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. 2013 Nov 22;8(11):e81218. doi: 10.1371/journal.pone.0081218

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© 2013 Lim et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) The density of dendritic spines was increased by application of recombinant IL-10. Recombinant proteins of IL-10 were applied to hippocampal neurons of DIV 8 and the density of dendritic spines was analyzed after one week. Means±SEM. n=31 dendrites for control (no IL-10), 31 for 2 ng/ml IL-10, 31 for 5 ng/ml IL-10, 31 for 10 ng/ml IL-10, 31 for 20 ng/ml IL-10. ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=15.66, p<0.0001. Scale bar, 10 μm.

(B) The number of excitatory synapses was increased by application of recombinant IL-10. The application of recombinant IL-10 increased the number of vGLUT. Means±SEM. n=34 dendrites for control (no IL-10), 35 for 2 ng/ml IL-10, 31 for 5 ng/ml IL-10, 31 for 10 ng/ml IL-10, 33 for 20 ng/ml IL-10. ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=16.33, p<0.0001.

(C) The number of inhibitory synapses was increased by application of recombinant IL-10. The application of recombinant IL-10 increased the number of vGAT. Means±SEM. n=32 dendrites for control (no IL-10), 28 for 2 ng/ml IL-10, 29 for 5 ng/ml IL-10, 27 for 10 ng/ml IL-10, 27 for 20 ng/ml IL-10. *p<0.05 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=2.960, p=0.0220.

(D) IL-10 was released from intact microglia without external stimuli. Microglia were plated on a porous cell culture insert, and incubated in a neuronal culture media. After 3~7 days, the amount of IL-10 released from the microglia was analyzed using an ELISA kit. An appreciable amount of IL-10 was released from microglia compared with controls (neuronal culture media only or hippocampal neuron only). **p<0.01 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=48.25, p=0.0013.

(E) Microglia and recombinant IL-10 use the same signal pathway for induction of synaptic formation. Addition of recombinant IL-10 did not additively enhance the induction of synaptic formation by microglia. Means±SEM. n=27 dendrites for control (no microglia no IL-10), 27 for the application of 1.0 × 10⁵ microglia, 28 for 5 ng/ml of IL-10, 27 for microglia and IL-10. ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=15.50, p<0.0001.