Limits...
P2Y12 expression and function in alternatively activated human microglia.

Moore CS, Ase AR, Kinsara A, Rao VT, Michell-Robinson M, Leong SY, Butovsky O, Ludwin SK, Séguéla P, Bar-Or A, Antel JP - Neurol Neuroimmunol Neuroinflamm (2015)

Bottom Line: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions.In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection.These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences (C.S.M.), Neuroscience, Memorial University of Newfoundland and Labrador, St. John's, Newfoundland, Canada; Neuroimmunology Unit (C.S.M., A.A., A.K., V.T.S.R., M.M.-R., S.Y.L., P.S., A.B.-O., J.P.A.), Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada; Center for Neurologic Diseases (O.B.), Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; and Department of Pathology and Molecular Medicine (S.K.L.), Queens University, Kingston, Ontario, Canada.

ABSTRACT

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation.

Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain.

Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection.

Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

No MeSH data available.


Related in: MedlinePlus

ADP-evoked intracellular calcium responses discriminate between human microglia and macrophages while increased ADP-evoked intracellular calcium responses are observed in M2-polarized human microglia(A) Averaged traces of intracellular calcium ([Ca2+]i) levels following a short application of ADP in fetal human microglia. The P2Y12 selective antagonist PSB0739 strongly blocks ADP-induced [Ca2+]i. (B) Quantification of ADP-evoked [Ca2+]i responses in the absence or presence of PSB0739. (C) ADP-evoked [Ca2+]i is minimal in human macrophages; however, the cells respond strongly to ATP. (D) Quantification of ADP- and ATP-induced [Ca2+]i transients. (E) Averaged traces of [Ca2+]i levels following application of ADP in unpolarized human fetal microglia and (F) comparison with responses to ADP in M2-polarized cells. (G) ADP-evoked [Ca2+]i responses in both unpolarized (Unpol.) and M2-polarized cells are blocked by the P2Y12 antagonist PSB0739, summary quantification. Error bars represent mean ± SEM; n = 14–35 cells/condition; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4370387&req=5

Figure 3: ADP-evoked intracellular calcium responses discriminate between human microglia and macrophages while increased ADP-evoked intracellular calcium responses are observed in M2-polarized human microglia(A) Averaged traces of intracellular calcium ([Ca2+]i) levels following a short application of ADP in fetal human microglia. The P2Y12 selective antagonist PSB0739 strongly blocks ADP-induced [Ca2+]i. (B) Quantification of ADP-evoked [Ca2+]i responses in the absence or presence of PSB0739. (C) ADP-evoked [Ca2+]i is minimal in human macrophages; however, the cells respond strongly to ATP. (D) Quantification of ADP- and ATP-induced [Ca2+]i transients. (E) Averaged traces of [Ca2+]i levels following application of ADP in unpolarized human fetal microglia and (F) comparison with responses to ADP in M2-polarized cells. (G) ADP-evoked [Ca2+]i responses in both unpolarized (Unpol.) and M2-polarized cells are blocked by the P2Y12 antagonist PSB0739, summary quantification. Error bars represent mean ± SEM; n = 14–35 cells/condition; ***p < 0.001.

Mentions: To further validate the presence of functional P2Y12 receptors at the surface of human microglia, prominent intracellular calcium ([Ca2+]i) transients were observed in human fetal microglia following a short application of ADP (figure 3, A and B). Since ADP is also an agonist of P2Y1 and P2Y13, the P2Y12-selective antagonist PSB0739 was used to assess the selective contribution of P2Y12 to ADP-evoked [Ca2+]i responses. Treatment with PSB0739 (10 µM) suppressed ADP-induced [Ca2+]i transients (figure 3, A and B), confirming P2Y12 as the primary ADP receptor on human microglia. In contrast, ADP-induced [Ca2+]i responses were small or absent in human MDMs, yet they responded to ATP, likely through Gq-coupled P2Y2 receptors (figure 3, C and D). To measure across species, similar experiments were performed using rat microglia and produced similar transients (figure e-1, B and C). Differential ADP-evoked [Ca2+]i responses were also observed between unpolarized, M1-polarized, and M2-polarized human microglia. Compared to unpolarized microglia, transients were significantly larger in the M2-polarized cells (figure 3, E–G). No differences between unpolarized and M1-polarized microglia were observed (figure e-2). In both unpolarized and M2-polarized cells, ADP-induced [Ca2+]i responses were blocked by a selective P2Y12 antagonist.


P2Y12 expression and function in alternatively activated human microglia.

Moore CS, Ase AR, Kinsara A, Rao VT, Michell-Robinson M, Leong SY, Butovsky O, Ludwin SK, Séguéla P, Bar-Or A, Antel JP - Neurol Neuroimmunol Neuroinflamm (2015)

ADP-evoked intracellular calcium responses discriminate between human microglia and macrophages while increased ADP-evoked intracellular calcium responses are observed in M2-polarized human microglia(A) Averaged traces of intracellular calcium ([Ca2+]i) levels following a short application of ADP in fetal human microglia. The P2Y12 selective antagonist PSB0739 strongly blocks ADP-induced [Ca2+]i. (B) Quantification of ADP-evoked [Ca2+]i responses in the absence or presence of PSB0739. (C) ADP-evoked [Ca2+]i is minimal in human macrophages; however, the cells respond strongly to ATP. (D) Quantification of ADP- and ATP-induced [Ca2+]i transients. (E) Averaged traces of [Ca2+]i levels following application of ADP in unpolarized human fetal microglia and (F) comparison with responses to ADP in M2-polarized cells. (G) ADP-evoked [Ca2+]i responses in both unpolarized (Unpol.) and M2-polarized cells are blocked by the P2Y12 antagonist PSB0739, summary quantification. Error bars represent mean ± SEM; n = 14–35 cells/condition; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4370387&req=5

Figure 3: ADP-evoked intracellular calcium responses discriminate between human microglia and macrophages while increased ADP-evoked intracellular calcium responses are observed in M2-polarized human microglia(A) Averaged traces of intracellular calcium ([Ca2+]i) levels following a short application of ADP in fetal human microglia. The P2Y12 selective antagonist PSB0739 strongly blocks ADP-induced [Ca2+]i. (B) Quantification of ADP-evoked [Ca2+]i responses in the absence or presence of PSB0739. (C) ADP-evoked [Ca2+]i is minimal in human macrophages; however, the cells respond strongly to ATP. (D) Quantification of ADP- and ATP-induced [Ca2+]i transients. (E) Averaged traces of [Ca2+]i levels following application of ADP in unpolarized human fetal microglia and (F) comparison with responses to ADP in M2-polarized cells. (G) ADP-evoked [Ca2+]i responses in both unpolarized (Unpol.) and M2-polarized cells are blocked by the P2Y12 antagonist PSB0739, summary quantification. Error bars represent mean ± SEM; n = 14–35 cells/condition; ***p < 0.001.
Mentions: To further validate the presence of functional P2Y12 receptors at the surface of human microglia, prominent intracellular calcium ([Ca2+]i) transients were observed in human fetal microglia following a short application of ADP (figure 3, A and B). Since ADP is also an agonist of P2Y1 and P2Y13, the P2Y12-selective antagonist PSB0739 was used to assess the selective contribution of P2Y12 to ADP-evoked [Ca2+]i responses. Treatment with PSB0739 (10 µM) suppressed ADP-induced [Ca2+]i transients (figure 3, A and B), confirming P2Y12 as the primary ADP receptor on human microglia. In contrast, ADP-induced [Ca2+]i responses were small or absent in human MDMs, yet they responded to ATP, likely through Gq-coupled P2Y2 receptors (figure 3, C and D). To measure across species, similar experiments were performed using rat microglia and produced similar transients (figure e-1, B and C). Differential ADP-evoked [Ca2+]i responses were also observed between unpolarized, M1-polarized, and M2-polarized human microglia. Compared to unpolarized microglia, transients were significantly larger in the M2-polarized cells (figure 3, E–G). No differences between unpolarized and M1-polarized microglia were observed (figure e-2). In both unpolarized and M2-polarized cells, ADP-induced [Ca2+]i responses were blocked by a selective P2Y12 antagonist.

Bottom Line: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions.In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection.These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

View Article: PubMed Central - PubMed

Affiliation: Division of BioMedical Sciences (C.S.M.), Neuroscience, Memorial University of Newfoundland and Labrador, St. John's, Newfoundland, Canada; Neuroimmunology Unit (C.S.M., A.A., A.K., V.T.S.R., M.M.-R., S.Y.L., P.S., A.B.-O., J.P.A.), Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada; Center for Neurologic Diseases (O.B.), Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; and Department of Pathology and Molecular Medicine (S.K.L.), Queens University, Kingston, Ontario, Canada.

ABSTRACT

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation.

Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain.

Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection.

Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

No MeSH data available.


Related in: MedlinePlus