Here, we demonstrated that antagonizing LPAR 1–3 with Ki16425 deteriorated specific behavioral symptoms and spinal demyelination after EAE induction, corresponding to increased cellular infiltrates (such as microglia, macrophage, Th1, and Th17 cells) and worsened BBB integrity. The mechanism underlying the deteriorated EAE was excessive oxidative stress via NOX2 and NOX3. Interestingly, agonizing LPAR 1/2 with 1-oleoyl-LPA improved neurological symptoms and representative pathophysiological characteristics of EAE. These results provide new mechanistic insights into how LPA 1–3 signaling contributes to EAE pathophysiology. Taken together, our findings suggest that agents that can regulate LPAR 1–3 might be used as therapeutics for treating MS.
LPARs are differentially expressed on most cell types within central and peripheral nervous tissues. They preferentially bind to saturated, monounsaturated, and polyunsaturated LPAs (14). Signal transduction through LPARs has been functionally linked to many neural processes, including cell proliferation, cell survival, apoptosis, morphological change, cell migration, and the production of other lipids such as prostaglandins through arachidonic acid conversion by cyclooxygenase-2 (14). Therefore, LPARs have been considered as novel targets in lipidomic-based therapeutics for neurological disorders (16). Many neurological disorders frequently accompany demyelination-associated signs and symptoms such as neuropathic pain, demyelinating neuropathies, and MS (21–23). Loss of LPAR1 can impair oligodendrocyte differentiation and myelination due to impaired intracellular transport of the proteolipid protein (PLP)/DM20 myelin protein in the mouse cerebral cortex (41). LPA1-null mutant mice have shown delayed Schwann cell-to-axon segregation, polyaxonal myelination by single Schwann cell, and thinner myelin sheaths via heterotrimeric G-alpha protein, Gαi, and small GTPase, Rac1 signaling (15). LPA2 deficient mice have shown enhanced motor skills and myelin sparing after spinal cord injury related to oligodendrocyte cell death by activating microglial LPA2 (42). These previous reports strongly suggest that investigating new signaling mechanisms in these disorders might be critical in the development of therapeutics to stimulate spontaneous remyelination and subsequent functional recovery. In the present study, treatment with Ki16425, an LPAR1-3 antagonist, impaired behavioral symptoms and spinal demyelination after EAElow induction (Figs. 2 and 3), whereas treatment with 1-oleoyl-LPA, an LPAR1/2 agonist, mitigated them after EAEhigh induction (Fig. 8). These results indicate that LPA signaling via LPARs, specifically LPAR 1–3, might play a pivotal role in MS pathology.
Levels of resident microglia activation and infiltration of monocyte-derived immune cells to the CNS are associated with neurodegeneration in both MS and EAE (4). Infiltrated immune cells are important contributors to the local chemical environment, releasing either anti-inflammatory growth factors or pro-inflammatory cytokines depending on their activation states. However, whether they have beneficial of detrimental roles remains controversial (4). BV-2 cells express LPAR 2, 3, 5, and 6, whereas primary murine microglia expresses LPAR 1, 2, 4, 5, and 6 (43). It has been shown that LPAR1 knockdown in the brain with its specific shRNA lentivirus can attenuate sepsis-induced microglia activation, morphological transformation, and proliferation, in agreement with the downregulation of TNF-α production by activating ERK1/2 in the brain and LPS-stimulated cells (44). On the other hand, LPAR1 antagonism reduced numbers and soma sizes of activated microglia. It also reduced microglial proliferation, in correspondence with reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-κB activation in the ischemic brain. Particularly, these LPAR1-drived proinflammatory responses have appeared in activated microglia because NF-κB activation occurs mainly in activated microglia (45). LPAR2 is constitutively expressed in the spinal cord parenchyma. Its transcripts are up-regulated after spinal cord injury, in part, by microglial cells (42). The demyelinating lesion triggered by intraspinal injection of LPA into the undamaged spinal cord was markedly reduced in the absence of LPAR2 (42). LPAR2 deficient mice have shown enhanced locomotor skills and myelin sparing after spinal cord injury (42). Thus, these previous reports suggest that LPAR1-3 has a novel function in microglial activation and that its mechanism could be involved in the pathogenesis of diverse neurological diseases related to microglial activation. Our previously study has shown that gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, can reduce 3-nitropropionic acid-induced striatal toxicity throught its antioxidant and anti-inflammatory activities. It downregulated microglial activation through LPA, whereas pre-antagonism with Ki16425 neutralized gintonin’s beneficial effects (46)(Jang et al., 2019). In the present study, LPAR 1–3 antagonist also increased microglial activation and infiltration of peripheral immune cells (macrophages) to demyelinating lesion following EAE induction, whereas LPAR 1/2 agonist inhibited them after EAEhigh induction (Fig. 8). Our findings suggest that LPAR 1–3 might play a critical role in the EAE pathology via microglial activation and peripheral immune cell infiltration into lesion.
During MS and EAE process, naive T cells primed by antigen presenting cells such as microglia, macrophages, and dendritic cells can differentiate into Th1, Th2, Th17, or Treg cells depending on the cytokine environment (31). Up to now, the role of LPA signaling in T cell differentiation is clearly unknown. In the current study, mild EAE did not significantly change the size of the spleen and the lymph nodes, the population (number) of CD4 cells or its major subsets, or the population of CD8 T cells in the spleen following EAElow induction (Fig. 4). However, LPAR1-3 antagonism clearly increased the size of the spleen and the lymph nodes and the population of CD4, Th1, and Th17 cells in the spleen associated with deteriorated EAElow symptoms and pathological features. However, LPAR1-3 antagonism did not significantly influence the population of CD8, Th2, or Treg cells in the spleen (Fig. 4). In the MS and EAE, peripheral autoreactive T cells can migrate across the disrupted BBB, attack myelin antigens, and induce demyelination in the CNS (5). Although the migration of autoreactive T cells is mediated by multi-step process of lymphocyte diapedesis through the BBB (5, 31), the role of LPA signaling in the process is largely unknown. LPA and LPA-generating enzyme autotaxin are constitutively expressed at high endothelial venules of lymph nodes. They are implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes (47). LPA signaling mediates the recruitment of leukocytes including CD3 T cells into unprimed and TNF-α-primed air pouches in a murine air pouch model of inflammation (48). LPAR5 is an inhibitory receptor that suppresses CD8 T-cell cytotoxic function via disruption of early TCR signaling (49). These reports strongly suggest that LPA signaling might have a critical role in T cell migration into demyelinating lesion of EAE. In the present study, mild EAE did not significantly increase the population of CD3+ T cell or major subsets of CD4 T cell in the spinal cord after EAElow induction (Fig. 4). LPAR1-3 antagonism clearly increased the population of CD3 (T), CD4 (Th), Th1, and Th17 cells in the spinal cord associated with deteriorated EAElow symptoms and pathological features (Fig. 4). However, LPAR1-3 antagonism did not significantly influence the population of CD8 (Tc), Th2, or Treg cells (Fig. 5). Such detrimental effect of LPAR1-3 antagonism could be supported by a previous similar report showing that LPAR2 deficiency mice induced more T-cells trafficked from the spleen to the spinal cord, leading to a defect in lymphocyte homing which was reflected by impaired clinical scores and stronger activation of microglia in the grey matter of spinal cords of EAE mice (18). Taken together, our findings suggest that LPA signaling via LPAR1-3 might have pivotal role in T cell differentiation in the secondary lymphatic organs and T cell migration into CNS after EAE induction.
The BBB consists of endothelial cells, pericytes, basal membrane, and foot process of astrocytes. It acts as structural and functional barrier to the crossing of peripheral immune cells (macrophages and T cells) into the CNS in vivo or cultured astrocytes expressing Lpar1-5 (50, 51). The LPA1-3 antagonist Ki16425 has abolished LPA-induced vasorelaxation (52). Cultured endothelial cells are known to express LPAR1-6 (53, 54). LPA signaling can promote the survival and proliferation of endothelial cells from a variety of sources (55), including brain microvascular bEND.3 cells (53). These reports suggest that LPAR antagonist might exert a negative effect on BBB maintenance. Here, we investigated the effect of LPAR1-3 antagonist Ki16425 on BBB integrity. LPAR1-3 antagonist Ki16425 upregulated protein expression levels of GFAP and PECAM as well as mRNA expression levels of ICAM-1 and VCAM-1 in spinal cords of EAElow mice, in agreement with impaired behavioral symptoms of EAElow (Fig. 5). These results suggest that LPAR1-3 antagonism might deteriorate EAE symptom associated with impaired BBB disruption caused by excessive astrocytic activation and increased expression levels of ICAM-1 and VCAM-1 in the spinal cord (Fig. 5).
Pathologically, NOX produces an excessive amount of ROS including hydrogen peroxide (H2O2), superoxide (O2•−), and hydroxyl (OH•) radicals (9). NOX2, NOX3, and NOX4 are the most prominently expressed NOX isotypes in the CNS. However, cellular and temporal expression profiles of these isotypes in injured and non-injured CNS are currently unclear (56). In the MS and EAE, excessive ROS production overwhelms antioxidant defenses and induces oxidative damage (e.g., lipid peroxidation, protein nitration) in endothelial cells of the BBB and the myelin sheath, thereby propagating neurodegeneration (7, 8). Activated microglia and infiltrated macrophages are responsible for ROS production in CNS lesions through upregulation of NOX2 (7, 8). Isolated microglia from NOX2 knock out mice show reduced oxidative stress-induced toxicity to oligodendrocytes. In addition, the mice are more resistant to EAE (9). NOX3 is expressed in neurons in the inner ear. Reduction of NOX3 exerts a protective effect in cochlear injury by reducing the level of oxidative stress (9). On the other hand, LPAR1 inhibitor AM095 treatment inhibits LPA-induced ROS production and NOX expression as well as LPA-induced toll like receptor 4 expression in mesangial cells and in the kidney of streptozotocin-induced diabetic mice (57). In addition, AM095 treatment suppressed LPA-induced pro-inflammatory cytokines through downregulation of phosphorylated NF-κBp65 and c-Jun N-terminal kinases in vitro and in the kidney of streptozotocin-induced diabetic mice (57). LPA signaling through LPAR3 increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs as well as a shorter lifespan (58). These results suggest that LPA or LPAR subtypes might exert significant positive or detrimental effects on neurodegeneration. Thus, we investigated the effect of LPAR1-3 antagonism on oxidative stress after EAElow induction in the present study. LPAR1-3 antagonist Ki16425 significantly increased protein expression levels of 4-HNE, mRNA expression levels of NOX2 and NOX3, and NADPH activities in spinal cords of EAElow mice compared to those in the EAElow group associated with the enhanced microglial activation and the increased microphage infiltration (Figs. 7). However, LPAR1/2 agonist 1-oleoyl-LPA significantly inhibited expression levels of ROS-associated markers in spinal cords of EAEhigh mice (Figs. 8). These results indicate that LPAR1-3 antagonism may induce oxidative stress via activation of NOX2 and NOX3 and that oxidative stress might lead to deterioration of EAE symptoms. Taken together, our findings indicate that regulation of NOX2 and NOX3 via LPAR 1–3 is a key contributor to MS and EAE.