Shahram Misaghi

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Glyburide, a sulfonylurea drug commonly used to treat type 2 diabetes, shuts down IL-1β secretion by preventing Cyropyrin activation.

Pannexin-1 Is Required for ATP Release during Apoptosis but Not for Inflammasome Activation

Apoptotic cell death is important for embryonic development, immune cell homeostasis, and pathogen elimination. Innate immune cells also undergo a very rapid form of cell death termed pyroptosis after activating the protease caspase-1. The hemichannel pannexin-1 has been implicated in both processes. In this study, we describe the characterization of pannexin-1–deficient mice. LPS-primed bone marrow-derived macrophages lacking pannexin-1 activated caspase-1 and secreted its substrates IL-1β and IL-18 normally after stimulation with ATP, nigericin, alum, silica, flagellin, or cytoplasmic DNA, indicating that pannexin-1 is dispensable for assembly of caspase-1–activating inflammasome complexes. Instead, thymocytes lacking pannexin-1, but not the P2X7R purinergic receptor, were defective in their uptake of the nucleic acid dye YO-PRO-1 during early apoptosis. Cell death was not delayed but, unlike their wild-type counterparts, Panx1−/− thymocytes failed to recruit wild-type peritoneal macrophages in a Transwell migration assay. These data are consistent with pannexin-1 liberating ATP and other yet to be defined “find me” signals necessary for macrophage recruitment to apoptotic cells.

Phosphorylation of NLRC4 is critical for inflammasome activation

NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4−/− macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd−/− macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1

ABSTRACT Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 β-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1C subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1C, suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G1-S transition.

DNA targets of AID evolutionary link between antibody somatic hypermutation and class switch recombination.

As part of the adaptive immune response, B cells alter their functional immunoglobulin (Ig) receptor genes through somatic hypermutation (SHM) and/or class switch recombination (CSR) via processes that are initiated by activation induced cytidine deaminase (AID). These genetic modifications are targeted at specific sequences known as Variable (V) and Switch (S) regions. Here, we analyze and review the properties and function of AID target sequences across species and compare them with non-Ig sequences, including known translocation hotspots. We describe properties of the S sequences, and discuss species and isotypic differences among S regions. Common properties of SHM and CSR target sequences suggest that evolution of S regions might involve the duplication and selection of SHM hotspots.

NLRP3 recruitment by NLRC4 during Salmonella infection.

By engineering a mutant mouse strain that preserves the scaffolding function of NLRC4, Dixit et al. show cooperativity between it and another NOD family sensor, NLRP3.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11‐derived dihydrofolate reductase deficient host cell

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR‐deficient DG44, and DUXB11‐based DHFR deficient CHO. Current Genentech commercial full‐length antibody products have all been produced in the DUXB11‐derived DHFR‐deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11‐derived DHFR‐deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11‐based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14‐day fed batch cultures in shake flasks. In contrast, the DUXB11‐based host produced ∼0.1 g/l for both antibodies in the same 14‐day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.

Increased Targeting of Donor Switch Region and IgE in Sγ1-Deficient B Cells

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sγ1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Sμ region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA–immunized mice and in anti-CD40 plus IL-4–stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sγ1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.

PILRα Negatively Regulates Mouse Inflammatory Arthritis

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRβ activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRβ, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα−/− mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chains core N‐linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8‐KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8‐KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII‐binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632–644.