Antipsychotic Drugs Attenuate Interleukin-6 Secretion by Microglia Via Dopamine Inhibition

Background: Antipsychotic drugs are commonly used for various neuropsychiatric disorders, such as psychotic disorders, mood disorders, and neuropsychiatric symptoms in dementia. Their mechanism of action is thought to be by modulation of neurotransmitter activity in the brain, mainly dopamine. It has been suggested that antipsychotic drugs may also exert anti-inflammatory properties. This study aimed to examine whether the modulating effect of antipsychotic drugs on neurotransmitters attenuates the inflammatory response of microglia cells. Methods: Levels of interleukin 6 (IL-6) were measured following activation of microglia cultures with lipopolysaccharides and treatment with antipsychotic drugs (risperidone, haloperidol, and clozapine), neurotransmitters (dopamine, serotonin, and acetylcholine), or a combination of dopamine and either haloperidol or clozapine. Results: Haloperidol and clozapine decreased IL-6 secretion by microglia cells when treated at a concentration of 10-5M. Interestingly, dopamine at a concentration of 1 μM increased IL-6 secretion by the microglia cells, while a concentration of 100 μM decreased it. The combination of dopamine (from 0.001 μM to 100 μM) with either haloperidol (10-5M or 10-8M) or clozapine (10-5M or 10-7M) attenuated IL-6 secretion in a bell-shaped curve with a peak at 1 μM. High concentrations of both haloperidol and clozapine decreased IL-6 secretion, while low concentrations modestly increased IL-6 levels. Conclusions: Our findings support anti-inflammatory properties of antipsychotic drugs, and suggest that their action is mediated via the inhibition of dopaminergic activity in microglia cells. The bell-shaped curve of IL-6 secretion by microglia might

suggest the presence of an "optimal zone" of operation for these cells that is mediated by dopamine. BACKGROUND Antipsychotic drugs are commonly used in the treatment of psychotic disorders, such as schizophrenia, as well as in the management of mood disorders [1,2], behavioral disturbances in children and adolescents [3], and neuropsychiatric symptoms in dementia patients (NPSD) [4].The first generation of antipsychotic drugs (also referred to as conventional or "typical" antipsychotics) has mainly antagonistic properties to the type two dopamine (D2) receptor in the brain, thus lowering dopaminergic activity that entails both therapeutic as well as adverse effects [5]. Newer (or "atypical") antipsychotic drugs were developed in order to decrease the rates of adverse effects, and they are mainly antagonistic to the D2 receptor as well as to the 5HT 2A serotonin receptors, although they also affect the muscarinic, adrenergic, and histaminergic receptors [5].
Neuroinflammation is a relatively new hypothesis for the pathophysiology of neuropsychiatric disorders. It suggests that aberrant immune activation is an additional key component for the development of these disorders (e.g., schizophrenia [6], mood disorders [7], autism [8], and dementia [9]). Various components of the immune system have been studied in humans, animal models, and in-vitro. Microglia and pro-inflammatory cytokines were suggested as key players in several studies [10][11][12]. Interleukin 6 (IL-6) has been often studied in this context, and it was suggested to play a central role in neuroinflammation [13][14][15].
Preliminary studies with an IL-6 antagonist (tocilizumab) as a treatment for schizophrenia have also been carried out [16,17].
Of great interest are studies that have shown that antipsychotic drugs have antiinflammatory properties [18][19][20], thus raising the possibility that their therapeutic effects are not based solely on their anti-dopaminergic/serotonergic properties [21].
It is not yet clear, however how antipsychotic drugs influence the underlying biological mechanism by which neuroinflammation appears to be driven [22].
The aim of this study was to examine whether there is a common pathway for the neurotransmitter blocking capacity of antipsychotic drugs and their immunemodulating abilities. We hypothesized that the anti-dopaminergic properties of the drugs will modulate the inflammatory response of microglia cells, as measured by their secretion of IL-6.

Microglia Cell Cultures
The N9 cell line (RRID CVCL_0452) was received from the laboratory Prof. Dr.

Cell Treatments
N9 cells were plated on 48-well tissue culture plates at a concentration of 1 × 10 5 cells/ml in all experiments. After 24 hours, neurotransmitters and/or antipsychotics were added to the medium with 0.1∝ g/ml lipopolysaccharide (LPS; Sigma-Aldrich).
After an additional 24 hours, the supernatants were removed and used for analysis of the IL-6 levels. Three experiments were conducted. In the first one, the N9 cells were treated with either haloperidol, risperidone, or clozapine at decreasing concentrations (10 − 5 , 10 − 6 , 10 − 8 , 10 − 10 , and 10 − 12 M). In the second one, the cells were treated with dopamine, serotonin, or acetylcholine in doses of 1, 10, and 100 ∝M. In the third experiment, the cells were treated by a combination of

Data Analyses
Statistical analyses were conducted using IBM SPSS Statistics for Windows v.20 (IBM Corp, Armonk, New York). Groups means were compared using one-way ANOVA followed by Scheffe post-hoc test.

Impact of Antipsychotic Drugs on Il-6 Secretion by Microglia
Based on the hypothesis that IL-6 plays a central role in neuroinflammation, we assessed the effect of various antipsychotics drugs on microglia cells in the neuroinflammatory microenvironment by activating the cells with LPS together with selected drugs. Although an overall significant difference was found for risperidone (F(5,11) = 4.07, P = 0.025), post-hoc analyses did not reveal any significant differences in IL-6 levels for specific concentrations of the drug (Table 1, Fig. 1A). In contrast, both haloperidol and clozapine significantly lowered IL-6 levels (F(5,11) = 14.30, P < 0.001 and F(5,12) = 13.99, P < 0.001 respectively). Both caused a significant decrease of IL-6 secretion by the N9 cells at a dose of 10 − 5 M (equivalent to 375.9 × 10 − 5 mg/ml for haloperidol and 326.8 × 10 − 5 mg/ml for clozapine), an effect that disappeared in lower doses (57.27 ± 9.02 vs. 103.15 ± 20.77, P = 0.012 for haloperidol and 67.27 ± 0.67 vs. 103.15 ± 20.77, P = 0.020 for clozapine.  Fig. 1B and 1C). The Impact of Neurotransmitters on IL-6 Secretion by Microglia

Antipsychotics and Neurotransmitters' Impact on IL-6 Secretion
We then sought to determine whether different concentrations of dopamine influence the effect of clozapine on microglial IL-6 secretion. The results showed that IL-6 secretion was significantly lower compared to the LPS control for most dopamine concentrations (0.001, 0.1 and 10 ∝M. F(6,12) = 11.47, P < 0.001) at a high dose of clozapine (10 − 5 M or 326.8 × 10 − 5 mg/ml), while the 1 ∝M and 100 ∝M doses yielded no significant differences (Table 2 and Fig. 3A).  Fig. 3A).
Interestingly, IL-6 levels for the dopamine 1∝M-treated cells were significantly higher compared to the cells treated at other dopamine concentrations (Table 2 and Fig. 3B).
Finally, we tested low-dose haloperidol (10 − 8 M equivalent to 375.9 × 10 − 8 mg/ml) and found that three dopamine concentrations showed significant differences compared to the LPS control (0.1, 1 and 10 ∝M), and that IL-6 levels were higher than the control (F(6,14) = 33.45, P < 0.001. Table 2 and Fig. 3B). Again, the cells treated with dopamine 1 ∝M exhibited significantly higher levels of IL-6 compared to the other treated cells (Table 2, Fig. 3B).

DISCUSSION
The aim of our study was to examine the hypothesis of there being a possible common pathway between the neurotransmitter-modulating and the antiinflammatory effects of antipsychotic drugs. Our findings are in line with previous studies that showed an anti-inflammatory effect of antipsychotic drugs [18][19][20]. The biologic mechanism of this effect is not altogether clear, and our results suggest that dopamine plays a central role in this process. The importance of dopamine as a mediator of inflammation has been demonstrated in several studies [25,26].
Investigations that aimed to identify the presence of dopamine receptors on various subpopulations of leukocytes found that the D2 receptor family is more prevalent than the D1 family and that monocytes have a higher density of D2 and D3 receptors compared to the other types [27]. Specifically, microglia cells were shown to have D1 and D2 receptors, and that the activation of these receptors regulates microglia cell activity [25]. In a study employing human monocyte-derived macrophages that were stimulated with different concentrations of dopamine (2 M and 20 M), an increase of IL-6, CCL2, CXCL8, and IL-10 secretion was found while the secretion of TNF declined. Lower concentrations of dopamine in that study (20 nM and 200 nM) affected only TNF , IL-6, and CCL2 secretion and they upregulated IL-10, albeit insignificantly. Therefore, the authors suggested that macrophages develop differential responses, depending upon their microenvironment, and that they are modulated by dopamine [28]. Similarly, our findings suggest that various concentrations of dopamine trigger different responses from microglia cells, which seem to be most active when dopamine concentration is in its physiologic range of ~ 1∝M [29]. The fact that haloperidol (an anti-psychotic drug with high affinity to the D2 receptor) had a much more significant effect on IL-6 secretion than clozapine (which has a lower affinity to the D2 receptor) supports the notion that the modulating effect of antipsychotic drugs on microglia is mediated by the D2 receptors.
IL-6 has been extensively studied in schizophrenia [13], NPSD [9], and other neuropsychiatric disorders [7,8]. It is considered to be a pro-inflammatory cytokine, but current studies indicated that IL-6 has more complex properties than had been thought in the past (e.g., that it can be anti-inflammatory as well as proinflammatory), and that it has a variety of physiologic roles in the central nervous system [13]. Due to its complex and varied role, and the fact that IL-6 is a major cytokine produced by microglia cells, it may be more prudent to view it as a marker of microglial cell activation in the context of this study. Our finding of the bellshaped curve of the resultant IL-6 levels support an "optimal" setpoint for microglia cell activity, and that a deviation from it might underlie pathologic processes.
Similarly, Yirmiya et al suggested that depression may be considered microgliopathies in which either microglial cell activation or decline underlies the depressive disorder [30]. Our findings propose that this hypothesis may also be applicable to other neuropsychiatric disorder, such as schizophrenia and dementia.
We had previously reported that neurotoxic microglia showed increased sensitivity to dopamine and had increased IL-6 secreted levels in Parkinson's disease [23,31].
The possibility also arises that the therapeutic role of antipsychotic drugs is due to their ability to attenuate the neuroinflammatory properties of microglia. An interesting finding in this context is that clozapine and haloperidol at low concentrations seemed to increase rather than decrease IL-6 secretion.   Interleukin 6 Mediated by Antipsychotic Drugs and Dopamine. The combined effect of antipsy