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. 2013 Aug;14(8):726-32.
doi: 10.1038/embor.2013.89. Epub 2013 Jul 2.

Adenosine-A3 receptors in neutrophil microdomains promote the formation of bacteria-tethering cytonemes

Ross Corriden  1 , Tim Self, Kathryn Akong-Moore, Victor Nizet, Barrie Kellam, Stephen J Briddon, Stephen J Hill
Affiliations

Adenosine-A3 receptors in neutrophil microdomains promote the formation of bacteria-tethering cytonemes

Ross Corriden et al. EMBO Rep. 2013 Aug.

Abstract

The A3-adenosine receptor (A3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A3AR ligand, we show that A3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A3ARs promote the formation of filipodia-like projections (cytonemes) that can extend up to 100 μm to tether and 'reel in' pathogens. Exposure to bacteria or an A3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A3AR-selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A3AR as a potential target for modulating their function.

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Conflict of interest statement

S.J.H. and B.K. are founders and directors of the University of Nottingham spin-out company that provided CA20065.

Figures

Figure 1
Figure 1
Endogenous A3ARs aggregate into plaque-like microdomains that regulate processes involving cytoskeletal remodelling. (A) Representative plots showing directional migration of neutrophils within fMLP gradients is inhibited by A3AR-selective antagonist MRS1334. (B) Quantification showing 51% reduction (***P<0.0001; Student’s t-test) of migration speed of MRS1334-treated cells (n=97–127 cells, three separate experiments). (C,D) Flow cytometry-based displacement experiments using CA200645 and unlabelled MRS1334 showed that the fluorescent ligand predominantly detected A3ARs. Data shown are means±s.e.m. of three separate experiments. A3AR, A3-adenosine receptor; fMLP, fMet-Leu-Phe.
Figure 2
Figure 2
A3AR activation promotes the extension of membrane projections from human neutrophils. (A) Confocal imaging of Vybrant DiO/CA200645-labelled human neutrophils revealed that A3AR plaques were frequently associated with membrane projections. Quantitative analysis of neutrophils isolated from the blood of three different healthy volunteers revealed that 69.3±6.7% of these projections were associated with A3AR plaques. (B) A Riveal Contrast Microscope (Quorum, Ontario, Canada) was used to show that these projections are also found on unstained, phorbol 12-myristate 13-acetate-treated human neutrophils. A3AR, A3-adenosine receptor.
Figure 3
Figure 3
The A3AR-selective antagonist MRS1334 and cytochalasin B inhibit A3AR agonist-stimulated cytoneme formation. (A) The A3AR-selective agonist 2-Cl-IB-MECA (1 μM at t=0 s) promotes the extension of cytonemes from neutrophils. Quantitative analysis revealed that 2-Cl-IB-MECA treatment significantly increases the percentage of cells exhibiting cytonemes (B) and the number of cytonemes/cell (C), effects inhibited by MRS1334 or cytochalasin B. Data shown are means±s.e.m. of 26–35 individual experiments; significance was determined via one-way analysis of variance with post-hoc Newman–Keuls tests. (D) ATRA-differentiated HL60 cells, which exhibited both A3AR plaques and plaque-associated cytonemes (merged image of a Vybrant DiO and CA200645-labelled HL60 cell), were transfected with the fluorescent F-actin probe LifeAct, confirming the presence of F-actin in these structures (inlay, white). **P<0.01, ***P<0.001. A3AR, A3-adenosine receptor; HBSS, Hank’s Buffered Salt Solution; 2-Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide.
Figure 4
Figure 4
Neutrophil cytonemes formed in response to E. coli exposure are capable of tethering and ‘reeling in’ bacteria for phagocytosis. (A) Time-lapse confocal imaging shows neutrophils (Vybrant DiO labelled; green) can capture E. coli (SYTO 83 labelled; orange) via extension of cytonemes, which are rapidly retracted for phagocytosis. (B) Imaging of CA200645-labelled neutrophils showed bacteria-tethering cytonemes are associated with A3AR plaques (red); SYTO 83-labelled E. coli are pseudocoloured blue. (C) Exposure of neutrophils to E. coli promotes cytoneme extension, an effect inhibited by MRS1334 (n=26 individual experiments). Data shown are means±s.e.m.; statistical significance was determined via repeated measures analysis of variance with post-hoc Newman–Keuls tests. *P<0.05, ***P<0.001. See also supplementary Videos S1, S2 online. A3AR, A3-adenosine receptor.
Figure 5
Figure 5
Neutrophil nanotubes enable the targeted, long-distance sampling/capture of pathogens. (A) Confocal imaging of Vybrant DiO-stained neutrophils that were exposed to SYTO 83-stained H. pylori revealed that cytonemes enable neutrophils to sample/capture bacteria up to 100 μm away from the cell. In addition, time-lapse imaging revealed that neutrophils are capable of simultaneously extending several cytonemes and capturing several pathogens at a time. (B) The spatial adaptation of cytonemes in relation to the position of motile bacteria (for example, the H. pylori cluster highlighted with white arrows) suggests that cytonemes are capable of adapting to changing spatio-temporal signals. See also supplementary Video S3 online. (C) Activation of A3ARs with 2-Cl-IB-MECA enhanced the ability of human neutrophils to phagocytose fluorescent S. aureus bioparticles. Data shown are means±s.e.m. of three individual experiments. Y axis shows relative intensity units. A3AR, A3-adenosine receptor; HBSS, Hank’s Buffered Salt Solution; 2-Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide.
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References

    1. Junger WG (2011) Immune cell regulation by autocrine purinergic signalling. Nat Rev Immunol 11: 201–212 - PMC - PubMed
    1. Elliott MR et al. (2009) Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461: 282–286 - PMC - PubMed
    1. Chen Y, Corriden R, Inoue Y, Yip L, Hashiguchi N, Zinkernagel A, Nizet V, Insel PA, Junger WG (2006) ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science 314: 1792–1795 - PubMed
    1. Kronlage M et al. (2010) Autocrine purinergic receptor signaling is essential for macrophage chemotaxis. Sci Signal 3: ra55. - PubMed
    1. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CCM, Beck PL, Muruve DA, Kubes P (2010) Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330: 362–366 - PubMed

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