A(H1N1) Vaccination Recruits T Lymphocytes To The Choroid Plexus For The Promotion Of Hippocampal Neurogenesis In Pregnant Mice

BackgroundFrom 1917 to 1919, European influenza epidemic, named Spanish flu, caused 20 million deaths globally. It is the most serious influenza epidemic situation in the world.From 1957 to 1958, Flu outbreak in China, it spread to all over the world, is considered as Asian flu, leads to 1 million deaths globally. From 1968 to 1969, Hong Kong flu epidemic leads to more than 700 thousand deaths all over the world. At 2009,a novel-three sources and recombinant H1N1 influenza virus broke out in Mexico and North America, and then spread worldwide, and has caused more than 280 thousand deaths all over the world. In this influenza epidemic situation, the main infection populations are young and middle-aged people(80% persons are younger than 65 years old), and the pregnant mothers are received more concerns. Therefore, pregnant women are recommended to immunize themselves with the inactivated influenza vaccine by the international commission on immunization. However, the vaccination rate is still at a low level in China. Though 2009 H1N1 flu epidemic has been well controlled, other subtype of influenza virus, such as H5N1, H7N9 flu virus still breaks out continuously or sporadically in the crowd and affects human’s health.Unfortunately, the influenza vaccination coverage of pregnant women is relativelylow; meanwhile, mental diseases are severely threatening the health of human beings with a noticeable increase in its incidence in infants and adulthood(15% in 2010,about 0.17 billion people). There are no definite evidences to contact both of them.However, the overall attack rates are higher in the influenza vaccination of pregnant women compared with the pregnant women without vaccination, implying of continuously high incidence in psychiatric diseases.In recent years it became apparent that the generalized perception of the brain and spinal cord as immune-privileged sites needs to be revisited. Alteration in adaptive immune response has a profound impact on adult hippocampal neurogenesis and cognition. Recent studies are mainly focused on immune cytokines and immune cells. Adaptive immune response affects the levels of cytokines in the periphery,activates monocyte-macrophages or T lymphocytes recruited through the BBB to the CP. The permeability is higher than other regions based on the CP consisting of loosened vascular cells and epithelial cells. Thus, immune cells permeate through BBB to the CP/CSF, secreting anti-inflammatory cytokines, IL-10 and IL-4, thereby regulating neurocognition and behavioral function.We have demonstrated that influenza vaccination during pregnant promote hippocampal neurogenesis and working memory abilities. We proposed influenza vaccination up-regulated the levels of BDNF and IGF-1, alleviated neuroinflammation, and eventually built a pro-neurogenic niche for growth and survival of neurons. However, how the adaptive immunity alters the microenvironment in the brain? The underlying mechanism remains unclear.Therefore, to determine the exact immune mechanism between adaptive immunity and the CNS, the present study was carried out using laser confocal microscopy,cerebral stereotaxic technique, FCM analysis, RT-PCR and so on. The present study aimed at explaining the mechanism of H1N1 influenza vaccination affecting neurogenesis and working memory, and providing data for the security of influenza vaccination during pregnancy.Materials and Methods1. Animal and VaccinationPregnant C57BL/6J mice of 8 weeks old(26-28g) were randomly assigned to six groups(n = 6 in each group). The presence of a vaginal plug was designated as gestational day 0(GD0). They were administered i.m. with split inactivated A(H1N1)Influenza Vaccine(AIV) of single dose for 3 μg /mouse respectively in the first trimester(G2.5), and the controls were treated with sterilized PBS. The split inactivated AIV was obtained from the Center for Disease Control and Prevention(CDC, Guangdong, China). The dose of vaccine is in accordance with our previous research. All animal are raised in SPF animal room in laboratory animal center of Zhongshan School of medicine.2. Specific humoral immune response immunized with flu vaccinesMouse blood was collected using the tail vein bleeding method before and 14 d after vaccination. The antibody titers were determined via a hemagglutination inhibition(HI) assay. We recorded the highest dilution that caused complete HI against four hemagglutination units(HAUs) of virus as the HI titer.3. Immunofluorescence staining and MBF Stereo InvestigatorImmunofluorescent staining detected T cells in the CP of the brain’s lateral and3 rd ventricle. For cell proliferation analysis, all of the mice received three Brd U(Sigma-Aldrich) injections(50 mg/kg, i.p., once every 2 h) at GD14. 2 h after the last injection, cell proliferation(Brd U+), neuronal progenitor cells(DCX+), neuronal stem cells(Nestin+), gliocyte proliferation(Brd U+/Iba-1+, Brd U+/GFAP+), neuronal differentiation(Brd U+/Neu N+), BDNF and IGF-1 expression levels were observed by immunofluorescence and analyzed using a Stereo Investigator stereological system(Micro Bright Field Inc., Williston, VT, USA).4. Neutralizing antibody and Minocycline treatment4.1 Cerebral stereotaxic techniqueThe GD7 mice vaccinated AIV were fixed in the stereotaxic instrument after anesthesia. Then 2μl anti-TCR antibodies or isotype control antibody Ig G2 a were injected to lateral ventricle using a microsyringe. BDNF and IGF-1 were delivered to lateral ventricle through a osmotic minipump for 7 days(50μg/kg, rates of infusion;1.0μl/h for anti-IGF-1 antibodies and 2μl/h for anti-BDNF antibodies, Alzet). The C57BL/6 lateral ventricle coordinate: 1.0 mm posterior to bregma, 0.75 mm left of midline and 2.5 mm ventral from the dura. The mice were administered for more than5 min and left the syringe in place for an additional 5 min.4.2 Anti-CD4 antibody injection via tail veinTo inhibit T cell function in the periphery, neutralizing anti-CD4 antibodies(Sigma-Aldrich, diluted 1:5) with PBS containing 0.1% BSA or control Ig G were intravenously(i.v.) injected twice(GD2.5 and GD7). The CD4+ T cells in the periphery were detected by FCM analysis.5. RT-PCRTotal RNA was extracted separately from the CP tissues and hippocampi,Messenger(m) RNA(2lg) was converted into c DNA using a Go Script TM c DNA Reverse Transcription Kit(Promega). Quantitative PCR reactions were performed using Trans Start Tip Green q PCR Super Mix to measure the m RNA levels of VCAM-1、ICAM-1、CXCL9、CXCL10、CCL5、IFN-gamma、IL-4、TNF-1、IL-6.All of the quantitative real-time PCR reactions were determined and analyzed using a Bio-Rad IQ5 Real-Time PCR System.6. ELISA assayThe hippocampal tissue was collected on GD14 in pregnant mice. The concentrations of BDNF and IGF-1 of hippocampal homogenates were measured by ELISA assay.7. Evens blue injectionA 2% solution of Evans Blue dye in 0.9% Na Cl was intraperitoneally injected into the Pre and Pre+AIV mice at a dose of 2 ml/kg 7 days after vaccination. 3 h later,All of the mice were perfused with cold 0.9% Na Cl. Brain tissue samples were homogenized in dimethylformamide and high speed centrifugation. The absorbance of the sample was measured at 620 nm using a microplate reader.8. TUNELOn GD14, TUNEL nuclear staining was performed using a Dead End TM Fluorometric TUNEL System(Promega, USA). Quantification of TUNEL+ cells was performed on a series of 5 randomly selected coronal brain sections. Each group has four mice.9. Eight arm maze testWorking memory and reference memory of control group, AIV group and AIV combined with anti-TCR Ab group were measured using an eight arm radial maze on GD11 to GD18.Results1. Specific humoral immune response after influenza vaccinationThe serum antibody titers of pregnant mice were kept at a baseline level before AIV vaccination. 14 d after vaccination, however, the antibody titer is significantly increased in AIV mice compared with the controls(p < 0.001), suggesting asuccessful immunization in AIV mice.2. The alteration in the T lymphocytes staining, neurogenesis,glial cell phenotype and neurotrophic factors expressionWe could detect TCR+ T lymphocytes in the CP, meninges of lateral ventricle,3rd ventricle in AIV mice. However, there are no T cells observed in the coronal slices of controls.On GD14, significantly more Brd U+ cells in the SGZ and SVZ, more DCX+ and Nestin+ cells in the SGZ were detected in the AIV mice, relative to the controls. There is no obvious difference of new microglia and astrocyte proliferation between the AIV mice and the controls 7 d after the Brd U injection(p > 0.05). Significantly more cells double labeled for Brd U and Neu N in the AIV mice 28 d later(p < 0.05. However, a large proportion of Iba-1+ cells in the hippocampus express IGF-1, moreover,increased expression of BDNF in the hippocampus of the AIV mice(p < 0.05).3. Regulation of T cell function affects hippocampal neurogenesis in Pre+AIV miceOn GD7, to block T cell function in the CP, we treated anti-TCR antibodies to the lateral ventricle. We find that depleting T cells in the CP eliminate the increase in the number of Brd U+, DCX+, Tbr2+ cells, and impair the working memory abilities,to the level of the controls(p < 0.05 for all).Neutralizing anti-CD4 antibodies or control Ig G were intravenously(i.v.)injected twice(GD2.5 and GD7), the num-bers of Brd U+, DCX+ and Tbr2+ cells were significantly decreased in the anti-CD4 antibody-treated mice compared with the Ig G antibody-treated mice(p < 0.05 for all).epithelial cells and cytokines in the hippocampusRT-PCR assay showed that AIV vaccination significantly up-regulated the levels of ICAM-1, VCAM-1, CCL5, CXCL9, CXCL10 relative to those of matched controls in the CP tissue(GD7, GD14 and GD21), presenting a trend of rise first then fall. We found the level of IFN-γ m RNA is continuously increased in AIV mice and a trend of rise first then fall for IL-4. However, it had dual effects on TNF-a and IL-6 m RNA levels.5. AIV vaccination regulate microglial M1/M2 polarization in pregnant miceRT-PCR assay showed AIV vaccination induced microglial M2 polarization,reflected by elevated basal expression of the M2 genes Arg and Ym1(p < 0.001);whereas, M2 polarized microglia could be prevented by anti-TCR antibodies and minocycline treatment.6. The effect of AIV vaccination on the expression of BDNF and IGF-1 in the hippocampusELISA assay showed that AIV vaccination significantly increased the expression of BDNF and IGF-1 in the hippocampus compared with the controls(BDNF, p < 0.05;IGF-1, p < 0.01).7. The effect of neutralizing antibody and minocycline treatment on hippocampal neuronal apoptosisTUNEL assay revealed that there were no obvious differences among all groups including AIV group, controls, antibody and minocycline treated group(p > 0.05).4. The expression of adhesion molecules, chemokines in CPConclusion1. Influenza vaccination during early pregnancy recruits T lymphocytes to the choroid plexus, which contribute to hippocampal neurogenesis and working memory abilities.2. Influenza vaccination during early pregnancy up-regulates the expression of adhesion molecules and chemokines in the CP epithelial cell.3. Infiltrative T lymphocytes in the CP promote hippocampal neurons expressing BDNF; induce M2-polarized microglia secreting IGF-1, elevate the levels of IL-4 and IFN-γ, decrease the levels of TNF-a and IL-6, eventually build a pro-neurogenic niche.4. Influenza vaccination during early pregnancy promotes hippocampal neurogenesis and working memory abilities.