Immunomodulatory Effects of Dopamine in Inflammatory Diseases

Dopamine (DA) receptor, a significant G protein-coupled receptor, is classified into two families: D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptor families, with further formation of homodimers, heteromers, and receptor mosaic. Increasing evidence suggests that the immune system can be affected by the nervous system and neurotransmitters, such as dopamine. Recently, the role of the DA receptor in inflammation has been widely studied, mainly focusing on NLRP3 inflammasome, NF-κB pathway, and immune cells. This article provides a brief review of the structures, functions, and signaling pathways of DA receptors and their relationships with inflammation. With detailed descriptions of their roles in Parkinson disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, this article provides a theoretical basis for drug development targeting DA receptors in inflammatory diseases.

INTRODUCTION Dopamine (DA), a member of a group of neurotransmitters called "catecholamines", relies on the conversion of tyrosine to L-DOPA by tyrosine hydroxylase (TH). Chromaffin cells in suprarenal glands and the intestine are the main sources of plasma dopamine. Other sources of dopamine are immune cells, peripheral nervous system, and central nervous system. Dopamine receptors (DRs) are mainly divided into D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors, which display different affinities for dopamine: D3R>D5R>D4R>D2R>D1R(Ki (nM) = 27, 228, 450, 1705, 2340, respectively) (1). As members of monoaminergic G proteincoupled receptor (GPCR) family, they not only regulate behavior, movement, and endocrine but are also important molecules connecting the nervous system and immune system. Integrating the knowledge acquired by those studies, evidences indicate that stimulation of low-affinity DRs are coupled to anti-inflammatory mechanisms, while high-affinity DRs have opposite effects ( Table 1).

STRUCTURE
DRs and other GPCRs can form homodimers as well as heteromers with receptors from other superfamilies.

Homodimers
It is a general physical property of the class-A GPCRs to form transient homodimerization (47), which can be confirmed by the minimal single functional unit consist of D2R homodimers and a G protein, mediated by direct association among receptors (48).

A2A-D2R Heteromers
The C-terminal tail of the A2AR and the intracellular loop3 of the D2R form antagonistic heteromers through direct electrostatic interactions in striatopallidal GABAergic neurons and glial cells (52), resulting in a rapid switch from D2R-Gi coupling toward b-arrestin2/Akt/GSK-3 signaling (53,54), and reducing the activity of the striatopallidal GABA/enkephalinmediated inhibition of the excitatory glutamate thalamocortical pathway through Ca2+ and cAMP/PKA pathway whose activation involves the phosphorylation of different PKA substrates, including DARPP-32, CREB, and a-amino-3hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) (55,56). In addition, A2AR agonists and antagonists produce the same allosteric modulation of D2R agonist binding when individually administered, the effect of which is offset when co-administered (57).

A1-D1R Heteromers
There exist antagonistic intramembrane A1-D1R interactions at the AC level in the dorsal, ventral striatum, and prefrontal cortex (59). A1R activation in the A1-D1R heteromers leads to an uncoupling of the D1 receptor to its Gs/olf protein, modulates D1R antagonist binding sites that cause a reduction of their affinity, and offsets D1R-induced GluA1 phosphorylation that facilitates AMPA glutamate transmission (60). In addition, A1R signaling inhibits excitatory synaptic transmission through the inhibition of glutamate release, ionotropic glutamate receptors, and neuronal excitability via inward rectifying, cAMP/PKA/ DARPP-32 pathway, as well as calcium-activated potassium channels (61,62).

H3-DR Heteromers
H3-D1R and H3-D2R heteromers, in which H3R potentiates the D2R-induced inhibition of the indirect pathway and inhibits the D1R-induced excitation of the direct pathway in AC level, modulate DA and GABA release (63) and subsequent locomotor activation. H3-D1R heteromers also couple to Gi to direct histaminergic input towards b-arrestin/MAPK pathway within the GABAergic neurons (64) with an increased phosphorylation of rpS6 and transient phosphorylation of GSK3b (65), and affect both rapid receptors signaling like Ca2+ mobilization and longer cell signaling pathways like p38, involving neuronal cell death (66). While H3-D2R heteromers regulate Akt-GSK3b, producing a more slowly developing dephosphorylation (65).

D3R-nAChR Heteromers
D3R-nAChR heteromers in DA neurons are the molecular unit involved in the induction of neurotrophic effects, neuroprotection, and inhibition of a-syn accumulation (78).

ROLES OF DOPAMINE IN INFLAMMATION NLRP3 Inflammasome
NLRP3 inflammasome is a group of intracellular multi-protein complexes, participating in the pathogenesis of a variety of diseases, including inflammatory bowel disease, gout, atherosclerosis, and Alzheimer's disease (83,84). It was found that DA can inhibit lipopolysaccharide (LPS)-induced activation of NLRP3 inflammasome and subsequent production of caspase-1 and IL-1b in a time-and dose-dependent manner through G protein pathway and b-arrestin-dependent pathway ( Figure 1).  D1-like DRs are coupled to the stimulatory G-subunit, Gas, whereas D2-like DRs, are coupled to the inhibitory G-subunit, Gai. D1-like DRs stimulate the activity of AC by activating Gas/ olf, thus promoting the production of cAMP, which directly binds to NLRP3, triggering the ubiquitination of NLRP3 NACHT and LRR domains with K48 ubiquitin chains by the E3 ubiquitin ligase membrane associated ring-CH-type finger 7, targeting NLRP3 to autophagy-mediated degradation (2,85).
b-arrestin is involved in the negative feedback regulation of inflammatory process in sepsis, asthma, rheumatoid arthritis, and other inflammatory diseases (86). b-arrestin2 recruited by D2R functions as the downstream protein of GPR120 and GPR40 to repress inflammasome activation via binding to NLRP3 in a concentration-dependent manner (87,88).

NF-kB Signaling
An inactive form of NF-kB consists of a three-subunit complex: two DNA-binding subunits of p50 and p65/RelA, and an inhibitory subunit called IkB ( Figure 1). Activation of NF-kB requires the phosphorylation and degradation of IkBa by ubiquitin-proteosome pathway, contributing to translocation of NF-kB dimer into the nucleus and the transcription of inflammatory genes, including cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) (89).
D1R participates in the regulation of inflammation through cAMP/PKA/NF-kB pathway. Elevated cAMP activates PKA and phosphorylates cAMP-response element binding protein (CREB), inhibiting p65/RelA and p50 activation as well as their DNA binding ability by delaying IKB-a degradation and competing for the KIX binding site on CREB binding protein (90).
D5R inhibits NF-kB signaling by mediating the negative regulation of ARRB2/PP2A on TRAF6-dependent signaling. A study found that D5R, via the EFD and IYX(X)I/L motifs in its CT and IC3 loop, respectively, can directly recruit TRAF6 and its negative regulator ARRB2, as well as downstream signaling proteins, such as TAK1, IKKs, and PP2A, to form a multiprotein complex, which impairs TRAF6-mediated activation of NF-kB (91).
D2R negatively regulates the NF-kB pathway. First, the pathway downstream D2R activation leads, via increasing the expression of PPP2R2C, to PP2A and Akt dephosphorylation, and the inhibition of the IKKa/IkBa/NF-kB pathway as well as the expression of NLRP3 mRNA (2,92). Second, D2R signaling increases the level of aB-crystallin, also called CRYAB, which is known as a small heat-shock protein with neuroprotective and anti-inflammatory activities (93). D2R agonists were found to promote a direct combination between CRYAB and NF-kB p65 (92) and enhance the interaction between CRYAB and STAT3, blocking its DNA binding activity (94).

T Cells
Dopamine dynamically regulates the immune response of T cells through DRs, depending on the concentrations of dopamine, the activation states of T cells, and the types and subtypes of T cells. Dopamine concentrations can be divided into three gradients: 10 nM, 1 mM, and 0.1 to 1 mM. Dopamine's optimal concentration for inducing a physiological and specific effect on resting T cells turns out to be low: 10 nM, in which dopamine activates normal resting/primeval effector cells or improves the continuous important cell function, and inhibits activated T cells. Dopamine at a concentration range of 0.1 to 10 mM still affects T cells, but the potency and specificity are lower. At a very high concentration of 0.1 to 1 mM, dopamine's effect is non-specific and even toxic (95).
Tregs are inhibitory T cells, mainly inhibiting the activity of Teffs. D1-like DRs on the surface of Tregs reduce their inhibitory activity, as well as the production of IL-10 and TGF-b (96), the effects of which are significantly attenuated in activated Tregs (97). Instead, activation of D1-like DRs in Teffs does not lead to self-inhibition.
According to cytokines produced during T cell activation, naive CD4+ T cells undergo differentiation into specific effector phenotypes, including Th1, Th2, and Th17. IFN-g and IL-12 induce Th1 differentiation, while IL-4 is the primary inducer of differentiation into Th2, and IL-6 and TGF-b together induce Th17 phenotype (98). In activated CD4+ T cells, D3R signaling, preferentially activated at lower dopamine concentrations, enhances the production of IFN-g (99) and reduces the synthesis of IL-4 and IL-10 and the expression of SOCS5 (100), a side-regulator of Th2 differentiation. Besides, D3R stimulating reduces cAMP level and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, resulting in enhanced activation of CD4+ T cells and Th1 differentiation (101).

Dendritic Cells
Dendritic cells (DCs) have been shown to synthesize and store dopamine, which is released to the original CD4 + T cells during DC-T cell interaction, thus affecting the differentiation of CD4+ T cells. The expression of D1R and D5R is more than that of D2R and D3R on the DC surface. D1-like DRs-mediated increase in cAMP promotes TH phosphorylation, thus boosting the synthesis of dopamine and Th2 differentiation, while D2-like DRs play an opposite role (95).
However, D5R and D2R have different effects. D5R signaling significantly enhances the production of LPS-induced IL-23 and IL-12 (44), inducing Th1/Th17 differentiation and the activity of B cell-activating transcription factor, increasing the expression of Th17 transcription factors, like ROR-gt (102). Besides, stimulation of D2R induces a significant human monocytederived DC-mediated Th2 differentiation and suppresses the secretion of inflammatory cytokines (103).

Monocytes and Macrophages
D2-like DRs participate in a cAMP-independent pathway to regulate macrophage phagocytic function, thus reducing the secretion of IL-2, IL-4, and IFN-g (104), the effect of which is concentration-dependent. Studies demonstrated that D2-like DRs decrease the production of TNF-a, IL-6, and CCL2 at 10 −8 M concentration (105), while increase the production of IL-6 significantly at 2 and 20 × 10 −6 M (106) concentration. Conversely, Gomez et al. proposed that another mechanism is to increase the expression of the Fcg receptor, thus increasing the phagocytosis of macrophages (107) (Figure 2). A study showed that D1R signaling blocks the function of LPS-activated macrophages and monocytes and production of inflammatory cytokines (108). Also, the elevated cAMP can indirectly activate CCAAT/enhancer-binding protein, together with which CREB/activating transcription factors are believed to be the major cause of IL-10 production by monocytes (109). However, D5R in monocytes obtained from MS patients involves the reduction of STAT3 activation, a transcription factor that limits the production of IL-12 and IL-23 (110). A study in HIV patients demonstrated that dopamine promotes the inflammatory phenotype of macrophages through D5R at physiological concentration and reduces the production of IL-10 (105).

INFLAMMATORY DISEASES Parkinson Disease
Parkinson disease (PD) is the second most prevalent central nervous system degenerative disease, characterized by slow and progressive loss of midbrain substantia nigra dopamine with the accumulation of a-synuclein in Lewy bodies and neuritis (111).

Astrocytes and Microglia
DRs expressed on astrocytes and microglia have been confirmed to participate in the pathogenesis of chronic nervous system inflammatory diseases. Increases in D1R and D4R, and decreases in D3R, D5R mRNA expression are showed in a study analyzing the mRNA expression of all five DRs in BV2 microglial cells in response to LPS (112). It is noteworthy that these antiinflammatory effects exerted by dopaminergic signaling in astrocytes are mediated by D1R and D2R, while D3R mediates the pro-inflammatory effects (8).

D1R
D1R signaling participates in the negative regulation of the activation of NLRP3 inflammasome, which can be assembled upon stimulation with accumulated endogenous metabolites such as fibrillar amyloid b and 25-hydroxycholesterol (113), the subsequent secretion of caspase-1 and IL-1b contributes to the destruction of dopaminergic neurons (114). A study reported that caspase-1 can process a-synuclein into a truncated, aggregation-prone form that facilitates its aggregation (115), thus participating in activation of NF-kB and expression of Toll-like receptors (TLRs).
Microglia can be divided into M1 type and M2 type phenotypes. M2 microglia promotes the release of antiinflammatory factors, such as TGF-b and IL-10, while M1 microglia plays an opposite role (116). Studies have shown that

D3R
Genetic deficiency of D3R, attenuated neuroinflammation and subsequent neurodegeneration on a murine model of PD induced by acute intoxication with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (121), related to the limited basal production of Fizz1 (8) and the acquisition of M1 phenotype. Besides, the high levels of IFN-g and TNF-a, secreted by D3R signalinginduced Th1 and Th17 differentiation, lead to M1 phenotype (122), confirmed by a study that compared with the control group, PD patients have increased Th1 cells and Th17 cells but decreased Tregs (123). However, experimental results showed decreased D3R expression on CD4+ T cells in the peripheral circulation of PD patients, which might be due to a compensatory mechanism attempting to reduce the inflammatory effect (124,125). Another plausible explanation was that CD4+ T cells with high D3R expression were specific for a-synuclein, thus these cells could only be detected at the site of inflammatory infiltration, instead of the peripheral circulation (124).

Striatum D3R
D3R expressed on striatal neurons can raise dopamine concentration, decrease a-Syn accumulation, enhance secretion of BDNF, ameliorate neuroinflammation, alleviate oxidative stress, and promote neurogenesis in the nigrostriatal pathway.
D3R increases the content of DA in the synaptic cleft by impeding DAT's reuptake of DA, inhibiting MAO to reduce DA decomposition, and promoting the release of DA by VMAT2. Further, D3R activation hinders the phosphorylation of a-Syn to inhibit fibril formation (126). Besides, D3R activation enhances autophagy-dependent degradation of toxic fibrils by modulating autophagy constituent proteins LC3 and autophagy-related protein Beclin1 (15). It was found that D1, D2, and possible D1-D2 receptor heteromers can activate BDNF receptors in striatal neurons (127). Also, D3R can jointly protect striatal neurons through its bi-directional regulation with BDNF, a high level of which may ameliorate symptoms in PD patients (128). Experiments have shown that D3R agonists can normalize glutathione (GSH) and GSH peroxidase levels in animal models of PD (17), thereby reducing ROS-induced damage, however, some studies showed no association.

Heteromers
Evidences for the existence of DR heteromers in the striatum (see Structure) provide novel targets in treating PD and other brain disorders. This section is a supplement to clinical implications of heteromers on treatment response and prognosis of PD.
Chronic administration of the histamine H3 receptor agonist immepip decreases L-Dopa-induced dyskinesias (28), while a combination of D2 agonists and inhibitors of endocannabinoid degradation improves parkinsonian motor deficits (131).
NMDAR antagonist MK-801 aggravates D1R-induced dyskinesias, while effectively reduces D2R-induced dyskinesias, the degree of which is of the same magnitude as the reduction of L-DOPA-induced dyskinesias (31).

Hippocampus
DRs are widely expressed in the hippocampus. D1R can upregulate Wnt/b-catenin signaling in the hippocampus of PD rats, leading to the enhanced NSC proliferation, long-term survival, and neuronal differentiation (33). Besides, D2Rdependent cross-talk modulates Wnt3a expression via Wnt/ b-catenin signaling and an evolutionarily conserved TCF/LEF site within the WNT3A promoter, thus modulating cell proliferation (135).

Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a group of chronic gastrointestinal inflammatory diseases including Crohn's disease (CD) and ulcerative colitis (UC). Dopamine in IBD can be produced from enteric nervous system, the intestinal epithelial layer, and certain immune cells. Interestingly, inflamed mucus from IBD patients show a significant reduction of dopamine, mainly related to reduced dopamine uptake and the number of sympathetic fibers interacting with the intestinal wall (136). Reduced intestinal dopamine levels [≈140 pg/ml in healthy individuals;≈45 pg/ml in CD and UC patients (137)] play a proinflammatory role by activating D3R and D5R. On one hand, D3R signaling depresses the immunosuppressive potency of Tregs, attenuates IL-10 production, and limits the acquisition of guttropism (34). On the other hand, Th1/Th17 differentiation, induced by increased D3R expression on intestinal CD4+ T cells, the induction of ROR-gt expression by D5R signaling in dendritic cells, and the increased IL-23 and IL-12 resulting from D5R signaling, contributes to the persistence of chronic inflammation (138).
Conversely, high concentrations of dopamine in the intestine of healthy people can stimulate D2R, promoting the production of IL-10, inhibiting intestinal motility and ulcer development (139), as well as playing a role of the negative regulator of VEGF-VEGFR2-mediated increase in vascular permeability (35,140,141), thus controlling the development of IBD.

Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease characterized by persistent inflammation of the joint synovium (142). Dysregulated immune signals, such as dopamine, control bone remodeling via affecting osteoclasts differentiation or the secretion of pro-inflammatory cytokines.

CD4+ T Cells
Dopamine released by DCs contributes to the Th17/Treg imbalance via the IL-6-Th17 axis and causes aggravation of synovial inflammation. A study showed that D2-like DRs agonist improves Th17/Treg imbalance by downregulating the expression of Th17-related pro-inflammatory cytokines but upregulating Treg-related anti-inflammatory cytokine expression (143), the effect of which can be suppressed by selective D2-like DRs antagonist.

Mast Cells
D3R on bone marrow-derived mast cells may negatively regulate LPS-induced TLR4 expression and its downstream production of TNF and other cytokines (38), thus effectively inhibiting the production of ROS and reducing joint inflammation in RA patients. With the increase in RA severity degree, D3R-positive MCs in the synovial fluid are gradually reduced, led by ROS production, reduced antioxidant capacity, reduced cell membrane stability, and increased sensitivity of membrane components to a damaging agent, which are negatively correlated with the level of MDA and protein carbonylation (144).

B Cells
B cells have unique bone action properties. D2R expression on B cells in RA patients is negatively correlated with disease activity (145), concerning the descending TNF-a level.

Fibroblasts
Synovial fibroblasts (SF) are resident cells of the intimal lining layer of synovial tissue. SFs have an intact endogenous dopamine system in which D1R is overexpressed, promoting the migration of RASF cells, leading to a strong increase of SF migration in young patients (146), and decreased release of IL-6 and IL-8. However, some experiments demonstrated that the inhibitory effect on IL-8 release is not significant (146). These findings suggest that DRs expressed on synovial fibroblasts in RA patients may mainly participate in cell migration rather than inflammatory processes.

Osteoclasts
Osteoclasts are tissue-specific macrophage polykaryons that arise from the differentiation of monocyte/macrophage precursor cells at or near the bone surface, whose maturation and activation are mainly related to the activation of the RANK signaling (147). It was found that dopamine significantly inhibits the formation of osteoclast in a dose-dependent manner, mainly related to the restraint of RANKL-mediated expression of c-Fos and NFATc1 in the preosteoclast by D2R-induced cAMP/PKA/CREB pathway (41).

Systemic Lupus Erythematosus
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the involvement of kidneys and brain, with under-expressed D2R and overexpressed D4R on peripheral blood mononuclear cells (PBMCs) (148).
D2R promotes the activation and differentiation of CD4+T cells by regulating the polarization of Treg (149). D2R agonist, such as bromocriptine, suppresses PRL secretion to decrease HPRL and to normalize the dopaminergic system in SLE, through the pertussis toxin (PTX)-sensitive Gi/o and PTXinsensitive Gz proteins, as well as a G protein-independent, barrestin/glycogen synthase kinase-3-dependent pathway (150,151).
Studies have shown that the stimulation of D4R on human T cells promotes quiescence (152), and overexpression of D4R in SLE patients may act as a compensatory mechanism to inhibit uncontrolled T cell proliferation, an important link in the pathogenesis of SLE.

Multiple Sclerosis
Multiple sclerosis (MS) is an inflammatory autoimmune disorder of central nervous system (CNS), correlated with Tregs dysfunction, enhanced Th1 and Th17 responses, and autoreactive B cell overactivity.

DCs
There is a decreased expression of D5R in PBMCs in untreated MS patients (153), and an increase in patients treated with IFN-b. Th cell subsets involved in the pathogenesis of MS include Th1 and Th17 lymphocytes (154). D5R expressed on DCs plays a role in MS by regulating Th1 and Th17 differentiation, gdT cell functions, and GM-CSF-producing CD4+T cells via STAT3/NF-kB/IL-6/12/23 pathway, and is correlated with disease severity (155).
Tregs D5R, which functions as a negative immunomodulator of TH and Tregs' inhibitory activity, is up-regulated in Tregs from untreated MS patients, resulting in neuronal damage and neuroinflammation (46). Besides, D3R expression in Tregs is unaltered in untreated MS patients but significantly decreases after IFN-b treatment. A recent study showed that increased D3R and D5R mRNA expression in Tregs may be associated with the risk of MS at twelve months (156).

CONCLUSIONS
Dopamine receptor, a significant G protein-coupled receptor, is classified into two families: D1-like DRs and D2-like DRs, with further formation of homodimers, heteromers, and receptor mosaic. Dysfunction of the systemic or local dopaminergic system during inflammation has been found in animal models and patients with various inflammatory diseases, such as Parkinson disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, indicating an important role that DRs play in inflammatory diseases. As described in this review, DRs regulate the release of inflammatory mediators and subsequent pathological processes by interacting with inflammasomes, inflammatory pathways, and immune cells, depending on different immune cells, receptor subtypes, and disease models. In conclusion, a comprehensive understanding of the relationship between DRs and inflammation will provide new insights into the inhibition of inflammatory responses by targeting dopamine receptors and ultimately contribute to the development of drugs to treat inflammatory diseases.

AUTHOR CONTRIBUTIONS
YFF and YL contributed to the conceptual design, writing, editing, and generation of figures for this manuscript. All authors contributed to the article and approved the submitted version.