Type 1 interferon suppresses expression and glucocorticoid induction of glucocorticoid-induced leucine zipper (GILZ).

SLE is a systemic multi-organ autoimmune condition associated with reduced life expectancy and quality of life. Glucocorticoids (GC) are heavily relied on for SLE treatment but are associated with detrimental metabolic effects. Type 1 interferons (IFN) are central to SLE pathogenesis and may confer GC insensitivity. Glucocorticoid-induced leucine zipper (GILZ) mediates many effects of GC relevant to SLE pathogenesis, but the effect of IFN on GC regulation of GILZ is unknown. We performed in vitro experiments using human PBMC to examine the effect of IFN on GILZ expression. JAK inhibitors tofacitinib and tosylate salt were used in vivo and in vitro respectively to investigate JAK-STAT pathway dependence of our observations. ChiP was performed to examine glucocorticoid receptor (GR) binding at the GILZ locus. Several public data sets were mined for correlating clinical data. High IFN was associated with suppressed GILZ and reduced GILZ relevant to GC exposure in a large SLE population. IFN directly reduced GILZ expression and suppressed the induction of GILZ by GC in vitro in human leukocytes. IFN actions on GILZ expression were dependent on the JAK1/Tyk2 pathway, as evidenced by loss of the inhibitory effect of IFN on GILZ in the presence of JAK inhibitors. Activation of this pathway led to reduced GR binding in key regulatory regions of the GILZ locus. IFN directly suppresses GILZ expression and GILZ upregulation by GC, indicating a potential mechanism for IFN-induced GC resistance. This work has important implications for the ongoing development of targeted GC-sparing therapeutics in SLE.

GILZ regulates type I interferon release and sequesters STAT1.

Glucocorticoids remain a mainstay of modern medicine due to their ability to broadly suppress immune activation. However, they cause severe adverse effects that warrant urgent development of a safer alternative. The glucocorticoid-induced leucine zipper (GILZ) gene, TSC22D3, is one of the most highly upregulated genes in response to glucocorticoid treatment, and reduced GILZ mRNA and protein levels are associated with increased severity of inflammation in systemic lupus erythematosus (SLE), Ulcerative Colitis, Psoriasis, and other autoimmune/autoinflammatory diseases. Here, we demonstrate that low GILZ permits expression of a type I interferon (IFN) signature, which is exacerbated in response to TLR7 and TLR9 stimulation. Conversely, overexpression of GILZ prevents IFN-stimulated gene (ISG) up-regulation in response to IFNα. Moreover, GILZ directly binds STAT1 and prevents its nuclear translocation, thereby negatively regulating IFN-induced gene expression and the auto-amplification loop of the IFN response. Thus, GILZ powerfully regulates both the expression and action of type I IFN, suggesting restoration of GILZ as an attractive therapeutic strategy for reducing reliance on glucocorticoids.

Non-apoptotic Cell Death Control of Neutrophil Extracellular Trap Formation.

Neutrophil extracellular traps (NETs) are networks of chromatin and microbicidal proteins released by neutrophils in response to infection and tissue damage. Although classically viewed as a discrete biochemical and cellular process in neutrophils, the effector pathways integrating diverse upstream activating signals to control the formation of NETs (NETosis) are poorly defined. Cell death is one such common unifying endpoint of neutrophils, with several bona fide non-apoptotic cell death agonists now described to initiate NETosis. Integrating these new genetic findings into our existing knowledge of NETosis will likely reveal varied cellular and biochemical processes controlling NET release and specific anti-microbial and inflammatory effector functions of NETs triggered by specific non-apoptotic cell death. To facilitate investigation of regulated cell death pathways in NETosis, we offer a detailed protocol for neutrophil purification from mouse bone marrow and human blood, analysis of NETs by flow cytometry, and validation by immunogold electron microscopy. Future studies may better define cell death-specific forms of NETosis and their influence on inflammation and autoimmunity.

Large-scale survey and database of high affinity ligands for peptide recognition modules.

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large-scale peptide-phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM-ligand complexes, indicating that a large majority of the phage-derived peptides are likely to target natural peptide-binding sites and could thus act as inhibitors of natural protein-protein interactions. The complete dataset has been assembled in an online database (http://www.prm-db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

A motive for killing: effector functions of regulated lytic cell death.

Immunological responses activated by pathogen recognition come in many guises. The proliferation, differentiation and recruitment of immune cells, and the production of inflammatory cytokines and chemokines are central to lifelong immunity. Cell death serves as a key function in the resolution of innate and adaptive immune responses. It also coordinates cell-intrinsic effector functions to restrict infection. Necrosis was formally considered a passive form of cell death or a consequence of pathogen virulence factor expression, and necrotic tissue is frequently associated with infection. However, there is now emerging evidence that points to a role for regulated forms of necrosis, such as pyroptosis and necroptosis, driving inflammation and shaping the immune response.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.

Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling.

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen ß-strands arranged into two ß-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

Structure and function of the SPRY/B30.2 domain proteins involved in innate immunity.

The SPRY domain is a protein interaction module found in 77 murine and ~100 human proteins, and is implicated in important biological pathways, including those that regulate innate and adaptive immunity. The current definition of the SPRY domain is based on a sequence repeat discovered in the splA kinase and ryanodine receptors. The greater SPRY family is divided into the B30.2 (which contains a PRY extension at the N-terminus) and "SPRY-only" sub-families. In this brief review, we examine the current structural and biochemical literature on SPRY/B30.2 domain involvement in key immune processes and highlight a PRY-like 60 amino acid region in the N-terminus of "SPRY-only" proteins. Phylogenetic, structural, and functional analyses suggest that this N-terminal region is related to the PRY region of B30.2 and should be characterized as part of an extended SPRY domain. Greater understanding of the functional importance of the N-terminal region in "SPRY only" proteins will enhance our ability to interrogate SPRY interactions with their respective binding partners.

TLR regulation of SPSB1 controls inducible nitric oxide synthase induction.

The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-ß (TRIF), which results in the transcription of a small array of genes, including IFN-ß. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.