The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G−F4/80−/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.

Toll-like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase family (IRAK-1, 2, M and 4). IRAK-1 and IRAK-2, known to form Myddosomes with MyD88-IRAK-4, mediate TLR7-induced TAK1-dependent NFjB activation. IRAK-M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK-M was also able to interact with MyD88-IRAK-4 to form IRAK-M Myddosome to mediate TLR7-induced MEKK3-dependent second wave NFjB activation, which is uncoupled from post-transcriptional regulation. As a result, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and IjBa), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NFjB activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.

SUMMARY Trans -autophosphorylation is among the most prevalent means of protein kinase activation, yet its molecular basis is poorly defined. In Toll-like receptor and interleukin-1 receptor signaling pathways, the kinase IRAK4 is recruited to the membrane proximal adapter MyD88 through death domain (DD) interactions, forming the oligomeric Myddosome and mediating NF-κB activation. Here we show that unphosphorylated IRAK4 dimerizes in solution with a Kd of 2.5 μM and that Myddosome assembly greatly enhances IRAK4 kinase domain (KD) autophosphorylation at sub-Kd concentrations. The crystal structure of the unphosphorylated IRAK4KD dimer captures a conformation that appears to represent the actual trans-autophosphorylation reaction, with the activation loop phosphosite of one IRAK4 monomer precisely positioned for phosphotransfer by its partner. We show dimerization is crucial for IRAK4 autophosphorylation in vitro and ligand-dependent signaling in cells. These studies identify a mechanism for oligomerization-driven allosteric autoactivation of IRAK4 that may be general to other kinases activated by autophosphorylation.

Mechanisms that degrade inflammatory mRNAs are well-known, however stabilizing mechanisms are poorly understood. Here we show that Act1, an interleukin-17 (IL-17) receptor complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, SBE (SEFIR-binding element), in the inflammatory chemokine Cxcl1 3’ UTR. mRNA-bound Act1 directs formation of three compartmentally-distinct protein-RNA complexes (RNPs) that regulate three disparate events in inflammatory mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P-bodies, and promoting translation. SBE RNA aptamers reduced IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3’ UTRs of select mRNAs to control receptor-mediated mRNA stabilization and translation during inflammation.

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

SUMMARYFungal infection stimulates the canonical C-type lectin receptors (CLRs) signaling pathway via Syk activation. Here we show that SHP-2 plays a crucial role in mediating CLRs-induced Syk activation. Genetic ablation of Shp-2 (Ptpn11) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated pro-inflammatory gene expression following fungal stimulation. Mechanistically, SHP-2 operates as a scaffold facilitating the recruitment of Syk to dectin-1 or FcRγ, through its N-SH2 domain and a previously unrecognized C-terminal ITAM motif. We demonstrate that DC-derived SHP-2 is crucial for the induction of IL-1β, IL-6 and IL-23, and anti-fungal TH17 cell responses to control Candida albicans infection. Together, these data reveal a mechanism by which SHP-2 mediates Syk activation in response to fungal infections

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Several components of the canonical pathway of response to lipopolysaccharide (LPS) are required for the EGF-dependent activation of NFκB. Conversely, the ability of Toll-like Receptor 4 (TLR4) to activate NFκB in response to LPS is impaired by down regulating EGF receptor (EGFR) expression or by using the EGFR inhibitor erlotinib. The LYN proto-oncogene (LYN) is required for signaling in both directions. LYN binds to the EGFR upon LPS stimulation, and erlotinib impairs this association. In mice, erlotinib blocks the LPS-induced expression of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and ameliorates LPS-induced endotoxity, revealing that EGFR is essential for LPS-induced signaling in vivo.