Extranuclear effects of thyroid hormones and analogs during development: An old mechanism with emerging roles.

Thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvß3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvß3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na+-dependent transport systems, such as glucose uptake, Na+/K+-ATPase, Na+/H+ exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na+/K+-ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

An emerging interplay between extracellular vesicles and cytokines.

Extracellular vesicles (EVs) are small membrane-bound particles that are naturally released from cells. They are recognized as potent vehicles of intercellular communication both in prokaryotes and eukaryotes. Because of their capacity to carry biological macromolecules such as proteins, lipids and nucleic acids, EVs influence different physiological and pathological functions of both parental and recipient cells. Although multiple pathways have been proposed for cytokine secretion beyond the classical ER/Golgi route, EVs have recently recognized as an alternative secretory mechanism. Interestingly, cytokines/chemokines exploit these vesicles to be released into the extracellular milieu, and also appear to modulate their release, trafficking and/or content. In this review, we provide an overview of the cytokines/chemokines that are known to be associated with EVs or their regulation with a focus on TNFα, IL-1ß and IFNs.

Thyroid Hormones Interaction With Immune Response, Inflammation and Non-thyroidal Illness Syndrome.

The interdependence between thyroid hormones (THs), namely, thyroxine and triiodothyronine, and immune system is nowadays well-recognized, although not yet fully explored. Synthesis, conversion to a bioactive form, and release of THs in the circulation are events tightly supervised by the hypothalamic-pituitary-thyroid (HPT) axis. Newly synthesized THs induce leukocyte proliferation, migration, release of cytokines, and antibody production, triggering an immune response against either sterile or microbial insults. However, chronic patho-physiological alterations of the immune system, such as infection and inflammation, affect HPT axis and, as a direct consequence, THs mechanism of action. Herein, we revise the bidirectional crosstalk between THs and immune cells, required for the proper immune system feedback response among diverse circumstances. Available circulating THs do traffic in two distinct ways depending on the metabolic condition. Mechanistically, internalized THs form a stable complex with their specific receptors, which, upon direct or indirect binding to DNA, triggers a genomic response by activating transcriptional factors, such as those belonging to the Wnt/ß-catenin pathway. Alternatively, THs engage integrin αvß3 receptor on cell membrane and trigger a non-genomic response, which can also signal to the nucleus. In addition, we highlight THs-dependent inflammasome complex modulation and describe new crucial pathways involved in microRNA regulation by THs, in physiological and patho-physiological conditions, which modify the HPT axis and THs performances. Finally, we focus on the non-thyroidal illness syndrome in which the HPT axis is altered and, in turn, affects circulating levels of active THs as reported in viral infections, particularly in immunocompromised patients infected with human immunodeficiency virus.

The involvement of plasmacytoid cells in HIV infection and pathogenesis.

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset that are specialized in type I interferon (IFN) production. pDCs are key players in the antiviral immune response and serve as bridge between innate and adaptive immunity. Although pDCs do not represent the main reservoir of the Human Immunodeficiency Virus (HIV), they are a crucial subset in HIV infection as they influence viral transmission, target cell infection and antigen presentation. pDCs act as inflammatory and immunosuppressive cells, thus contributing to HIV disease progression. This review provides a state of art analysis of the interactions between HIV and pDCs and their potential roles in HIV transmission, chronic immune activation and immunosuppression. A thorough understanding of the roles of pDCs in HIV infection will help to improve therapeutic strategies to fight HIV infection, and will further increase our knowledge on this important immune cell subset.

Staphylococcus aureus Esx Factors Control Human Dendritic Cell Functions Conditioning Th1/Th17 Response.

The opportunistic pathogen Staphylococcus aureus (S. aureus) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (ΔesxA), esxB (ΔesxB), or both genes (ΔesxAB). The S. aureus mutant lacking only the EsxA protein (ΔesxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, ΔesxAB, and ΔesxB strains. When the mutant carrying the esxB deletion (ΔesxB) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from ΔesxB-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4+ T cells as compared with those conditioned with supernatants derived from wild type USA300-, ΔesxAB-, and ΔesxA-infected cultures. Although, the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus.

TRAIL is a key target in S-phase slowing-dependent apoptosis induced by interferon-beta in cervical carcinoma cells.

Interferon (IFN)-beta induces S-phase slowing and apoptosis in human papilloma virus (HPV)-positive cervical carcinoma cell line ME-180. Here, we show that apoptosis is a consequence of the S-phase lengthening imposed by IFN-beta, demonstrating the functional correlation between S-phase alteration and apoptosis induction. In ME-180 cells, where p53 function is inhibited by HPV E6 oncoprotein, IFN-beta effects on cell cycle and apoptosis occur independently of p53. The apoptosis due to IFN-beta is mediated by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a manner dependent on the S-phase deregulation. IFN-beta appears to increase TRAIL expression both directly at the mRNA level and indirectly by augmenting surface protein levels as a consequence of the induced S-phase cell accumulation. Moreover, the alteration of the S-phase due to IFN-beta promotes TRAIL-dependent apoptosis by potentiating cell sensitivity to TRAIL, possibly through induction of a proapoptotic NF-kappaB activity and TRAIL-R2 receptor expression. Interestingly, IFN-beta-induced TRAIL-dependent apoptotic events strongly differ in the requirement of caspase activity. These results show that IFN-beta may induce an apoptotic response by deregulating cell cycle. Understanding the linkage between these mechanisms appears to be of primary importance in the search for new IFN-based therapeutic strategies to circumvent cancer disease or improve clinical outcome.

TNF-related apoptosis-inducing ligand (TRAIL) as a pro-apoptotic signal transducer with cancer therapeutic potential.

The powerful inducer of apoptosis Apo2L/TNF-related apoptosis-inducing ligand (TRAIL) has generated exciting promise as a potential tumour specific cancer therapeutic agent, since it selectively induces apoptosis in transformed versus normal cells. Interferons (IFNs) are important modulators of TRAIL expression, thus the ligand appears to play an important role in surveillance against viral infection and malignant transformation. In the light of the emerging importance of TRAIL in cancer therapy, we will discuss the molecular basis of the cooperation of TRAIL and IFNs or chemotherapeutic drugs. In particular, we will focus on the data known to date concerning the biochemical pathways leading to TRAIL-induced apoptosis in specific cancer cells and warranting further work to enable the investigation in cancer patients.

IRF-1 as a negative regulator of cell proliferation.

Numerous evidence has demonstrated the involvement in growth control of interferon (IFN) regulatory factor-1 (IRF-1), which shows tumor suppressor activity. IRF-1 is a well-studied member of the IRF transcription factors that reveals functional diversity in the regulation of cellular response by activating expression of a diverse set of target genes, depending on the cell type and on the specific stimuli. IRF-1 gene rearrangements may be a crucial point in the pathogenesis of some cancer types. Furthermore, different aspects of the tumor suppressor function of IRF-1 may be explained, at least in part, by the observations that IRF-1 is a regulator of cell cycle and apoptosis and that its inactivation accelerates cell transformation. Studies on gene knockout mice contributed greatly to the clarification of these multiple IRF-1 functions. We summarize our current knowledge of the antigrowth effect of IRF-1, focusing also on a more general involvement of IRF-1 in mediating negative regulation of cell growth induced by numerous cytokines and other biologic response modifiers.

Exogenous nef induces proinflammatory signaling events in murine macrophages.

Despite the fact that murine cells are not permissive for human immunodeficiency virus type 1 (HIV-1) infection, several investigators have constructed transgenic (Tg) mice to model HIV-1-induced diseases to overcome this restriction. The generation of Tg mice expressing selected HIV-1 genes revealed that Nef harbors a major disease determinant. HIV-1 Nef protein is a molecular adapter able to interact with several cellular partners, interfering with cellular functions. The phenotype of Nef Tg mice was extensively characterized regarding in vivo development of AIDS-like disease and the effects of Nef expression in T lymphocytes, but the functions eventually corrupted by Nef in monocytes and macrophages were less studied. Nef treatment of human monocyte-derived macrophages induces the internalization of the protein and modulates the production and secretion of different chemokines and cytokines by activating specific intracellular signaling pathways (i.e., NF-κB, MAPK, and IRF3). Therefore we set up an in vitro murine macrophage-based model using stabilized cell lines and primary peritoneal macrophages, and treated them with recombinant myristoylated Nef(SF2) (recNef). Like human cells, murine macrophages responded to Nef treatment, activating IKK-α and IKK-ß, JNK, and p38 MAP kinases. Activation of the NF-κB pathway is mandatory for the synthesis and release of a pool of cytokines and chemokines, including IFN-ß, that induce tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1, STAT-2, and STAT-3, in an autocrine and paracrine manner, confirming that murine macrophages respond to Nef similarly to human ones. These data extend the results previously obtained in human primary macrophages, allowing the use of murine cells in culture to study signaling events modulated by Nef in myeloid-derived cells. In particular, it may be feasible to use macrophages derived from mice knocked out in specific signaling intermediates to obtain greater insight into the mechanism of Nef-induced effects.

Interferon-ß induces cellular senescence in cutaneous human papilloma virus-transformed human keratinocytes by affecting p53 transactivating activity.

Interferon (IFN)-ß inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-ß treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-ß appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-ß treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative ΔNp73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer.

HMGB1 and cord blood: its role as immuno-adjuvant factor in innate immunity.

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4(+) T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2.

BACKGROUND: HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNß to activate STAT1, -2 and -3. METHODOLOGY/PRINCIPAL FINDINGS: Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. CONCLUSIONS: Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNß.

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon.

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.