CIS is a potent checkpoint in NK cell-mediated tumor immunity.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15.

The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

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.

Cortical Layer Inversion and Deregulation of Reelin Signaling in the Absence of SOCS6 and SOCS7.

Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.

Suppressor of cytokine signaling 4 (SOCS4) protects against severe cytokine storm and enhances viral clearance during influenza infection.

Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.

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.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

The anti-inflammatory actions of IL-4 in human monocytes are not mediated by IL-10, RP105 or the kinase activity of RIPK2.

The anti-inflammatory actions of IL-4 in activated human monocytes may reflect transcriptional regulation of genes involved in TLR signaling pathways. Tailored gene arrays were conducted to profile the expression of 84 genes central to TLR-mediated signal transduction in human monocytes treated with the TLR4 ligand, LPS, with or without IL-4. In the first 3h, IL-4 down-regulated mRNA levels of LPS-induced inflammatory cytokines and chemokines, without altering mRNA levels of TLRs, TLR-related signaling molecules or multiple transcription factors. The down-regulation of inflammatory genes by IL-4 was preceded by an early up-regulation of IL-10 mRNA and protein and mRNA for receptor-interacting serine-threonine kinase 2 (RIPK2), the TLR homolog, RP105, and c-Maf, a transcription factor required for IL-10 gene expression. However, IL-4 still suppressed LPS-induced TNFα production in bone-marrow derived macrophages from IL10(-/-) mice, and in the presence of a neutralizing antibody to IL-10 in human monocytes. The up-regulation of RIPK2 and RP105 mRNA by IL-4 occurred independently of IL-10. IL-4 maintained the ability to suppress LPS-induced TNFα and enhance IL-10 production in the presence of RIPK2 kinase inhibitors. Further, IL-4 failed to up-regulate expression of RP105 at the cell surface. In conclusion, the anti-inflammatory actions of IL-4 occur independently of IL-10, RP105, and the kinase activity of RIPK2.

The anti-inflammatory effects of interleukin-4 are not mediated by suppressor of cytokine signalling-1 (SOCS1).

While it is known that the anti-inflammatory effects of interleukin (IL)-4 require new protein synthesis, the exact mechanisms by which IL-4 suppresses the production of pro-inflammatory cytokines by human monocytes and macrophages is unclear. IL-4 rapidly induced suppressor of cytokine signalling-1 (SOCS1) mRNA and protein, which peaked at 60 min, much earlier than lipopolysaccharide (LPS)-induced SOCS1 mRNA and protein which were consistently maximal 4 hr post-exposure. SOCS1 is a molecule generally considered to be induced for negative feedback of inflammatory processes. We investigated whether the early induction of SOCS1 by IL-4 was responsible for the suppression of LPS-induced tumour necrosis factor (TNF)-alpha production by IL-4. IL-4 suppressed LPS-induced TNF-alpha in freshly isolated monocytes at the level of transcription but acted by a different, possibly translational, mechanism in monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF). Despite different modes of regulation by IL-4, the kinetics and magnitude of induction of SOCS1 mRNA and protein by IL-4 in the two cell types were identical. There was no significant difference in the suppression by IL-4 of LPS-induced TNF-alpha production by bone-marrow derived macrophages from wild-type mice, Ifngamma(-/-) mice and mice lacking SOCS1 (Socs1(-/-)Ifngamma(-/-)). These data suggest that SOCS1 is not involved in the suppression of LPS-induced TNF-alpha production by IL-4.

Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling.

Influenza virus infections have a significant impact on global human health. Individuals with suppressed immunity, or suffering from chronic inflammatory conditions such as COPD, are particularly susceptible to influenza. Here we show that suppressor of cytokine signaling (SOCS) five has a pivotal role in restricting influenza A virus in the airway epithelium, through the regulation of epidermal growth factor receptor (EGFR). Socs5-deficient mice exhibit heightened disease severity, with increased viral titres and weight loss. Socs5 levels were differentially regulated in response to distinct influenza viruses (H1N1, H3N2, H5N1 and H11N9) and were reduced in primary epithelial cells from COPD patients, again correlating with increased susceptibility to influenza. Importantly, restoration of SOCS5 levels restricted influenza virus infection, suggesting that manipulating SOCS5 expression and/or SOCS5 targets might be a novel therapeutic approach to influenza.

DNAM-1 expression marks an alternative program of NK cell maturation.

Natural killer (NK) cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling and functional studies that the activating receptor DNAM-1 (CD226) identifies two distinct NK cell functional subsets: DNAM-1(+) and DNAM-1(-) NK cells. DNAM-1(+) NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1(-) NK cells that differentiate from DNAM-1(+) NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression.

Innate immunodeficiency following genetic ablation of Mcl1 in natural killer cells.

The cytokine IL-15 is required for natural killer (NK) cell homeostasis; however, the intrinsic mechanism governing this requirement remains unexplored. Here we identify the absolute requirement for myeloid cell leukaemia sequence-1 (Mcl1) in the sustained survival of NK cells in vivo. Mcl1 is highly expressed in NK cells and regulated by IL-15 in a dose-dependent manner via STAT5 phosphorylation and subsequent binding to the 3'-UTR of Mcl1. Specific deletion of Mcl1 in NK cells results in the absolute loss of NK cells from all tissues owing to a failure to antagonize pro-apoptotic proteins in the outer mitochondrial membrane. This NK lymphopenia results in mice succumbing to multiorgan melanoma metastases, being permissive to allogeneic transplantation and being resistant to toxic shock following polymicrobial sepsis challenge. These results clearly demonstrate a non-redundant pathway linking IL-15 to Mcl1 in the maintenance of NK cells and innate immune responses in vivo.

Analysis of Suppressor of Cytokine Signalling (SOCS) gene expression by real-time quantitative PCR.

The Suppressor of Cytokine Signalling (SOCS) proteins are a family of negative regulators characterized by a central SH2 domain and C-terminal SOCS box motif. Cytokine Inducible SH2-containing protein (CIS), SOCS1, 2 and 3 are rapidly upregulated in response to cytokine stimulation and act to inhibit JAK/STAT signalling by a variety of mechanisms. The expression of SOCS proteins provides a level of specificity in the control of signalling, with SOCS proteins differentially upregulated in response to individual cytokines and in various cell-types. Real-time reverse transcription (RT) quantitative polymerase chain reaction (RT-qPCR) is an established technique for quantifying mRNA in biological samples, measuring the relative expression of genes of interest and identifying single nucleotide polymorphisms. Here we describe the use of SYBR(®) Green I RT-qPCR to quantify the relative expression level of SOCS mRNA in murine bone marrow-derived macrophages (BMDM). The approach can be universally applied to different cell types and various tissues.

Suppressor of Cytokine Signaling (SOCS) 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1.

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.

The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation.

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.

SPRY domain-containing SOCS box protein 2: crystal structure and residues critical for protein binding.

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent beta-sandwich, consisting of two seven-stranded beta-sheets wrapped around a long loop that extends from the centre strands of the inner or concave beta-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.