The pattern and level of cytokines secreted by Th1 and Th2 lymphocytes of syphilitic patients correlate to the progression of the disease

The results of our previous work indicated that cell-mediated immune response, of importance in protection against Treponema pallidum, is distinctly inhibited at certain periods of syphilitic infection. Considering that cytokines, produced by Th1 lymphocytes, take part in this response and that their secretion may be regulated by cytokines of Th2 lymphocytes, we examined if, and in which stages of syphilis, such a regulation may exist. In this study we have examined the ability of cells to produce IL-2, IFN and TNF (Th1 or Th1 like cytokines) as well as IL-6 and IL-10 (Th2 or Th2 like cytokines). It was found that cells of syphilitic patients were able to produce IL-2, IFN, TNF, IL-10 and weakly IL-6 already in primary seronegative syphilis. At the same stage of syphilis, but seropositive, ability of Th1 lymphocytes to produce cytokines reached the highest values, whereas the cells producing IL-10 lost this ability. The cells producing IL-6 and MIF had the highest ability in secondary early syphilis. In this stage of syphilis again slightly increased the ability of cells to secrete IL-10, which reached the highest value in early latent syphilis. The growing ability to produce IL-6 and IL-10 was accompanied with a diminished production of IL-2, IFN and TNF nearly in all stages of syphilis. Only MIF, in contrast to other cytokines, was produced in late syphilis without distinct changes. The greatest suppression of Th1 lymphocytes to produce cytokines and cells to secretion of MIF was found in early latent syphilis when the level of IL-10 in cell culture supernates was the highest. High ability of Th2 lymphocytes to cytokines secretion in late syphilis and low ability of Th1 ones, which are very important for cell-mediated immune response, may be the reason for facilitating T. pallidum multiplication and development of latent stages of disease despite presence of immunologically competent cells.     

Inducible adeno-associated virus-mediated IL-2 gene therapy prevents autoimmune diabetes

IL-2 and TGF-β1 play key roles in the immunobiology of Foxp3-expressing CD25(+)CD4(+) T cells (Foxp3(+)Treg). Administration of these cytokines offers an appealing approach to manipulate the Foxp3(+)Treg pool and treat T cell-mediated autoimmunity such as type 1 diabetes. However, efficacy of cytokine treatment is dependent on the mode of application, and the potent pleiotropic effects of cytokines like IL-2 may lead to severe side effects. In the current study, we used a gene therapy-based approach to assess the efficacy of recombinant adeno-associated virus vectors expressing inducible IL-2 or TGF-β1 transgenes to suppress ongoing β cell autoimmunity in NOD mice. Intramuscular vaccination of recombinant adeno-associated virus to 10-wk-old NOD female mice and a subsequent 3 wk induction of IL-2 was sufficient to prevent diabetes and block the progression of insulitis. Protection correlated with an increased frequency of Foxp3(+)Treg in the periphery as well as in the draining pancreatic lymph nodes and islets. IL-2 induced a shift in the ratio favoring Foxp3(+)Treg versus IFN-γ-expressing T cells infiltrating the islets. Induction of IL-2 had no systemic effect on the frequency or activational status of T cells and NK cells. Induction of TGF-β1 had no effect on the Foxp3(+)Treg pool or the progression of β cell autoimmunity despite induced systemic levels of activated TGF-β1 that were comparable to IL-2. These results demonstrate that inducible IL-2 gene therapy is an effective and safe approach to manipulate Foxp3(+)Treg and suppress T cell-mediated autoimmunity and that under the conditions employed, IL-2 is more potent than TGF-β1.     

The ability of peripheral blood mononuclear cells of rabbits infected with Treponema pallidum to produce IL-2

Abstract It was previously found that the cell-mediated immune response involved in protection against Treponema pallidum is distinctly suppressed during some periods in the course of syphilis infection in rabbits. This may be a result of the weak ability of cells to produce Interleukin-2 (IL-2) as well as of IL-2 absorption. The ability of peripheral blood mononuclear cells (PBMC) of syphilitic rabbits to produce IL-2 develops within the first two weeks after infection reaching a maximum in about the eleventh week. In infection of longer duration, this capability was distinctly lowered. This low level of activity (no higher than in PBMC of normal rabbits) was maintained for 31 weeks. The ability of PBMC to absorb IL-2, in parallel with its production, was found at the same time in the course of syphilis infection (7–11 weeks). In long-lasting syphilis (more than 12 weeks) both abilities seem to be inhibited. Sera of syphilitic rabbits were found to have a higher level of IL-2 inhibitor than those of normal rabbits. Only in syphilis lasting 9 to 11 weeks, when the production of IL-2 was the greatest, was the level of IL-2 inhibitor nearly the same as in normal rabbit sera. In syphilis lasting longer, the increased level of inhibitor was accompanied by a decreased ability of cells to produce IL-2. These findings suggest that IL-2 inhibitor may be bound to IL-2 or IL-2 receptor on T lymphocytes and in this way would lead to weakening of T cell function and resistance against Treponema pallidum infection.     

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment

TGF-β is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-β on pDC, and to our knowledge, no study has assessed the capacity of TGF-β-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-β-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-β/Smad signal, because TGF-β treatment induced Smad2 phosphorylation in pDC and blockade of TGF-β signaling with the SD208 TGF-βRI kinase inhibitor abrogated Th17 commitment induced by TGF-β-treated pDC. Moreover, TGF-β mRNA synthesis and active TGF-β release were induced in TGF-β-treated pDC and anti-TGF-β Ab blocked Th17 commitment. Unexpectedly, TGF-β treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-β-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-β-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-β-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-β-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.     

Role of TGF-β and IL-6 in dendritic cells,Treg and Th17 mediated immune response during experimental cerebral malaria

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor β (TGF-β), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-β are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-β treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-β and IL-6 neutralization. Interestingly anti-TGF-β reduces CD11c⁺CD8⁺ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-β, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-β and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The breakdown of immune tolerance (regulatory T [Treg] cells and suppressor cytokines) plays an important role in ITP pathophysiology, especially in refractory ITP. Bone marrow–derived mesenchymal stem cells (BM-MSCs) show immunomodulatory properties and have been extensively utilized for autoimmune diseases. However, it has not been fully elucidated how BM-MSCs affect ITP. In this study, we explore the therapeutic mechanism of BM-MSCs on ITP in mice. Dose-escalation passive ITP mice were inducted by injection of MWReg30. BALB/c mice were randomly divided into two groups: ITP with BM-MSC transplantation and ITP controls. The serum levels of cytokines (interleukin 10 [IL-10] and transforming growth factor-β1 [TGF-β1]) were examined by enzyme-linked immunosorbent assays. The frequency of Treg cells in both peripheral blood and spleen mononuclear cells was analyzed by flow cytometry, and the forkhead box P3 (Foxp3) messenger RNA (mRNA) level was measured by real-time polymerase chain reaction. After BM-MSC treatment, the platelet (PLT) counts were significantly elevated. Meanwhile, cytokines (TGF-β1 and IL-10), the ratios of Treg cells, and the Foxp3 mRNA expression level were significantly higher in the BM-MSC group. Our results show that BM-MSCs can improve PLT counts mainly by secreting suppressive cytokines and upregulating Tregs, which may provide new therapeutic potential for human ITP.     

Nitric oxide modulates TGF-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development

TGF-β can induce Foxp3(+) inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-β activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-β in both IL-12-independent and -dependent fashions by augmenting IFN-γ-activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-β-directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-β and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-β to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity.     

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.     

Pulmonary CCL18 recruits human regulatory T cells

CCL18 is both a constitutively expressed and an inducible chemokine, whose role in the inflammatory reaction is poorly known. The aim of this study was to evaluate whether CCL18 has the capacity to attract human T cells with a regulatory function (regulatory T cells [Treg]). Results from chemotaxis assays performed on different types of Treg showed that CD4(+)CD25(+)CD127(low) cells, but neither T regulatory type 1 clones nor Treg differentiated in vitro with anti-CD3/CD46 mAbs, were recruited by CCL18 in a dose-dependent manner. CCL18-recruited memory CD4(+) T cells were enriched in CD25(high), CD25(+)CD127(low), latency-associated peptide/TGF-β1, and CCR4-expressing T cells, whereas there was no enrichment in Foxp3(+) cells as compared with controls. Stimulated CCL18-recruited memory T cells produced significantly increased amounts of the regulatory cytokines IL-10 and TGF-β1, as well as IL-4, but not IFN-γ and IL-17. Cell surface CCL18 binding was found predominantly on IL-10(+) (26.3 ± 5.8%) and on a few latency-associated peptide/TGF-β1(+) (18.1 ± 1.9%) and IL-4(+) (14.5 ± 2.9%) memory T cells. In an in vivo model of SCID mice grafted with human skin and reconstituted with autologous PBMCs, the intradermal injection of CCL18 led to the cutaneous recruitment of CD4(+), CD25(+), and IL-10(+) cells, but not Foxp3(+) cells. Furthermore, CCL18-recruited memory T cells inhibited the proliferation of CD4(+)CD25(-) effector T cells through an IL-10-dependent mechanism. These data suggest that CCL18 may contribute to maintaining tolerance and/or suppressing deleterious inflammation by attracting memory Tregs into tissues, particularly in the lung, where it is highly and constitutively expressed.     

Regulation of T cell activation by TLR ligands

Regulatory T cells (Treg) maintain peripheral tolerance and play a critical role in the control of the immune response in infection, tumor defense, organ transplantation and allergy. CD4(+)CD25(high) Treg suppress the proliferation and cytokine production of CD4(+)CD25(-) responder T cells. The suppression requires cell-cell-contact and/or production of inhibitory cytokines like IL-10 or TGF-β. The current knowledge about the regulation of Treg suppressive function is limited. Toll-like receptors (TLR) are widely expressed in the innate immune system. They recognize conserved microbial ligands such as lipopolysaccharide, bacterial lipopeptides or viral and bacterial RNA and DNA. TLR play an essential role in innate immune responses and in the initiation of adaptive immune responses. However, certain TLR are also expressed in T lymphocytes, and the respective ligands can directly modulate T cell function. TLR2, TLR3, TLR5 and TLR9 act as costimulatory receptors to enhance proliferation and/or cytokine production of T-cell receptor-stimulated T lymphocytes. In addition, TLR2, TLR5 and TLR8 modulate the suppressive activity of naturally occurring CD4(+)CD25(high) Treg. The direct responsiveness of T lymphocytes to TLR ligands offers new perspectives for the immunotherapeutic manipulation of T cell responses. In this article we will discuss the regulation of Treg and other T cell subsets by TLR ligands.     

Subfamily I Treponema pallidum repeat protein family: sequence variation and immunity

A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates.While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.     

变应性鼻炎患者外周血中IL-27和Treg细胞的相关性分析

目的探讨外周血IL-27和CD4^＋CD25^＋调节性T细胞（Treg）在变应性鼻炎（AR）发病机制中的作用。方法2012年3月至7月,收集AR患者32例（AR组）和20例健康志愿者（对照组）外周血,采用流式细胞术（FCM）检测外周血中Treg细胞比例;ELISA检测血清中IL-27、IL-10和TGF-β1的水平。结果AR组Treg细胞百分率[（1．75±0．56）％]明显低于对照组[（4．76±1．75）％],两组比较的差异有统计学意义（P〈0．01）。AR组IL-27、IL-10和TGF-β1的水平分别为（24．43±16．36）pg／ml、（14．29±6．16）pg／ml、（0．34±0．04）pg／ml,均低于对照组（44．09±13．12）pg／ml、（31．32±21．20）pg／ml、（O．49±0．06）pg／ml,两组之间的差异有统计学意义（P〈0．01）。AR患者外周血中IL-27和Treg细胞百分率、IL-10、TGF-β1存在正相关（r分别为0．825,0．646,0．517,P〈0．01）,Treg细胞百分率和IL-10、TGF-β1存在正相关（r=0.622,0．738,P〈0．01）,IL-10和TGF-β1无相关性（r=0．304,P〉0．05）结论AR患者外周血中IL-27水平降低,Treg细匏百分率降低及其主要分泌因子IL-10、TGF-β1水平降低,且IL-27与Treg细胞百分率、IL-10、TGF-β1水平呈正相关,提示在AR发病中IL-27对Treg细胞可能具有免疫调节作用。     

The ability of peripheral blood mononuclear cells (PBMC) of syphilitic patients to produce IL-2

Abstract The cell-mediated immune response of importance in protection against Treponema pallidum , is distinctly suppressed in some stages of the disease. This may be a result of decreased ability of cells to produce IL-2, or IL-2 absorption by different factors. The experiments were designed to evaluate the ability of peripheral blood mononuclear cells (PBMC) of patients with different stages of syphilis to produce IL-2, and to investigate the causes which could possibly limit its activity. The ability of the PBMC of syphilitic patients to produce IL-2 develops at the beginning of the disease, reaching a maximum in primary seropositive syphilis. In the next stages of the disease this capability is distinctly lowered. The lowest was in malignant syphilis and tabes dorsalis, i.e. during severe disease. Absorption of adherent cells from PBMC increased the ability of lymphocytes to produce IL-2. The highest level of this interleukin was observed at the stages of the disease where suppression was the deepest. Sera of both control and syphilitic patients contained IL-2 inhibitor. Its level was the highest in early and late latent syphilis where no symptoms of disease were present. In all syphilitic sera a distinctly elevated level of soluble IL-2 receptors (sIL-2R) was also found. Its high level was noted in sera of patients in which PBMC had the weakest ability to produce IL-2. These findings suggest that sIL-2R may be bound to IL-2 and in this way would lead to weakening of T cell function and of resistance against Treponema pallidum infection.     

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis.

The surface of Treponema pallidum subsp. pallidum (T. pallidum), the etiologic agent of syphilis, appears antigenically inert and lacks detectable protein, as judged by immunocytochemical and biochemical techniques commonly used to identify the outer membrane (OM) constituents of gram-negative bacteria. We examined T. pallidum by freeze-fracture electron microscopy to visualize the architecture of its OM. Treponema phagedenis biotype Reiter (T. phagedenis Reiter), a nonpathogenic host-associated treponeme, and Spirochaeta aurantia, a free-living spirochete, were studied similarly. Few intramembranous particles interrupted the smooth convex and concave fracture faces of the OM of T. pallidum, demonstrating that the OM of this organism is an unusual, nearly naked lipid bilayer. In contrast, the concave fracture face of the OM of S. aurantia was densely covered with particles, indicating the presence of abundant integral membrane proteins, a feature shared by typical gram-negative organisms. The concentration of particles in the OM concave fracture face of T. phagedenis Reiter was intermediate between those of T. pallidum and S. aurantia. Similar to typical gram-negative bacteria, the OM convex fracture faces of the three spirochetes contained relatively few particles. The unique molecular architecture of the OM of T. pallidum can explain the puzzling in vitro properties of the surface of the organism and may reflect a specific adaptation by which treponemes evade the host immune response.