The Relationship Between Vitamin D And Daly Of Malaria In Africa And Its Mechanism For Prevention Of Cerebral Malaria Immunity

Objective:Malaria is a global zoonotic disease caused by an infected Anopheles and almost 3.4 billion people are at the risk of infection.Currently there are still more than 90countries with malaria attack,and cerebral malaria is one of the most serious complication of malaria.In sub-Saharan Africa,the morbidity and mortality among children is high,and malnutrition is recognized as one of the key contributing factors.Recent research shows that vitamin D deficiency is a common malnutrition problem in Africa.The public would mistakenly believe that the deficiency of VD should only occur in the region above 35degrees north latitude and south latitude,while those living in Africa are close to the equator,there will be no shortage of VD due to adequate light.However,realistic studies show that there is a problem of VD deficiency even in areas with sufficient sunshine.In addition to sources of vitamin D depending on sun explosure,the lack of dietary vitamin D may be another important factor.VD levels in serum were significantly lower in children with severe malaria than in healthy children.At present,more and more researchers begin to pay attention to the basic research of Vitamin D,and consider that it can exert its anti-infection function by regulating the immune system.The molecular and cellular mechanisms of cerebral include the increase of proinflammatory cytokines expression,the upregulation of NF-κB pathway induced by endothelial activation,the activation of glial cells and the isolation of infected erythrocytes,monocytes and platelets in brain capillary.Recent studies have found that calcitriol has anti-malarial ability in vitro.Studies have reported that in mice infected with P.b.,the therapeutic use of vitamin D after infection can reduce the infection rate and mortality of mice infected with cerebral malaria.So we conducted a review based on the UN Food and Agriculture Organization of the United Nations（FAO）database,the United States Department of Agriculture（USDA）,the Global Burden of Disease（GBD）,systematically conducted studies on the relationship between the level of vitamin D consumed by seven kinds of food ingredients in sub-Saharan Africa and the incidence of DALY,YLL and YLD caused by malaria in 38 African countries,as well as the immunomodulatory effects and related mechanisms of preventative treatment of VD on ECM models,and the immunomodulatory effects and and related mechanisms of VD on macrophage RAW264.7,aiming to provide a new theory for the effective prevention and treatment of malaria so as to develop more effective anti-malarial strategies.Methods:1.The level of vitamin D consumed by food in sub-Saharan AfricaWe calculate the content of VD in seven kinds of food ingredients,and calculate the daily average amount of vitamin D in seven kinds of food consumption in each country.The results were correlated with DALY of malaria in these countries and divided into high and low dose groups for comparative analysis DALY of malaria.2.mice infection and the observation of parasitemiaMice were infected by intraperitoneal injection of 1×10⁶ P.b ANKA parasitic erythrocytes（pRBC）.After the infection,the tail vein blood samples were taken and stained with Giemsa stain.Parasitemia was determined by light microscopy.Mortality was monitored daily and clinical ECM scores was counted to define whether the mice are infected of cerebral malaria.3.VD treatment of miceC57BL/6 mice were randomly divided into 4 groups:uninfected group,PbA group（PbA）,PbA+Oil group as a control group,preventative treatment of Vitamin D group（VD+PbA）.Mice in preventative treatment of Vitamin D group（VD+PbA）were orally administered a daily dosage of 50μg/kg of VD for 5 consecutive days before PbA infection.At the same time,PbA groups received the same volume of soybean oil at the identical time points as the VD+PbA groups.4.Blood-brain barrier permeability test in miceOn the fifth day after the mice were infected by Pb,the mice were injected of 200μl 2%Evans blue dye via tail vein in each group.One hour later,the heart was infused with normal saline,and the brain was taken and photographed immediately after the mice were sacrificed.The mice brains were placed in centrifuge tubes,2 ml of formamide was added in each tube,200μl of the supernatant was taken and placed in a 96-well plate after centrifuged and incubation.The absorbance at the wavelength of 630 nm was measured and the EB content was calculated.5.Culture of macrophage RAW264.7 in vitro The experiment contained four groups:Normol group with only culture medium was added,VD group with effective concentration of VD（10 nM VD）and LPS group with LPS（1μg/ml）as control groups.10nM of VD and 1μg/ml of LPS were added to each well in LPS+VD groups.The supernatant and cells were collected after all groups were cultured in triplicate for 48 h at 37°C in a humidified 5%CO₂ incubator.6.RNA extraction and Real-time RT-PCRRNA was extracted from the brain,spleen and macrophage RAW264.7.After removing the remaining genomic DNA with PrimeScriptTMRT reagent kit with gDNA Eraser kit,the RNA was reverse transcribed into cDNA.Real-time RT-PCR reaction was performed with the obtained cDNA product as a template.The 2^-ΔΔCT method was used to quantify the relative gene expression of each group.7.ELISAMice serum,spleen culture supernatant,brain and macrophage RAW264.7 culture supernatants were tested by ELISA kit（R&D Systems,Minneapolis,MN,USA）.The experiment was operated under the instruction of direction.8.Flow cytometryAfter the preparation of single cell suspension of mice spleen cells,brain cells,macrophages,stain them.Flow cytometric analysis was performed using a FACS Calibur,and data were analyzed with the FlowJo software.9.Statistical AnalysisAll data are presented as mean±standard deviation（x±s）.Statistical significance was analyzed using one way ANOVA by Prism 7.The comparison between groups was performed using q test.Student’s t-test was performed to analyze the differences between groups and intra-group,and Kaplan-Meyer method was used to analyze the survival time.A value of P<0.05 was considered significant.Results:1.African children are in a state of severe vitamin D deficiencyVitamin D levels per capita from food consumption in 38 African countries is very low.The vitamin D levels was significantly negatively correlated with DALY,Death and YLL in these countries.The average value of vitamin D in 30 countries is below 2μg（80IU）/day,which is much lower than the recommended values of WHO.Only eight countries were above 5μg/day.According to the above two groups of vitamin D levels,DALY,YLL and YLD in the two groups were compared,and the results had significant differences.2.Preventative treatment of Vitamin D was effective at raising the serum 25（OH）D3 levels of miceOn day 5 p.i.,the mice were sacrificed to determine the content of VD in serum.The results showed that there was a significant difference in the level of VD between PbA-infected groups（PbA）and preventative treatment of Vitamin D groups（VD+PbA）,and Vitamin D status varied greatly between the mice.3.Preventative treatment of Vitamin D protects mice against CMThere are no differences on mice in symptoms,survival,incidence of cerebral malaria,parasitemia and other aspects between PbA-infection groups（PbA）and the vehicle control（PbA+oil）,but there are significant differences in all aspects between PbA-infection groups and preventative treatment of Vitamin D groups（VD+PbA）.4.Preventative treatment of Vitamin D reduces brain pathologyThe integrity of blood-brain barrier in PbA-infected groups（PbA）was obviously destroyed,with significant EB extravasation,but only a small amount of EB extravasation was seen in preventative treatment of Vitamin D groups（VD+PbA）.5.Preventative treatment of Vitamin D can significantly reduce T cells trafficking to the brainOn day 5 p.i.,the levels of CXCR3,CXCL9 and CXCL10 in the brain of both groups were significantly increased.The expression levels of chemokines CXCR3,CXCL9 and CXCL10 were significantly reduced in preventative treatment of Vitamin D groups（VD+PbA）in brain tissue.6.Preventative treatment of Vitamin D dampens the Th1 immune respons and decreases the levels of IFN-γand TNFThe proportion of Th1-type cells in the mice splenocytes of preventative treatment of Vitamin D groups（VD+PbA）was significantly decreased.In serum and spleen culture supernatants,the expression levels of IFN-γand TNF-αin preventative treatment of Vitamin D groups（VD+PbA）were significantly lower than those in PbA-infection groups（PbA）on 5 p.i..Compared with PbA-infection groups（PbA）,the mRNA expression levels of TNF-γand IFN-γin splenocytes in preventative treatment of Vitamin D groups（VD+PbA）were significantly increased.7.Preventative treatment of Vitamin D stimulates expansion of Tregs and increases IL-10and TGF-βsecretionOn day 3 p.i.,the number of Tregs in the splenocytes of preventative treatment of Vitamin D groups（VD+PbA）was significantly lower than that of PbA-infection groups（PbA）.Compared with PbA-infection groups（PbA）,the levels of TGF-βin mice serum and IL-10and TGF-βin spleen culture supernatant of preventative treatment of Vitamin D groups（VD+PbA）were significantly higher than that of PbA-infection groups（PbA）on day 5p.i..8.Preventative treatment of Vitamin D inhibits DC differentiation,maturation and functionThe absolute number and proportion of myeloid DCs（mDCs）and plasma DCs（pDC）of PbA-infection group（PbA）were slightly higher than that of preventative treatment of Vitamin D groups（VD+PbA）on day 5 p.i..The number and proportion of costimulatory molecules MHC-II of DCs in preventative treatment of Vitamin D groups（VD+PbA）were slightly decreased compared to PbA-infection groups（PbA）.In addition,the expression of costimulatory molecules TLR4 and TLR9 were both decreased in preventative treatment of Vitamin D groups（VD+PbA）to a certain extent.9.Preventative treatment of Vitamin D promotes the secretion of IL-1,IL-6 and IL-12It is shown that the levels of IL-1,IL-6 and IL-12 in serum and spleen supernatant and mRNA expression levels of IL-1,IL-6 and IL-12 in splenocytes of preventative treatment of Vitamin D groups（VD+PbA）were significantly higher than those of PbA-infection group（PbA）on day 3 p.i.and day 5 p.i..It is similar to the results of stimulation of RAW264.7 macrophages in vitro.10.Preventative treatment of Vitamin D inhibits the expression of NF-κBOn day 3 p.i.,the mRNA expression of NF-κB in splenocytes of preventative treatment of Vitamin D groups（VD+PbA）was significantly decreased compared with PbA-infection group（PbA）.11.VD inhibits TLR4,TLR9 and CD40 expression of macrophages RAW264.7Compared with LPS groups,the expression of CD40 and TLR9 in VD+LPS groups was decreased significantly.The mRNA expression of TLR4 and TLR9 in VD+LPS groups（VD concentration was 10nM）was also decreased significantly compared with LPS groups.12.VD can inhibit macrophage RAW264.7 Th1-type inflammatory cytokines secretion When VD+LPS were simultaneously added to the culture medium,the expression level of TNF-αwas found to be significantly lower than that of the LPS.In addition,the mRNA levels of TNF-αin macrophages RAW264.7 cultured with VD+LPS were significantly decreased compared with those in LPS groups（1μg/ml）.13.VD can inhibit macrophage RAW264.7 lymphocyte stimulationThe results of flow cytometry showed that LPS stimulated the expression of CD80,CD86and MHC-II on macrophages RAW264.7.When cultured with VD+LPS,we found that the expression levels of CD80 and CD86 were significantly lower than that of LPS groups（1μg/ml）,and the mRNA expression of MHC-II in VD+LPS groups was significantly decreased as well compared with LPS groups.Conclusions:Vitamin D levels per capita from overall food consumption in 38 African countries is only4μg/day.And 30 of countries daily consumption of vitamin D is less than 2μg（80IU）/day.Much lower than the WHO recommended 5μg（200IU）/day and the American Academy of Medical Sciences recommended 1-70 year-old vitamin D daily intake of 15μg（600IU）,70 years of age and above 800IU.This is enough to prove that children in Africa are in a state of serious inadequate vitamin D.Our research on vitamin D and DALY shows that vitamin D is a protective factor for malaria.Preventative treatment of Vitamin D can significantly prevent the occurrence of CM in P.b ANKA infected C57BL/6 mice,as well as reduce the mortality of mice and improve the pathological changes of experimental cerebral malaria.VD can inhibit the differentiation,maturation and activation of DCs,affect the number of splenic DCs,decrease the expression of CD80,CD86 and MHC-II on macrophages RAW264.7,and then inhibit the ability of presenting antigen.Preventative treatment of Vitamin D can also inhibit TLR4,TLR9 and CD40 expression of macrophages RAW264.7,reduce the affection of NF-κB pathway,and inhibit macrophage secretion of IL-1,IL-6,IL-12 and Th1 type inflammatory cytokines such as TNF-α,to further reduce the stimulation of lymphocytes by antigen-presenting cells.Preventative treatment of Vitamin D can inhibit Th1 immune response,reduce the level of IFN-γand TNF,increase the number of Tregs and increase the secretion of IL-10 and TGF-β,and inhibit the inflammatory reaction.In addition,Preventative treatment of Vitamin D inhibits NF-κB expression,and may further suppress the inflammatory response.Thereby reducing the expression of chemokines CXCL9,CXCL10 and ICAM-1,VCAM-1 in the brain,then reducing the accumulation of CD8⁺T cells in the brain and improving the integrity of the blood-brain barrier,ultimately preventing the occurrence of cerebral malaria.