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Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2β). CK2 promotes cancer progression by activating the NF-κB, PI3K/AKT/mTOR, and JAK/STAT pathways, and also is critical for immune cell development and function. The potential involvement of CK2 in CD8 + T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8 + T cell activation. CK2α deficiency results in impaired CD8 + T cell activation and proliferation upon TCR stimulation. Furthermore, CK2α is involved in CD8 + T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly, using a mouse Listeria monocytogenes infection model, we demonstrate that CK2α is required for CD8 + T cell expansion, maintenance, and effector function in both primary and memory immune responses. Collectively, our study implicates CK2α as an important regulator of mouse CD8 + T cell activation, metabolic reprogramming, and differentiation both in vitro and in vivo., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury., Methods: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury., Results: Analyses of single-cell RNA-seq data of CD45 + immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease., Conclusions: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease., (Copyright © 2022 by the American Society of Nephrology.)

The University of Alabama at Birmingham academic medical center (UAB AMC) had achieved great success and growth during the 50 years since its founding. However, the challenging and more competitive environment of the 2000s left the UAB AMC on a downward trajectory. The UAB AMC had to overcome difficult internal cultural and structural barriers that stood in the way of the transformational change needed to remain competitive. Competition rather than collaborative and strategic financial investment were the primary cultural barriers for the UAB AMC, while people were the primary structural barrier. Leadership identified 5 steps that were critical for the transformation that occurred between 2013 and 2018: alignment of leadership; creating a compelling and credible shared vision; identifying cultural and structural barriers; creating a thoughtful, data-driven intervention; and improved communication and accountability. Following these steps enabled the UAB AMC to transform its institutional structure and culture. As a result, the UAB AMC thrived, returning to substantial growth in research and clinical care. UAB AMC School of Medicine grew by $100 million in National Institutes of Health funding and moved up 10 spots in ranking. In 2018, UAB Hospital had 10 specialties ranked by U.S. News & World Report, 7 more than in 2013. This article outlines the approach taken and provides a conceptual framework for other AMCs eager to transform their structure and culture and position themselves for growth., (Copyright © 2021 by the Association of American Medical Colleges.)

Protein kinase CK2 (also known as Casein Kinase 2) is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory CK2β subunits. CK2 is overexpressed and overactive in B cell acute lymphoblastic leukemia and diffuse large B cell lymphomas, leading to inappropriate activation of the NF-κB, JAK/STAT, and PI3K/AKT/mTOR signaling pathways and tumor growth. However, whether CK2 regulates normal B cell development and differentiation is not known. We generated mice lacking CK2α specifically in B cells (using CD19-driven Cre recombinase). These mice exhibited cell-intrinsic expansion of marginal zone B cells at the expense of transitional B cells, without changes in follicular B cells. Transitional B cells required CK2α to maintain adequate BCR signaling. In the absence of CK2α, reduced BCR signaling and elevated Notch2 signaling activation increased marginal zone B cell differentiation. Our results identify a previously unrecognized function for CK2α in B cell development and differentiation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Objective: To determine the activation status and cytokine profiles of CD4 + T cells, CD8 + T cells, and CD19 + B cells from patients with early-stage Parkinson disease (PD) compared with healthy controls (HCs)., Methods: Peripheral blood samples from 41 patients with early-stage PD and 40 HCs were evaluated. Peripheral blood mononuclear cells were analyzed by flow cytometry for surface markers and intracellular cytokine production. Correlations of immunologic changes and clinical parameters were analyzed., Results: Adaptive immunity plays a role in the pathogenesis of PD, yet the contribution of T cells and B cells, especially cytokine production by these cells, is poorly understood. We demonstrate that naive CD4 + and naive CD8 + T cells are significantly decreased in patients with PD, whereas central memory CD4 + T cells are significantly increased in patients with PD. Furthermore, IL-17-producing CD4 + Th17 cells, IL-4-producing CD4 + Th2 cells, and IFN-γ-producing CD8 + T cells are significantly increased in patients with PD. Regarding B cells, we observed a decrease in naive B cells and an increase in nonswitched memory and double-negative B cells. As well, TNF-α-producing CD19 + B cells were significantly increased in patients with PD. Notably, some of the changes observed in CD4 + T cells and B cells were associated with clinical motor disease severity., Conclusions: These findings suggest that alterations in the adaptive immune system may promote clinical disease in PD by skewing to a more proinflammatory state in the early-stage PD patient cohort. Our study may shed light on potential immunotherapies targeting dysregulated CD4 + T cells, CD8 + T cells, and CD19 + B cells in patients with PD., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Increasing evidence supports the role of brain and systemic inflammation in the etiology of Parkinson disease (PD). We used gene expression profiling to examine the activation state of peripheral blood monocytes in 18 patients with early, untreated PD and 16 healthy control (HC) subjects. Monocytes were isolated by negative selection, and gene expression studied by RNA-seq and gene set enrichment analysis. A computational model that incorporated case/control status, sex, and the interaction between case/control status and sex was utilized. We found that there was a striking effect of sex on monocyte gene expression. There was inflammatory activation of monocytes in females with PD, with enrichment of gene sets associated with interferon gamma stimulation. In males, the activation patterns were more heterogeneous. These data point to the importance of systemic monocyte activation in PD, and the importance of studies which examine the differential effects of sex on pathophysiology of the disease.

Background: Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM., Methods: We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5)., Results: Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances., Conclusions: The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD), is characterized by chronic inflammation of the gastrointestinal tract and associated with aberrant CD4 + T-helper type 1 (Th1) and Th17 responses. Protein kinase 2 (CK2) is a conserved serine-threonine kinase involved in signal transduction pathways, which regulate immune responses. CK2 promotes Th17 cell differentiation and suppresses the generation of Foxp3 + regulatory T cells. The function of CK2 in CD4 + T cells during the pathogenesis of CD is unknown. We utilized the T cell-induced colitis model, transferring CD45RB hi -naive CD4 + T cells from CK2α fl/fl controls and CK2α fl/fl dLck-Cre mice into Rag1 -/- mice. CD4 + T cells from CK2α fl/fl dLck-Cre mice failed to induce wasting disease and significant intestinal inflammation, which was associated with decreased interleukin-17A-positive (IL-17A + ), interferon-γ-positive (IFN-γ + ), and double-positive IL-17A + IFN-γ + CD4 + T cells in the spleen and colon. We determined that CK2α regulates CD4 + T cell proliferation through a cell-intrinsic manner. CK2α is also important in controlling CD4 + T cell responses by regulating NFAT2, which is vital for T cell activation and proliferation. Our findings indicate that CK2α contributes to the pathogenesis of colitis by promoting CD4 + T cell proliferation and Th1 and Th17 responses, and that targeting CK2 may be a novel therapeutic treatment for patients with CD.

To understand the origins of the infant gut microbial community, we have used a published metagenomic dataset of the faecal microbiome of mothers and their related infants at early (4, 7 and 21 days) and late times (6-15 months) following birth. Using strain-tracking analysis, individual-specific patterns of microbial strain sharing were found between mothers and infants following vaginal birth. Overall, three mother-infant pairs showed only related strains, while 12 infants of mother-infant pairs contained a mosaic of maternal-related and unrelated microbes. Analysis of a second dataset from nine women taken at different times of pregnancy revealed individual-specific faecal microbial strain variation that occurred in seven women. To model transmission in the absence of environmental microbes, we analysed the microbial strain transmission to F1 progenies of human faecal transplanted gnotobiotic mice bred with gnotobiotic males. Strain-tracking analysis of five different dams and their F1 progeny revealed both related and unrelated microbial strains in the mother's faeces. The results of our analysis demonstrate that multiple strains of maternal microbes, some that are not abundant in the maternal faecal community, can be transmitted during birth to establish a diverse infant gut microbial community., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Liver kinase B1 (LKB1) is a ubiquitously expressed kinase involved in the regulation of cell metabolism, growth, and inflammatory activation. We previously reported that a single nucleotide polymorphism in the gene encoding LKB1 is a risk factor for multiple sclerosis (MS). Since astrocyte activation and metabolic function have important roles in regulating neuroinflammation and neuropathology, we examined the serine/threonine kinase LKB1 in astrocytes in a chronic experimental autoimmune encephalomyelitis mouse model of MS. To reduce LKB1, a heterozygous astrocyte-selective conditional knockout (het-cKO) model was used. While disease incidence was similar, disease severity was worsened in het-cKO mice. RNAseq analysis identified Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in het-cKO mice relating to mitochondrial function, confirmed by alterations in mitochondrial complex proteins and reductions in mRNAs related to astrocyte metabolism. Enriched pathways included major histocompatibility class II genes, confirmed by increases in MHCII protein in spinal cord and cerebellum of het-cKO mice. We observed increased numbers of CD4+ Th17 cells and increased neuronal damage in spinal cords of het-cKO mice, associated with reduced expression of choline acetyltransferase, accumulation of immunoglobulin-γ, and reduced expression of factors involved in motor neuron survival. In vitro, LKB1-deficient astrocytes showed reduced metabolic function and increased inflammatory activation. These data suggest that metabolic dysfunction in astrocytes, in this case due to LKB1 deficiency, can exacerbate demyelinating disease by loss of metabolic support and increase in the inflammatory environment., (© 2019 Wiley Periodicals, Inc.)

Background: Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12-14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified., Methods: We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation., Results: Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11c lo ) to R2a (CD11c hi ) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease., Conclusions: These data uncover an important pathogenic role for resident macrophages during rapid cyst progression., (Copyright © 2019 by the American Society of Nephrology.)

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model, Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE, however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to G-CSF, and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis., (© 2018 Wiley Periodicals, Inc.)

Although it has historically been studied in the context of cancer, recent literature has highlighted the importance of the highly conserved serine/threonine kinase casein kinase II (CK2) in inflammatory disorders. Most strikingly, CK2 is a major regulator of the Th17-Treg axis relevant to many T cell-driven autoimmune disorders including multiple sclerosis (MS)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CK2 is a highly conserved and pleiotropic serine/threonine kinase that promotes many prosurvival and proinflammatory signaling pathways, including PI3K/Akt/mTOR and JAK/STAT. These pathways are essential for CD4 + T cell activation and polarization, but little is known about how CK2 functions in T cells. In this article, we demonstrate that CK2 expression and kinase activity are induced upon CD4 + T cell activation. Targeting the catalytic activity of CK2 using the next-generation small molecule inhibitor CX-4945 in vitro significantly and specifically inhibited mouse and human Th17 cell differentiation while promoting the generation of Foxp3 + regulatory T cells (Tregs). These findings were associated with suppression of PI3K/Akt/mTOR activation and STAT3 phosphorylation upon CX-4945 treatment. Furthermore, we demonstrate that CX-4945 treatment inhibits the maturation of Th17 cells into inflammatory IFN-γ-coproducing effector cells. The Th17/Treg axis and maturation of Th17 cells are major contributing factors to the pathogenesis of many autoimmune disorders, including multiple sclerosis. Using a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, we demonstrate that in vivo administration of CX-4945 targets Akt/mTOR signaling in CD4 + T cells and the Th17/Treg axis throughout disease. Importantly, CX-4945 treatment after disease initiation significantly reduced disease severity, which was associated with a significant decrease in the frequency of pathogenic IFN-γ + and GM-CSF + Th17 cells in the CNS. Our data implicate CK2 as a regulator of the Th17/Treg axis and Th17 cell maturation and suggest that CK2 could be targeted for the treatment of Th17 cell-driven autoimmune disorders., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α') and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133 + BTICs compared to CD133 - cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133 + BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

Glioblastoma is the most common and aggressive malignant brain tumor in adults. The existence of glioblastoma stem cells (GSCs) or stem-like cells (stemloids) may account for its invasiveness and high recurrence. Rac proteins belong to the Rho small GTPase subfamily which regulates cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as therapeutic targets for glioblastoma, especially for GSCs or stemloids, we examined the potential roles of Rac1, Rac2 and Rac3 on the properties of tumorspheres derived from glioblastoma cell lines. Tumorspheres are thought to be glioblastoma stem-like cells. We showed that Rac proteins promote the STAT3 and ERK activation and enhance cell proliferation and colony formation of glioblastoma stem-like cells. Knockdown of Rac proteins reduces the expression of GSC markers, such as CD133 and Sox2. The in vivo effects of Rac proteins in glioblastoma were further studied in zebrafish and in the mouse xenotransplantation model. Knocking-down Rac proteins abolished the angiogenesis effect induced by the injected tumorspheres in zebrafish model. In the CD133+-U373-tumorsphere xenotransplanted mouse model, suppression of Rac proteins decreased the incidence of tumor formation and inhibited the tumor growth. Moreover, knockdown of Rac proteins reduced the sphere forming efficiency of cells derived from these tumors. In conclusion, not only Rac1 but also Rac2 and 3 are important for glioblastoma tumorigenesis and can serve as the potential therapeutic targets against glioblastoma and its stem-like cells.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are diseases with high mortality. Macrophages and neutrophils are responsible for inflammatory responses in ALI and ARDS, which are characterized by excessive production of proinflammatory mediators in bronchoalveolar lavage fluid (BALF) and plasma. Aberrant activation of the JAK/STAT pathway is critical for persistent inflammation in many conditions such as infection and autoimmunity. Given the importance of the STAT3 transcription factor in activating macrophages and neutrophils and augmenting inflammation, we investigated the therapeutic potential of inhibiting STAT3 activity using the small-molecule STAT3 inhibitor, LLL12. Our results demonstrate that LPS induces STAT3 activation in macrophages in vitro and in CD45 + CD11b + cells from BALF in the LPS-induced ALI model in vivo. LLL12 treatment inhibits LPS-induced lung inflammation in the ALI model, which is accompanied by suppression of LPS-induced STAT3 activation and an inhibition of macrophage and inflammatory cell infiltration in lung and BALF. LLL12 treatment also suppresses expression of proinflammatory genes including IL-1β, IL-6, TNF-α, iNOS, CCL2, and MHC class II in macrophages and inflammatory cells from BALF and serum as determined by ELISA. Furthermore, hyperactivation of STAT3 in LysMCre-SOCS3 fl/fl mice accelerates the severity of inflammation in the ALI model. Both pre- and post-LPS treatment with LLL12 decrease LPS-induced inflammatory responses in mice with ALI. Importantly, LLL12 treatment attenuates STAT3 phosphorylation in human peripheral blood mononuclear cells induced by plasma from patients with ARDS, which suggests the feasibility of targeting the STAT3 pathway therapeutically for patients with ALI and ARDS.

Astrocytes are intimately involved in immunological and inflammatory events occurring in the central nervous system (CNS), due to their ability to secrete and respond to a large number of immunoregulatory cytokines/chemokines such as IL-1β, IL-6, IL-8, IL-10, IL-17, IL-27, TNF-α, TGF-β, IFN-γ, IFN-β, CCL2, CCL3, CCL5, CXCL10, and CXCL12. Although expression of cytokines and chemokines is limited in the normal CNS, elevated expression of these proteins, as seen in disease entities such as multiple sclerosis (MS), HIV-1 associated neurocognitive disorders (HAND), Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), contributes to the development of inflammation and neuronal demise in these diseases. As a potent source of cytokines and chemokines, astrocytes play a pivotal role in the type and extent of neuroinflammatory responses. Astrocytes can be stimulated in vitro to produce numerous cytokines/chemokines, which are secreted and detected in supernatants by a technique known as enzyme-linked immunosorbent assay (ELISA). In this chapter, we describe our experience using ELISAs to detect and quantify cytokines and chemokines secreted by stimulated murine astrocytes, specifically IL-6 and CXCL10.

Malignant gliomas are diffusively infiltrative and remain among the deadliest of all cancers. NF-κB is a transcription factor that mediates cell growth, migration and invasion, angiogenesis and resistance to apoptosis. Normally, the activity of NF-κB is tightly regulated by numerous mechanisms. However, in many cancers, NF-κB is constitutively activated and may function as a tumor promoter. Herein, we show that in gliomas, NF-κB is constitutively activated and the levels of cIAP2, Bcl-2, Bcl-xL and Survivin are elevated. These genes are regulated by NF-κB and can inhibit apoptosis. To understand the potential role of NF-κB p65 in suppressing apoptosis, we generated human glioma cell lines that inducibly express shRNA molecules specific for p65. We demonstrate that in the absence of p65, TNF-α induced cIAP2 expression is significantly reduced while the levels of Bcl-2, Bcl-xL and Survivin are not affected. These data suggest that of these genes, only cIAP2 is a direct target of p65, which was confirmed using RT-PCR and chromatin immunoprecipitation (ChIP) assays. By reducing the levels of p65 and/or cIAP2 levels, we demonstrate that the levels of RIP poly-ubiquitination are reduced, and that p65-deficient glioma cells are more sensitive to the cytotoxic effects of TNF-α. Specifically, in the presence of TNF-α glioma cells lacking p65 and/or cIAP2 showed cellular proliferation defects and underwent cell death. These data suggest that NF-κB and/or cIAP2 may be therapeutically relevant targets for the treatment of malignant gliomas.

HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-beta suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-kappaB-dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-beta at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-beta on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1-infected brain, such as transactivator of transcription, inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

Elevated levels of Oncostatin M (OSM), an interleukin-6 family cytokine, have been observed in multiple sclerosis (MS), HIV-associated neurocognitive disorder (HAND), and glioblastoma (GBM); however, its effects within the CNS are not well understood. OSM regulates gene expression primarily by activating the JAK/STAT, NF-kappaB, and/or MAPK pathways, in a cell-type specific manner. In our studies, OSM induces the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) from microglia in an NF-kappaB-dependent manner. This expression also partially requires the intermediate production of TNF-alpha and subsequent NF-kappaB activation via TNF-R1. We also demonstrate that OSM-induced TNF-alpha production from microglia is neurotoxic. The IL-12 family member, IL-27, suppresses OSM-mediated TNF-alpha and iNOS expression at the transcriptional level by inhibiting activation of the NF-kappaB pathway, and rescues the neurotoxicity induced by OSM-stimulated microglia. These studies are the first to demonstrate the proinflammatory effects of OSM in microglia, and also identify IL-27 as a novel inhibitor of inflammatory processes in these cells., (Copyright 2010 Wiley-Liss, Inc.)

TGF-beta, together with IL-6 and IL-21, promotes Th17 cell development. IL-6 and IL-21 induce activation of STAT3, which is crucial for Th17 cell differentiation, as well as the expression of suppressor of cytokine signaling (SOCS)3, a major negative feedback regulator of STAT3-activating cytokines that negatively regulates Th17 cells. However, it is still largely unclear how TGF-beta regulates Th17 cell development and which TGF-beta signaling pathway is involved in Th17 cell development. In this report, we demonstrate that TGF-beta inhibits IL-6- and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT3 activation in naive CD4(+)CD25(-) T cells. TGF-beta inhibits IL-6-induced SOCS3 promoter activity in T cells. Also, SOCS3 small interfering RNA knockdown partially compensates for the action of TGF-beta on Th17 cell development. In mice with a dominant-negative form of TGF-beta receptor II and impaired TGF-beta signaling, IL-6-induced CD4(+) T cell expression of SOCS3 is higher whereas STAT3 activation is lower compared with wild-type B6 CD4(+) T cells. The addition of a TGF-beta receptor I kinase inhibitor that blocks Smad-dependent TGF-beta signaling greatly, but not completely, abrogates the effect of TGF-beta on Th17 cell differentiation. Our data indicate that inhibition of SOCS3 and, thus, enhancement of STAT3 activation is at least one of the mechanisms of TGF-beta promotion of Th17 cell development.

The NF-kappaB family mediates immune and inflammatory responses. In many cancers, NF-kappaB is constitutively activated and induces the expression of genes that facilitate tumorigenesis. ING4 is a tumor suppressor that is absent or mutated in several cancers. Herein, we demonstrate that in human gliomas, NF-kappaB is constitutively activated, ING4 expression is negligible, and NF-kappaB-regulated gene expression is elevated. We demonstrate that an ING4 and NF-kappaB interaction exists but does not prevent NF-kappaB activation, nuclear translocation, or DNA binding. Instead, ING4 and NF-kappaB bind simultaneously at NF-kappaB-regulated promoters, and this binding correlates with reductions in p65 phosphorylation, p300, and the levels of acetylated histones and H3-Me3K4, while enhancing the levels of HDAC-1 at these promoters. Using a knockdown approach, we correlate reductions in ING4 protein levels with increased basal and inducible NF-kappaB target gene expression. Collectively, these data suggest that ING4 may specifically regulate the activity of NF-kappaB molecules that are bound to target gene promoters.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-gamma induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins; however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-gamma-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-gamma inducible expression of CIITA, and statins inhibit IFN-gamma-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-gamma induction of the CIITA gene is regulated by the transcription factors STAT-1alpha, interferon regulatory factor (IRF)-1 and upstream stimulatory factor (USF)-1. We previously reported that statins inhibit constitutive STAT-1alpha expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1alpha and IRF-1 to the endogenous CIITA promoter IV (pIV). The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA polymerase II (Pol II), p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene.

Interleukin-8 (IL-8) is a key component of the innate immune response because of its ability to recruit inflammatory cells to sites of inflammation. Although the effects of IL-8 are largely beneficial, aberrant expression of IL-8 is known to contribute to a number of pathologic states. Interferon-beta (IFN-beta), an antiviral cytokine, is known to inhibit the expression of IL-8, although the exact mechanism by which this occurs has yet to be elucidated. In this study, we dissect the role of each member of the IFN-stimulated gene factor 3 (ISGF3) signaling complex in contributing to IFN-beta inhibition of IL-8 gene expression. To date, no IFN-stimulated response element (ISRE) (the DNA binding target for ISGF3) has been identified within the promoter region of the IL-8 gene. We conclude, through use of cell lines deficient for ISGF3 components, that all three members of this complex, Stat1, Stat2, and IFN regulatory factor-9 (IRF-9), are required for IFN-beta-mediated inhibition of IL-8 expression. In contrast to positive signaling by ISGF3 to activate gene expression, we find that the transactivation domains of Stat1 and Stat2 are not essential to IFN-beta inhibition of IL-8. Taken together, these data define the role of the ISGF3 members in IFN-beta inhibitory signaling.

Matrix metalloproteinase-9 (MMP-9) displays a preference for a broad range of substrates including extracellular matrix proteins and cytokines. MMP-9 plays an important role in physiological processes, as well as in inflammatory diseases and numerous cancers. Interferon-beta is a pleiotropic cytokine with antiviral, antiproliferative and immunomodulatory activities. Interferon-beta positively regulates gene expression, predominantly through the Janus kinase-signal transducer and activator of transcription (STAT) pathway. However, little is known about the mechanisms used by interferon-beta to negatively regulate gene expression. In the present study, we show that interferon-beta inhibits MMP-9 gene expression at the transcriptional level. Using cell lines deficient in three components of the interferon-beta-activated interferon-stimulated gene factor 3 (ISGF3) complex (i.e. STAT-1, STAT-2 and interferon regulatory factor 9), the results of our study indicate that all three members are required for interferon-beta inhibition. Chromatin immunoprecipitation assays demonstrate that interferon-beta reduces recruitment of transcriptional activators and coactivators, such as nuclear factor kappa B p65, Sp1, CREB-binding protein and p300, to the MMP-9 promoter, and decreases the degree of histone acetylation at the MMP-9 promoter. This occurs in the absence of an association of the ISGF3 complex with the MMP-9 promoter. Taken together, these data define the role of interferon-beta and the ISGF3 members in suppressing MMP-9 gene expression.

CD40, a member of the TNF receptor superfamily, is critical for productive immune responses. Macrophages constitutively express CD40 at low levels, which are enhanced by IFN-gamma. IFN-gamma-induced CD40 expression involves activation of STAT-1alpha as well as NF-kappaB activation through an autocrine response to IFN-gamma-induced TNF-alpha production. Statins are 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, which exert anti-inflammatory effects independent of their cholesterol-lowering actions. Herein, we describe that simvastatin (SS) inhibits IFN-gamma-induced CD40 expression via the suppression of STAT-1alpha expression. This results in diminished STAT-1alpha recruitment to the CD40 promoter upon IFN-gamma treatment, in addition to reduced RNA Polymerase II recruitment and diminished levels of H3 and H4 histone acetylation. SS-mediated inhibition of STAT-1alpha occurs through suppression of constitutive STAT-1alpha mRNA and protein expression. The inhibitory effect of SS on CD40 and STAT-1alpha is dependent on HMG-CoA reductase activity, as the addition of mevalonate reverses the inhibitory effect. In addition, CD40 and/or STAT-1alpha expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, a specific inhibitor of Rho family protein prenylation, indicating the involvement of small GTP-binding proteins in this process. Collectively, these data indicate that SS inhibits IFN-gamma-induced CD40 expression by suppression of STAT-1alpha, and altering transcriptional events at the CD40 promoter.

IFN-gamma is an immunomodulatory cytokine and uses the STAT-1alpha transcription factor to mediate gene expression. The promyelocytic leukemia (PML) protein regulates transcription as an activator or repressor, depending on the gene under investigation. Herein, we examined the influence of PML on IFN-gamma signaling, using PML wild-type (Pml(+/+)) and deficient (Pml(-/-)) mouse embryonic fibroblasts (MEF). Pml(-/-) MEF exhibit enhanced IFN-gamma-induced STAT-1alpha transcriptional activity compared with Pml(+/+) cells. Moreover, reconstitution of PML in Pml(-/-) MEF reduced STAT-1alpha transcriptional activity to levels comparable to Pml(+/+) MEF. Numerous endogenous IFN-gamma-regulated genes were up-regulated in Pml(-/-) MEF compared with Pml(+/+) MEF. IFN-gamma-mediated STAT-1alpha DNA-binding activity was enhanced in Pml(-/-) cells compared with Pml(+/+) cells. Lastly, IFN-gamma enhanced the formation of a PML-STAT-1alpha complex in the nucleus. These data suggest a novel function for PML in the IFN-gamma signaling pathway by inhibiting STAT-1alpha DNA binding and transcriptional activity.

IL-8 is a chemokine that recruits migrating neutrophils and leukocytes to areas of inflammation. In noninflamed tissue, IL-8 expression is low but can be rapidly induced by proinflammatory cytokines. Typically, inflammation and transient IL-8 expression are beneficial. However, some diseases are characterized by excessive inflammation and high levels of IL-8. Previous studies have shown that IFN-beta can inhibit the expression of IL-8, although the mechanism is unknown. Using chromatin immunoprecipitation assays, we define the IL-8 transcriptional program in the absence or presence of inducing stimuli and/or inhibition by IFN-beta. In the absence of stimuli, the IL-8 promoter is acetylated but negatively regulated by corepressor proteins. Upon PMA stimulation, the levels of these corepressors are reduced and the promoter is rapidly bound and activated by transcription factors, including NF-kappaB p65, C/EBPbeta, and c-Fos. In addition, RNA polymerase II is recruited to the IL-8 promoter to initiate transcription. However, in the presence of both PMA and IFN-beta, there are diminished levels of histone acetylation, reduced levels of transcription factors such as NF-kappaB p65 and RNA polymerase II, and an increased presence of corepressor proteins such as histone deacetylases 1 and 3 and silencing mediator of retinoic acid and thyroid hormone receptors. IFN-gamma-inducible protein-10 and MCP-1 genes, also regulated by NF-kappaB, are unaffected by IFN-beta, and IFN-beta does not prevent the activation, nuclear migration, or binding of NF-kappaB p65 to the kappaB element of the IFN-gamma-inducible protein-10 promoter. As such, these data show that the inhibitory effects of IFN-beta are specific to the IL-8 promoter.

Costimulation between T cells and antigen-presenting cells is required for adaptive immune responses. CD40, a costimulatory molecule, is expressed in macrophages and microglia. The aberrant expression of CD40 is involved in human diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease. CD40 expression is induced by a variety of stimuli, including IFN-gamma and lipopolysaccharide (LPS). In this study, we describe the molecular basis by which IFN-beta, a cytokine with immunomodulatory properties, regulates CD40 gene expression. IFN-beta induces CD40 expression in macrophages and microglia at the transcriptional level, and GAS elements in the CD40 promoter are required for IFN-beta-induced CD40 promoter activity. The critical role of signal transducers and activators of transcription-1alpha (STAT-1alpha) in this response was confirmed by utilizing primary microglia from STAT-1alpha deficient mice. IFN-beta induces suppressor of cytokine signaling-1 (SOCS-1) gene expression, which inhibits cytokine signaling by inhibiting activation of STAT proteins. The ectopic expression of SOCS-1 abrogates IFN-beta-mediated STAT-1alpha activation and inhibits IFN-beta-induced CD40 expression. IFN-beta-induced recruitment of STAT-1alpha and RNA Pol II and permissive histone modifications on the CD40 promoter are also inhibited by SOCS-1 overexpression. These novel results indicate that IFN-beta-induced SOCS-1 plays an important role in the negative regulation of IFN-beta-induced CD40 gene expression.

Oncostatin M (OSM), a cytokine of the interleukin-6 (IL-6) family, can either promote or inhibit cell growth in various normal and tumor cells. We addressed the effects of exogenous OSM on the proliferation and invasion of human astroglioma cells. In addition, we investigated one of the possible mechanisms involved: modulation of matrix metalloproteinase (MMP) expression and enzymatic activity. We found that OSM inhibited the proliferation of two human astroglioma cell lines (CH235-MG and U87-MG), and that this effect was not due to apoptosis. The inhibitory effect of OSM on proliferation was mediated through the gp130/OSMRbeta receptor complex. To extend these findings, we analyzed the effects of OSM on primary tumor cells from glioblastoma patients. OSM suppressed the proliferation of primary glioblastoma cells, but not that of normal astrocytes. Interestingly, OSM did not suppress astroglioma cell invasion. This may be due to the differential regulation of MMPs by OSM. We found that OSM inhibited the constitutive expression of MMP-2, while MMP-9 expression was enhanced in astroglioma cell lines. We conclude that OSM inhibits proliferation of human astroglioma cells and primary glioblastoma cells via the gp130/OSMRbeta receptor complex. However, OSM does not affect the invasive capacity of the astroglioma cells, which may be due to the divergent effects of OSM on MMP-2 and MMP-9 expression. Collectively, these findings suggest a complex role for OSM in astroglioma biology., (2005 Wiley-Liss, Inc.)

CD40 is expressed on various immune cells, including macrophages and microglia. Aberrant expression of CD40 is associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Interaction of Toll-like receptor-4 (TLR4) with the Gram-negative bacteria endotoxin lipopolysaccharide (LPS) results in the induction of an array of immune response genes. In this study, we describe that LPS is a strong inducer of CD40 expression in macrophages and microglia, which occurs at the transcriptional level and involves the activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 1alpha (STAT-1alpha). LPS-induced CD40 expression involves the endogenous production of the cytokine interferon-beta (IFN-beta), which contributes to CD40 expression by the activation of STAT-1alpha. Blocking IFN-beta-induced activation of STAT-1alpha by IFN-beta-neutralizing antibody reduces LPS-induced CD40 gene expression. Furthermore, LPS induces acetylation and phosphorylation of histones H3 and H4 and the recruitment of NF-kappaB, STAT-1alpha, and RNA polymerase II on the CD40 promoter in vivo in a time-dependent manner, all events important for CD40 gene transcription. These results indicate that both LPS-induced NF-kappaB activation and endogenous production of IFN-beta that subsequently induces STAT-1alpha activation play critical roles in the transcriptional activation of the CD40 gene by LPS.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in aspects of immune regulation, cell proliferation, and host defense mechanisms directed toward various cancers. Some of the biological functions of IFN-gamma are achieved through inhibition of gene expression, although the mechanisms by which IFN-gamma suppresses gene transcription are poorly understood. Herein, we demonstrate the molecular basis by which IFN-gamma mediates suppression of the matrix metalloproteinase-9 (MMP-9) gene. IFN-gamma-activated signal transducer and activator of transcription-1alpha (STAT-1alpha) suppresses MMP-9 gene transcription, which is dependent on phosphorylation of tyrosine 701 but not phosphorylation of serine 727. The coactivator cyclic AMP response element-binding protein-binding protein (CBP) is an important component of induction of MMP-9 gene transcription. IFN-gamma induces the in vivo association of STAT-1alpha and CBP and decreases the association of CBP to the MMP-9 promoter. IFN-gamma does not influence the stability of CBP nor does IFN-gamma affect chromatin-remodeling events on the MMP-9 promoter. IFN-gamma inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II to the MMP-9 promoter. These findings indicate that IFN-gamma/STAT-1alpha exert their inhibitory effects by affecting multiple aspects of MMP-9 gene transcription.

Class II major histocompatibility complex (MHC) proteins are important for specific recognition of foreign antigens by the immune system. Previously we showed that 17beta-estradiol (E2) down-regulates class II MHC expression by attenuation of histone acetylation and cAMP response element binding protein (CREB)-binding protein recruitment to the class II MHC promoter. Estrogen signals through nuclear receptors to mediate genomic effects; however, estrogen is also known to mediate rapid nongenomic effects. Our observation that ER antagonists fail to prevent E2 inhibition of class II MHC expression suggests that E2 is signaling in a nonclassical manner. We find that E2, as well as the antiestrogens tamoxifen (TAM) and ICI 182,780 (ICI), inhibit class II MHC expression through activation of the c-Jun N-terminal kinase (JNK) pathway. Pharmacological JNK inhibitors reverse the inhibitory effects of E2, TAM, and ICI on class II MHC expression. E2, TAM, and ICI activate the JNK pathway and subsequently activate c-Jun and activating transcription factor-2 transcription factors. Our results demonstrate that blocking E2 activation of the JNK signaling pathway prevents estrogen-mediated attenuation of histone acetylation and CREB-binding protein recruitment to the class II MHC promoter. Collectively, these findings demonstrate that the JNK signaling pathway is necessary for E2-mediated inhibition of class II MHC expression.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases whose aberrant expression are correlated with tumor invasion and angiogenesis. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive expression of MMP-2 in tumor cells; however, the regulatory mechanisms of MMP-2 expression are not well understood. We investigated the involvement of Brg-1, the ATPase subunit of the SWI/SNF complex, in human MMP-2 gene transcription. Reconstitution of Brg-1 enhances MMP-2 transcription in Brg-1-deficient SW-13 cells. Chromatin immunoprecipitation assay demonstrates that Brg-1 is required for recruitment of Sp1, AP-2, and polymerase II to the MMP-2 promoter, whereas the binding of Sp3 to the MMP-2 promoter is decreased upon Brg-1 reconstitution. Furthermore, Sp1 interacts with Brg-1 in vivo. Restriction enzyme accessibility assays indicate that accessibility of the MMP-2 promoter region is not changed in the absence or presence of Brg-1. These results illustrate the connection between the SWI/SNF complex and optimal expression of MMP-2 and highlight the critical function of Brg-1 in regulating the recruitment of Sp1, Sp3, AP-2, and polymerase II to the MMP-2 promoter.

Major histocompatibility complex (MHC) class II proteins are important for the initiation of immune responses and are essential for specific recognition of foreign antigens by the immune system. Regulation of class II MHC expression primarily occurs at the transcriptional level. The class II transactivator protein is the master regulator that is essential for both constitutive and interferon-gamma-inducible class II MHC expression. Estrogen [17beta-estradiol (17beta-E2)] has been shown to have immunomodulatory effects. In this study, we show that 17beta-E2 down-regulates interferon-gamma inducible class II MHC protein levels on brain endothelial cells, as well as other cell types (astrocytes, fibrosacroma cells, macrophages). The inhibitory effects of 17beta-E2 on class II MHC expression are not due to changes in class II transactivator mRNA or protein levels, rather, 17beta-E2 mediates inhibition at the level of class II MHC gene expression. We demonstrate that 17beta-E2 attenuates H3 and H4 histone acetylation and cAMP response element binding protein-binding protein association with the class II MHC promoter, suggesting that 17beta-E2 inhibits class II MHC expression by a novel mechanism involving modification of the histone acetylation status of the class II MHC promoter.

Resistance to Fas-mediated apoptosis contributes to tumor evasion from the host immune system and enables tumors to mediate alternative responses such as inflammation and angiogenesis. In this study, we investigated the molecular mechanisms of the resistance to Fas-mediated apoptosis and sensitization to Fas-induced cell death by IFN-gamma in human astrocytoma cells. To address this, we investigated the expression of thirty-three genes related to the Fas signal transduction pathways using RNase protection assay in five different human astrocytoma cells. Patterns of expression of these genes were similar between different cell lines and did not correlate with sensitivity to Fas-mediated cell death. Treatment with IFN-gamma increased the mRNA expression of caspases-1, -4 and -7 in addition to those of Fas and TRAIL in a time- and dose-dependent manner. Studies using specific caspase inhibitors showed that Fas-induced cell death was mediated by caspases-1, -3 and 8 in the Fas-sensitive human astrocytoma cell lines, CRT-J and U87-MG. We further demonstrated that these caspases were proteolytically cleaved upon Fas ligation in these cells. Interestingly, caspase-1 protein expression but not that of caspase-3 nor -8 was up-regulated by IFN-gamma only in Fas-sensitive CRT-J cells but not in Fas-resistant U373-MG cells. These results collectively suggest that caspase-1, along with caspases-3 and -8, mediate Fas-induced cell death in human astrocytoma cells, and post-transcriptional regulation of caspase-1 may determine the responsiveness to IFN-gamma-induced sensitization to Fas-mediated apoptosis.

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast's gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1beta and a twofold greater IL-1beta-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1beta. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1beta and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1beta-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1beta bioactivity and autocrine IL-1beta-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.

Tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD) is essential in recruiting signaling molecules to the TNFR1 receptor complex. Interferon-gamma (IFN-gamma) is a potent activator of macrophages and uses signal transducer and activator of transcription 1-alpha (STAT1-alpha) for signal transduction. Here we demonstrate that IFN-gamma induces the formation of a nuclear-localized TRADD-STAT1-alpha complex. IFN-gamma-mediated STAT1-alpha phosphorylation was prolonged in cells with reduced TRADD expression. Moreover, we noted an increase in IFN-gamma-mediated STAT1-alpha DNA-binding activity, nuclear presence and transcriptional potential in the TRADD knockdown cells. These data indicate that TRADD may be involved in IFN-gamma signaling by forming a complex with STAT1-alpha within the nucleus and regulating IFN-gamma-mediated STAT1-alpha activation.

TNF-alpha and IFN-gamma cooperate in the activation of macrophages. TNF-alpha-dependent activation of NF-kappaB is stronger in the presence of IFN-gamma. STAT-1alpha associates with TNFR1 in TNF-alpha-treated cells, and this association attenuates TNF-alpha-mediated NF-kappaB activation. We hypothesized that nuclear localization of STAT-1alpha due to IFN-gamma signaling would preclude it from being recruited to the TNFR1 and therefore enhance TNF-alpha-induced NF-kappaB activation. In the RAW264.7 macrophage cell line, TNF-alpha treatment indeed recruits STAT-1alpha to the TNFR1, and this association is abrogated when cells are exposed to IFN-gamma. TNF-alpha treatment induces a more robust activation of NF-kappaB in STAT-1alpha-deficient cells, and restoration of STAT-1alpha inhibits TNF-alpha-dependent NF-kappaB activation. Our results suggest that a receptor-proximal level of cross-talk exists between these two cytokine pathways: IFN-gamma limits STAT-1alpha availability to the TNFR1 by depleting STAT-1alpha from the cytoplasm, thus allowing for optimal NF-kappaB activation upon TNF-alpha ligation.

Oncostatin M (OSM), a cytokine of the interleukin-6 family, is expressed in rheumatoid arthritis, multiple sclerosis, multiple myeloma, and other inflammatory and neoplastic conditions. Prostaglandin E(2) (PGE(2)), an eicosanoid also associated with inflammation and cancer, has recently been shown to induce OSM expression. We report here that OSM in turn induces PGE(2) production by astrocytes and astroglioma cells. More importantly, in combination with the inflammatory mediators IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, OSM exhibits a striking synergy, resulting in up to 50-fold higher PGE(2) production by astrocytes, astroglioma, and neuroblastoma cell lines. Enhanced PGE(2) production by OSM and IL-1beta treatment is explained by their effect on cyclooxygenase-2 (COX-2), an enzyme that catalyzes the committed step in PGE(2) synthesis. Of the enzymes involved in PGE(2) biosynthesis, only COX-2 mRNA and protein levels are synergistically amplified by OSM and IL-1beta. Nuclear run-on assays demonstrate that OSM and IL-1beta synergistically upregulate transcription of the COX-2 gene, and the mRNA stability assay indicates that COX-2 mRNA is posttranscriptionally stabilized by OSM and IL-1beta. To effect synergy on the PGE(2) level, OSM signals in part through its gp130/OSMRbeta receptor, since neutralizing antibodies against gp130 and OSMRbeta, but not LIFRbeta, decrease PGE(2) production in response to OSM plus IL-1beta. SB202190 and U0126, inhibitors of p38 MAPK and ERK1/2 activation, respectively, inhibit IL-1beta and OSM upregulation of COX-2 and PGE(2), indicating that these MAPK cascades are utilized by both stimuli. This mechanism of PGE(2) amplification may be active in brain pathologies where both OSM and IL-1beta are present, such as glioblastomas and multiple sclerosis., (Copyright 2003 Wiley-Liss, Inc.)

Chloroquine, an antimalarial lysosomotropic base, is known for its anti-inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti-inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS-induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS-induced expression of pro-inflammatory cytokines such as lymphotoxin (LT)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta and IL-6 in human astroglial cells, while the same treatment suppressed LPS-induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro-inflammatory cytokines in a dose- and time-dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor-kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti- or pro-inflammatory responses in the CNS depending on the cellular context.

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.

Oncostatin-M (OSM), a pluripotent cytokine of the interleukin-6 (IL-6) family, is produced in a number of inflammatory conditions. Known sources of OSM include monocytes-macrophages and T-cells. Here we present microglia, the resident macrophages of the brain, as a source of OSM in the CNS. In this context, we describe a novel inducer of OSM, prostaglandin E(2) (PGE(2)). PGE(2) induces OSM expression in microglia, monocytes, and macrophages of human and murine origin. PGE(2) induction of OSM is mimicked by cholera toxin, an activator of stimulatory G (G(s))-proteins; by forskolin, an activator of adenylate cyclase; and by the cAMP analog, dibutyryl-cAMP. PGE(2) induction of OSM gene expression is inhibited by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, by the protein kinase A (PKA) inhibitor H-89, and by a dominant-negative PKA construct. These data indicate that PGE(2) signals via G(s)-protein-coupled receptor(s), adenylate cyclase, and PKA to induce OSM expression. Accordingly, other activators of cAMP signaling such as norepinephrine and PGE(1) induce OSM. The ability of PGE(2) to induce OSM expression was tested under more physiological conditions, using cocultures of astrocytes and monocytes. Treatment of the cocultures with IL-1beta or tumor necrosis factor-alpha (TNF-alpha) results in production of PGE(2) and OSM. PGE(2) produced in the cocultures is responsible for OSM induction, because pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, as well as depletion of PGE(2), abrogate OSM expression induced by IL-1beta or TNF-alpha. These data suggest that in the CNS, OSM may be produced through collaboration of astrocytes and macrophages-microglia.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 expression on antigen-presenting cells (including macrophages and microglia) is crucial for T-cell activation. Aberrant expression of CD40 has been associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We have recently shown that the cytokine interferon (IFN)-gamma is the most potent inducer of CD40 expression in macrophages and microglia, and this induction is mediated by the IFN-gamma-activated transcription factor STAT-1alpha and constitutively expressed PU.1 and/or Spi-B. In this study, we have discovered that a major component of IFN-gamma-induced CD40 expression involves the endogenous production of the cytokine TNF-alpha. The inclusion of anti-TNF-alpha-neutralizing antibody significantly inhibits IFN-gamma-induced CD40 mRNA and CD40 promoter activity. IFN-gamma-induced CD40 protein expression is attenuated in TNF-alpha-deficient microglia and can be restored with exogenous TNF-alpha. Site-directed mutagenesis studies demonstrate that three of the four NF-kappaB elements in the CD40 promoter are required for IFN-gamma-induced CD40 promoter activity. IFN-gamma treatment leads to the activation of NF-kappaB in a time-dependent manner, which is inhibited in the presence of anti-TNF-alpha-neutralizing antibody. These results indicate that IFN-gamma-induced TNF-alpha production and subsequent NF-kappaB activation are integral parts of the mechanism of IFN-gamma-induced CD40 expression.