Patients with IBD have higher rates of psychiatric disorders, such as depression and anxiety; however, the mechanisms underlying a link between intestinal inflammation and depressive-like symptoms are largely unknown. In this study, we investigated the effect of EF-2001 in IBD-like physiological changes and depressive-like behavior in DSS-treated mice. Chronic administration of EF-2001 prevented such changes. In addition, EF-2001 attenuated the increase of inflammatory cytokines in the rectum and hippocampus, attenuated the reduction of neurogenesis in the hippocampus, and facilitated the NFκB p65/XIAP pathway in the hippocampus of DSS-treated mice. This is the first report that the antidepressant effect of EF-2001 may involve hippocampal neuroprotection via decreased inflammatory cytokine expression in the rectum and hippocampus as well as apoptotic cell death regulation via inhibition of caspase-3 activity through facilitation of the NFκB p65/XIAP pathway in the hippocampus.
IBD, including Crohn’s disease and ulcerative colitis, is a chronic relapsing condition characterized by intestinal damage (barrier disruption, altered microbiota) and high levels of inflammation such as elevated inflammatory cytokines [
43,
46,
47]. Cytokines induce extensive inflammation in the colon, which has a negative impact on epithelial cells, resident and recruited immune cells, and stromal cells [
44]. Specifically, inflammation can cause damage to epithelial cells, and activate and recruit immune and stromal cells, ultimately leading to non-resolving chronic inflammation and the development of IBD [
44]. Moreover, during acute or chronic inflammation, inflammatory cytokines can induce the development of depression [
48,
49]. Clinical studies have reported that patients with IBD often exhibit obsessive–compulsive disorder, panic disorder, depression, and anxiety [
4‐
7]. DSS treatment induces colonic epithelial cell lesions and intestinal inflammation, including elevated inflammatory cytokines, via epithelial cell toxicity, increased intestinal permeability, and macrophage activation [
9]. It has been suggested that
E. faecalis improves colitis by increasing interleukin-10 (IL-10), a factor that inhibits cytokine synthesis, in colonic epithelial cells [
50,
51]. The present study showed that EF-2001 reduced inflammatory cytokines in the rectum (Fig.
5). This effect was similar to that of steroids, which are commonly prescribed for treating IBD [
52]. Recently, other researchers have reported that EF-2001 protects dinitrobenzene sulfonic acid-induced colitis, a chemically induced colitis model, via a decrease in inflammatory cytokines [
30]. Thus, the anti-inflammatory effect of EF-2001 may also be related to reduced inflammatory cytokines. Peripheral inflammation is a risk factor for developing mood or psychotic disorders, such as depression [
53‐
56], and may affect hippocampal neurogenesis, including the proliferation, differentiation, and survival of newborn neurons [
54,
55]. Adult neurogenesis occurs in two main regions of the brain, one of which is the subgranular zone of the DG [
57,
58]. In the present study, we observed that DSS treatment significantly increased TNF-α and IL-6 levels in the rectum and hippocampus. A previous study has reported that DSS treatment significantly increases rectal TNF-α and IL-6 levels [
59]. Interestingly, hippocampal TNF-α and IL-6 mRNA in DSS-treated mice were unchanged compared with controls (Fig.
6). Depression is closely associated with altered inflammation [
16], manifested by increased expression of inflammatory cytokines such as TNF-α and IL-6 [
17]. Neuroinflammatory factors, such as TNF-α and IL-6, can negatively affect many stages of neurogenesis in the adult mammalian brain, including the proliferation, differentiation, and survival of newborn neurons [
18‐
20,
54,
55]. Therefore, cytokine-induced reduction of neurogenesis might establish a key link between inflammation and depression. In this study, DSS-treated mice showed a significant decrease in neurogenesis in the DG, consistent with a previous study [
12]. Likewise, DeCarolis and Eisch reported a reduction in neurogenesis in the hippocampus of patients with depression [
60]. These findings suggest that DSS-induced depressive-like behavior may be associated with the reduction of neurogenesis in the DG via the release of inflammatory cytokines derived from peripheral inflammation. Moreover, several studies have suggested that antidepressant effects are critically dependent on intact adult neurogenesis and may be mediated by the enhancement of neurogenesis in the hippocampal DG [
21,
23,
24]. We observed that administration of EF-2001 significantly attenuated the enhancements of rectal and hippocampal inflammation and reduction of newborn neurons in the hippocampus of DSS-treated mice. Therefore, we suggest that the antidepressant effect and enhanced neurogenesis observed upon EF-2001 administration are partly independent effects, resulting from the EF-2001-mediated reduction of peripheral inflammation. Although the mechanism by which pro-inflammatory cytokines reduce neurogenesis is not fully understood, we believe that inflammatory cytokines in peripheral tissue might be key mediators of this process.
In human neutrophils, the activation of NFκB p65 seems to control spontaneous apoptosis and anti-apoptotic effects. Unexpectedly, we found that the activation of NFκB p65-positive cells were increased by EF-2001 administration in water- and DSS-treated groups compared with control groups. Moreover, we found that activation of NFκB p65 was localized to hippocampal astrocytes and microglia; however, the mechanisms underlying this activation remain unclear. We hypothesized that β-glucan may be associated with activation of NFκB p65 via TLR2. β-Glucan activates TLR2 and increases TLR2 expression via NFκB p65 activation [
61‐
63]. Interestingly, we found that hippocampal TLR2 was activated by EF-2001, which was localized in all cell types, including immature and mature neurons, astrocytes, and microglia (Fig.
9). We did not assess the migration of EF-2001 or β-glucan into the brain; we will examine them in a future study. Modulation of this pathway most likely regulates the balance between pro- and anti-apoptotic factors [
64], thus affecting neutrophil survival. In this study, EF-2001 attenuated DSS-induced neuroinflammation and we hypothesized that NFκB p65 may play a role in anti-apoptosis. The activation of NFκB p65 inhibits apoptosis via a mechanism involving upregulation of various anti-apoptotic genes, such as cellular FLICE-inhibitory protein, Bcl-xL, and XIAP [
26,
27]. XIAP, a key member of the inhibitors of apoptosis protein family, can inhibit apoptosis by directly binding to the initiator caspases: caspase-3, -7, and -9 [
65]. Moreover, hippocampal XIAP regulates synaptic plasticity, which is associated with the development of depression [
66,
67]. In this study, XIAP was significantly increased by EF-2001. Further, XIAP was localized in hippocampal microglia. Moreover, cleaved caspase-3, which is crucial in the process of apoptosis and contributes to the irreversible stage of apoptosis [
68], was significantly increased in the hippocampus of DSS-treated mice. In contrast, EF-2001 suppressed the increased levels of cleaved caspase-3 in the hippocampus of DSS-treated mice. These results suggested that EF-2001 might partly modulate apoptosis via regulation of the microglial NFκB p65/XIAP pathway and caspase-3 in the hippocampus of DSS-treated mice.