The Role Of CAMP/PKA Signaling Pathway In Dendritic Cell That Regulates Treg Homeostasis

Dendritic cells (DCs) are the members of innate immune system of the host. They are highly effective antigen-presenting cells, which can perceive and capture external stimuli,decide whether activate the host immune defenses and start what kind of immune defense.DCs could regulate the immune response through certain signaling pathways. As immune suppressive cells, Treg play a key role in balancing the immune response and immune tolerance. It has been known that DC could regulate the homeostasis of Treg. However,through what kind of molecular pathway is still unknown.Here, we used the mice model Cbl-b-/-C-cblf/f mice and Rlαf/f mice which already exist in the laboratory, and constructed the DC conditional knock-out mice. The results showed that both cAMP/PKA and Cbl signaling pathway in DC play role in regulation of Treg homeostasis. Then we focus the role of cAMP/PKA signaling pathway in DC, and the mechanism of Treg homeostasis regulation.1 Both cAMP/PKA and Cbl signaling pathways in dendritic cells are involved in Treg homeostasis regulationIn order to know which signaling pathway was involved in DC regulating Treg homeostasis, we use the Rlαf/fmice and Cbl-b-/-C-cblf/f mouse model to construct to DC knock-out mice. When R1α knocked out in DC, the R subunit in PKA lost the inhibitory function to C subunit, mimicing the activation of cAMP/PKA signaling pathway, thus appears a unique group of MHCⅡhiCD11clow DC in spleen. Because the similar with the migration DC group in lymph nodes, we call this group of DC "S2DC".In addition, when DC knocked out Cbl-b/C-cbl, although the population and total cell number of DC do not change so much,the ration of CD8+DC v.s.CD11b+DC has been converted. The ration of CD8+DC v.s. CD11b+DC in WT mice is 2:7, however, in KO mice is 7:2. More importantly, both of these knock-out mice have Treg proliferation in lymphoid organs, suggesting that both cAMP/PKA signaling pathway and Cbl signaling pathways play role in DC regulating Treg homeostasis.2 The activation of cAMP/PKA signaling pathway in DC will affect its development and functionPathogen Infection and host immune response are inseparable. Because of this, we use R1αDCKO mice as a tool, to study the mechanism of cAMP/PYKA signaling pathway in DC regulating Treg. It is meaningful in the study of pathogen causing the host immune defense.In the last chapter, we already know that when cAMP/PKA signaling pathway activated in DC, DC cell will assemble in lymphoid organs, and at the same time, Treg also showed an extensive proliferation in lymphoid organs in R1αDCKO mice, suggesting that cAMP/PKA signaling pathway plays a role in DC regulating Treg homeostasis. In this chapter,we focus on how cAMP/PKA signaling pathway affects the development and function of DC. The reason of DC expansion in lymphoid organs could be due to either producing more in bone marrow or migration more from non-lymphoid tissue. First we found that preDC in bone marrow has no significant difference between WT and RlaDCKO mice. And using FLT-3L in vitro culture BMDC has also no difference between WT and R1αDCKO mice, indicating that DC assembling in lymphoid organs is not because generation more in bone marrow.After the "danger signals",such as inflammation, received by peripheral non-lymphoid tissues DC, they will move to lymphoid organs. In this process, DC maturation, chemokine receptor CCR7 expression levels will be upregulated, which means CCR7 plays a key role in the migration of DC function. By flow cytometry, we found that the skin cDC proportion in RlaDCKO deceased to 1/3 of the WT mice, while the skin and spleen CCR7 expression levels were significantly higher than in WT mice. It was suggested that the activation of cAMP/PKA signaling pathway in DC, as if mimic DC simulated by"danger signals", and therefore regulated CCR7 expression, so that a large amount of peripheral non-lymphoid tissues DC migrate to the lymphoid organs, gathered in the spleen and even the form S2DC.3 Dendritic cells regulate Treg homeostasis through cAMP/PKA signaling pathway in R1α knock out miceIn last chapter we already know that the expansion of DC in lymphoid organs in due to up-regulating the expression level of CCR7 since cAMP/PKA signaling pathway activated in DC. In this chapter we are interested in how DC affects Treg proliferation, through directly or indirectly regulation? And what is the relationship between this regulation and cAMP/PKA signaling pathways. We first observed that R1αDCKO mice have high levels of IL-2 production. IL-2 is the most important cytokine required for Treg generation and proliferation. In vivo blocking of IL-2 generation could decrease Treg population which expansion in spleen of R1αDCKO mice. This suggested that the expansion Treg in R1αDCKO mice is IL-2 dependent. Further studies showed that the excess IL-2 in R1αDCKO mice is due to excessive secretion of CD4+T cell. CD4+T cells produced IL-2 after stimulation by antigen. In vitro antigen presentation experiments confirmed that, S2DC of R1αDCKO mice have a stronger antigen-presenting ability, thereby stimulating the corresponding OTII CD4+T cells to secrete more IL-2. This is because S2DC express higher levels of MHCII.And MHCII is a necessary molecular structure that could use by DC presenting antigens to CD4+T cells. Inhibit S2DC MHCII expression in vitro could block S2DC antigen-presenting ability, thus inhibit the production of IL-2.In summary, tha activation of cAMP/PKA signaling pathway in DC, as being"dangerous signal" instruction, upregulated CCR7 expression and trigger DC in peripheral non-lymphoid tissue migrate to lymphoid organs, so that gathered a group of MHCIIhiDC in spleen, which have a stronger antigen-presenting ability to stimulate CD4+T cells produced more IL-2, affect Treg homeostasis, massive growth performance.4 Dendritic cells regulate Treg cell homeostasis through cAMP/PKA signaling pathway in physiological conditionsDC could affect Treg homeostasis through cAMP/PKA signaling pathway during pathogen infection. In addition to this, DC could also regulate Treg homeostasis through cAMP/PKA signaling pathway under normal physiological state. UV irradiation is a normal physiological phenomenon. As universal environmental stimuli, UV irradiation could affect the function of antigen-presenting cells. DC is the most important antigen-presenting cells in the skin. Study showed that ultraviolet radiation can affect the distribution of skin DC,thus affecting downstream of the immune response, induced activation of Treg. This suggested that DC can affect Treg homeostasis. In our work, we use ultraviolet irradiation mice model successfully mimic DC migration and Treg proliferation. Although DC could regulate Treg has been confirmed in recent years by a variety of experiments, however, the specific regulatory mechanism remains unclear. Sensing external signals through body needs a certain signal transduction pathways. The study confirmed that ultraviolet exposure to the skin, can stimulate the body to produce large amounts of prostaglandin E2 (pGE2).pGE2 could send signals into the cell by binding to its G-protein coupled receptor EP in the cell membrane. Since EP belongs to the upstream of cAMP/PKA pathway, suggesting that pGE2 generated after UV irradiation could go through cAMP/PKA pathway.In order to prove that ultraviolet radiation cause skin DC migration, draining lymph nodes Treg proliferation through cAMP/PKA pathway, we block pGE2 production in vivo in ultraviolet radiation model. And it has been found that blocking pGE2 in vivo after ultraviolet radiation, the proliferation of Treg will be declined, which is the first time to demonstrate ultraviolet irradiation induce DC migration and Treg proliferation could be partially through PKA / c AMP pathway.5 (Research in China) Function of Tran, the transcriptional regulator of Streptococcus suis Serotype 2Streptococcus suis (SS) is an important zoonotic pathogen causing a variety of life-threatening infections in pigs and humans. Tran, a novel transcriptional regulator which was identified to be an infection-related factor in S. suis serotype 2 using suppression subtractive hybridization (SSH), has been reported by our group. In this study, a tran deletion mutant was constructed to compare with the wild-type ZY05719 in some biological characteristics. It is suggested that longer chains and relatively slower growth could be observed in tran deletion mutants. In order to identify gene transcription profiles,microarray analysis was performed. It indicated that the inactivation of Tran led to 130 differentially expressed genes spread throughout the genome. Among these, 21 genes were upregulated, and 109 genes were downregulated. Most of the differentially expressed genes were involved in bacterial metabolism, such as the phosphotransferase system (PTS), and heat shock proteins. In the case of glucose scarcity, the growth characteristics of tran deletion mutants was impacted significantly, meanwhile Atran mutant was highly sensitive to environmental stresses. Moreover, cell adherence decreased by 22.2%, and virulence in zebrafish declined to more than 5 times in Atran mutants. These data demonstrate the role of Tran in regulation in S. suis serotype 2, that is affect bacterial virulence by influencing bacterial metabolism and stress tolerance of external environment.Streptococcus suis infected the host, could cause the host innate and adaptive immune responses. Although the molecular mechanism remains unclear, lots of identified virulence factors showed related with infection and host interactions. For exemple, Capsular polysaccharide (CPS), which is the most important virulence factor, can resist phagocytosis of host immune cell. Recent studies showed that adenylate synthetase Ssads may also be a virulence factor. It can convert the nucleotides of the host to adenosine. Once adenosine accumulated in the cell, they will combine with the A2a, which belongs to G protein-coupled receptors on the cell surface. Then start downstream cAMP/PKA signaling pathway, thus inhibit leukocyte activation pathway of bacterial adhesion sensitivity, reduce oxidation and plasmin function to undermine the host immune system. This indicates that S.suis serotype 2 could also infect the host cell through GPCR signaling pathway.