Stress can enhance or suppress immune functions depending on a variety of factors. Our previous studies observed that Toll-like receptor 2 (TLR2) participates in chronic restraint stress-induced immune dysfunction. However, the mechanism by which TLR2 prevents immune suppression remains elusive. Our investigation found that stimulation of TLR2 by peptidoglycan (PGN) significantly attenuates splenocyte apoptosis and markedly blocks alterations of anti-apoptotic and apoptotic proteins. Activation of TLR2 inhibits chronic stress-reduced phosphorylation of c-Jun N-terminal kinase (JNK) and diminishes chronic stress-induced up-regulation of corticosterone production. Additionally, our data show that chronic stress causes a dramatic decrease of cytokine IL-2 level but an increase of IL-4 and IL-17 in CD4+ T cells. Interestingly, PGN could block these alterations of cytokine levels. Collectively, our studies demonstrate that stimulation of TLR2 attenuates chronic stress-induced immune suppression by modulating apoptosis-related proteins and immunoregulatory agents.

Stress can enhance or suppress immune functions depending on a variety of factors. Our previous studies observed that Toll-like receptor 2 (TLR2) participates in chronic restraint stress-induced immune dysfunction. However, the mechanism by which TLR2 prevents immune suppression remains elusive. Our investigation found that stimulation of TLR2 by peptidoglycan (PGN) significantly attenuates splenocyte apoptosis and markedly blocks alterations of anti-apoptotic and apoptotic proteins. Activation of TLR2 inhibits chronic stress-reduced phosphorylation of c-Jun N-terminal kinase (JNK) and diminishes chronic stress-induced up-regulation of corticosterone production. Additionally, our data show that chronic stress causes a dramatic decrease of cytokine IL-2 level but an increase of IL-4 and IL-17 in CD4+ T cells. Interestingly, PGN could block these alterations of cytokine levels. Collectively, our studies demonstrate that stimulation of TLR2 attenuates chronic stress-induced immune suppression by modulating apoptosis-related proteins and immunoregulatory agents.

Stress, either psychological or physical, can have a dramatic impact on the immune system. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. We have reported that stress modulates the immune response in a TLR4-dependent manner. However, the mechanisms underlying TLR4-mediated signaling in stress modulation of immune system have not been identified. Here, we demonstrate an essential role for the TLR4-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling. PI3K inhibition by inhibitors wortmannin or LY294002 abrogated protection of stress-induced immune suppression in TLR4-deficient mice compared with TLR4-deficient mice that did not receive the inhibitors. The mechanisms by which PI3K are increased in the TLR4-deficient lymphocytes may involve increased phosphorylation of Akt as well as increased phosphorylation of glycogen synthase kinase-3β (GSK-3β). The stress-mediated suppression of T help 1 (Th1) cytokine and increased production of Th2 cytokine was greatly reduced in TLR4 deficient mice compared with the wild type mice. Moreover, inhibition of PI3K diminished protection of the above Th1 and Th2 changes caused by stress in TLR4-deficient mice compared with non-stressed mice and the wild type mice. Our data demonstrated that TLR4 negatively regulates PI3K activity in wild type mice, leading to the observed the stress-induced immune response. The higher levels of PI3K prevent TLR4 deficient mice from the stress-induced immune response. Therefore, stress modulates the immune system through TLR4-mediated PI3K/Akt signaling.

Physical or psychological chronic stress can suppress the immune system. However, the mechanisms remain to be elucidated. We investigated the effect of hematopoietic stem-progenitor cells (HSPCs) on chronic stress-induced the alterations of immune responses. We demonstrate that HSPCs prevents stress-induced lymphocyte apoptosis. Moreover, we also demonstrate that the protective effect of HSPCs on stress-induced lymphocyte reduction exerts by steroid hormones. Furthermore, we reveal that chronic stress-induced T cell-mediated immune responses contributes to the protective effect of HSPCs. These results indicate that HPSCs might offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.

The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. Previous research in our laboratory showed that integrin αDβ2 is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, a key ligand for αDβ2 within the tissue is yet to be identified, since αDβ2 does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. There is evidence that macrophages adhere to CEP-modified albumin in αDβ2-dependent manner. We continued the advancement of the proposed hypothesis that non-conserved, basic amino acids of integrin αDβ2 located near the MIDAS site of the I-domain are responsible for binding to CEP. αD I-domain and generated I-domain mutants: H272(D), K297(Q) and K309(N) were used to map the ligand binding site between integrin and CEP. Using site-directed mutagenesis, mutant αD I-domains were generated with minimal amino acid substitutions. Protein-protein binding reveals that the generated mutation of K297(Q) on the I-domain demonstrates the strong reduction of binding, while H272(D) and K309(N) did not have a significant effect on integrin binding properties. Therefore, lysine 297 located in I-domain of integrin αD, is a critical amino acid for αDβ2 binding to CEP-modified proteins. The identification of a binding site for CEP-modified proteins within αDβ2 will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.

Aflatoxin B1 and T-2 toxin are mycotoxins, which produce their immunotoxic effects by affecting nonspecific and acquired immunity in different species. The mechanisms of their immunotoxicity are still obscure. Cytokines are the key signaling molecules during the immune response. In this study, expression of macrophage-produced cytokines Interleukin-lα (IL-lα), tumor necrosis factor (TNF), and IL-6, and lymphocyte-produced cytokines IL-2, interferon y (IFNy), and IL-3 was measured at the mRNA and protein levels, after in vitro activation with mitogens in AFB1-and T-2-toxin-exposed mice. Significant changes in the organ weights, especially in the mice exposed to a high dose of T-2 toxin, and no effect in AFB1-exposed mice were observed. ConA-induced production of IL-2, IFNy, and IL-3 mRNA and protein levels in AFB1-exposed mice showed a decrease in low dose groups (significant for IL-2 mRNA), but no change at other doses. However, in T-2-toxin-treated animals, there was a significant induction of IL-2 and IFNy mRNA in high and low doses and of IL-3 mRNA at the medium dose. The protein levels of IL-2 and IFNy did not follow the mRNA levels in high dose and the protein levels of IL-3 were significantly increased in medium and low doses. LPS-induced IL-lα and TNF mRNA and protein levels in AFB1-exposed mice were suppressed at the high dose while mRNA levels of both cytokines were increased significantly in the low and medium doses. Low and medium doses of AFB1 also significantly decreased IL-lα protein levels and the high dose decreased IL-6 protein. In T-2 toxin-treated mice, no significant difference in mRNA levels of these cytokines was observed but a general pattern of significant suppression of their protein levels (except IL-lα at medium dose) showed that both toxins regulate the cytokine expression differently. Based on the above discussed results and others, AFB1 may alter cell-mediated immunity by affecting the communication between macrophages and T lymphocytes through inhibiting the macrophage-producing cytokines. T-2 toxin-induced immunosuppression may be due not only to the inhibition of macrophage-producing cytokines, but also to the lack of effector cells to respond to the cytokines (IL-2, IFNy, and IL-3).

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第12923号 / 論工博第4116号 / 新制||工||1625(附属図書館) / 32133 / (主査)教授 木村 俊作, 教授 跡見 晴幸, 教授 岩田 博夫 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19035号 / 農博第2113号 / 新制||農||1031(附属図書館) / 学位論文||H27||N4917(農学部図書室) / 31986 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 宮川 恒, 教授 西田 律夫, 教授 間藤 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13454号 / 論医博第2247号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 杉田 昌彦, 教授 生田 宏一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Adaptive and innate immune responses play a critical role in the protection against extracellular or intracellular pathogens. The function of these two types of immune responses is coordinated by CD4+ T-helper (Th) cells. Depending on the cytokine environment, Th progenitor (Thp) cells differentiate into three functionally different effector subsets. T-helper-1 (Th1) cells which mediate cell-mediated immunity, T-helper-2 (Th2) which orchestrates humoral immunity and T-helper-17 (Th17) cells key players in autoimmunity response. Cytokine induced transcription factors that are differentially expressed in Th cells are required for the development and commitment to a specific Th lineage. The population of Th2 cells can be subdivided in subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. Very limited information is available about the regulation of cytokine production in this array of Th2 cells. We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN-regulatory factor (IRF)-4, a transcription factor expressed in lymphocytes and macrophages. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. To investigate the role of IRF4 in cytokine heterogeneity, Th2 cells were infected with retrovirus expressing IRF4. The cells overexpressing IRF4 secreted significantly higher levels of IL-10 and IL-4 compared to cells infected with a control vector at the same time the level of IL-9 decreases. To understand the mechanism by which IRF4 regulates IL-10 expression in various Th2 cell subpopulations we used co-immunoprecipitation assays to determine transcription factors that interact with IRF4. Our data shows that PU.1, IRF4 and NFATc2 form a complex in Th2 nuclear extract. We also demonstrated by ChIP assay that IRF4 directly binds the Il10 and Il4 loci in a time dependent manner. The role of these protein-protein and protein-DNA complexes and their contribution towards Th2 heterogeneity will be further defined. Understanding the regulation of the anti-inflammatory cytokine IL-10 in Th2 cells may give us a tool to control inflammation.