The TGF-ß superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation.

Th17 cells are known to exert pathogenic and non-pathogenic functions. Although the cytokine transforming growth factor ß1 (TGF-ß1) is instrumental for Th17 cell differentiation, it is dispensable for generation of pathogenic Th17 cells. Here, we examined the T cell-intrinsic role of Activin-A, a TGF-ß superfamily member closely related to TGF-ß1, in pathogenic Th17 cell differentiation. Activin-A expression was increased in individuals with relapsing-remitting multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. Stimulation with interleukin-6 and Activin-A induced a molecular program that mirrored that of pathogenic Th17 cells and was inhibited by blocking Activin-A signaling. Genetic disruption of Activin-A and its receptor ALK4 in T cells impaired pathogenic Th17 cell differentiation in vitro and in vivo. Mechanistically, extracellular-signal-regulated kinase (ERK) phosphorylation, which was essential for pathogenic Th17 cell differentiation, was suppressed by TGF-ß1-ALK5 but not Activin-A-ALK4 signaling. Thus, Activin-A drives pathogenic Th17 cell differentiation, implicating the Activin-A-ALK4-ERK axis as a therapeutic target for Th17 cell-related diseases.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.

Transcriptomic Differences Underlying the Activin-A Induced Large Osteoclast Formation in Both Healthy Control and Fibrodysplasia Ossificans Progressiva Osteoclasts.

Fibrodysplasia Ossificans Progressiva (FOP) is a very rare genetic disease characterized by progressive heterotopic ossification (HO) of soft tissues, leading to immobility and premature death. FOP is caused by a mutation in the Activin receptor Type 1 (ACVR1) gene, resulting in altered responsiveness to Activin-A. We recently revealed that Activin-A induces fewer, but larger and more active, osteoclasts regardless of the presence of the mutated ACVR1 receptor. The underlying mechanism of Activin-A-induced changes in osteoclastogenesis at the gene expression level remains unknown. Transcriptomic changes induced by Activin-A during osteoclast formation from healthy controls and patient-derived CD14-positive monocytes were studied using RNA sequencing. CD14-positive monocytes from six FOP patients and six age- and sex-matched healthy controls were differentiated into osteoclasts in the absence or presence of Activin-A. RNA samples were isolated after 14 days of culturing and analyzed by RNA sequencing. Non-supervised principal component analysis (PCA) showed that samples from the same culture conditions (e.g., without or with Activin-A) tended to cluster, indicating that the variability induced by Activin-A treatment was larger than the variability between the control and FOP samples. RNA sequencing analysis revealed 1480 differentially expressed genes induced by Activin-A in healthy control and FOP osteoclasts with p(adj) < 0.01 and a Log2 fold change of ≥±2. Pathway and gene ontology enrichment analysis revealed several significantly enriched pathways for genes upregulated by Activin-A that could be linked to the differentiation or function of osteoclasts, cell fusion or inflammation. Our data showed that Activin-A has a substantial effect on gene expression during osteoclast formation and that this effect occurred regardless of the presence of the mutated ACVR1 receptor causing FOP.

Heterogeneity of the bone marrow niche in patients with myeloproliferative neoplasms: ActivinA secretion by mesenchymal stromal cells correlates with the degree of marrow fibrosis.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

Two-day-treatment of Activin-A leads to transient change in SV-HFO osteoblast gene expression and reduction in matrix mineralization.

Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin-A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression profile. A single 2-day treatment of Activin-A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A-treated osteoblasts responded with transient change in gene expression, in a 2-wave-fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1-2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin-A. In summary, 2-day treatment with Activin-A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes.

Activin-A co-opts IRF4 and AhR signaling to induce human regulatory T cells that restrain asthmatic responses.

Type 1 regulatory T (Tr1) cells play a pivotal role in restraining human T-cell responses toward environmental allergens and protecting against allergic diseases. Still, the precise molecular cues that underlie their transcriptional and functional specification remain elusive. Here, we show that the cytokine activin-A instructs the generation of CD4+ T cells that express the Tr1-cell-associated molecules IL-10, inducible T-Cell costimulator (ICOS), lymphocyte activation gene 3 protein (LAG-3), and CD49b, and exert strongly suppressive functions toward allergic responses induced by naive and in vivo-primed human T helper 2 cells. Moreover, mechanistic studies reveal that activin-A signaling induces the activation of the transcription factor interferon regulatory factor (IRF4), which, along with the environmental sensor aryl hydrocarbon receptor, forms a multipartite transcriptional complex that binds in IL-10 and ICOS promoter elements and controls gene expression in human CD4+ T cells. In fact, IRF4 silencing abrogates activin-A-driven IL10 and ICOS up-regulation and impairs the suppressive functions of human activin-A-induced Tr1-like (act-A-iTr1) cells. Importantly, using a humanized mouse model of allergic asthma, we demonstrate that adoptive transfer of human act-A-iTr1 cells, both in preventive and therapeutic protocols, confers significant protection against cardinal asthma manifestations, including pulmonary inflammation. Overall, our findings uncover an activin-A-induced IRF4-aryl hydrocarbon receptor (AhR)-dependent transcriptional network, which generates suppressive human Tr1 cells that may be harnessed for the control of allergic diseases.

Dendritic Cells: Critical Regulators of Allergic Asthma.

Allergic asthma is a chronic inflammatory disease of the airways characterized by airway hyperresponsiveness (AHR), chronic airway inflammation, and excessive T helper (Th) type 2 immune responses against harmless airborne allergens. Dendritic cells (DCs) represent the most potent antigen-presenting cells of the immune system that act as a bridge between innate and adaptive immunity. Pertinent to allergic asthma, distinct DC subsets are known to play a central role in initiating and maintaining allergen driven Th2 immune responses in the airways. Nevertheless, seminal studies have demonstrated that DCs can also restrain excessive asthmatic responses and thus contribute to the resolution of allergic airway inflammation and the maintenance of pulmonary tolerance. Notably, the transfer of tolerogenic DCs in vivo suppresses Th2 allergic responses and protects or even reverses established allergic airway inflammation. Thus, the identification of novel DC subsets that possess immunoregulatory properties and can efficiently control aberrant asthmatic responses is critical for the re-establishment of tolerance and the amelioration of the asthmatic disease phenotype.

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.

Activin-A in the regulation of immunity in health and disease.

The TGF-ß superfamily of cytokines plays pivotal roles in the regulation of immune responses protecting against or contributing to diseases, such as, allergy, autoimmunity and cancer. Activin-A, a member of the TGF-ß superfamily, was initially identified as an inducer of follicle-stimulating hormone secretion. Extensive research over the past decades illuminated fundamental roles for activin-A in essential biologic processes, including embryonic development, stem cell maintenance and differentiation, haematopoiesis, cell proliferation and tissue fibrosis. Activin-A signals through two type I and two type II receptors which, upon ligand binding, activate their kinase activity, phosphorylate the SMAD2 and 3 intracellular signaling mediators that form a complex with SMAD4, translocate to the nucleus and activate or silence gene expression. Most immune cell types, including macrophages, dendritic cells (DCs), T and B lymphocytes and natural killer cells have the capacity to produce and respond to activin-A, although not in a similar manner. In innate immune cells, including macrophages, DCs and neutrophils, activin-A exerts a broad range of pro- or anti-inflammatory functions depending on the cell maturation and activation status and the spatiotemporal context. Activin-A also controls the differentiation and effector functions of Th cell subsets, including Th9 cells, TFH cells, Tr1 Treg cells and Foxp3+ Treg cells. Moreover, activin-A affects B cell responses, enhancing mucosal IgA secretion and inhibiting pathogenic autoantibody production. Interestingly, an array of preclinical and clinical studies has highlighted crucial functions of activin-A in the initiation, propagation and resolution of human diseases, including autoimmune diseases, such as, systemic lupus erythematosus, rheumatoid arthritis and pulmonary alveolar proteinosis, in allergic disorders, including allergic asthma and atopic dermatitis, in cancer and in microbial infections. Here, we provide an overview of the biology of activin-A and its signaling pathways, summarize recent studies pertinent to the role of activin-A in the modulation of inflammation and immunity, and discuss the potential of targeting activin-A as a novel therapeutic approach for the control of inflammatory diseases.

Heterogeneity of Stem Cells in the Thyroid.

Identification of thyroid stem cells in the past few years has made important contributions to our understanding of the cellular and molecular mechanisms that induce tissue regeneration and repair. Embryonic stem (ES) cells and induced-pluripotent stem cells have been used to establish reliable protocols to obtain mature thyrocytes and functional follicles for the treatment of thyroid diseases in mice. In addition, the discovery of resident thyroid progenitor cells, along with other sources of stem cells, has defined in detail the mechanisms responsible for tissue repair upon moderate or severe organ injury.In this chapter, we highlight in detail the current state of research on thyroid stem cells by focusing on (1) the description of the first experiments performed to obtain thyroid follicles from embryonic stem cells, (2) the identification of resident stem cells in the thyroid gland, and (3) the definition of the current translational in vivo and in vitro models used for thyroid tissue repair and regeneration.

Dendritic cells conditioned by activin A-induced regulatory T cells exhibit enhanced tolerogenic properties and protect against experimental asthma.

BACKGROUND: Previously, we demonstrated that regulatory T (Treg) cells induced by the cytokine activin-A suppress TH2-mediated allergic responses and linked airway disease. Still, the effects of activin-A-induced regulatory T (Act-A-iTreg) cells on the regulation of dendritic cell (DC)-driven allergic inflammation remain elusive. OBJECTIVE: Here we investigated whether Act-A-iTreg cells can modulate DC responses and endow them with enhanced tolerogenic functions. METHODS: Using adoptive cell transfer studies in mouse models of allergic airway disease, we examined the effects of Act-A-iTreg cells on DC phenotype, maturation status, and TH2 cell priming potential. Genome-wide gene expression profiling characterized the transcriptional networks induced in tolerogenic DCs by Act-A-iTreg cells. The ability of DCs conditioned by Act-A-iTreg cells (Act-A-iTreg cell-modified DCs) to protect against experimental asthma, and the mechanisms involved were also explored. RESULTS: Act-A-iTreg cell-modified DCs exhibited a significantly impaired capacity to uptake allergen and stimulate naive and TH2 effector responses on allergen stimulation in vivo accompanied by markedly attenuated inflammatory cytokine release in response to LPS. Gene-profiling studies revealed that Act-A-iTreg cells dampened crucial TH2-skewing transcriptional networks in DCs. Administration of Act-A-iTreg cell-modified DCs ameliorated cardinal asthma manifestations in preventive and therapeutic protocols through generation of strongly suppressive forkhead box P3+ Treg cells. Finally, programed death protein 1/programmed death ligand 1 signaling pathways were essential in potentiating the generation of DCs with tolerogenic properties by Act-A-iTreg cells. CONCLUSION: Our studies reveal that Act-A-iTreg cells instruct the generation of a highly effective immunoregulatory circuit encompassing tolerogenic DCs and forkhead box P3+ Treg cells that could be targeted for the design of novel immunotherapies for allergic disorders.

Comparative proteomic profiling of human osteoblast-derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization.

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell-secreted ECMs have become of interest as they reproduce tissue-architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast-derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast-differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non-ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast-differentiating MSCs with Activin-A. Extracellular proteins were upregulated in Activin-A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC-secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.

Mechanisms involved in enhancement of osteoclast formation by activin-A.

Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-ß (TGF-ß) family is known to be present in bone tissues and an important regulator in osteoclastogenesis with SMAD-mediated signaling being crucial for inducing osteoclast differentiation. In the present study, we examined the effect and underlying mechanisms of activin-A on osteoclast formation in vitro culture systems. Activin-A enhanced osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW 264.7 cells induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and/or macrophage stimulating factor (M-CSF). We also found that activin-A stimulated bone resorption and actin ring formation induced by RANKL and/or M-CSF. Furthermore, activin-A enhanced RANKL-induced expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis, thereby increasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, and cathepsin K. Blockage of receptor binding by follistatin, an activing-binding protein suppressed the activin-A-mediated stimulation of NFATc1. In addition, activin-A increased RANKL-induced c-fos expression without significantly affecting the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment of the cells with a specific inhibitor of SMAD2/3 attenuated the activin-A-induced expression of NFATc1 and co-immunoprecipitation assay revealed that treatment with activin-A increased physical interaction of phosphorylated-c-fos and phosphorylated-SMAD2 protein induced by RANKL. These results suggest that activin-A enhances RANKL-induced osteoclast formation mediated by interaction of c-fos and smad2/3.

Activin-A causes Hepatic stellate cell activation via the induction of TNFα and TGFß in Kupffer cells.

BACKGROUND & AIMS: TGFß superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased. METHODS: We studied the effect of activin-A on mouse primary Kupffer cells (KCs) and Hepatic Stellate cells (HSCs) and the levels of activin-A and its inhibitor follistatin in the serum of patients from a large panel of liver diseases. RESULTS: Activin-A is expressed by mouse hepatocytes, HSCs and Liver Sinusoid Endothelial cells but not KCs. Each cell type expresses different activin receptor combinations. HSCs are unresponsive to activin-A due to downregulation/desensitization of type-II activin receptors, while KCs respond by increasing the expression/production of TNFα και TGFß1. In the presence of KCs or conditioned medium from activin-A treated KCs, HSCs switch to a profibrogenic phenotype, including increased collagen and αSMA expression and migratory capacity. Incubation of activin-A treated KC conditioned medium with antibodies against TNFα and TGFß1 partially blocks its capacity to activate HSCs. Only patients with alcoholic liver diseases and NASH cirrhosis have significantly higher activin-A levels and activin-A/follistatin ratio. CONCLUSIONS: Activin-A may induce fibrosis in NASH and alcoholic cirrhosis via activation of KCs to express pro-inflammatory molecules that promote HSC-dependent fibrogenesis and could be a target for future anti-fibrotic therapies.

Mechanism of chimeric vaccine stimulation of indoleamine 2,3-dioxygenase biosynthesis in human dendritic cells is independent of TGF-ß signaling.

Cholera toxin B subunit fusion to autoantigens such as proinsulin (CTB-INS) down regulate dendritic cell (DC) activation and stimulate synthesis of DC immunosuppressive cytokines. Recent studies of CTB-INS induction of immune tolerance in human DCs indicate that increased biosynthesis of indoleamine 2,3-dioxygenase (IDO1) may play an important role in CTB-INS vaccine suppression of DC activation. Studies in murine models suggest a role for transforming growth factor beta (TGF-ß) in the stimulation of IDO1 biosynthesis, for the induction of tolerance in DCs. Here, we investigated the contribution of TGF-ß superfamily proteins to CTB-INS induction of IDO1 biosynthesis in human monocyte-derived DCs (moDCs). We show that CTB-INS upregulates the level of TGF-ß1, activin-A and the TGF-ß activator, integrin αvß8 in human DCs. However, inhibition of endogenous TGF-ß, activin-A or addition of biologically active TGF-ß1, and activin-A, did not inhibit or stimulate IDO1 biosynthesis in human DCs treated with CTB-INS. While inhibition with the kinase inhibitor, RepSox, blocked SMAD2/3 phosphorylation and diminished IDO1 biosynthesis in a concentration dependent manner. Specific blocking of the TGF-ß type 1 kinase receptor with SB-431542 did not arrest IDO1 biosynthesis, suggesting the involvement of a different kinase pathway other than TGF-ß type 1 receptor kinase in CTB-INS induction of IDO1 in human moDCs. Together, our experimental findings identify additional immunoregulatory proteins induced by the CTB-INS fusion protein, suggesting CTB-INS may utilize multiple mechanisms in the induction of tolerance in human moDCs.

Activins and their related proteins in colon carcinogenesis: insights from early and advanced azoxymethane rat models of colon cancer.

BACKGROUND: Activin-A may exert pro- or anti-tumorigenic activities depending on cellular context. However, little is known about its role, or the other mature activin proteins, in colorectal carcinoma (CRC). This study measured the expression of activin ßA- & ßB-subunits, activin type IIA & IIB receptors, smads 2/3/4/6/7 and follistatin in CRC induced by azoxymethane (AOM) in rats. The results were compared with controls and disseminated according to the characteristics of histopathological lesions. METHODS: Eighty male Wistar rats were allocated into 20 controls and the remaining were equally divided between short 'S-AOM' (15 weeks) and long 'L-AOM' (35 weeks) groups following injecting AOM for 2 weeks. Subsequent to gross and histopathological examinations and digital image analysis, the expression of all molecules was measured by immunohistochemistry and quantitative RT-PCR. Activin-A, activin-B, activin-AB and follistatin were measured by ELISA in serum and colon tissue homogenates. RESULTS: Colonic pre-neoplastic and cancerous lesions were identified in both AOM groups and their numbers and sizes were significantly (P < 0.05) greater in the L-AOM group. All the molecules were expressed in normal colonic epithelial cells. There was a significantly (P < 0.05) greater expression of ßA-subunit, IIB receptor and follistatin in both pre-neoplastic and cancerous tissues. Oppositely, a significant (P < 0.05) decrease in the remaining molecules was detected in both AOM groups. Metastatic lesions were only observed within the L-AOM group and were associated with the most significant alterations of all molecules. Significantly higher concentrations of activin-A and follistatin and lower activin-AB were also detected in both groups of AOM. Tissue and serum concentrations of activin-A and follistatin correlated positively, while tissue activin-AB inversely, and significantly with the numbers and sizes of colonic lesions. CONCLUSIONS: Normal rat colon epithelial cells are capable of synthesising, controlling as well as responding to activins in a paracrine/autocrine manner. Colonic activin systems are pathologically altered during tumorigenesis and appear to be time and lesion-dependent. Activins could also be potential sensitive markers and/or molecular targets for the diagnosis and/or treatment of CRC. Further studies are required to illustrate the clinical value of activins and their related proteins in colon cancer.

Activin-A promotes the development of goat isolated secondary follicles in vitro.

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 µm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

Neutrophil-derived Activin-A moderates their pro-NETotic activity and attenuates collateral tissue damage caused by Influenza A virus infection.

Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-A-deficient animal model (S100a8-Cre/Inhba fl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response.

ActivinA modulates B-acute lymphoblastic leukaemia cell communication and survival by inducing extracellular vesicles production.

Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

Monocyte Chemoattractant Protein-1 (MCP-1), Activin-A and Clusterin in Children and Adolescents with Obesity or Type-1 Diabetes Mellitus.

Inflammation plays a crucial role in diabetes and obesity through macrophage activation. Macrophage chemoattractant protein-1 (MCP-1), activin-A, and clusterin are chemokines with known roles in diabetes and obesity. The aim of this study is to investigate their possible diagnostic and/or early prognostic values in children and adolescents with obesity and type-1 diabetes mellitus (T1DM). METHODS: We obtained serum samples from children and adolescents with a history of T1DM or obesity, in order to measure and compare MCP-1, activin-A, and clusterin concentrations. RESULTS: Forty-three subjects were included in each of the three groups (controls, T1DM, and obesity). MCP-1 values were positively correlated to BMI z-score. Activin-A was increased in children with obesity compared to the control group. A trend for higher values was detected in children with T1DM. MCP-1 and activin-A levels were positively correlated. Clusterin levels showed a trend towards lower values in children with T1DM or obesity compared to the control group and were negatively correlated to renal function. CONCLUSIONS: The inflammation markers MCP-1, activin-A, and clusterin are not altered in children with T1DM. Conversely, obesity in children is positively correlated to serum MCP-1 values and characterized by higher activin-A levels, which may reflect an already established systematic inflammation with obesity since childhood.