Toll-like receptor 4, Toll-like receptor 7 and Toll-like receptor 9 agonists enhance immune responses against blood-stage Plasmodium chabaudi infection in BALB/c mice

BackgroundToll-like receptor (TLR) signals play vital roles during the blood-stage of malaria infections. However, the roles of TLR agonists in the regulation of immune responses and the development of protective immunity to malaria remain poorly understood.MethodBALB/c mice were pre-treated with TLR4, TLR7 and TLR9 agonists, followed by infection with Plasmodium chabaudi. After infection, splenic dendritic cells (DCs), Th1 cells and programmed death-1 (PD-1) expressed on Th1 cells, as well as regulatory T cells (Tregs) were analyzed by flow cytometry. The levels of IFN-γ, TNF-α, TGF-β and IL-10 in splenocytes and IgG1 and IgG2a in serum were measured by ELISA.ResultAdministration of TLR4, TLR7 and TLR9 agonists prior to infection improved disease outcomes. All TLR agonists promoted DC activation, and the proportions of Th1 cells increased. In TLR4, TLR7 and TLR9 agonist treated groups the levels of pro-inflammatory cytokines IFN-γ and TNF-α were elevated, and IgG1 and IgG2a serum levels were also significantly increased. TLR4, TLR7 and TLR9 agonists diminished the activation of Tregs and down-regulated the anti-inflammatory cytokines TGF-β and IL-10. Finally, PD-1 expressed on Th1 cells were decreased in TLR4, TLR7 and TLR9 agonist treated groups compared with control groups.ConclusionTLR4, TLR7 and TLR9 agonists activated DC-mediated innate immune responses and adaptive immune response, which against the blood-stage of Plasmodium and might be applied to malaria protection and treatment.     

左旋精氨酸通过上调Th1应答介导抗疟保护性免疫

目的探讨左旋精氨酸(L-Arginine,L-Arg)对致死型约氏疟原虫(Plasmodium yoelii 17XL,P.y17XL)感染BALB/c小鼠Th1免疫应答的调节效应。方法于P.y17XL感染前7天,连续每日给予BALB/c小鼠L-Arg(1.5 g.kg-1体质量)灌胃预处理;动态观察各组感染小鼠原虫血症水平和生存率;于感染后d 0、d 3和d 5分别提取小鼠脾细胞,流式细胞术检测BALB/c小鼠CD4+CD69+T细胞、F4/80+CD36+巨噬细胞数量;ELISA法检测脾细胞培养上清中IFN-γ的分泌水平,Griess反应检测脾细胞培养上清中NO含量。结果与NC组比较,L-Arg能够降低感染小鼠的原虫血症水平,延长生存时间。L-Arg组CD4+CD69+T细胞数量出现有意义的增加,IFN-γ分泌水平明显提高,F4/80+CD36+巨噬细胞数量出现明显升高,NO的分泌也明显增加。结论在感染早期L-Arg处理可诱导Th1免疫应答的有效建立,明显遏制P.y17XL感染早期红内期疟原虫的感染进程。     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

Upregulation of PD-1 on CD4+CD25+T cells is associated with immunosuppression in liver of mice infected with Echinococcus multilocularis

Alveolar echinococcosis is a zoonotic disease caused by Echinococcus multilocularis (E. multilocularis) infection. The relationship between PD-1/PD-L1 pathway and Tregs at different stages of E. multilocularis infection has rarely been reported. This study aims to investigate the role of PD-1/PD-L1 in immunosuppression of Tregs in E. multilocularis infection. Hematoxylin-eosin staining, flow cytometry, immunohistochemistry, quantitative RT-PCR analysis, cytometric bead array and MTT assay were used to analyze liver pathological changes, percentages of PD-1⁺ Tregs and PD-L1⁺ dendritic cells (DCs), expression levels of PD-1, PD-L1 and Foxp3, levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and proliferation of lymphocytes. During middle-late stage (day 30 to day 330) the percentages of PD-1⁺ Tregs and PD-L1⁺ DCs together with levels of Foxp3, IL-10 and TGF-β increased significantly and maintained at high level. The expression of PD-1 and PD-L1 was increased with the enlarging erosion of E. multilocularis, and was mainly distributed in hepatic sinus, fibrous wall of alveolar hydatid and germinal layer around foci of infection. PD-1/PD-L1 promoted the secretion of IL-10 and TGF-β. Our results indicate that engagement of the PD-1 and PD-L1 correlates with inhibition of T-cell effector function, cytokine secretion and proliferation. High expression of PD-1/PD-L1 may play an important role in stimulating CD4⁺CD25⁺ T cells, and maintaining peripheral tolerance and immune evasion during chronic infection of E. multilocularis.     

地塞米松对P.y17XL感染DBA/2小鼠早期免疫应答的影响

目的探讨地塞米松对约氏疟原虫(Plasmodium yoelii 17XL,P.y17XL)感染DBA/2小鼠早期免疫应答的调节效应机制。方法 6～8周、雌性DBA/2小鼠,随机分为实验组和对照组,经腹腔接种1×106P.yl7XL寄生的红细胞,实验组于感染后第1和2天尾静脉给予地塞米松处理。动态观察各组感染小鼠原虫血症水平和生存率;于感染后第0天、3天和5天分别提取小鼠脾细胞,流式细胞术检测树突状细胞(DCs)亚群(髓样树突与浆样树突)百分比及功能分子的表达,CD4+CD25+Foxp3+T细胞占CD4+T细胞总数的百分比;ELISA法检测脾细胞培养上清中IFN-γ、IL-10的分泌水平;Griess反应检测脾细胞培养上清中一氧化氮(NO)含量。结果与正常感染组相比,地塞米松处理组:①生存率明显降低;②感染后第3和5天脾细胞中两种树突状细胞亚群水平以及DCs表面MHCⅡ类分子和共刺激分子CD86的表达水平显著下降,而CD4+CD25+Foxp3+T细胞仅于感染后第3天降低,而第5天之后则明显升高;③脾细胞培养上清中IFN-γ、IL-10、NO的产生水平均明显降低。结论地塞米松由于显著抑制DBA/2小鼠抵御P.y17XL感染保护性免疫应答的建立,从而致其死亡。     

Differential immune modulation by deoxynivalenol (vomitoxin) in mice

Mycotoxin deoxynivalenol (DON), a secondary metabolite produced by Fusarium fungi, is a contaminant in wheat, barley, and corn worldwide. It has been suggested that DON exhibits toxicity in various organs. Due to the lack of immunotoxicity data for DON, we investigated the differential immunomodulatory effects of DON in mice. DON was orally administered to female BALB/c mice at a dose of 0, 0.5, or 2 mg/kg body weight for 14 days and various immunotoxicity tests were performed with standard protocols. The population of CD19⁺ and CD11c⁺ cells in the spleen and mesenteric lymph node (MLN) and of F4/80⁺ cells in the spleen was significantly decreased in DON-treated mice, whereas the level of CD8⁺ and CD4⁺CD25⁺Foxp3⁺ cells in the spleen and CD4⁺ T cells in MLN was significantly increased. In intra-epithelial lymphocytes (IELs) of the small intestine, the population of CD4 ⁺ and CD19⁺ cells was increased but that of CD8⁺ cells was decreased. Levels of CD4 ⁺ and CD8⁺ cells were decreased in lamina propria lymphocytes (LPLs) of small intestine; however, the level of CD4⁺ and CD8⁺ lymphocytes was increased but that of CD19⁺ cells was decreased in Peyer's patches lymphocytes (PPLs). Normalized expression of TLR4 in spleen, TLR9 in PPs, and TLR2, TLR3 and TLR4 in MLNs was significantly decreased, whereas expression of TLR5 and TLR9 was increased in spleen. The concentration of IgA and IgE was decreased and increased, respectively, in serum; however, the mucosal IgA level was significantly increased in the duodenum. Levels of IFN-γ, IL-2, IL-4, and IL-6 were significantly increased in serum. Furthermore, DON induced apoptosis in spleen, MLNs, and PPs, and DON-induced apoptosis was promoted by increased expression of Bax and decreased expression of Bcl-2. The autophagy genes Atg5 and Beclin-1 were up-regulated in spleen but down-regulated in MLN. After priming of the RAW 264.7 macrophage cell line with different TLR ligands, DON exposure differentially modulated IL-1β, IL-10, and TNF-α production. These results indicate that DON can cause various immunomodulatory effects in mice, creating a milieu that might allow invasion by other microorganisms.     

Alloantigen specific T regulatory cells in transplant tolerance

CD4⁺CD25⁺Foxp3⁺T cells are regulatory/suppressor cells (Treg) that include non-antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve CD4⁺CD25⁺Treg develop into specific Tregs is unknown. We have studied DA rats tolerant to fully allogeneic PVG cardiac grafts that survived with out immunosuppression for over 100 days and identified the cellular basis of alloantigen specific tolerance. Key observations from our studies will be reviewed including how CD4⁺CD25⁺Tregs were first identified and the cytokine dependence of CD4⁺T cells that transfer alloantigen specific transplant tolerance which died in culture unless stimulated with both cytokine rich ConA supernatant and specific donor alloantigen. Both the tolerant CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell populations are required to transfer tolerance, yet alone the CD4⁺CD25⁻ T cell effect rejection. Tolerance transfer occurs with a low ratio of CD4⁺CD25⁺T cells (< 1:10), whereas to induce tolerance with naive CD4⁺CD25⁺T cells requires both a ratio of > 1:1 and is not alloantigen specific.Recent findings on how naïve CD4⁺CD25⁺T cells developed into two separated pathways of alloantigen specific Tregs, by culturing them with alloAg with either IL-2 or IL-4 and donor alloantigen are described. IL-2 enhances IFN-γR and IL-5 mRNA while IL-4 induced a reciprocal profile with de novo IL-5Rα and increased IFN-γ mRNA expression. Both IL-2 and IL-4 alloactivated CD4⁺CD25⁺Tregs within 3–4 days of culture can induce alloantigen specific tolerance at ratios of 1:10. Long term, CD4⁺CD25⁺T cells from tolerant hosts given IL-2 cultured cells have increased IL-5 and IFN-γR mRNA; whereas hosts given IL-4 cultured cells had enhanced IL-5Rα mRNA expression and IL-5 enhanced their proliferation to donor but not third party alloAg.These findings suggest that Th1 and Th2 responses activate two pathways of alloantigen specific Tregs that can mediate transplant tolerance but are dependent upon cytokines produced by ongoing Th1 and/or Th2 immune responses.     

Rapamycin inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells

Reciprocal differentiation of immunosuppressive CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) and proinflammatory IL-17-producing cells (Th17) from naïve CD4 cells is contingent upon the cytokine environment. Using MACS-purified CD4 cells, we found that rapamycin and cyclosporine A (CsA) potently inhibited the TGFβ and IL-6-induced generation of IL-17-producing cells. Intriguingly, rapamycin promoted, while CsA markedly inhibited, TGFβ-mediated generation of Tregs. The aforementioned effects of rapamycin and CsA were also observed for Flow-sorted CD4⁺CD25⁻ T cells, indicating that the effect of these two immunosuppressive agents was based on their action on de novo generation of Tregs and Th17 cells from naïve CD4 cells. Our observation suggests a distinct mode of immunosuppressive action and tolerance induction by rapamycin and CsA. The capacity of rapamycin to generate immunosuppressive Tregs and to suppress differentiation of pathogenic Th17 cells furthers our understanding of the basis for the therapeutic immunosuppressive effects of rapamycin in patients with autoimmune diseases and allo-transplantation reactions.     

Immune modulatory effects of the foodborne contaminant citrinin in mice

The mycotoxin citrinin can cause mycotoxic nephropathy, cytotoxicity and genotoxicity. To investigate the immune modulatory effects, CTN was orally administered to female BALB/c mice at the dose of 1, 5, or 10 mg/kg body weight for 14 days, and several immunotoxicity tests were performed. The populations of F4/80⁺ cells and CD19⁺ cells were significantly decreased in spleen and MLN. In MLN, CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ cell populations were increased. CD8 ⁺ cells were increased but CD19⁺ cells were decreased in intra-epithelial, lamina propria and Peyer’s patches lymphocytes. In a cell proliferation assay, along with the increased proliferative capacities of ConA-induced splenocytes and MLN cells, IFN-γ production was increased. The expression of TLR 2 was increased in spleen, but TLR 3 expression in MLN was decreased. The level of serum IgM was reduced. Furthermore, apoptosis was induced in spleen, MLN and Peyer’s patches and promoted by the change in the ratio of Bax/Bcl-2 activities. Autophagy gene Atg5 and Beclin-1 were up-regulated in spleen. The expressions of IL-1β, IL-10, and TNF-α were inhibited in murine macrophage cells pre-exposed with TLR ligands. These results indicate that CTN has multiple immune modulatory effects in mice that may alter normal functions of immune system.     

Dendritic cells treated with a prostaglandin I2 analog,iloprost, promote antigen-specific regulatory T cell differentiation in mice

Iloprost, a stable prostaglandin I₂ (PGI₂) analog, can inhibit allergic inflammation in an ovalbumin (OVA)-induced asthma model via inhibition of airway dendritic cell (DC) function. However, the underlying mechanism of PGI₂ signaling-mediated immunosuppression remains unclear. This study explored whether iloprost-treated DCs can suppress inflammation by promoting antigen-specific regulatory T cell (Treg) differentiation through PGI₂-G-protein-coupled receptor (IP). We established an allergic lung inflammation model using a hydrogel biomaterial delivery system and observed that iloprost significantly suppressed OVA-induced Th2 lung inflammation and increased the frequency of OVA-specific Tregs in vivo. We further observed that iloprost-treated DCs displayed tolerogenic characteristics, including low inflammatory cytokine (IL-12, TNF-α, IL-6, IL-23) expression levels, high anti-inflammatory cytokine (IL-10) production, and a semimature phenotype. In addition, iloprost-treated DCs increased OVA-specific CD4⁺Foxp3⁺ T cell differentiation from naïve T cells in an IP-dependent pathway in vitro and in vivo. Blocking experiments showed that iloprost-treated DCs promoted Treg differentiation, at least in part, through programmed death ligand 1 (PD-L1), whereas iloprost-induced PD-L1 expression in DCs was through the IP receptor. Furthermore, iloprost treatment suppressed DC-mediated airway inflammation and increased the frequency of OVA-specific Tregs through PD-L1 in vivo. Taken together, these results show that PGI₂-IP signaling mediated by iloprost in DCs may lead to immune tolerance, suggesting that the PGI₂ analog has the potential to be applied therapeutically for tolerogenic DC immunotherapy in autoimmune diseases or allergic asthma.     

Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation

α7 nAChRs expressed on immune cells regulate antigen-specific antibody and proinflammatory cytokine production. Using spleen cells from ovalbumin (OVA)-specific T cell receptor transgenic DO11.10 mice and the α7 nAChR agonist GTS-21, investigation of (1) antigen processing-dependent and (2) -independent, antigen presenting cell (APC)-dependent, naïve CD4⁺ T cell differentiation, as well as (3) non-specific APC-independent, anti-CD3/CD28 mAbs-induced CD4⁺ T cell differentiation, revealed the differential roles of α7 nAChRs expressed on T cells and APCs in the regulation of CD4⁺ T cell differentiation. GTS-21 suppressed OVA-induced antigen processing- and APC-dependent differentiation into regulatory T cells (Tregs) and effector T cells (Th1, Th2 and Th17) without affecting OVA uptake or cell viability. By contrast, GTS-21 upregulated OVA peptide-induced antigen processing-independent T cell differentiation into all lineages. During anti-CD3/CD28 mAbs-induced T cell differentiation in the presence of polarizing cytokines, GTS-21 promoted wild-type T cell differentiation into all lineages, but did not affect α7 nAChR-deficient T cell differentiation. These results demonstrate (1) that α7 nAChRs on APCs downregulate T cell differentiation by inhibiting antigen processing and thereby interfering with antigen presentation; and (2) that α7 nAChRs on T cells upregulate differentiation into Tregs and effector T cells. Thus, the divergent roles of α7 nAChRs on APCs and T cells likely regulate the intensity of immune responses. These findings suggest the possibility of using α7 nAChR agonists to harvest greater numbers of Tregs and Th1 and Th2 cells for adoptive immune therapies for treatment of autoimmune diseases and cancers.     

Triptolide regulates T cell-mediated immunity via induction of CD11c dendritic cell differentiation

Triptolide(TPT) isolated from one of the Chinese herbs, Tripterygium wilfordii Hook. F. (TWHF), are known to have a variety of immunomodulatory activities. This study was performed to investigate the effect of TPT on the differentiation of splenic DCs and its influence on T cell-mediated immunity regarding to DC subsets CD11c^lowI-a/e^lowCD45RB⁺(CD11c^low DCs) and CD11c^highI-a/e^highCD45RB^- (CD11c^high DCs) in male C57BL/6 mice spleens in vitro. The percentage of CD11c^low DCs was significantly increased after treatment with TPT compared to their counterparts (CD11c^high DCs). It was found that unlike the gradually decreasing interleukin (IL)-12 secretion of CD11c^high DCs induced by TPT, CD11c^low DCs showed a obvious dose-dependent response between the increasing of IL-10 production and TPT stimulation. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4⁺ T cells + CD11c^high DCs or CD11c^low DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically. These data demonstrated that TPT might induce the differentiation of splenic DCs to CD11c^low DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro.     

Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)-based vaccine development

We utilized ferritin protein cage nanoparticles (FPCN) as antigen delivery nanoplatforms for DC-based vaccine development and investigated DC-mediated antigen-specific immune responses. Antigenic peptides, OT-1 (SIINFEKL) or OT-2 (ISQAVHAAHAEINEAGR) which are derived from ovalbumin, were genetically introduced either onto the exterior surface or into the interior cavity of FPCN. FPCN carrying antigenic peptides (OT-1-FPCN and OT-2-FPCN) were effectively delivered to DCs and processed within endosomes. Delivered antigenic peptides, OT-1 or OT-2, to DCs successfully induced antigen-specific CD8⁺ or CD4⁺ T cell proliferations both in vitro and in vivo. Naïve mice immunized with OT-1-FPCN efficiently differentiated OT-1 specific CD8⁺ T cells into functional effector cytotoxic T cells resulting in selective killing of antigen-specific target cells. Effective differentiation of proliferated OT-2 specific CD4⁺ T cells into functional CD4⁺ Th1 and Th2 cells was confirmed with the productions of IFN-γ/IL-2 and IL-10/IL-13 cytokines, respectively.From the Clinical EditorIn this study, the authors utilized ferritin protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development and investigated DC-mediated antigen-specific immune responses using strong model antigens derived from ovalbumin, suggesting potential future clinical applicability of this or similar techniques.     

Effects of human umbilical cord-derived mesenchymal stem cells on anterior chamber-associated immune deviation

Introduction of antigen into anterior chamber (AC) induces a deviant immune response termed anterior chamber-associated immune deviation (ACAID) that protects the eye from inflammatory destruction consequent to a systemic immune response. Mesenchymal stem cells (MSCs) can modulate a variety of immune responses. However, the effects of systemic administration of MSCs on ACAID have not been explored. In this study, C57BL/6 mice were inoculated with ovalbumin in the AC to induce ACAID, control group received AC injection of solvent alone. Immediately after the AC injection, the mice were injected through the tail vein with human Umbilical Cord-derived MSCs (hUC-MSC) or phosphate buffer saline. All animals were subcutaneously immunized with ovalbumin one week later. Delayed-type hypersensitivity assay was performed another week following immunization. The splenic monocytes were then isolated, cultured and stimulated with ovalbumin. Levels of IL-10, TGF-β, and IFN-γ in culture media were measured by ELISA. The frequencies of CD4⁺CD25⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells (Tregs) were determined by flow cytometry. The results showed that the AC inoculation of ovalbumin induced significantly less ear swelling than controls, confirming the establishment of ACAID. MSCs potentiated IL-10 and TGF-β production, further suppressed IFN-γ secretion from splenic monocytes in ACAID mice, and enhanced expansion of CD4⁺CD25⁺Foxp3⁺ and CD8⁺Foxp3⁺ Tregs isolated from the spleen of ACAID mice. Therefore, our study, for the first time, provides clear evidence that systemic administration of MSCs augments cytokine production and Treg expansion from ACAID spleens, which may contribute to promotion and maintenance of ACAID.     

Estradiol enhances capacity of TLR-matured splenic dendritic cells to polarize CD4+ lymphocytes into IL-17/GM-CSF-producing cells in vitro

There are little data on modulatory effects of estrogens on rat dendritic cell (DC) responses to inflammatory stimuli, and consequently their ability to activate and polarize CD4+ T lymphocyte-mediated immune responses. Splenic conventional DCs from young female Albino Oxford rats were activated in vitro with LPS (TLR4 agonist) or R848 (TLR7/8 agonist) in the presence and absence of 17β-estradiol (E2), and their allostimulatory and CD4+ lymphocyte polarizing ability in mixed leukocyte culture (MLC) were studied. Irrespective of the E2 presence, LPS and R848 up-regulated the expression of MHC II on DCs, so they exhibited enhanced allostimulatory capacity in co-culture with CD4+ lymphocytes. On the other hand, E2 promoted stimulatory action of both TLRs on OX62+ DC IL-23 production, augmented their stimulatory effects on IL-6 and IL-1β production, but diminished their enhancing effects on the expression IL-10 and IL-27 by DCs. Consequently, in MLC, OX62+ DCs activated/matured in the co-presence of E2 and either LPS or R848 increased the levels of IL-17, the signature Th17 cell cytokine, when compared with those activated/matured in the absence of E2. GM-CSF levels were also increased in these MLC. Given that the expression of IL-7 mRNA was diminished in DCs activated/matured in the co-presence of E2 and TLR, this increase most likely did not reflect enhanced differentiation of Th cells producing GM-CSF only (Th-GM).ConclusionsE2 augments capacity of LPS- and R848-activated/matured DCs from young rat spleen to induce differentiation of IL-17- and GM-CSF-producing cells.     

CD4+ Foxp3+ regulatory T cells induced by TGF-β, IL-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation

Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4⁺CD25⁻ T cells to differentiate into CD4⁺CD25⁺Foxp3⁺ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation.     

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU).MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4⁺T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA.ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4⁺T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4⁺T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls.ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4⁺T cells and attenuated disease severity in EAU mice.