The Role Of Wnt7a/?-catenin In Neurogenesis Promoted By Remodeling Th1 Immunity Induced By BCG Serum

Background Neurogenesis occurs in local microenvironments or neurogenic niches, especially the subventricular zone(SVZ) of the lateral ventricles and the subgranular zone(SGZ) of the hippocampus. Hippocampal neurogenesis is indispensable for hippocampal-dependent learning and memory. Accumulating evidences reveal that immune activation can modulate brain development in early life. Prenatal Influenza vaccination contributes to neurogenesis and behavioral function in offspring involving alteration of varieties of cytokines. The modulation of periphery cytokines by glatiramer acetate treatment is capable of repairing the cognitive deficits triggered by cranial irradiation. By contrast, central nervous system inflammation caused by LPS had a destructive effects on memory, neural plasticity and neurogenesis in early life and led to post-synaptic inhibitory and excitatory changes during early phases of synaptogenesis. Strong evidences have demonstrated neurogenesis can be bi-directionally regulated by different factors, ranging from cytokines, neurotrophins, growth factors to transcription factors, paracrine signaling molecules on individual stages of neuronal development. In addition, many cytokines exert dual effect during neural development and function at all stages.It is demonstrated that Wnt/?-catenin signaling can be regulated by inflammation in CNS. And Wnt/?-catenin signaling is closely related to CNS. Wnt7 a, as one of Wnt families,is essential for multiple steps of neurogenesis, ranging from neural stem cell self-renewal and neural progenitor cell cycle progression to neuronal differentiation and maturation? Bacille Calmette-Guerin(BCG) is an attenuated live bovine tuberculosis bacillus involved in M. tuberculosis and is administered to neonates worldwide against tuberculosis(TB). In clinical trial, BCG shows variable efficacy(0-80%) in preventing adult pulmonary tuberculosis while high efficacy in preventing childhood tuberculosis. There are some evidences to support that BCG induces immune response against TB. Immune response induced by BCG also has been extensively stuied. The stimulation by BCG treatment in infants results in a complex pattern of cytokine expression through induction T cells, including pro-inflammatary cytokines: IFN-??TNF-??IL-2 and IL-6; Th2 cytokines: IL-4?IL-5 and IL-13; the regulatory cytokine IL-10; the TH17 cytokine IL-17. And neonatal immune response with BCG shifts to Th1 type. It has been reported that important function of immune response by BCG is imitively associated with center neual system. In MPTP mouse model of Parkinson's disease(PD), BCG vaccination prior to exposure of MPTP restrict reduction of striatal dopamine(DA) and dopamine transporter(DAT), providing neuroprotection in PD. The postnatal period is a crucial phase on the central nervous(CNS) system development and behavior later in life. We have demonstrated that neonatal BCG vaccination promotes the dendritic development of hippocampal neurons and synaptic plasticity in addition to an increase of neurogenesis and behavior. The improvement of neurogenesis and behavior regulated by BCG is attributed to cytokines alteration in serum and hippocampus. It has been confirmed that immune response to BCG vaccination produces a complex pattern of cytokines in periphery of infants, including IFN-?, TNF-?, IL-4, et al, the greatest increase in cytokine induced by BCG vaccines compared to unvaccinated infants is found with IFN-?. In addition, rodent neonates injected with cytokines exhibit various behavioral abnormalities.The cytokines of serum and hippocampus triggered by BCG is considered to be the chief culprit in enhancement of synaptic plasticity and neurogenesis and behavior in early life. Therefore, the objective of our study was to determine if the BCG-induced cytokines in periphery/serum directly affected hippocampal neurogenesis and cognitive function in neonatal mice. In this study, BCG-serum(BS) was identified, and transferred to the neonatal mice to determine whether BS could remodel the immune internal environment to influence neurogenesis and cognition.Materials and Methods1. Animals and Vaccination C57BL/6 male mice(Pd7) and male newborn pups of C57BL/6(Pd0) were purchased from the Laboratory Animal Center of Sun Yat-sen University. Mice(Pd7) were randomly injected intraperitoneally(i.p.) with BCG(0.1 mg/50 ?l) at a single dose of 50 ?l/mouse containing 105 colony forming units(CFU)(BCG mice). Controls were treated with equivalent sterile PBS(PBS mice). Newborn mice(Pd1) were designedly treated with BCG-Serum or PBS-serum(PS)(20?l/per injection) by intraperitoneally injection seven times over 19 days.2. Serum collection, identification and administration Serum was obtained from mice injected with BCG or PBS via orbital-blood collection, the BCG-Serum(BS) and PBS-serum(PS) were identified to determine cytokines alteration by ELISA. Newborn mice(Pd1) were designedly treated with BCG-Serum or PBS-serum(PS)(20?l/per injection) by intraperitoneally injection seven times over 19 days.3. Early physical development The influence of vaccination on early physical development were recorded. We analyzed the difference of mean body mass of each group.4. Intracerebroventricular(ICV) delivery ICV delivery was performed with recombinant Dickkopf-1(DKK1) in neonatal mice treated with BS at Pd21 and used to block Wnt7a/?-catenin signaling pathway.5. Behavioral tests analysis 5.1 Open-field test(OFT) It was performed to measure spontaneous activity of neonatal mice in the novel environment. 5.2 Morris water maze(MWM) It was used to measure the hippocampal-dependent spatial learning and memory ability in neonatal mice.6. Enzyme Linked Immunosorbent Assay(ELISA) Both blood and brain tissue samples were collected to detect the concentrations of IFN-?, IL-4, TNF-? and IL-6 in serum and hippocampus at Pd25 by ELISA assay.7. Immunofluorescence staining and quantification Mice were administrated single i.p. with Brd U(50mg/kg, Sigma) at Pd25. Cell proliferation(Brd U+), neuronal differentiation(Brd U+/DCX+, Brd U+/Neu N+), gliocyte proliferation(Brd U+/Iba-1+, Brd U+/GFAP+), expression levels of brain-derived neurotrophic factor(BDNF), Wnt7 a, ?-catenin, active ?-catenin were measured by immunofluorescence. Brd U+ cells, nestin+ cells and Brd U double labelled cells in DG were counted bilaterally with the stereo investigator stereology system. Image J software was used to evaluate semi-quantitatively relative immunoactivity of BDNF, Wnt7 a, ?-catenin, active ?-catenin.8. Western Blot(WB) Hippocampal tissue samples were obtained from each mouse at PD25, the expression levels of Wnt7 a signaling pathway protein, including Wnt7 a, ?-catenin, active ?-catenin, Cyclin D1 and Ngn2 were detected by WB assay. Relative protein levels were calculated as the ratio of target protein to tubulin.Results1. T helper 1(Th1) polarized cytokines predominate in BS The levels of IFN-? and IL-4 in BCG mice were significantly increased(P < 0.05), BCG mice displayed a significant decrease in TNF-? and IL-6 levels compared with PBS mice(P < 0.05). BS exhibited a higher ratio of IFN-? to IL-4(P < 0.05), indicating that BCG induced Th1 immune response.2. BS induces a Th1 polarized systemic environment in neonatal mice There were no significant difference in body weight. The levels of IFN-? and IL-4 were significantly up-regulated in serum of BS group when compared with PS group(IFN-?: P < 0.001, IL-4: P < 0.05). By contrast, the levels of TNF-? and IL-6 in BS group were significantly decreased when compared with those in PS group(TNF-?: P < 0.05, IL-6: P < 0.05). There was a higher ratio of IFN-? to IL-4 in BS group compared with PS group in serum(P < 0.05) and hippocampus(P < 0.05). The results suggest that BS remodels Th1 polarized systemic environment.3. Behavioral Detection 3.1 Open-field test Inhibition of Wnt7a/?-catenin pathway significantly reduced the levels of locomotion in center(P < 0.001) and total(P < 0.001) in BS group and PS group. The time in center during 30 min(0-30min)(P < 0.001) and the first 20min(0-20min)(P <0.001) were significantly decreased in r DKK1-treated BS group compared to BS group and r DKK1-treated PS group compared to PS group. No difference in locomotion activity of periphery(P > 0.05) and the center time of the last 10min(20-30min)(P > 0.05) could be seen. The results displayed that BS enhances the exploration and adaptability by Wnt7 a pathway in neonatal mice. 3.2. Morris water maze In the acquisition phase, all groups improved their performance across successive five days, and there was significant difference in escape latencies among mice(P < 0.001). BS group showed a trend of shorter latencies compared to PS group. Inhibition of Wnt7a/?-catenin pathway significantly increased latencies in BS group and PS group. In the probe phase, the percentages of time spent(P < 0.001) and distance travelled(P < 0.001) in the target quadrant were significantly higher in BS group than those in PS group. r DKK1-treated BS group compared to BS group(Time: P < 0.001, Distance: P < 0.001) and r DKK1-treated PS group compared to PS group(Time: P < 0.001, Distance: P < 0.001) showed lower percentages of time spent and distance travelled. In the reversal phase, BS group spent less time than PS group on one day 7 and day 8(P > 0.05), latencies were increased in r DKK1-treated BS group compared to BS group and r DKK1-treated PS group compared to PS group. There was no significant difference among the four groups in swimming speed(P > 0.05). The findings revealed that BS promoted learning and memory ability by Wnt7 a pathway in neonatal mice.4. Neurogenesis and nestin-positive cells in hippocampal DG For assessment of neurogenesis in hippocampal DG, our data displayed that BS group significantly elevated Brd U+ cells(P < 0.001), Brd U+/DCX+ cells(cells that co-localized Brd U and DCX)(P < 0.001), Brd U+/Neu N+ cells(cells double labeled for Brd U and Neu N)(P < 0.01) and nestin+ cells(P < 0.01) in the DG compared with PS group. The results indicated that BS promoted hippocampal neurogenesis in neonatal mice. Blocking Wnt7a/?-catenin pathway significantly by treatment of DKK1eliminated the increase in Brd U+ cells(P < 0.001), Brd U+/DCX+ cells(P < 0.001), Brd U+/Neu N+ cells(P < 0.01) and nestin+ cells(P < 0.01) in BS group and PS group. The findings indicated that Wnt7a/?-catenin pathway played a vital role in BS-induced neurogenesis in hippocampal DG.5. BS activates canonical Wnt7a/?-catenin pathway Wnt7 a signaling pathway, including Wnt7 a, ?-catenin active ?-catenin, Cyclin D1 and Ngn2 was investigated at Pd25. Western blot analysis displayed that BS group showed significantly increased expression of Wnt7a(2.87-fold), ?-catenin(3.03-fold), active ?-catenin(1.95-fold), Cyclin D1(2.17-fold) and Ngn2(2.22-fold) compared with PS group(Wnt7a: P < 0.001, ?-catenin: P < 0.001, active ?-catenin: P < 0.001, Cyclin D1: P < 0.001, Ngn2: P < 0.001). Wnt7 a, ?-catenin, active ?-catenin, Cyclin D1 and Ngn2 were significantly decreased in r DKK1-treated BS group in comparison with BS group, 0.31-fold, 0.60-fold, 0.64-fold, 0.51-fold and 0.36-fold respectively at Pd25(Wnt7a: P < 0.001, ?-catenin: P < 0.001, active ?-catenin: P < 0.001, Cyclin D1: P < 0.001, Ngn2: P < 0.001). Similarly, Wnt7a(0.61-fold), ?-catenin(0.31-fold), active ?-catenin(0.41-fold), Cyclin D1(0.52-fold) and Ngn2(0.41-fold) were significantly decreased in r DKK1-treated PS group in comparison with PS group(Wnt7a: P < 0.001, ?-catenin: P < 0.001, active ?-catenin: P < 0.001, Cyclin D1: P < 0.001, Ngn2: P < 0.001). The findings suggested that BS activates canonical Wnt7a/?-catenin pathway in hippocampus. Canonical Wnt7a/?-catenin immunoactivity in DG was measured using IF test, BS group showed higher Wnt7a/?-catenin immunoactivity than PS group, but Blocking Wnt7a/?-catenin pathway significantly by treatment of DKK1 reduced immunoactivity level in BS group and PS group, it is consistent with WB analysis. Co-staining of Wnt7 a and nestin was shown by IF analyses, confirming expression of Wnt7 a in NSCs.6. BS elevates hippocampal BDNF, the effect can be prevented by DKK1 IF analysis showed higher Brain-derived neurotrophic factor(BDNF) and IGF-1 immunoactivity in BS group as compared with PS group. An increase of BNDFimmunoactivity in BS group was attenuated by treatment of Wnt7 a antagonist DKK1, but IGF-1 not. And BNDF immunoactivity in r DKK1-treated PS group was lower than PS group. Brd U+/IBa-1+ cells were identified, and there were no significant differences among the four groups(P > 0.05). Neu N+ and IBa-1+cells expressed BDNF and IGF-1, respectively. It is consistent with that BS group showed higher BDNF(P < 0.001) and IGF-1(P < 0.01) protein level by ELISA. However, the up-regulation of BDNF protein expression was significantly reversed by Wnt7 a inhibitor DKK1 in BS group(P < 0.001), but IGF-1 not(BS VS BS+DKK1: P > 0.05). BNDF protein expression in r DKK1-treated PS group was significantly reduced relative to PS group(P < 0.001), but no difference of IGF-1 was observed between r DKK1-treated PS group and PS group(PS VS PS+DKK1: P > 0.05).Conclusion 1. T helper 1(Th1) polarized cytokines predominate in BS. The serum of mice vaccinated by BCG contained increased IFN-? and IL-4 and decreased TNF-? and IL-6 compared with control, which indicated the Th1 polarized cytokines predominated in BS. And we found a higher ratio of IFN-? to IL-4 in BS, suggesting that BCG induced Th1 immune response in mice. 2. Th1 polarized serum remodels a Th1 polarized systemic environment in the neonatal mice.Neonatal mice administrated with Th1 polarized serum showed increased IFN-? and IL-4 and decreased TNF-? and IL-6 in serum and hippocampus of BS mice when compared with PS mice, in addition to a higher Th1/Th2 cytokines ratio in BS mice than PS mice. 3. Remolding Th1 polarized systemic environment by BCG serum treatment promotes cognitive function in neonatal mice. Our results showed superior exploratory in OFT and spatial ability in MWM task respectively in BS group compared with those in PS group.4. Remolding Th1 polarized systemic environment by BCG serum treatment increaseshippocampal neurogenesis and nestin-positive cells. Our results disclosed an increaseof proliferating newborn cells, DCX-positive cells, mature neurons and nestin-positive cells in BS group compared with those in PS group.5. Remolding Th1 polarized systemic environment by BCG serum treatment activates Wnt7a/?-catenin signaling pathway in hippocampus DG. Inhibition of Wnt7a/?-catenin signaling decreases signaling protein levels in addition to impairement of neurogenesis and cognitive function.