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. 2020 Jan 23;11(1):50.
doi: 10.1038/s41419-020-2249-y.

Intravenous immunoglobulin mediates anti-inflammatory effects in peripheral blood mononuclear cells by inducing autophagy

Affiliations

Intravenous immunoglobulin mediates anti-inflammatory effects in peripheral blood mononuclear cells by inducing autophagy

Mrinmoy Das et al. Cell Death Dis. .

Abstract

Autophagy plays an important role in the regulation of autoimmune and autoinflammatory responses of the immune cells. Defective autophagy process is associated with various autoimmune and inflammatory diseases. Moreover, in many of these diseases, the therapeutic use of normal immunoglobulin G or intravenous immunoglobulin (IVIG), a pooled normal IgG preparation, is well documented. Therefore, we explored if IVIG immunotherapy exerts therapeutic benefits via induction of autophagy in the immune cells. Here we show that IVIG induces autophagy in peripheral blood mononuclear cells (PBMCs). Further dissection of this process revealed that IVIG-induced autophagy is restricted to inflammatory cells like monocytes, dendritic cells, and M1 macrophages but not in cells associated with Th2 immune response like M2 macrophages. IVIG induces autophagy by activating AMP-dependent protein kinase, beclin-1, class III phosphoinositide 3-kinase and p38 mitogen-activated protein kinase and by inhibiting mammalian target of rapamycin. Mechanistically, IVIG-induced autophagy is F(ab')2-dependent but sialylation independent, and requires endocytosis of IgG by innate cells. Inhibition of autophagy compromised the ability of IVIG to suppress the inflammatory cytokines in innate immune cells. Moreover, IVIG therapy in inflammatory myopathies such as dermatomyositis, antisynthetase syndrome and immune-mediated necrotizing myopathy induced autophagy in PBMCs and reduced inflammatory cytokines in the circulation, thus validating the translational importance of these results. Our data provide insight on how circulating normal immunoglobulins maintain immune homeostasis and explain in part the mechanism by which IVIG therapy benefits patients with autoimmune and inflammatory diseases.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. IVIG induces autophagy in peripheral blood mononuclear cells.
a PBMCs from healthy donors were cultured with IVIG (10 or 25 mg/ml) or HSA for 24 h. Cells were treated with bafilomycin (Baf) for last 45 min. Levels of LC3-II and the fold changes in LC3-II levels (mean ± SEM, n = 4 donors) based on the densitometry analysis of western blots are presented. **P < 0.01; ***P < 0.001; ns not significant; by one-way ANOVA with Dunnett’s multiple comparison test. b Induction of autophagy flux by IVIG. PBMCs of healthy donors were cultured with IVIG (25 mg/ml) for 0, 18, or 24 h. At the end of treatment, cells were treated with or without Baf for 45 min. Autophagy flux (∆LC3-II) was quantified by subtracting the band intensity of LC3-II in the presence of Baf from that of absence of Baf. Representative blot of two different donors is shown.
Fig. 2
Fig. 2. IVIG induces autophagy in monocytes, dendritic cells and M1 macrophages but not in M2 macrophages.
ad Peripheral blood monocytes (a), monocyte-derived DCs (b), M1 macrophages (c) and M2 macrophages (d) were cultured with IVIG for 24 h. Levels of LC3-II and fold changes in the LC3-II levels (mean ± SEM, n = 4 donors) are presented. Starvation condition (Stv) was used as a positive control for autophagy. Images are cropped for the presentation. **P < 0.01; ***P < 0.001; ns not significant; by one-way ANOVA with Dunnett’s multiple comparison test.
Fig. 3
Fig. 3. Transmission electron microscopic analysis of autophagic organelles induced by IVIG in dendritic cells.
a, b Lipopolysaccharide-stimulated monocyte-derived human DCs were cultured for 24 h in the absence (a) or presence of IVIG (b). The cells were then processed for the transmission electron microscopy to visualize autophagic organelles. Arrows show autophagosomes. (scale bar = 100 nm).
Fig. 4
Fig. 4. IVIG-induced autophagy in monocytes is associated with induction of class III PI3K, beclin-1, p-AMPK and p-p38MAPK and inhibition of p-mTOR.
a Monocytes were cultured with IVIG (25 mg/ml) or HSA for 24 h. Expression of p-mTOR, mTOR, p-p38MAPK, p38MAPK, p-AMPK, AMPK, beclin-1 and class III PI3K was analyzed by western blot. Representative blots of four experiments by using monocytes from different donors are shown. bf Densitometry analysis of fold changes (mean ± SEM, n = 4) in the expression of p-mTOR (b), p-p38MAPK (c), p-AMPK (d), beclin-1 (e), and class III PI3K (f). *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant; as determined by one-way ANOVA Dunnett’s multiple comparison test.
Fig. 5
Fig. 5. Inhibition of Class III PI3K, p38MAPK and AMPK kinases abrogates IVIG-induced autophagy in monocytes.
ac Peripheral blood monocytes were pretreated with a Class III PI3K inhibitor: 3MA, b p38MAPK inhibitor: SB202190 and c AMPK inhibitor: Compound C, followed by IVIG treatment for 24 h. Levels of LC3-II by western blot and densitometry analyses of fold changes (mean ± SEM; n = 4 donors) are presented. Images are cropped for the presentation. d, e THP-1 cells were either cultured alone or treated with IVIG or HSA for 24 h. In other set of experiments, cells were pretreated with respective inhibitors followed by IVIG treatment for 24 h. The cells were then stained with anti-MAP1LC3I/II primary antibody followed by Alexa Fluor® 488-conjugated secondary antibody. Nuclei were stained with DAPI. Representative fluorescent images and percent (mean ± SEM; n = 3) of LC3-punctated cells are shown. Bars indicate magnifications (×63. Scale bars = 5 µm). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; as determined by one-way ANOVA Dunnett’s (ac) or Tukey’s multiple comparison test (e).
Fig. 6
Fig. 6. IVIG-induced autophagy is F(ab′)2-dependent but sialylation-independent, and requires endocytosis of IgG.
a, b Monocytes were cultured with IVIG (25 mg/ml), equimolar concentration of F(ab′)2 or desialylated IVIG for 24 h. Levels of LC3-II were analyzed by western blot. Representative blots of four different donors and fold changes in the LC3-II levels based on densitometry analysis of western blots (mean ± SEM, n = 4 donors) are presented. Images are cropped for the presentation. c Monocytes were pretreated with cytochalasin D (Cyto D) followed by IVIG or rapamycin (Rapa) treatment for 24 h. Levels of LC3-II by western blot and densitometry analysis of fold changes (mean ± SEM; n = 3 donors) are shown. *P < 0.05; **P < 0.01; ns not significant; as determined by one-way ANOVA with Dunnett’s multiple comparison test.
Fig. 7
Fig. 7. Induction of autophagy is critical for the anti-inflammatory actions of IVIG.
LPS-stimulated DCs were pretreated with 3MA followed by culture with IVIG (25 mg/ml) for 24 h. Amounts of IL-8, IL-1β and IL-6 (mean ± SEM; n = 7 donors) in the cell-free supernatants were analyzed by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001; as determined by one-way ANOVA with Tukey’s multiple comparison test.
Fig. 8
Fig. 8. IVIG therapy in inflammatory myopathy patients induces autophagy.
a Levels of LC3-II in PBMC of myopathy patients before (pre-IVIG) and post-IVIG therapy as analyzed by western blot (five patients, P1−P5). b Fold changes in the LC3-II levels based on the densitometry analysis of western blots (mean ± SEM, n = 5 patients) **P < 0.01 two-tailed Mann–Whitney test. c Changes in the levels (pg/ml) of inflammatory cytokines (IL-8, TNF-α, IL-1β and IL-17A) in the plasma of myopathy patients following IVIG therapy.

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