The Mechanism Of Bacillus Subtilis Spore Induced Intestinal Mucosal Immune Memory Cells

The intestinal mucosa is the primary exposure and entry site of majority pathogenic microorganisms.Oral immunization is a commonly employed route for inducing local immunity and blocking the invasion of pathogen.However,oral immunization has some disadvantages such as short duration of immunity and low efficiency.Thus,the application of oral immunization is limited by the short-term persistence of the immunity,which has become a bottleneck restricting mucosal immune.In addition,effective mucosal immune responses can elicit memory T cells against pathogen through the retention and persistence of T cells in the intestine.Studies have shown that mucosal immunopotentiator can significantly enhance the mucosal immune response,but there is no systematic study on how to improve immune memory.Although tissue-resident memory T cells(TRM)is an important first line of defense against infection in mucosal tissues,their function in intestinal immunization remains to be investigated.Recently,Bacillus subtilis spore has received extensive attention and research due to its strong immune stimulation effect.The main mechanism of Bacillus subtilis spore is that it can stimulate dendritic cells(DCs)maturation and effectively enhance mucosal and systemic immune responses.So,what is the effect of Bacillus subtilis spores on immune memory cells?The exact mechanism is unclear.Therefore,we explored whether the Bacillus subtilis spore as potential immunopotentiator assisted PEDV WIV in enhancing the immunity by upregulating memory CD4+CD8+T cells via oral immunization in piglets.And the related mechanisms were further studied by using the mouse oral immunization model.Secondly,the effect of Bacillus subtilis spore on DCs activation and maturation was studied in vivo and in vitro.Thirdly,the effects of Bacillus subtilis spore combined with H9N2 WIV also enhanced the levels of local mucosal and systemic immune responses.Finally,our data demonstrated that Bacillus subtilis spore promoted the immune response by inducing intestinal memory T cells formation,which were associated with upregulation of ICAM-1 expression in DCs.Our research is divided into the following three parts in detail:1.Bacillus subtilis spores combined with PEDV whole inactivated virus induced mucosal immunityWe discussed the immune effect and related mechanism of piglets after oral bacillus subtilis spores plus whole inactivated porcine epidemic diarrhea virus(PEDV WIV)model.In the intestine,long-term persistence of immunity is related to CD4+CD8+memory T-cells.In this study,piglets were orally immunized with Bacillus subtilis spores plus PEDV WIV,followed by booster oral immunization.Initially,the results showed that Bacillus subtilis spore plus PEDV WIV enhanced the anti-PEDV capability on mucosal surfaces,as evidenced by plaque reduction neutralization tests in serum and intestinal fluid.Elevated antigen-specific IgG titers in the serum and IgA titers in saliva,feces and nasal washing liquid were also observed.Meanwhile,Bacillus subtilis spore plus PEDV WIV increased the area of Peyer's patches and the number of intraepithelial lymphocytes in the ileum of piglets.Similarly,the percentage of CD4+CD8+ memory T-cells were upregulated and proliferation ability of antigen-specific memory T-cell was strengthened in intestinal mucosal-associated lymphocytes,which was accompanied with increased expression of CCR9 after oral immunization with Bacillus subtilis spores plus PEDV WIV.In addition,the activation of memory T-cells is correlated with the increased mRNA expression of Toll-like receptor 2 and 4,as well as interleukin-6 and induced by Bacillus subtilis spore.Collectively,the study provided further insight into the potential immunopotentiator ability of Bacillus subtilis spore to assist PEDV WIV in the potentiation of immunity by upregulating memory CD4+CD8+ T cells via oral immunization.2.Bacillus subtilis spore induced dendritic cell activation and maturationActivation and maturation of dendritic cells(DCs)which are powerful antigen presenting cell(APC)in the mucosa after phagocytosis of pathogenic microorganisms are the primary conditions for the formation of immune memory.The objective of this study was to evaluate the stimulatory effects of Bacillus subtilis spore on DCs and to verify its ability to enhance the immune response by DCs maturation.The results demonstrated that Bacillus subtilis spore can adhere to the nasal epithelium and be taken up by DCs in the nasal mucosa,thereby inducing DCs maturation and resulting in increased CD80,CD86,CD40 and MHCII expression and cytokine secretion.In conclusion,an effective immune response may result from the uptake of H9N2 by DCs in the nasal mucosa,thereby stimulating DCs maturation and migration to cervical lymph nodes to initiate immune response.In addition,a DCs-driven Th1/Th2 differentiation experiment was performed.In coculture system,Bacillus subtilis spore induced DCs to form TEDs across ECs in vivo and in vitro.Thus,we assessed DCs maturation in the presence of an epithelial barrier and stimulation by Bacillus subtilis spore had the powerful capacity to increase the expression of CD40 and CD80 and the release of cytokines such as TNF-? and IL-1?.3.The mechanism of Bacillus subtilis spore induced immunologic memory cellsThrough studying the activation effect of bacillus subtilis spores on dendritic cells(DCs),we further used the mouse model of oral mmunization with bacillus subtilis spores plus oral H9N2 whole inactivated virus(H9N2 WIV)to detect the level of local mucosal immune response and the mechanism of immune memory cell formation.Here,we reported that the levels of local mucosal and systemic immune responses were enhanced through oral immunization with H9N2 WIV plus spore.Firstly,the levels of sIgA in the intestinal tract and the lung and the levels of IgG,IgGl and IgG2a in serum were significantly increased in mice administered H9N2 WIV plus spore compared to mice administered H9N2 WIV alone.Subsequently,H9N2 WIV plus spore led to the generation of CD103+CD69+ TRM,which was independent of circulating T cells during the immune period.Meanwhile,Bacillus subtilis spore as an efficient immunopotentiator upregulated intercellular adhesion molecule-1(ICAM-1)expression on DCs in vitro and in vivo.Furthermore,the proportion of HA-tetramer+ TRM cells was severely curtailed when ICAM-1 expression was suppressed,which was also dependent on HA-loaded DCs.Taken together,our data demonstrated that Bacillus subtilis spore promoted the immune response by stimulating TRM,which were associated with activation of ICAM-1 in DCs.In conclusion,in model of piglet oral immunization,this study demonstrated that Bacillus subtilis spore assist PEDV WIV in the potentiation of immunity by upregulating memory CD4+CD8+ T cells via oral immunization in piglets.In addition,Bacillus subtilis spore can stimulate DCs maturation accompanied by effectively providing an important basis for inducing effective immune response and a premise for establishing mucosal immune memory.Furthermore,Bacillus subtilis spore as a mucosal immunopotentiator induced the formation of TRM,which is dependent on ICAM-1 expression of DCs.Finally,these results indicated that Bacillus subtilis spores,as mucosal immune enhancer,provided a new idea for the study of mucosal immune memory induction,and provided a new target and theoretical basis for the development of mucosal immune vaccine.