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Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Clausen BH, Lambertsen KL, Dagnæs-Hansen F, Babcock AA, von Linstow CU, Meldgaard M, Kristensen BW, Deierborg T, Finsen B - Acta Neuropathol. (2016)

Bottom Line: The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1β, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex.The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke.Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloewsvej 25, 5000, Odense C, Denmark. bclausen@health.sdu.dk.

ABSTRACT
Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/β identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1β to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1β, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

No MeSH data available.


Related in: MedlinePlus

IL-1Ra, IL-1α and IL-1β are expressed by largely different microglial subsets. a Flow cytometric quantification of CD11b+CD45dim microglia and CD11b+CD45high leukocytes in non-lesioned C56BL/6 controls (Ctl) and in C56BL/6 mice 6, 12 and 24 h after pMCAo (left), n = 4–8/group, with dot plots (right) showing flow cytometric profiles and the isotype quadrant from a mouse with 24 h survival. b, c Flow cytometric quantification of IL-1Ra+, IL-1α+, and IL-1β+ CD11b+CD45dim microglia (b) and CD11b+CD45high leukocytes (c), n = 4/group. Statistical data are presented as mean ± SD (Kruskal–Wallis test with Dunns post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001. dDot plot (left) with isotype quadrants showing gated CD11b+CD45dim microglia expressing either IL-1Ra or IL-1β 24 h after pMCAo. ePie charts show the percentage of gated CD11b+CD45dim microglia co-expressing either IL-1Ra and IL-1α or IL-1β and IL-1α, with no co-expression of IL-1Ra and IL-1β 24 h after pMCAo (co-expression is indicated as percentages outside the pie charts). f–k IHC double-fluorescence staining showing IL-1Ra and IL-1β (f, i), IL-1Ra and IL-1α (g, j), and IL-1β and IL-1α (h, k) in the peri-infarct 24 h after pMCAo. IL-1Ra and IL-1β are expressed in spatially segregated microglia (f, i, from the same section). Scale bars 10 µm (f, i), 50 µm (g, h), 10 µm (j, k), and 10 µm (inserts)
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Fig3: IL-1Ra, IL-1α and IL-1β are expressed by largely different microglial subsets. a Flow cytometric quantification of CD11b+CD45dim microglia and CD11b+CD45high leukocytes in non-lesioned C56BL/6 controls (Ctl) and in C56BL/6 mice 6, 12 and 24 h after pMCAo (left), n = 4–8/group, with dot plots (right) showing flow cytometric profiles and the isotype quadrant from a mouse with 24 h survival. b, c Flow cytometric quantification of IL-1Ra+, IL-1α+, and IL-1β+ CD11b+CD45dim microglia (b) and CD11b+CD45high leukocytes (c), n = 4/group. Statistical data are presented as mean ± SD (Kruskal–Wallis test with Dunns post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001. dDot plot (left) with isotype quadrants showing gated CD11b+CD45dim microglia expressing either IL-1Ra or IL-1β 24 h after pMCAo. ePie charts show the percentage of gated CD11b+CD45dim microglia co-expressing either IL-1Ra and IL-1α or IL-1β and IL-1α, with no co-expression of IL-1Ra and IL-1β 24 h after pMCAo (co-expression is indicated as percentages outside the pie charts). f–k IHC double-fluorescence staining showing IL-1Ra and IL-1β (f, i), IL-1Ra and IL-1α (g, j), and IL-1β and IL-1α (h, k) in the peri-infarct 24 h after pMCAo. IL-1Ra and IL-1β are expressed in spatially segregated microglia (f, i, from the same section). Scale bars 10 µm (f, i), 50 µm (g, h), 10 µm (j, k), and 10 µm (inserts)

Mentions: A quantitative assessment of microglial versus leukocyte-derived IL-1Ra, IL-1α and IL-1β was performed by flow cytometry separating CD11b+ cells into CD11b+CD45dim microglia and CD11b+CD45high leukocytes (Fig. 3a) [10, 40, 58]. As previously shown [10, 40], the number of recruited CD11b+CD45high leukocytes gradually increased from 6 to 24 h after pMCAo in C57BL/6 mice, whereas the number of CD11b+CD45dim microglia overall remained constant (Fig. 3a). IL-1Ra, IL-1α and IL-1β were expressed by both CD11b+CD45dim microglia (Fig. 3b) and CD11b+CD45high leukocytes (Fig. 3c), however, with significant differences in baseline and temporal expression profiles (Figs. 3b, S3a–c). Interestingly, there were significantly higher numbers of IL-1Ra+ compared to IL-1α+ and IL-1β+ microglia in non-lesioned control mice and at 6 h after pMCAo, but not at 12 h, when both IL-1α+ and IL-1β+ microglia had increased compared to controls (Fig. 3b). By 24 h, the proportion of IL-1Ra+ microglia reached 34 ± 11 % [mean ± SD, (n = 4)] of the gated CD11b+CD45dim population, while IL-1α+ reached 9 ± 1 % [mean ± SD, (n = 4)] and IL-1β+ reached 18 ± 5 % [mean ± SD, (n = 4)].Fig. 3


Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Clausen BH, Lambertsen KL, Dagnæs-Hansen F, Babcock AA, von Linstow CU, Meldgaard M, Kristensen BW, Deierborg T, Finsen B - Acta Neuropathol. (2016)

IL-1Ra, IL-1α and IL-1β are expressed by largely different microglial subsets. a Flow cytometric quantification of CD11b+CD45dim microglia and CD11b+CD45high leukocytes in non-lesioned C56BL/6 controls (Ctl) and in C56BL/6 mice 6, 12 and 24 h after pMCAo (left), n = 4–8/group, with dot plots (right) showing flow cytometric profiles and the isotype quadrant from a mouse with 24 h survival. b, c Flow cytometric quantification of IL-1Ra+, IL-1α+, and IL-1β+ CD11b+CD45dim microglia (b) and CD11b+CD45high leukocytes (c), n = 4/group. Statistical data are presented as mean ± SD (Kruskal–Wallis test with Dunns post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001. dDot plot (left) with isotype quadrants showing gated CD11b+CD45dim microglia expressing either IL-1Ra or IL-1β 24 h after pMCAo. ePie charts show the percentage of gated CD11b+CD45dim microglia co-expressing either IL-1Ra and IL-1α or IL-1β and IL-1α, with no co-expression of IL-1Ra and IL-1β 24 h after pMCAo (co-expression is indicated as percentages outside the pie charts). f–k IHC double-fluorescence staining showing IL-1Ra and IL-1β (f, i), IL-1Ra and IL-1α (g, j), and IL-1β and IL-1α (h, k) in the peri-infarct 24 h after pMCAo. IL-1Ra and IL-1β are expressed in spatially segregated microglia (f, i, from the same section). Scale bars 10 µm (f, i), 50 µm (g, h), 10 µm (j, k), and 10 µm (inserts)
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Fig3: IL-1Ra, IL-1α and IL-1β are expressed by largely different microglial subsets. a Flow cytometric quantification of CD11b+CD45dim microglia and CD11b+CD45high leukocytes in non-lesioned C56BL/6 controls (Ctl) and in C56BL/6 mice 6, 12 and 24 h after pMCAo (left), n = 4–8/group, with dot plots (right) showing flow cytometric profiles and the isotype quadrant from a mouse with 24 h survival. b, c Flow cytometric quantification of IL-1Ra+, IL-1α+, and IL-1β+ CD11b+CD45dim microglia (b) and CD11b+CD45high leukocytes (c), n = 4/group. Statistical data are presented as mean ± SD (Kruskal–Wallis test with Dunns post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001. dDot plot (left) with isotype quadrants showing gated CD11b+CD45dim microglia expressing either IL-1Ra or IL-1β 24 h after pMCAo. ePie charts show the percentage of gated CD11b+CD45dim microglia co-expressing either IL-1Ra and IL-1α or IL-1β and IL-1α, with no co-expression of IL-1Ra and IL-1β 24 h after pMCAo (co-expression is indicated as percentages outside the pie charts). f–k IHC double-fluorescence staining showing IL-1Ra and IL-1β (f, i), IL-1Ra and IL-1α (g, j), and IL-1β and IL-1α (h, k) in the peri-infarct 24 h after pMCAo. IL-1Ra and IL-1β are expressed in spatially segregated microglia (f, i, from the same section). Scale bars 10 µm (f, i), 50 µm (g, h), 10 µm (j, k), and 10 µm (inserts)
Mentions: A quantitative assessment of microglial versus leukocyte-derived IL-1Ra, IL-1α and IL-1β was performed by flow cytometry separating CD11b+ cells into CD11b+CD45dim microglia and CD11b+CD45high leukocytes (Fig. 3a) [10, 40, 58]. As previously shown [10, 40], the number of recruited CD11b+CD45high leukocytes gradually increased from 6 to 24 h after pMCAo in C57BL/6 mice, whereas the number of CD11b+CD45dim microglia overall remained constant (Fig. 3a). IL-1Ra, IL-1α and IL-1β were expressed by both CD11b+CD45dim microglia (Fig. 3b) and CD11b+CD45high leukocytes (Fig. 3c), however, with significant differences in baseline and temporal expression profiles (Figs. 3b, S3a–c). Interestingly, there were significantly higher numbers of IL-1Ra+ compared to IL-1α+ and IL-1β+ microglia in non-lesioned control mice and at 6 h after pMCAo, but not at 12 h, when both IL-1α+ and IL-1β+ microglia had increased compared to controls (Fig. 3b). By 24 h, the proportion of IL-1Ra+ microglia reached 34 ± 11 % [mean ± SD, (n = 4)] of the gated CD11b+CD45dim population, while IL-1α+ reached 9 ± 1 % [mean ± SD, (n = 4)] and IL-1β+ reached 18 ± 5 % [mean ± SD, (n = 4)].Fig. 3

Bottom Line: The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1β, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex.The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke.Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloewsvej 25, 5000, Odense C, Denmark. bclausen@health.sdu.dk.

ABSTRACT
Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/β identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1β to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1β, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

No MeSH data available.


Related in: MedlinePlus