Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b+ DCs

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40−/−, Batf3−/−, and IL-17A−/− mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3−/− mice, lacking CD8α⁺ DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b⁺ DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.     

Autocrine transforming growth factor-β1 promotes in vivo Th17 cell differentiation

TGF-β1 is a regulatory cytokine that has an important role in controlling T?cell differentiation. T?cell-produced TGF-β1 acts on T?cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGF-β1-producing T?cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T?cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation, resulting in almost complete protection from EAE. Furthermore, differentiation of T?cells both in?vitro and in?vivo demonstrated that TGF-β1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in?vivo. These findings reveal an essential role for activated T?cell-produced TGF-β1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases.     

Effects of 17β-estradiol and its isomer 17α-estradiol on learning in rats with chronic cholinergic deficiency in the brain

It was shown for the first time that estrogens 17β- and 17α-estradiols compensate impaired cognitive functions in rats with partial chronic deprivation of cholinergic functions in the central nervous system induced by intracerebral administration of selective cholinergic neurotoxin AF64A. 17β-Estradiol produced strong dose-dependent changes in the weights of hormone-sensitive endocrine glands, while 17α-estradiol did not affect the weight of the gonads and slightly influenced (in high concentration) the weights of the adrenal glands and thymus. The positive effects of exogenous 17β- and 17α-estradiols on cognitive functions are due to their antioxidant properties, rather than due to specific action on hormone-sensitive endocrine glands. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 5, pp. 525–527, May, 2000     

SIRPα on CD11c+ cells induces Th17 cell differentiation and subsequent inflammation in the CNS in experimental autoimmune encephalomyelitis

Signal regulatory protein α (SIRPα) is expressed predominantly on type 2 conventional dendritic cells (cDC2s) and macrophages. We previously showed that mice systemically lacking SIRPα were resistant to experimental autoimmune encephalomyelitis (EAE). Here, we showed that deletion of SIRPα in CD11c⁺ cells of mice (Sirpa^ΔDC mice) also markedly ameliorated the development of EAE. The frequency of cDCs and migratory DCs (mDCs), as well as that of Th17 cells, were significantly reduced in draining lymph nodes of Sirpa^ΔDC mice at the onset of EAE. In addition, we found the marked reduction in the number of Th17 cells and DCs in the CNS of Sirpa^ΔDC mice at the peak of EAE. Whereas inducible systemic ablation of SIRPα before the induction of EAE prevented disease development, that after EAE onset did not ameliorate the clinical signs of disease. We also found that EAE development was partially attenuated in mice with CD11c⁺ cell-specific ablation of CD47, a ligand of SIRPα. Collectively, our results suggest that SIRPα expressed on CD11c⁺ cells, such as cDC2s and mDCs, is indispensable for the development of EAE, being required for the priming of self-reactive Th17 cells in the periphery as well as for the inflammation in the CNS.     

Plasticity of the murine spleen T-cell cholinergic receptors and their role in in vitro differentiation of naïve CD4 T cells toward the Th1, Th2 and Th17 lineages

Acetylcholine (ACh) regulates vital functions of T cells by acting on the nicotinic and muscarinic classes of cholinergic receptors, nAChR and mAChRs, respectively. This study was performed in murine splenic T cells. In freshly isolated CD4 and CD8 T cells, we detected mRNAs encoding α5, α9, α10, β1, β2, β4 nAChR subunits and M?, M?, M? and M? mAChR subtypes, whereas α2 was detected only in CD8 T cells. In vitro activation of CD4 T cells through T-cell receptor (TCR)/CD3 cross-linking was associated with the appearance of α4 and α7, upregulation of α5, α10, β4, M? and M? and downregulation of α9 and β2, whereas in vitro activation of CD8 T cells also featured the appearance of α4 and α7, as well as upregulation of α2, α5, β4, M? and M?, and downregulation of α10, β1, β2 and M?. In vitro polarization toward T helper (Th) 1 lineage was associated with a decrease of β2, β4 and M? expression; that toward Th2 cells with downregulation of α9 and M?, and upregulation of M? and M?; and that toward Th17 phenotype with downregulation of α9, α10, β2 and M? mAChR. Polarized T cells also expressed α4, but not α1, α2, α3, α6, β3 or M?. To determine the role of cholinergic receptors in mediating the immunoregulatory action of autocrine/paracrine ACh, we analyzed the effects of nicotinic and muscarinic agonists±antagonists on cytokine production in the CD4+CD62L+ T cells co-stimulated via TCR/CD3 cross-linking. The nicotinergic stimulation upregulated interferon-γ (IFN-γ) and downregulated interleukin (IL)-17 secretion, whereas the muscarinic stimulation enhanced IL-10 and IL-17 and inhibited INF-γ secretion. These results demonstrated plasticity of the T-cell cholinergic system.     

Immunohistological detection of the β subunit of prolyl 4-hydroxylase in rat and mini pig lungs with radiation-induced pulmonary fibrosis

Polyclonal and monoclonal antibodies to the subunit of prolyl 4-hydroxylase, the protein disulphide isomerase, were used to compare the pulmonary cells in 13 normal and in 20 fibrotic rat and mini-pig lungs made fibrotic by X-ray irradiation, using the ABC immunoperoxidase technique. In normal lungs, prominent staining of Clara cells and type II pneumocytes and weaker reactivity with alveolar macrophages, fibroblasts, endothelial and smooth muscle cells were detectable. In pulmonary disease, in which interstitial fibrosis was the characteristic feature, the immunoreactivity was increased in both the epithelial and interstitial cells. Type I pneumocytes remained negative. In the early stages of disease (3 to 4 weeks after irradiation) when little morphological alteration was seen, capillary endothelial cells had already become immunoreactive. These results underline the complex involvement and interaction of different lung cell populations in the process of pulmonary fibrogenesis.     

Involvement of Transforming Growth Factor β and its Type 1 Receptor in the Development of Breast Cancer

Introduction of recent achievements of molecular biology into clinical practice contributes significantly to both prevention and early detection of breast cancer in women and development of new approaches, including genotyping, to screening for breast cancer. Screening for genetic polymorphisms in genes TGFβ1 and TGFβR1 reveals carriers of sporadic breast cancer. This provides an opportunity to reassess the role and place of preventive measures, also including surgical method, in the fight against breast cancer.     

Pyruvate dehydrogenase E1α subunit genes in the mouse: Mapping and comparison with human homologs

Two gene loci for the E1 subunit of the pyruvate dehydrogenase (PDH) complex have been mapped in the mouse by in situ hybridization. One locus maps to the X chromosome in the region F3–F4, the other to chromosome 19, in band B close to the centromere. This arrangement is exactly comparable to the situation in man where there is an X-linked PDH E1 locus and an autosomal locus on chromosome 4. Comparison of the regional localization of the human and mouse X-linked PDH E1 genes provides further information concerning sites of rearrangement of segments of the X chromosome during mammalian evolution. The human autosomal PDH E1 gene is a processed gene, which lacks the introns that are present in the X-linked gene. It codes for a testis-specific E1 subunit that is only expressed after the onset of spermatogenesis. The comparative mapping results in the mouse suggest that the genetic organization and pattern of expression of the two PDH E1 genes is the same in the two species.     

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of α2-adrenergic and A1-adenosine agonists,insensitive to pertussis toxin and dependent on extracellular calcium

The accumulation of cyclic adenosine 3,5-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A₁ agonist of adenosine (-)-N ⁶-(R-phenylisopropyl) adenosine (PIA), an ₂-adrenergic agonist clonidine (CLO), and prostaglandin E₂ (PGE₂). The negative regulation elicited by PGE₂ was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 M PGE₂ with either 0.1 M PIA or 1 M CLO led to an inhibition of the response to AVP (80.0±3.5%, SEM, N=7 and 92.6±0.8%, N=5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE₂ in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 g/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE₂. PGE₂-induced inhibition was prevented in a calcium-free medium [0 Ca²⁺+0.1 mM [ethylene-bis (oxyethylenenitrilo)] tetraacetate (EGTA)]: values were 67.0 ±2.1% and 5.8±8.7% (± SEM) in 2 mM Ca²⁺ and 0 Ca²⁺ medium, respectively, N=7. When applied to Fura-2-loaded OMCD, 0.3 M PGE₂ increased intracellular calcium concentration ([Ca²⁺]_i) with a peak phase (in 2 mM or 0 Ca²⁺ medium) followed by a plateau phase (observed only in 2 mM Ca²⁺ medium). It is concluded that: (1) in the rat OMCD, PGE₂, PIA and CLO act on the same AVP-sensitive cell, (2) PGE₂ induces a cumulative inhibition on the cAMP level when combined with other inhibitors by a mechanism insensitive to pertussis toxin, (3) the presence of extracellular calcium is a prerequisite condition to observe PGE₂-induced inhibition, and (4) the inhibition by PGE₂ might be linked to its capacity of increasing [Ca²⁺]_i.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Anti-TNF therapy in patients with rheumatoid arthritis decreases Th1 and Th17 cell populations and expands IFN-γ-producing NK cell and regulatory T cell subsets

The aim of this work was to study the effect of anti-TNF treatment on CD4+ Th1, Th17 and regulatory T cells (Tregs), together with CD8+ T cells and NK cells from rheumatoid arthritis (RA) patients. For this purpose, 18 RA patients received adalimumab during 16 weeks and their peripheral blood lymphocytes were assessed by flow cytometry at the beginning and at the end of the study. We found that the proportion of Th17 cells was directly correlated with Th1 cells, but inversely correlated with IFN-γ-producing NK cells. A decrease was observed in Th1, Th17 cells and IFN-γ-producing CD8+ T cells by anti-TNF therapy. Conversely, the proportion of Tregs increased, as did the percentage of IFN-γ-producing NK cells. We postulate that a rise in IFN-γ production due to recovery of NK cells’ function, together with expanded Tregs, contribute to decrease the Th17 response in anti-TNF-treated RA patients.     

The critical role of IL-12 and the IL-12Rβ2 subunit in the generation of pathogenic autoreactive Th1 cells

Experimental Allergic Encephalomyelitis (EAE) is a demyelinating disease of the central nervous system which is an animal model for the human autoimmune disease, multiple sclerosis. EAE is mediated by CD4⁺ T cells and the T cells responsible for disease induction produce Th1 cytokines. IL-12 produced by monocytes and dendritic cells is the most critical factor which influences the development and differentiation of pathogenic autoreactive Th1 cells. Here, we review our recent studies on the critical contributions of IL-12 and the IL-12Rβ2 subunit to the generation of autoreactive effector cells which mediate EAE. In addition, we discuss the potential contribution of IL-18 to the upregulation of the IL-12/IL-12Rβ2 pathway and the contribution of the suppressor cytokines, IL-4 and IL-10, in downregulating this pathway. Collectively, our studies demonstrate that the IL-12/IL-12Rβ2 pathway is a critical intermediary in the process of Th1 differentiation which can be both positively or negatively regulated. This pathway remains an attractive immunotherapeutic target for blockade of function with inhibitory reagents or downregulation by Th2 cytokines.     

Effects of interleukin-18 on airway inflammation and the Th1/Th2 cytokine imbalance in guinea pig asthmatic model

Withrecent development of molecular biology andimmunology, more and more evidences suggestthat the Th1/Th2imbalanceis one of theimportantaspect of pathogenesisin bronchial asthma [1-3] .And modulation of the Th1/Th2 balance to controlthe airway inflammations has been proved to be ahot-spot inthe study of immune therapyfor bron-chial asthma [4-6] .Interleukin-18 (IL-18) , anewly discovered cytokine withregulatory effect onimmuno-competent cells ,has beeninvestigated byvarious authors for the …     

Growth and differentiation factor-5 (GDF-5) in the human intervertebral annulus cells and its modulation by IL-1ß and TNF-α in vitro

Growth and differentiation factor-5 (GDF-5) is a member of the TGF-ß superfamily which regulates cell division and differentiation. GDF-5 attracted high interest because of its role in skeletal development, especially in cartilaginous sites. Little is known, however, about the role of GFD-5 in disc cell biology. The present work demonstrated the immunohistologic presence of GDF-5 in human outer and inner annulus tissue. Microarray analysis of annulus cells showed significant upregulation of GDF-5 expression in herniated vs. non-herniated lumbar discs (2.14-fold change, p = 0.021). In vitro three-dimensional culture studies challenged human annulus cells with IL-1ß and TNF-α, two proinflammatory cytokines known to be elevated in the human degenerating disc. Exposure resulted in significant downregulation of GDF-5 during both TNF-α exposure (5.83-fold change, p = 0.044) and IL-1ß exposure (3.38-fold change, p = 0.015). In vitro findings suggest that the degenerating disc milieu, with high proinflammatory cytokine levels, may limit expression of GDF-5, resulting in limited regenerative capacity of the intact disc.     

The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers

Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.     

Bacterial load and inflammation in fetal tissues is not dependent on IL-17a or IL-22 in 10–14 day pregnant mice infected with Listeria monocytogenes

In this study, we first assessed the effect of intragastric infection of pregnant mice with Listeria monocytogenes on relative expression of select genes associated with T cell subsets. Relative gene expression was moderately increased in placental tissues for IFNγ, IL-4, IL-17a, IL-22, CD3, and FoxP3. To assess the roles of IL-17a and IL-22 in resistance to listeriosis during pregnancy, we compared the severity of maternal and fetal infection in IL-17a^(−/−), IL-22^(−/−), and IL-17a^(−/−)/IL-22^(−/−) mice with that of wild type C57BL/6 mice. Intragastric infection with modest numbers of bacterial cells (10⁵ CFU) caused reproducible maternal and fetal infection in all four mouse strains. We recovered greater numbers of CFU from the bloodstream of pregnant IL-22^(−/−) mice than pregnant wild type mice. Otherwise we found no significant difference in bacterial load in maternal or fetal tissues (spleen, liver, fetoplacental units) from pregnant IL-17a^(−/−), IL-22^(−/−), or IL-17a^(−/−)/IL-22^(−/−) or wild type mice. Nor did we observe histopathologic differences in severity of inflammation in maternal or fetal tissues from the various groups of mice. Although IL-17a and IL-22 are up-regulated in placental tissue, our study suggests that antibacterial resistance and the host inflammatory response are not dependent on IL-17a or IL-22 during infection of mice with L. monocytogenes at 10–14 days of gestation.