Studies On Immune And Microbial Barriers Characteristics And Proteomics Of ALNA In Bactrian Camels

The aggregated lymphoid nodules area(ALNA)in the abomasum is a special organized lymphoid tissue discovered only in Bactrian camels at present.This study aimed to explore the characteristics associated with mucosal immune homeostasis of ALNA of Bactrian camels.Twenty-seven Alashan Bactrian camels were divided into the following five age groups: fetus(10–13 months of gestation),young(1–2 years),pubertal(3–5 years),middle-aged(6–16 years)and old(17–20 years).And the study was divided into following four aspects:First,IgA~+ and IgG~+ cells in the lamina propria of ALNA were observed and analyzed using immunohistochemcal and statistical techniques.The results showed that,in ALNA,the distribution of IgA~+ and IgG~+ cells were diffuse,and only a few were in subepithelium dome(SED)and most of them in non-SED,except which were not observed in fetus periods.Meanwhile,there were significantly more IgA~+ cells than IgG~+ cells in SED from the young to the middle aged group,but which reversed in old group(P<0.05).However,the aging significantly decreased the densities of two positive cells populations in non-SED(P<0.05);in SED,there were no significant differences between the densities of IgA~+ and IgG~+ cells,but which were both significantly lower in old group than those in young group(P<0.05).The results demonstrated that,in mucosal effector sites,the aging significantly decreased the densities of two cells populations and impacted on the defense barriers formed by Ig A and Ig G,but had no impact on the scattered characteristics.In inductive sites,the aging dramatically declined their densities,and they should have close relationships with immune memory.Second,the age impact on neonatal Fc receptor(FcRn)expressions were observed and analysed in detail with histology,immunohistochemistry,micro-image analysis and statistical methods.The results showed that the FcRn was expressed in mucosal epithelial cells of ALNA from the fetus to the old group,although the expression level rapidly declined in old group;moreover,after the ALNA maturated,the FcRn expression level in the non-follicle-associated epithelium(non-FAE)was significantly higher than that in FAE(P<0.05).In addition,the FcRn was also expressed in the vessel endothelium,smooth muscle tissue,and macrophages and dendritic cells(DCs)of secondary lymphoid follicles(s LFs).It was demonstrated that FcRn was mainly expressed in non-FAE,the effector sites,although which was expressed in FAE,the inductive sites for mucosal immunity.And it was also expressed in DCs and macrophages in s LFs of all ages of Bactrian camels.Third,based on the first and second studying results,the pubertal with the mostdeveloped structure of ALNA were chose.Based on the high throughput sequencing of 16 S r DNA,the diversity of microorganisms on the ALNA mucosa was detected and the difference between those on PPs mucosa.The results showed that there were high bacterial diversities on ALNA mucosa.There were clustered into 18 phylum,30 classes,61 orders,61 families and 68 genus.The dominant bacteria were the Bacteroidetes,Firmicutes,Verrucomicrobia and Proteobacteria,and there were no differences among the different parts;however,the abundances of the Fibrobacteres,Elusimicrobia and Spirochaetes were all significant higher on RMFR and LMFR mucosa than those on PPs mucosa.Meanwhile,the abundance of Fusobacteria on PPs mucosa was significantly higher than on RMFR and LMFR mucosa.In the family classification level,Prevotellaceae,Ruminococcaceae,Lachnospiraceae,Veillonellaceae,BS11,Victivallaceae,RF16,Bacteroidaceae,Elusimicrobiaceae,Mogibacteriaceae,Clostridiaceae,Peptostreptococcaceae and Fusobacteriaceae were common dominant bacteria and there were no significantly differences among different groups.However,the abundances of the Fibrobacteraceae,Erysipelotrichaceae,Campylobacteraceae,Spirochaetaceae,R4-45 B,R4-41 B and Dethiosulfovibrionaceae were all significant higher on RMFR and LMFR mucosa than on PPs mucosa.Meanwhile,the abundance of Enterobacteriaceae,Verrucomicrobiaceae,Rikenellaceae,Barnesiellacea,Odoribacteracea,Alcaligenaceae,Deferribacteraceae,Peptococcaceae and Turicibacteraceae on PPs mucosa was significantly higher than on RMFR and LMFR mucosa.It was suggested that the bacterial diversities colonized on the mucosa of RFMR,LMRF and PPs have some differences in family classifications although it was almost no difference in phylum classification.It was laid a foundation for the further VII study of the relationship between the mucosal immune function of ALNA and microbial flora colonized on it.Finally,based on i TRAQ technique,the total protein and differential protein expressed in the reticular mucosal folds region(RMFR),longitudinal mucosal folds region(LMFR)and ileal Peyer’s patches(PPs)were quantitatively analyzed,and GO,COG and KEGG were annotated and enriched.The results showed that the total protein number was 2513,among which 60 differential proteins were identified between the RMFR and LMFR,among which 37 were up-regulated and 23 were down-regulated.There were 67 differential proteins between the RNFR and PPs,which increased 38,down 19;and LFMR and PPs between 111 different proteins,which raised 67,down 44.The total protein was analyzed by KEGG pathways,and these proteins were annotated that the pathways associated with immunization mainly include cell skeletal regulation and immune cell differentiation,adhesion,migration,FcγR-regulated phagocytosis,antigen capturing and processing.The differential proteins were enriched by the metabolic pathways of the KEGG database,mainly comparing the key proteins in the following pathways: proteins involved in the process of presenting antigenic processes,including MHC-II,β2m,CD4,MHC-I,CD8,CD74(T cells),T cell lymphocyte invasion and metastasis factor 1(Tiam1),actin multidrug regulatory molecule(Rho A),phosphatidyl(PI3K),serine threonine kinase(AKT)and ornithinereleasing protein(Ras);related proteins in the T cell-B cell receptor pathway,including CD21,CD22,guanine acid substituting factors(VAV),CD40 L and CD45;and the main adhesion molecule α4β7 and ligands VCAM-1 and MAd CAM.It was proved that expression levels of the key proteins such as antigen uptake,processing,treatment,T,B cell activation and lymphocyte chemotaxis and homing within RMFR and LMFR of ALNA and PPs were similar,but there were also a little local differences.This study demonstrated that the maintenance of the mucosal immune homeostasis in ALNA showed some basic characteristics as follows:(1)IgA~+ and IgG~+ cells with scattered distribution mainly in the effector sites was one of the outstanding characteristics,which could play a crucial role in forming an integral immune defective barrier;(2)In addition to SIg A main effective molecule of the mucosal immunity,Ig G also has the capacity involved in the maintenance of the mucosal immune homeostasis,meanwhile,aging could reduce the IgA~+ and IgG~+ cell densities and FcRn expression levels;(3)The dominant microbial flora colonized in the ALNA mucosa was the same as that in the ileal PPs mucosa,but there were some differences in its constituent structures;(4)There is a great similarity that the protein expression profiles between ALNA and PPs,however,there are some differences in the expression levels of related proteins such as cytoskeleton regulation and antigen uptakes.It provides a basis for studying the effect of senescence on the immune homeostasis of gastric mucosa,and also laid a foundation for further revealing the position of the ALNA in the gastrointestinal immune system.