Nuclear Pore Permeabilization Is a Convergent Signaling Event in Effector-Triggered Immunity.

Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction.

Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens.

Neutrophils are critical for antifungal defense, but the mechanisms that clear hyphae and other pathogens that are too large to be phagocytosed remain unknown. We found that neutrophils sensed microbe size and selectively released neutrophil extracellular traps (NETs) in response to large pathogens, such as Candida albicans hyphae and extracellular aggregates of Mycobacterium bovis, but not in response to small yeast or single bacteria. NETs were fundamental in countering large pathogens in vivo. Phagocytosis via dectin-1 acted as a sensor of microbe size and prevented NET release by downregulating the translocation of neutrophil elastase (NE) to the nucleus. Dectin-1 deficiency led to aberrant NET release and NET-mediated tissue damage during infection. Size-tailored neutrophil responses cleared large microbes and minimized pathology when microbes were small enough to be phagocytosed.

Crosstalk between nucleocytoplasmic trafficking and the innate immune response to viral infection.

The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response.

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage.

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.

Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells.

The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1 cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H2O2 or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1ß, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1 cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H2O2. Moreover, SNP or H2O2 exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1 cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1ß. Thus, hypoxia not only increases RAGE expression in THP-1 cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression.

ß-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity.

The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that ß-arrestins negatively regulate Toll signaling in two different ways. ß-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of ß-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. ß-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that ß-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

Mycobacterium tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1/2 signaling.

Crucial to the pathogenesis of the tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis is its ability to subvert host immune defenses to promote its intracellular survival. The mammalian cell entry protein 3E (Mce3E), located in the region of difference 15 of the M. tuberculosis genome and absent in Mycobacterium bovis bacillus Calmette-Guérin, has an essential role in facilitating the internalization of mammalian cells by mycobacteria. However, relatively little is known about the role of Mce3E in modulation of host innate immune responses. In this study, we demonstrate that Mce3E inhibits the activation of the ERK1/2 signaling pathway, leading to the suppression of Tnf and Il6 expression, and the promotion of mycobacterial survival within macrophages. Mce3E interacts and colocalizes with ERK1/2 at the endoplasmic reticulum in a DEF motif (an ERK-docking motif)-dependent manner, relocates ERK1/2 from cytoplasm to the endoplasmic reticulum, and finally reduces the association of ERK1/2 with MEK1 and blocks the nuclear translocation of phospho-ERK1/2. A DEF motif mutant form of Mce3E (F294A) loses its ability to suppress Tnf and Il6 expression and to promote intracellular survival of mycobacteria. Inhibition of the ERK1/2 pathway in macrophages using U0126, a specific inhibitor of the ERK pathway, also leads to the suppressed Tnf and Il6 expression and the enhanced intracellular survival of mycobacteria. Taken together, these results suggest that M. tuberculosis Mce3E exploits the ERK1/2 signaling pathway to suppress host innate immune responses, providing a potential Mce3E-ERK1/2 interface-based drug target against M. tuberculosis.

Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3.

Accurate cellular localization plays a crucial role in the effective function of most signaling proteins, and nuclear trafficking is central to the function of transcription factors. IFN regulatory factor (IRF)3 is a master transcription factor responsible for the induction of type I IFN, which plays a crucial role in host antiviral innate immune responses. However, the mechanisms for control and regulation of IRF3 nuclear import largely remain to be elucidated. In our study, we identified a bipartite nuclear localization signal (NLS) in IRF3, with two interdependent basic clusters separated by a 7-aa linker. Our study further demonstrated that the bipartite NLS of IRF3 is also critical for IRF3 DNA-binding activity, indicating that the two functions of this region are integrated, which is in contrast to other IRFs. Furthermore, the IFN bioassay and infection studies suggest that IRF3 NLS is essential to the IRF3-mediated IFN responses and antiviral immunity. Overall, our results reveal a previously unrecognized bipartite NLS for IRF3 that contains both DNA-binding activity and nuclear import function, and they shed light on the regulatory mechanisms of IRF3 activation and IRF3-mediated antiviral responses.

mTOR Inhibition Per Se Induces Nuclear Localization of FOXP3 and Conversion of Invariant NKT (iNKT) Cells into Immunosuppressive Regulatory iNKT Cells.

CD1d-restricted activation of invariant NKT (iNKT) cells results in the abundant production of various types of cytokines and the subsequent modulation of immune responses. This has been shown to be relevant in several clinical disorders, including cancer, autoimmunity, and graft tolerance. Although it is well known that the suppressive function of regulatory T cells is critically dependent on the FOXP3 gene, FOXP3 can also be expressed by conventional human T cells upon activation, indicating the lack of specificity of FOXP3 as a marker for suppressive cells. In this study, we report that the mammalian target of rapamycin (mTOR) inhibitor rapamycin and IL-10, but not TGF-ß, can induce FOXP3 expression in iNKT cell lines. Importantly, however, FOXP3(+) iNKT cells only acquired suppressive abilities when cultured in the presence of the mTOR inhibitor rapamycin. Suppression of responder T cell proliferation by FOXP3(+) iNKT cells was found to be cell contact-dependent and was accompanied by a reduced capacity of iNKT cells to secrete IFN-γ. Notably, imaging flow cytometry analysis demonstrated predominant nuclear localization of FOXP3 in suppressive FOXP3(+) iNKT cells, whereas nonsuppressive FOXP3(+) iNKT cells showed a predominance of cytoplasmically localized FOXP3. In conclusion, whereas IL-10 can enhance FOXP3 expression in iNKT cells, mTOR inhibition is solely required for promoting nuclear localization of FOXP3 and the induction of suppressive FOXP3(+) iNKT cells.

Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.

IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37-mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1ß upon LPS stimulation. IL-37-expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37-neutralizing antibodies before LPS challenge. In IL-37-expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation.

Additive anti-inflammatory effects of corticosteroids and phosphodiesterase-4 inhibitors in COPD CD8 cells.

BACKGROUND: CD8 lymphocytes play an important role in the pathogenesis of COPD. Corticosteroids and phosphodiesterase 4 (PDE4) inhibitors are anti-inflammatory drugs used for COPD treatment. Little is known of the combined effect of these drugs on COPD CD8 cells. We studied the effect of corticosteroid combined with PDE4 inhibitors on cytokine release form circulating and pulmonary CD8 cells, and on glucocorticoid (GR) nuclear translocation. METHODS: The effect of dexamethasone alone and in combination with the PDE4 inhibitors roflumilast and GSK256066 on cytokine release from circulating and pulmonary CD8 cells was measured. The effect of the compounds on nuclear translocation of GR and cyclic AMP-responsive element-binding protein (CREB) was studied using immunofluorescence. RESULTS: Dexamethasone inhibited cytokine release from COPD CD8 cells in a concentration dependent manner. PDE4 inhibitors enhanced this anti-inflammatory effect in an additive manner. PDE4 inhibitors did not increase corticosteroid induced GR nuclear translocation. PDE4 inhibitors, but not corticosteroid, increased phospho-CREB nuclear translocation. CONCLUSION: The combination of corticosteroids and PDE4 inhibitors results in an additive anti-inflammatory effect in COPD CD8 cells. This enhanced anti-inflammatory effect could translate to important clinical benefits for patients with COPD.

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

Noncanonical NF-κB activation mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast cancer.

Immunotherapy for cancer treatment is achieved through the activation of competent immune effector cells and the inhibition of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). Although MDSCs have been shown to contribute to breast cancer development, the mechanism underlying MDSC-mediated immunosuppression is unclear. We have identified a poorly differentiated MDSC subset in breast cancer-suppressing T cell function through STAT3-dependent IDO upregulation. In this study we investigated the mechanisms underlying aberrant expression of IDO in MDSCs. MDSCs were induced by coculturing human CD33(+) myeloid progenitors with MDA-MB-231 breast cancer cells. Increased STAT3 activation in MDSCs was correlated with activation of the noncanonical NF-κB pathway, including increased NF-κB-inducing kinase (NIK) protein level, phosphorylation of cytoplasmic inhibitor of NF-κB kinase α and p100, and RelB-p52 nuclear translocation. Blocking STAT3 activation with the small molecule inhibitor JSI-124 significantly inhibited the accumulation of NIK and IDO expression in MDSCs. Knockdown of NIK in MDSCs suppressed IDO expression but not STAT3 activation. RelB-p52 dimers were found to directly bind to the IDO promoter, leading to IDO expression in MDSCs. IL-6 was found to stimulate STAT3-dependent, NF-κB-mediated IDO upregulation in MDSCs. Furthermore, significant positive correlation between the numbers of pSTAT3(+) MDSCs, IDO(+) MDSCs, and NIK(+) MDSCs was observed in human breast cancers. These results demonstrate a STAT3/NF-κB/IDO pathway in breast cancer-derived MDSCs, which provides insight into understanding immunosuppressive mechanisms of MDSCs in breast cancer.

6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation.

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell-based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4(+) T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.

Human memory, but not naive, CD4+ T cells expressing transcription factor T-bet might drive rapid cytokine production.

We found that after stimulation for a few hours, memory but not naive CD4(+) T cells produced a large amount of IFN-γ; however, the mechanism of rapid response of memory CD4(+) T cells remains undefined. We compared the expression of transcription factors in resting or activated naive and memory CD4(+) T cells and found that T-bet, but not pSTAT-1 or pSTAT-4, was highly expressed in resting memory CD4(+) T cells and that phenotypic characteristics of T-bet(+)CD4(+) T cells were CD45RA(low)CD62L(low) CCR7(low). After short-term stimulation, purified memory CD4(+) T cells rapidly produced effector cytokines that were closely associated with the pre-existence of T-bet. By contrast, resting naive CD4(+) T cells did not express T-bet, and they produced cytokines only after sustained stimulation. Our further studies indicated that T-bet was expressed in the nuclei of resting memory CD4(+) T cells, which might have important implications for rapid IFN-γ production. Our results indicate that the pre-existence and nuclear mobilization of T-bet in resting memory CD4(+) T cells might be a possible transcriptional mechanism for rapid production of cytokines by human memory CD4(+) T cells.

Lipopolysaccharide activates Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway and proinflammatory response in human pericytes.

Pericytes and mesenchymal stem cells (MSCs) are ontogenically related, and in fact, no significant phenotypic differences could be observed by flow cytometry. Transcriptome analysis of human pericytes and MSCs revealed that 43 genes were up-regulated more than 10-fold in pericytes compared with MSCs. Identification of Toll-like receptor 4 (TLR4) as one of the most abundant RNA species in pericytes with respect to MSCs and confirmation of TLR4 expression on the cell surface led us to obtain a comprehensive overview of the expression program of lipopolysaccharide (LPS)-stimulated pericytes. Transcriptional profiling of LPS-treated cells revealed that 22 genes were up-regulated more than 5-fold. Of them, 10 genes encoded chemokines and cytokines (CXCL10, CCL20, IL8, CXCL1, IL6, CCL2, IL1B, CXCL2, IL1A, and CXCL6), and three genes encoded adhesion molecules (ICAM1, VCAM1, and SELE). LPS induced nuclear translocation of the transcription factor NF-κB in stimulated pericytes. Moreover, inhibition of NF-κB activation by SC-514 blocked LPS-induced up-regulation of a subset of chemokine genes, confirming the key role of NF-κB in LPS signaling in pericytes. At the protein level, we assessed the secretion of the proinflammatory cytokines and chemokines IL-6, IL-8, CXCL1, CXCL2, CXCL3, and CCL2 not only after LPS treatment but also in HMGB1-stimulated pericytes. Up-regulation of the adhesion molecules ICAM-1 and VCAM-1 resulted in an increased adhesion of peripheral blood leukocytes to an LPS-treated pericyte monolayer. The role of pericytes in the inflammatory context has been scarcely addressed; according to these results, pericytes should be considered as active players in the inflammatory cascade with potential physiopathological implications.

Intact type I Interferon production and IRF7 function in sooty mangabeys.

In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-ß mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

Immune activation of human brain microvascular endothelial cells inhibits HIV replication in macrophages.

There is limited information about the role of blood-brain barrier (BBB) endothelial cells (ECs) in the central nervous system (CNS) and their innate immunity against HIV. We examined whether brain ECs can be immunologically activated to produce antiviral factors that inhibit HIV replication in macrophages. Human brain microvascular ECs expressed functional toll-like receptor 3 (TLR3) that could be activated by polyinosinic-polycytidylic acid (PolyI:C), resulting in the induction of endogenous interferon-ß (IFN-ß) and IFN-λ. The TLR3 activation of ECs also induced the phosphorylation of interferon regulatory transcription factor 3 (IRF3) and IRF7, the key regulators of IFN signaling pathway. When supernatant (SN) of PolyI:C-activated EC cultures was applied to infected macrophage cultures, HIV replication was significantly suppressed. This SN action of ECs on HIV was mediated through both IFN-ß and IFN-λ because antibodies to their receptors could neutralize the SN-mediated anti-HIV effect. The role of IFNs in EC-mediated anti-HIV activity is further supported by the observation that treatment with SN from EC cultures induced the expression of IFN-stimulated genes (ISGs: ISG56, OAS-1, and MxA) in macrophages. These observations indicate that brain microvascular ECs may be a key regulatory bystander, playing a crucial role in the BBB innate immunity against HIV infection.