The activation of cerebral cortical and cerebellar microglias increased by sera or IgG from children with OMS and NB
Besides cerebellum, emerging evidence has shown that the cerebral cortex has structural and functional changes in OMS patients, for most OMS patients have neurological handicaps in cerebral functions, such as deficits in attention, memory and language [4, 32]. Additionally, brain imaging of OMS patients shows changes in the cerebrum. Cerebral cortical thickness is reduced across the motor and visual areas in patients with pediatric OMS [33]. A patient with OMS revealed significant nodular enhancing lesions at gray–white junction of bilateral cerebral hemispheres by magnetic resonance imaging [34]. Another patient showed decreased metabolism in the bilateral occipital lobes and increased functional connectivity, including the primary and motion-sensitive visual cortex [35]. Therefore, both rat cerebral cortical and cerebellar microglias were exposed to sera or the IgG fraction isolated form sera of children with OMS and NB.
The expression of CD11b, a marker of microglial activation, was upregulated in cerebral cortical microglias incubated with sera from children with OMS and NB (3.41 ± 0.32 ng/mg total protein OMS+NB, 0.99 ± 0.09 ng/mg total protein NB, 1.09 ± 0.12 ng/mg total protein Healthy control, p< 0.001 vs NB, p< 0.001 vs Healthy control), whereas CD11b concentration was not statistically changed by sera of children with only NB at least under our experimental conditions (Fig. 1a). Moreover, IgG isolated from sera upregulated CD11b expression from 0.91 ± 0.10 ng/mg total protein in the NB group and 0.88 ± 0.07 ng/mg total protein in the healthy control group to 2.95 ± 0.23 ng/mg total protein in the OMS+NB group (p< 0.001 vs NB, p< 0.001 vs Healthy control, Fig. 1c). With respect to cerebellar microglias, the concentration of CD11b was also increased after incubation with sera or IgG from the OMS+NB group compared with the NB or healthy control group (Fig. 1b, d). However, neither commercially available human IgG nor IgG from children with JIA or anti-NMDAR encephalitis had a significant impact on the concentration of CD11b (Fig. 1c, d), suggesting that upregulation of CD11b induced by serum IgG from children with OMS and NB is not simply induced by increased dose of IgG, is not common to all diseases with IgG, and is not common to all autoantibody-mediated disorders of the CNS. In addition, no alteration of CD11b concentration was observed after treatment with the IgG-free fraction (Fig. 1e, f), which suggested that the upregulation of CD11b induced by sera mainly depends on the IgG fraction.
To further investigate the effects of serum IgG from children with OMS and NB on microglial activation, the concentrations of proinflammatory cytokines (including IL-1β, IL-6, TNF-α and MCP-1) and NO in the media of cerebral cortical and cerebellar microglias were detected by ELISA. The releases of IL-1β, IL-6, TNF-α and MCP-1 from cerebral cortical microglias exposed to OMS+NB sera were elevated (IL-1β: 105.4 ± 10.48 pg/ml OMS+NB, 43.99 ± 4.29 pg/ml NB, 39.83 ± 5.64 pg/ml Healthy control, p< 0.001 vs NB, p< 0.001 vs Healthy control; IL-6: 107.8 ± 11.20 pg/ml OMS+NB, 61.61 ± 3.67 pg/ml NB, 49.11 ± 5.53 pg/ml Healthy control, p< 0.01 vs NB, p< 0.001 vs Healthy control;TNF-α: 169.1 ± 14.32 pg/ml OMS+NB, 100.3 ± 9.15 pg/ml NB, 90.24 ± 6.76 pg/ml Healthy control, p< 0.001 vs NB, p< 0.001 vs Healthy control; MCP-1: 294.1 ± 9.30 pg/ml OMS+NB, 181.7 ± 7.93 pg/ml NB, 184.7 ± 9.59 pg/ml Healthy control, p< 0.001 vs NB, p< 0.001 vs Healthy control), whereas incubation with NB sera had no such effect (Fig. 2a1). Furthermore, the levels of IL-1β, IL-6, TNF-α and MCP-1 secreted from cerebral cortical microglias were increased to 109.6 ± 12.09 pg/ml, 133.2 ± 11.28 pg/ml, 225.3 ± 9.84 pg/ml and 324.2 ± 6.68 pg/ml after incubation with OMS+NB IgG, compared with NB IgG or healthy control IgG (p< 0.001 vs NB, p< 0.001 vs Healthy control, Fig. 2b1). In cerebellar microglias, similar impacts of sera and IgG were observed (Fig. 2c1, d1). Consistent with the results of cytokines, NO expression was also improved in the media of cerebral cortical (Fig. 2a2, b2) and cerebellar microglias (Fig. 2c2, d2) treated with OMS+NB sera or IgG. Together with the aforementioned CD11b expression, these results suggested that the activation of cultured cerebral cortical and cerebellar microglias was upregulated by serum IgG from children with OMS and NB.
In order to explore whether serum IgG-enhanced activation is specific to microglias or common to glial cells in the CNS, we detected the activation of cerebral cortical and cerebellar astrocytes. Using the marker of astrocytic activation, we found that the expression of GFAP was not significantly changed in cerebral cortical (Fig. 3a, c) and cerebellar astrocytes (Fig. 3b, d) incubated with sera or IgG from the OMS+NB group compared with the NB group and healthy control group at least under our experimental conditions, suggesting that the enhancement of activation induced by IgG from children with OMS and NB is specific to microglias, but not astrocytes.
Previously we have revealed that serum IgG from children with OMS and NB induces cytolysis in cultured neurons [6], whether serum IgG impacts the cytolysis of microglias or astrocytes needs further study. We observed that neither the cytolysis of cerebral cortical microglias (Fig. 4a, c) and cerebellar microglias (Fig. 4b, d) treated with OMS+NB sera or IgG, nor the cytolysis of cerebral cortical astrocytes (Fig. 4e, g) and cerebellar astrocytes (Fig. 4f, h) treated with OMS+NB sera or IgG was statistically changed, which suggested that preincubation with sera or IgG from children with OMS and NB specially upregulates microglial activation, rather than the cytolysis of microglias and astrocytes at least under our experimental conditions.
The cytolysis of neurons induced by conditioned media from microglias treated with OMS+NB IgG
It has been established that activated microglias contribute to neuron death by secreting various neurotoxic molecules in multiple disorders [8, 17] and serum IgG from patients with OMS and NB enhances neuronal death [6, 7, 36], we therefore explored whether neuronal cytolysis is enhanced by microglial activation in OMS. After getting rid of the remaining IgG, serum IgG-treated microglia conditioned media were collected and replaced the culture media of neurons. As expected, incubation with conditioned media from cerebral cortical and cerebellar microglias increased the cytolysis of neurons in the same brain regions (Cerebral cortical neuron: 31.95 ± 1.09% OMS+NB, 13.45 ± 1.12% NB, 14.53 ± 1.08% Healthy control; Cerebellar neuron: 31.21 ± 1.25% OMS+NB, 10.50 ± 1.27% NB, 12.75 ± 1.34% Healthy control; p< 0.001 vs NB, p< 0.001 vs Healthy control, Figure 5a, b). Moreover, conditioned media from cerebral cortical microglias to cerebellar neurons or the exchanged situation had similar results (Cerebral cortical neuron: 25.67 ± 2.71% OMS+NB, 9.00 ± 1.21% NB, 9.85 ± 1.95% Healthy control; Cerebellar neuron: 27.24 ± 2.55% OMS+NB, 11.79 ± 1.06% NB, 10.03 ± 2.86% Healthy control; p< 0.001 vs NB, p< 0.001 vs Healthy control, Fig. 5c, d). In contrast, we observed no alteration in the cytolysis of neurons treated with conditional media from astrocytes (Fig. 5e, f). Taken together, these results suggested that conditioned media from cerebral cortical or cerebellar microglias rather than astrocytes induce the cytolysis of neurons.
The effects of inhibitors or activators of NO/sGC/PKG pathway on conditioned media-induced neuronal cytolysis
To investigate the mechanisms of cytolysis induced by conditioned media from microglias treated with OMS+NB IgG, we focus on the NO/sGC/PKG pathway, for its role in neuronal death is well documented [21-23] and the production of NO from microglias was raised by OMS+NB IgG (Fig. 2a2-d2). The cytolysis of cerebral cortical and cerebellar neurons induced by conditioned media was alleviated by pretreatment with the NO synthesis inhibitor 7-NINA before OMS+NB IgG to microglias (Cerebral cortical neuron: 6.50± 1.46% 7-NINA, OMS+NB vs 26.91 ± 1.45% Saline, OMS+NB, p <0.001; 26.91 ± 1.45% Saline, OMS+NB vs 8.36 ± 1.10% Saline, NB, p <0.001; Cerebellar neuron: 11.30 ± 3.14% 7-NINA, OMS+NB vs 28.72 ± 2.43% Saline, OMS+NB, p <0.001; 28.72 ± 2.43% Saline, OMS+NB vs 11.50 ± 1.83% Saline, NB, p <0.001; Fig. 6a, b). Similar to 7-NINA, pretreatment with the sGC inhibitor ODQ or the PKG inhibitor Rp-8Br-PET-cGMP ameliorated the cytolysis of cerebral cortical and cerebellar neurons (Fig. 6c-f).
Moreover, the activators of NO synthesis and NO-activated intracellular pathway were also examined. The cytolysis of cerebral cortical neurons was exaggerated after incubation with conditioned media from microglias pretreated with the NO-donor SNAP 30 min before OMS+NB IgG (45.05 ± 1.74% SNAP, OMS+NB vs 26.09 ± 2.08% DMSO, OMS+NB, p <0.001; 26.09 ± 2.08% DMSO, OMS+NB vs 7.12 ± 0.98% DMSO, NB, p <0.001; Fig. 7a), and the cytolysis of cerebellar neurons was exacerbated by treatment with SNAP before OMS+NB IgG (51.51 ± 2.55% SNAP, OMS+NB vs 35.27 ± 1.87% DMSO, OMS+NB, p <0.001; 35.27 ± 1.87% DMSO, OMS+NB vs 8.06 ± 1.21% DMSO, NB, p <0.001; Fig. 7b). Pretreatment with the activator of sGC or PKG, namely YC-1 or 8Br-cGMP, also exacerbated cerebral cortical and cerebellar neuronal cytolysis induced by conditioned media from microglias stimulated with OMS+NB IgG (Fig. 7c-f). In addition, the effects of these above-mentioned activators were at least partly blocked by pretreatment with minocycline, an inhibitor of microglia, in cerebral cortical and cerebellar neurons exposed to OMS+NB IgG-treated microglia conditioned media (Fig. 7a-f). Together with the data of inhibitors, these results suggested that the NO/sGC/PKG cascade plays a vital role in neuronal cytolysis induced by conditioned media from microglias treated with serum IgG from children with OMS and NB, which depends on the activation of microglias.
The expression of proinflammatory cytokines from microglias incubated with serum IgG from OMS+NB children (Fig. 2a1-d1) and in CSF of OMS children [37] were upregulated, which raises the possibility that cytokines may have similar effects on neuronal cytolysis. Unexpectedly, our results showed that the cytolysis of cerebral cortical and cerebellar neurons induced by conditioned media from microglias preincubated with OMS+NB IgG was not influenced by IL-1β (Cerebral cortical neuron: 32.89 ± 1.70% IL-1β, OMS+NB vs 33.23 ± 1.37% 0.1% BSA, OMS+NB, p >0.05; 33.23 ± 1.37% 0.1% BSA, OMS+NB vs 10.65± 1.76% 0.1% BSA, NB, p <0.001; Cerebellar neuron: 27.55 ± 2.32% IL-1β, OMS+NB vs 31.26 ± 1.88% 0.1% BSA, OMS+NB, p >0.05; 31.26 ± 1.88% 0.1% BSA, OMS+NB vs 11.48 ± 1.39% 0.1% BSA, NB, p <0.001; Fig. 8a, b). Also, enhanced cytolysis of cerebral cortical and cerebellar neurons was not affected by IL-6, TNF-α or MCP-1 (Fig. 8c-h). These results suggested that the application of cytokines does not have similar effects as NO on the cytolysis of neurons induced by IgG-treated microglia conditioned media.
The activation of cerebral cortical and cerebellar microglias by the Fc fragment of serum IgG rather than the Fab fragment
IgG contains the Fab fragment and the Fc fragment. The Fab fragment combines with the targeted antigens, while the Fc fragment interacts with Fcγ receptor (FcγR), which can be expressed on the surface of microglias and other immune effector cells, mediating immune reactions in the brain. In order to explore whether increased microglial activation depends on the Fab fragment or the Fc fragment of serum IgG, we detected the effects of the Fab fragment on the concentrations of CD11b and NO in cerebral cortical and cerebellar microglias. The results showed that the expression of CD11b and the release of NO was not significantly changed in cerebral cortical and cerebellar microglias incubated with the Fab fragment from the OMS+NB group compared with the NB group and healthy control group (Fig. 9a, b). Consistently, the release of NO was not significantly changed in cerebral cortical and cerebellar microglias (Fig. 9c, d). These results suggesting that microglial activation induced by IgG from children with OMS and NB may depend on the Fc fragment of IgG, but not the Fab fragment.