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. 2015 Feb;144(2):206-17.
doi: 10.1111/imm.12368.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors

Affiliations

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors

Charlotte Admyre et al. Immunology. 2015 Feb.

Abstract

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Keywords: chemotaxis; inflammation; neutrophils; oligonucleotide.

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Figures

Figure 1
Figure 1
Oligonucleotides (ODN) reduce surface expression of CXCR1 and CXCR2 on human polymorphonuclear neutrophils (PMN). PMN from healthy blood donors were stimulated with 0·5, 10 μm or 25 μm of ODN or with medium alone (untreated) for 3 hr. Cells were subsequently harvested and analysed for CXCR1 (a and b) or CXCR2 (c) surface expression by flow cytometry. (a) FACS histograms show the expression of CXCR1 of PMN incubated with 25 μmIDX9054 or IDX9031 compared with medium alone. The fold changes in mean fluorescence intensity (MFI) of CXCR1+ (b) or CXCR2+ (c) CD66b+ PMN were calculated by normalizing the MFI of corresponding untreated cells to 1 (dotted line). Results are presented as mean values ± SEM (n = 5–9). *P < 0·05, **P < 0·01 and ***P < 0·001 by one-way analysis of variance with Dunnett's post hoc correction versus untreated cells.
Figure 2
Figure 2
The reduction in CXCR1 surface expression starts already after 15 min of oligonucleotide (ODN) stimulation. (a) Polymorphonuclear neutrophils (PMN) from healthy blood donors were stimulated with 10 μm of the ODN IDX9059 or with medium alone (untreated) for 15 min, 30 min, 1 hr, 2 hr or 3 hr. Cells were subsequently harvested and fixed at each time-point and analysed for CXCR1 surface expression by flow cytometry. The fold changes in mean fluorescence intensity (MFI) of CXCR1+ CD66b+ PMN was calculated by normalizing the MFI of corresponding untreated cells to 1. (b) PMN from healthy blood donors were stimulated with 10 μm of the ODN IDX9059 for 3 or 20 hr, after which the expression of CXCR1 was analysed with flow cytometry. Results are presented as mean values ± SEM (n = 4). ***P < 0·001 by two-way analysis of variance with Bonferroni's post hoc correction versus untreated cells.
Figure 3
Figure 3
The decrease in CXCR1 surface expression can be mediated through both Toll-like receptor 9 (TLR9) -dependent and -independent mechanisms and do not involve MAC-1, interleukin-8 (IL-8) or tumour necrosis factor-α (TNF-α). Polymorphonuclear neutrophils (PMN) from healthy blood donors were pre-incubated for 30 min with 0·5, 5 or 10 μg/ml of Chloroquine (a) (n = 4) or with a monoclonal anti-MAC1 antibody (b) (n = 3) before being stimulated with 10 μm of oligonucleotides (ODN) or with medium alone (untreated) for 3 hr. Cells were subsequently harvested and analysed for CXCR1 surface expression by flow cytometry. The fold changes in mean fluorescence intensity (MFI) of CXCR1+ CD66b+ PMN were calculated by normalizing the MFI of corresponding untreated cells to 1 (dotted line). (c) PMN from healthy blood donors were stimulated with 10 μm of the ODN IDX9059 or with medium alone (untreated) for 20 hr after which the supernatants were collected and analysed for the presence of IL-8 using cytometric bead array (n = 4). Results are presented as mean values ±SEM. *P < 0·05 by one-way analysis of variance with Dunnett's post hoc correction versus untreated cells.
Figure 4
Figure 4
Oligonucleotides (ODN) decrease surface expression of the leukotriene B4 (LTB4) receptor BLT1. Human polymorphonuclear neutrophils (PMN) from healthy blood donors were stimulated with 0·5, 10 or 25 μm of ODN or with medium alone (untreated) for 3 hr. Cells were subsequently harvested and analysed for BLT1 surface expression by flow cytometry. (a) FACS histograms show the expression of BLT1 of PMN incubated with 25 μmIDX9054 compared with medium alone. The fold change in mean fluorescence intensity (MFI) of BLT1+ CD66b-positive PMN was calculated by normalizing the MFI of corresponding untreated cells to 1 (dotted line). Results are presented as mean ± SEM (n = 5). **P < 0·01 by one-way analysis of variance with Dunnett's post hoc correction versus untreated cells.
Figure 5
Figure 5
Oligonucleotides (ODN) inhibit interleuin-8 (IL-8) and leukotriene B4 (LTB4) -induced chemotaxis. (a) Human polymorphonuclear neutrophils (PMN) from healthy blood donors were pre-incubated with 0·5, 10 or 25 μm of ODN or with medium alone [untreated (untr)] for 1 hr after which the cells were investigated for their ability to migrate towards IL-8 in the presence of free ODN (n = 3) (a) or free oligonucleotides were washed away and the cells were investigated for their ability to migrate towards IL-8 (n = 5) (b) or LTB4 (n = 4–6) (c) in a chemotaxis assay for 3 hr. As a control for specificity, a monoclonal antibody against IL-8 (α-IL-8) was included in the assay (b). Results are presented as the mean number of migrated PMN ± SEM. *P < 0·05 and **P < 0·01 by one-way analysis of variance with Dunnett's post hoc correction versus untreated cells. (d,e) The mean fluorescence intensity (MFI) of CXCR1 (d), CXCR2 (e) and BLT1 (f) surface expression on PMN after stimulation with ODN were plotted against the number of PMN that migrated towards IL-8 (d and e) or LTB4 (f) in the chemotaxis assay. The curve fit (r2) is specified in the figures. ***P < 0·001 by linear regression analysis.
Figure 6
Figure 6
IDX9059 inhibit leucocyte migration in vivo. Leucocyte migration was analysed in vivo by chemotactic intravital microscopy in the cremaster muscle of mice. Cells per field before (a) and after (b) addition of platelet activating factor (PAF) were analysed using time-lapse video recordings. The animals were given IDX9059 subcutaneously, 50 μg/100 μl/mouse, c. 20 min before induction of inflammation. R = rolling cells, A = adhering cells and T = transmigrated cells. Results are presented as mean ± SEM,n = 4. *P < 0·05 by Student's t-test.
Figure 7
Figure 7
IDX9059 inhibit polymorphonuclear neutrophils (PMN) migration in a mouse model of ovalbumin (OVA) -induced airway inflammation. Mice were sensitized to OVA by intraperitoneal injection of OVA/alum on days 0 and 12. On days 23, 26, 30 and 33, mice were challenged in the lungs by inhalation of aerosolized OVA. At days 16 and 21, 50 μg/animal of IDX9059 were instilled intranasally. PBS was used as control for the ODN treatment. Mice that were not immunized with OVA but treated with PBS and exposed to OVA aerosol were included as controls, as well as mice that were immunized with OVA but not exposed to OVA aerosol (no aerosol). The total numbers of leucocytes, PMN and lymphocytes were analysed in bronchoalveolar lavage fluid 48 hr after the last OVA aerosol and are presented as mean ± SEM. *P < 0·05, **P < 0·01 and ***P < 0·001 by one-way analysis of variance using Dunnett's post hoc correction compared with PBS-treated control mice.

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