Neuronal synapse formation induced by microglia and interleukin 10 - PubMed (original) (raw)

Neuronal synapse formation induced by microglia and interleukin 10

So-Hee Lim et al. PLoS One. 2013.

Abstract

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Neuronal synapse formation induced by microglia.

(A) Microglia were prepared from mixed glia without astrocytes. The prepared microglia were stained with antibodies of CD11b/c (red) and GFAP (green). CD11b/c and GFAP are specific markers of microglia and astrocytes respectively. The numbers of cells stained with antibodies of CD11b/c (microglia) or GFAP (astrocytes) were counted, and the purity of microglia was analyzed. Scale bar, 10 μm. (B) An RT-PCR assay was performed to determine the purity of microglia prepared from mixed glia. The expressions of Iba-1 (a specific marker of microglia) and GFAP (a specific marker of astrocytes) were analyzed. (C) The density of dendritic spines was increased by application of microglia. Rat cultured hippocampal neurons were transfected at days in vitro (DIV) 7 with pSuper.neo-gfp, and then immunostained using anti-GFP antibodies at DIV 15 for analysis. Microglia plated on a porous cell culture insert were applied to hippocampal neurons of DIV 8. The density of dendritic spines increased when microglia was applied. Means±SEM. _n_=48 dendrites for no microglia, 48 for 0.25 × 105 microglia, 48 for 0.5 × 105 microglia, 48 for 1.0 × 105 microglia. **p<0.01 and ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, _F_=9.915, p<0.0001. Scale bar, 10 μm. (D) The number of excitatory synapses was increased by application of microglia. The cultured hippocampal neurons were immunostained using antibodies of GFP (green) and vGLUT, the excitatory synaptic marker (red). The addition of microglia increased the number of vGLUT. Means±SEM. _n_=40 dendrites for no microglia, 40 for 0.25 × 105 microglia, 40 for 0.5 × 105 microglia, 40 for 1.0 × 105 microglia. ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=8.206, p<0.0001. Scale bar, 10 μm. (E) The number of inhibitory synapses was increased by application of microglia. Antibodies of GFP (green) and vGAT, the inhibitory synaptic marker (red), were used for the immunostaining of hippocampal neurons. The addition of microglia increased the number of vGAT. Means±SEM. _n_=40 dendrites for no microglia, 40 for 0.25 × 105 microglia, 40 for 0.5 × 105 microglia, 40 for 1.0 × 105 microglia. *p<0.05 and ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, _F_=31.81, p<0.0001. Scale bar, 10 μm.

Figure 2

Figure 2. Neuronal synapse formation induced by IL-10 released from microglia.

(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.

Figure 3

Figure 3. IL-10 receptors expressed on hippocampal neurons.

(A) Expression of IL-10 receptor mRNAs in hippocampal neurons. The expression of the IL-10 receptor was identified using RT-PCR. The mRNAs of IL-10 receptor α and β were expressed in the hippocampal neurons. IL-10 receptor α was expressed mainly in hippocampal neurons of DIV 7. (1, cultured hippocampal neurons at DIV 7; 2, cultured hippocampal neurons at DIV 15; 3, mixed glial culture at DIV 7; 4, mixed glial culture at DIV 15; 5, microglia) Quantification (DIV 15 neuron/ DIV 7 neuron): IL-10 receptor α, 0.61; IL-10 receptor β, 1.06. (B) Expression of IL-10 receptor proteins in cultured hippocampal neurons. Similar to the expression of mRNA, the IL-10 receptor α protein was expressed mainly in neurons of DIV 7. Anti-IL-10 receptor α antibodies (0.5 μg/ml, Santa Cruz, sc-985) were used for western blotting [27]. Quantification (DIV 15 neuron/ DIV 7 neurons): IL-10 receptor α, 0.73. (C) Expression of IL-10 receptor proteins in the developing rat brains. The IL-10 receptor α proteins were expressed mainly in the developing brains of embryonic and early postnatal days (E18~P3). Quantification of IL-10 receptor α: E18, 0.30; P1, 0.27; P3, 1.0; P7, 0.22; P14, 0.20; P3W, 0.17; P6W, 0.15 (E, embryonic days; P, postnatal days). (D) Images of IL-10 receptor expressions in cultured hippocampal neurons. Hippocampal neurons of DIV 6 were stained with antibodies of IL-10 receptor α (5 μg/ml, Santa Cruz, sc-985) (red) and MAP2 (the neuronal marker, green) after treatment with 4% formaldehyde and then -20 °C methanol. Hippocampal neurons expressed IL-10 receptor proteins comparable to spinal neurons or cortical neurons. (E) The induction of synaptic formation by microglia was antagonized by the neutralizing antibody of IL-10 receptor α. When anti-mouse IL-10 receptor α antibody was applied to the co-culture of mouse microglia and mouse hippocampal neurons, the density of dendritic spines was significantly decreased compared with the control (without anti-IL-10 receptor antibody). Means±SEM. _n_=30 dendrites for no microglia without anti-IL-10 receptor antibody, 29 for the application of microglia only, 29 for no microglia with anti-IL-10 receptor antibody only, 29 for microglia with anti-IL-10 receptor antibody. ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=17.35, p<0.0001. (F) The induction of synaptic formation via microglia was not antagonized by the blocking antibody of TNFα. When anti-rat TNFα antibody was applied to the co-culture of rat microglia and rat hippocampal neurons, the density of dendritic spine was not decreased compared with control (without anti-TNFα antibody). Means±SEM. _n_=27 dendrites for no microglia without anti-TNFα antibody, 28 for the application of microglia only, 29 for no microglia with anti-TNFα receptor antibody only, 27 for microglia with anti-TNFα antibody. **p<0.01 and ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, _F_=9.104, p<0.0001.

Figure 4

Figure 4. Neuronal synapse formation attenuated by the knockdown of IL-10 receptors.

(A) The expressions of IL-10 receptors attenuated by shRNA in hippocampal neurons. The lentivirus expressing shRNA of IL-10 receptor α was applied to the cultured hippocampal neurons of DIV 3, and then RT-PCR was performed at DIV 7. The expression of IL-10 receptor α was decreased significantly by the application of shRNA. Quantification (shRNA/vector only): 15 A.U. shRNA, 0.79; 20 A.U. shRNA, 0.49; 25 A.U. shRNA, 0.20 (A.U., arbitrary unit). (B) Attenuated synaptic formation by application of shRNA of IL-10 receptor α. The induction of synaptic formation by microglia or recombinant IL-10 was dramatically attenuated when shRNA of IL-10 receptor α was applied to hippocampal neurons. Means±SEM. _n_=29 dendrites for vector only (shVec), 28 for shRNA of IL-10 receptor α #1 (shRNA#1), 26 for shRNA of IL-10 receptor α #2 (shRNA#2), 29 for the application of microglia only, 30 for microglia with shRNA#1, 26 for microglia with shRNA#2, 29 for the application of recombinant IL-10 (5 ng/ml) only, 30 for IL-10 with shRNA#1, 27 for IL-10 with shRNA#2. ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, _F_=23.04, p<0.0001. Scale bar, 10 μm.

Figure 5

Figure 5. IL-1β antagonized IL-10 in neuronal synapse formation.

(A) The induction of synaptic formation by microglia was attenuated when microglia were pretreated with lipopolysaccharide (LPS). Means±SEM. _n_=29 dendrites for no microglia, 29 for non-treated microglia, 30 for microglia pretreated with 1 ng/ml LPS, 30 for microglia pretreated with 10 ng/ml LPS, and 29 for microglia pretreated with 100 ng/ml LPS. ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=8.785, p<0.0001. (B) Increased expressions of cytokine mRNAs after LPS treatment. An RT-PCR was performed after the microglia were treated with LPS at indicated concentrations. The mRNAs of IL-1β and TNFα were increased significantly by 1 ng/ml LPS, but the expression of IL-10 mRNA was not convincingly increased by 1 ng/ml LPS. Quantification (LPS treated/not treated): IL-10 by 1 ng/ml LPS, 1.57; IL-10 by 10 ng/ml LPS, 4.13; IL-10 by 100 ng/ml LPS, 4.06; IL-1β by 1 ng/ml LPS, 19.2; IL-1β by 10 ng/ml LPS, 28.0; IL-1β by 100 ng/ml LPS, 25.8; TNFα by 1 ng/ml LPS, 4.26; TNFα by 10 ng/ml LPS, 5.07; TNFα by 100 ng/ml LPS, 4.48. (C) The induction of synaptic formation by IL-10 was attenuated when IL-1β was applied together with IL-10. Recombinant proteins of IL-10 or IL-1β were applied to the cultured hippocampal neurons of DIV 8 and the density of dendritic spines was analyzed after one week. IL-1β antagonized the induction of synaptic formation by IL-10. Means±SEM. _n_=30 dendrites for control (no IL-10 no IL-1β), 28 for 5 ng/ml IL-10, 27 for 10 ng/ml IL-10, 26 for 2 ng/ml IL-1β, 28 for 5 ng/ml IL-1β, 29 for 10 ng/ml IL-1β, 29 for 5 ng/ml IL-10 plus 5 ng/ml IL-1β, 30 for 10 ng/ml IL-10 plus 10 ng/ml IL-1β. **p<0.01 and ***p<0.001 by the Newman-Keuls multiple comparison test after application of one-way ANOVA, _F_=14.67, p<0.0001. Scale bar, 10 μm.

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National Research Foundation of Korea (NRF) grant funded by the Korea government (2012R1A2A2A02014520 and 2009-0087354 to J.R.L.) and a grant from KRIBB research initiative program (to J.R.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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