Interferon regulatory factor 3 plays an anti-inflammatory role in microglia by activating the PI3K/Akt pathway

<p>Abstract</p> <p>Background</p> <p>Microglia are the principal cells involved in the innate immune response in the CNS. Activated microglia produce a number of proinflammatory cytokines implicated in neurotoxicity but they also are a major source of anti-inflammatory cytokines, antiviral proteins and growth factors. Therefore, an immune therapy aiming at suppressing the proinflammatory phenotype while enhancing the anti-inflammatory, growth promoting phenotype would be of great benefit. In the current study, we tested the hypothesis that interferon regulatory factor 3 (IRF3), a transcription factor required for the induction of IFNβ following TLR3 or TLR4 activation, is critical to the microglial phenotype change from proinflammatory to anti-inflammatory, and that this phenotype change can be greatly facilitated by IRF3 gene transfer.</p> <p>Methods</p> <p>Cultures of primary human fetal microglia were transduced with IRF3 using recombinant adenovirus (Ad-IRF3) and subjected to microarray analysis, real-time PCR, immunoblotting and ELISA to determine inflammatory gene expression. Two different types of immune stimuli were tested, the TLR ligands, poly IC (PIC) and LPS, and the proinflammatory cytokines, IL-1/IFNγ. In addition, the role of the PI3K/Akt pathway was examined by use of a pharmacological inhibitor, LY294002.</p> <p>Results</p> <p>Our results show that Ad-IRF3 suppressed proinflammatory genes (IL-1α, IL-1β, TNFα, IL-6, IL-8 and CXCL1) and enhanced anti-inflammatory genes (IL-1 receptor antagonist, IL-10 and IFNβ) in microglia, regardless of the cell stimuli applied. Furthermore, Ad-IRF3 activated Akt, and LY294002 reversed the effects of Ad-IRF3 on microglial inflammatory gene expression. pAkt was critical in LPS- or PIC-induced production of IL-10 and IL-1ra. Significantly, microglial IFNβ protein production was also dependent on pAkt and required both Ad-IRF3 and immunological stimuli (PIC > IL-1/IFNγ). pAkt played much less prominent and variable roles in microglial proinflammatory gene expression. This anti-inflammatory promoting role of PI3K/Akt appeared to be specific to microglia, since astrocyte proinflammatory gene expression (as well as IFNβ expression) required PI3K/Akt.</p> <p>Conclusions</p> <p>Our results show a novel anti-inflammatory role for the PI3K/Akt signaling pathway in microglia. They further suggest that IRF3 gene therapy could facilitate the microglial phenotype switch from proinflammatory ("M1-like") to anti-inflammatory and immunomodulatory ("M2-like"), in part, by augmenting the level of pAkt.</p

Regulation of Progranulin Expression in Human Microglia and Proteolysis of Progranulin by Matrix Metalloproteinase-12 (MMP-12)

Background: The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia. Goal: In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells. Methods: Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNc), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined. Results: Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNc, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation

Aberrant Expression of Interleukin-1β and Inflammasome Activation in Human Malignant Gliomas

<div><p>Objective</p><p>Glioblastoma is the most frequent and malignant form of primary brain tumor with grave prognosis. Mounting evidence supports that chronic inflammation (such as chronic overactivation of IL-1 system) is a crucial event in carcinogenesis and tumor progression. IL-1 also is an important cytokine with species-dependent regulations and roles in CNS cell activation. While much attention is paid to specific anti-tumor immunity, little is known about the role of chronic inflammation/innate immunity in glioma pathogenesis. In this study, we examined whether human astrocytic cells (including malignant gliomas) can produce IL-1 and its role in glioma progression.</p><p>Methods</p><p>We used a combination of cell culture, real-time PCR, ELISA, western blot, immunocytochemistry, siRNA and plasmid transfection, micro-RNA analysis, angiogenesis (tube formation) assay, and neurotoxicity assay.</p><p>Results</p><p>Glioblastoma cells produced large quantities of IL-1 when activated, resembling macrophages/microglia. The activation signal was provided by IL-1 but not the pathogenic components LPS or poly IC. Glioblastoma cells were highly sensitive to IL-1 stimulation, suggesting its relevance <i>in vivo</i>. In human astrocytes, IL-1β mRNA was not translated to protein. Plasmid transfection also failed to produce IL-1 protein, suggesting active repression. Suppression of microRNAs that can target IL-1α/β did not induce IL-1 protein. Glioblastoma IL-1β processing occurred by the NLRP3 inflammasome, and ATP and nigericin increased IL-1β processing by upregulating NLRP3 expression, similar to macrophages. RNAi of annexin A2, a protein strongly implicated in glioma progression, prevented IL-1 induction, demonstrating its new role in innate immune activation. IL-1 also activated Stat3, a transcription factor crucial in glioma progression. IL-1 activated glioblastoma-conditioned media enhanced angiogenesis and neurotoxicity.</p><p>Conclusions</p><p>Our results demonstrate unique, species-dependent immune activation mechanisms involving human astrocytes and astrogliomas. Specifically, the ability to produce IL-1 by glioblastoma cells may confer them a mesenchymal phenotype including increased migratory capacity, unique gene signature and proinflammatory signaling.</p></div

Glioma releases neurotoxic substances.

<p>(A, B) GBM2 conditioned media (CM) were prepared by incubating cells with medium alone (Ctr), IL-1ra (1 µg/ml), IL-1α (10 ng/ml) or both, as indicated (right panel, B). Control cultures were exposed to fresh medium (Ctr medium) or recombinant IL-1α (left panel, A). Human fetal mixed neuronal cultures were exposed to CM and neurotoxicity was measured by trypan blue exclusion at 72 h post incubation. Arrows indicate the examples of dead (blue) cells. (C) Number of trypan blue+ cells are counted in each conditions and data are pooled from three independent experiments. *** p<0.001, **p<0.01, * p<0.005 Neurotoxic activity was induced by glioma secretome (activated or non-activated), as well as recombinant IL-1α. IL-1ra completed reversed neurotoxicity in glioma CM.</p

miR-132, miR-212 or the proteasome inhibitor lactacystin do not affect the expression of IL-1 in human astrocytes.

<p>Human astrocytes were transfected with specific or control anti-miR inhibitors (10 nM) for 48 h, and then stimulated with IL-1α for 24 h. (A) The expression of miR-132 was quantified by TaqMan real-time RT-PCR. Specific anti-miRs but not control anti-miR suppress miR-132 expression. (B) The culture supernatants were examined for the presence of IL-1β protein by sensitive ELISA with a lower detection limit of 3.9 pg/ml. There was no detectable IL-1β protein production in any of the human astrocyte cultures examined. (C) The effect of the proteasome inhibitor lactacystin on astrocyte IL-1β was examined. Astrocytes were treated with lactacystin at indicated concentrations with or without IL-1α, then cell lysates were subjected to ELISA after 24 h. IL-1β protein was undetectable under any conditions. Mean ± SD from triplicate cultures.</p

IL-1-activated U87 secretome promotes angiogenesis <i>in vitro</i>.

<p>Tube formation assay was performed using the BD BioCoat Angiogenesis System-Endothelial Cells Tube Formation Matrigel Matrix 96-well plate as described in the Materials and Methods. U87 conditioned media (CM) were prepared by incubating cultures with medium alone (Ctr), IL-1β (10 ng/ml), or IL-1β plus IL-1ra (1 µg/ml). HUVEC at 2.5×10⁴ cells were suspended in U87 CM and then added to the plates for 18 h at 37°C. Cells were then fixed in 4% PFA and viewed by phase-contrast microscopy. (A) Representative photography. (B) The number of closed network of vessel-like tubes was counted from three experiments. Data are mean ± SD ***p<0.001.</p

IL-1 expression in patient-derived glioblastoma cells.

<p>Three patient-derived glioma cell lines (one low grade astrocytoma and two GBM cells) were tested for IL-1 protein production spontaneously (Ctr) and following IL-1/IFNγ stimulation. ELISA was performed with cell lysates (intracellular = i.c.) and culture supernatants (secreted = s). (A) IL-1 protein production was seen in GBM cells only. In one of them (GBM2), IL-1 production occurred spontaneously (Ctr) and was potentiated by IL-1 (± IFNγ). A portion of IL-1 was secreted. (B) Dose-dependent induction of IL-1: production of IL-1β was examined in GBM2 cells after stimulation with different concentrations (0.1 pg/ml to 100 ng/ml) of IL-1α. (C) IL-1β immunostain of unstimulated GBM2 cells reveals scattered positive cells, while preimmune IgG (negative control) showed no positive cells. Data shown are mean ± SD (n = 3), ***p<0.001, **p<0.01, *p<0.05 vs. control (Ctr).</p