines in the RPMI controls of all 8 donors tested were 1791 (719–4825 pg/mL), 3522 (1044–7232 pg/mL), 3181 (1839–7434 pg/mL), and 9840 (3732–14,230 pg/mL) for conditions A to D, respectively. The boxes represent 50% of the data, and the line is the median value where upper and lower whiskers present the upper and lower 25% of the data, respectively. The significance of differences is shown as p-value < 0.01 (**).

Figure 6

The fold changes in the production of TNF-α (A) and IL-6 (B) in the cells trained with α-bIgG only (5 µg/mL) only, bIgG alone (3 µg/mL), or bIgG: α-bIgG ICs compared to untrained monocytes (RPMI) upon stimulation with LPS and also TNF-α (C) and IL-6 (D) production after R848 stimulation. The average (range) of cytokines in the RPMI controls of all 8 donors tested were 1740 (719–4825 pg/mL), 3279 (1044–7232 pg/mL), 3508 (1839–7434 pg/mL), and 8961 (3732–14,230 pg/mL) for conditions A to D, respectively. The boxes represent 50% of the data, and the line is the median value where upper and lower whiskers present the upper and lower 25% of the data, respectively.