Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown.
We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients.
We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes.
We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation–related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients.
We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.
Rhinovirus; asthma; asthma exacerbation; atopy; interferon; innate immunity; cytokine; TH2 inflammation; suppressor of cytokine signaling; AA, Atopic asthma; BAL, Bronchoalveolar lavage; BEC, Bronchial epithelial cell; CISH, Cytokine-inducible SH2-containing protein; GFP, Green fluorescent protein; ISG, Interferon-stimulated gene; ISRE, Interferon-stimulated response element; KC, Keratinocyte-derived chemokine; LIX, LPS-induced CXC chemokine; NANA, Nonatopic nonasthmatic; NF-κB, Nuclear factor κB; NLS, Nuclear localization sequence; polyI:C, Polyinosinic-polycytidylic acid; SOCS, Suppressor of cytokine signaling; SOCS1wt, Full-length wild-type human SOCS1; STAT, Signal transducer and activator of transcription; STRA, Severe therapy-resistant atopic asthma
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 β-strands arranged into two β-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 (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.
B30.2; E3 ligase; interferon; oestrogen-responsive finger protein (Efp); retinoic acid-inducible gene-I (RIG-I); tripartite motif (TRIM); tripartite motif-containing 25 (TRIM25)
The discovery of the Suppressor Of Cytokine Signaling (SOCS) family of proteins has resulted in a significant body of research dedicated to dissecting their biological functions and the molecular mechanisms by which they achieve potent and specific inhibition of cytokine and growth factor signaling. The Australian contribution to this field has been substantial, with the initial discovery of SOCS1 by Hilton, Starr and colleagues (discovered concurrently by two other groups) and the following work, providing a new perspective on the regulation of JAK/STAT signaling. In this review, we reflect on the critical discoveries that have lead to our current understanding of how SOCS proteins function and discuss what we see as important questions for future research.
SOCS; SOCS box; cytokine; receptor; JAK
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.
The suppressor of cytokine signaling proteins are key regulators of immunity. As yet there is no described biological role for SOCS4, despite its broad expression in cells of the immune system. Given the important role of other SOCS proteins in controlling the immune response, we have generated SOCS4-mutant mice and used a mouse influenza infection model to investigate the biological function of SOCS4. We demonstrate that mice lacking SOCS4 rapidly succumb to infection with a pathogenic H1N1 influenza virus and are hypersusceptible to infection with the less virulent H3N2 strain. This is the first demonstration of a functional phenotype in SOCS4-deficient mice. Our study reveals that in SOCS4-deficient animals, there is a dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This is associated with impaired trafficking of virus-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. Understanding the regulation of the inflammatory response to influenza is particularly relevant given the current climate concerning pandemic influenza outbreaks.
The UK Clinical Aptitude Test (UKCAT) was introduced to facilitate widening participation in medical and dental education in the UK by providing universities with a continuous variable to aid selection; one that might be less sensitive to the sociodemographic background of candidates compared to traditional measures of educational attainment. Initial research suggested that males, candidates from more advantaged socioeconomic backgrounds and those who attended independent or grammar schools performed better on the test. The introduction of the A* grade at A level permits more detailed analysis of the relationship between UKCAT scores, secondary educational attainment and sociodemographic variables. Thus, our aim was to further assess whether the UKCAT is likely to add incremental value over A level (predicted or actual) attainment in the selection process.
Data relating to UKCAT and A level performance from 8,180 candidates applying to medicine in 2009 who had complete information relating to six key sociodemographic variables were analysed. A series of regression analyses were conducted in order to evaluate the ability of sociodemographic status to predict performance on two outcome measures: A level ‘best of three’ tariff score; and the UKCAT scores.
In this sample A level attainment was independently and positively predicted by four sociodemographic variables (independent/grammar schooling, White ethnicity, age and professional social class background). These variables also independently and positively predicted UKCAT scores. There was a suggestion that UKCAT scores were less sensitive to educational background compared to A level attainment. In contrast to A level attainment, UKCAT score was independently and positively predicted by having English as a first language and male sex.
Our findings are consistent with a previous report; most of the sociodemographic factors that predict A level attainment also predict UKCAT performance. However, compared to A levels, males and those speaking English as a first language perform better on UKCAT. Our findings suggest that UKCAT scores may be more influenced by sex and less sensitive to school type compared to A levels. These factors must be considered by institutions utilising the UKCAT as a component of the medical and dental school selection process.
Medical student selection; Educational attainment; Aptitude tests; UKCAT; Socio-economic factors
Measures used for medical student selection should predict future performance during training. A problem for any selection study is that predictor-outcome correlations are known only in those who have been selected, whereas selectors need to know how measures would predict in the entire pool of applicants. That problem of interpretation can be solved by calculating construct-level predictive validity, an estimate of true predictor-outcome correlation across the range of applicant abilities.
Construct-level predictive validities were calculated in six cohort studies of medical student selection and training (student entry, 1972 to 2009) for a range of predictors, including A-levels, General Certificates of Secondary Education (GCSEs)/O-levels, and aptitude tests (AH5 and UK Clinical Aptitude Test (UKCAT)). Outcomes included undergraduate basic medical science and finals assessments, as well as postgraduate measures of Membership of the Royal Colleges of Physicians of the United Kingdom (MRCP(UK)) performance and entry in the Specialist Register. Construct-level predictive validity was calculated with the method of Hunter, Schmidt and Le (2006), adapted to correct for right-censorship of examination results due to grade inflation.
Meta-regression analyzed 57 separate predictor-outcome correlations (POCs) and construct-level predictive validities (CLPVs). Mean CLPVs are substantially higher (.450) than mean POCs (.171). Mean CLPVs for first-year examinations, were high for A-levels (.809; CI: .501 to .935), and lower for GCSEs/O-levels (.332; CI: .024 to .583) and UKCAT (mean = .245; CI: .207 to .276). A-levels had higher CLPVs for all undergraduate and postgraduate assessments than did GCSEs/O-levels and intellectual aptitude tests. CLPVs of educational attainment measures decline somewhat during training, but continue to predict postgraduate performance. Intellectual aptitude tests have lower CLPVs than A-levels or GCSEs/O-levels.
Educational attainment has strong CLPVs for undergraduate and postgraduate performance, accounting for perhaps 65% of true variance in first year performance. Such CLPVs justify the use of educational attainment measure in selection, but also raise a key theoretical question concerning the remaining 35% of variance (and measurement error, range restriction and right-censorship have been taken into account). Just as in astrophysics, ‘dark matter’ and ‘dark energy’ are posited to balance various theoretical equations, so medical student selection must also have its ‘dark variance’, whose nature is not yet properly characterized, but explains a third of the variation in performance during training. Some variance probably relates to factors which are unpredictable at selection, such as illness or other life events, but some is probably also associated with factors such as personality, motivation or study skills.
Medical student selection; Undergraduate performance; Postgraduate performance; Educational attainment; Aptitude tests; Criterion-related construct validity; Range restriction; Right censorship; Grade inflation; Markov Chain Monte Carlo algorithm
Most UK medical schools use aptitude tests during student selection, but large-scale studies of predictive validity are rare. This study assesses the United Kingdom Clinical Aptitude Test (UKCAT), and its four sub-scales, along with measures of educational attainment, individual and contextual socio-economic background factors, as predictors of performance in the first year of medical school training.
A prospective study of 4,811 students in 12 UK medical schools taking the UKCAT from 2006 to 2008 as a part of the medical school application, for whom first year medical school examination results were available in 2008 to 2010.
UKCAT scores and educational attainment measures (General Certificate of Education (GCE): A-levels, and so on; or Scottish Qualifications Authority (SQA): Scottish Highers, and so on) were significant predictors of outcome. UKCAT predicted outcome better in female students than male students, and better in mature than non-mature students. Incremental validity of UKCAT taking educational attainment into account was significant, but small. Medical school performance was also affected by sex (male students performing less well), ethnicity (non-White students performing less well), and a contextual measure of secondary schooling, students from secondary schools with greater average attainment at A-level (irrespective of public or private sector) performing less well. Multilevel modeling showed no differences between medical schools in predictive ability of the various measures. UKCAT sub-scales predicted similarly, except that Verbal Reasoning correlated positively with performance on Theory examinations, but negatively with Skills assessments.
This collaborative study in 12 medical schools shows the power of large-scale studies of medical education for answering previously unanswerable but important questions about medical student selection, education and training. UKCAT has predictive validity as a predictor of medical school outcome, particularly in mature applicants to medical school. UKCAT offers small but significant incremental validity which is operationally valuable where medical schools are making selection decisions based on incomplete measures of educational attainment. The study confirms the validity of using all the existing measures of educational attainment in full at the time of selection decision-making. Contextual measures provide little additional predictive value, except that students from high attaining secondary schools perform less well, an effect previously shown for UK universities in general.
Medical student selection; Educational attainment; Aptitude tests; UKCAT; Socio-economic factors; Contextual measures
Since its discovery two decades ago, the activation of the JAK/STAT pathway by numerous cytokines and growth factors has resulted in it becoming one of the most well studied intracellular signalling networks. The field has progressed from the identification of the individual components, to high-resolution crystal structures of both JAK and STAT, and an understanding of the complexities of the molecular activation and deactivation cycle which results in a diverse, yet highly specific and regulated pattern of transcriptional responses. While there is still more to learn, we now appreciate how disruption and de-regulation of this pathway can result in clinical disease and look forward to adoption of the next generation of JAK inhibitors in routine clinical treatment.
JAK; STAT; signalling; cytokine; receptor; SOCS
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 mechanism by which Suppressor of Cytokine Signaling-3 (SOCS3) negatively regulates cytokine signaling has been widely investigated using over-expression studies in cell lines and is thought to involve interactions with both the gp130 receptor and JAK1. Here, we compare the endogenous JAK/STAT signaling pathway downstream of Leukemia Inhibitory Factor (LIF) signaling in wild type (WT) Embryonic Stem (ES) cells and in ES cells lacking either the entire Socs3 gene or bearing a truncated form of SOCS3 (SOCS3DSB) lacking the C-terminal SOCS box motif (SOCS3DSB/DSB). In SOCS3DSB/DSB cells phosphorylated JAK1 accumulated at much higher levels than in WT cells or even cells lacking SOCS3 (SOCS3-/-). In contrast enhanced activation of STAT3 and SHP2 was seen in SOCS3-/- cells. Size exclusion chromatography of cell extracts showed that in unstimulated cells, JAK1 was exclusively associated with receptors but following cytokine stimulation hyperphosphorylated JAK1 (pJAK1) appeared to dissociate from the receptor complex in a manner independent of SOCS3. In WT and SOCS3DSB/DSB cells SOCS3 was associated with pJAK1. The data suggest that dissociation of activated JAK1 from the receptor results in separate targeting of JAK1 for proteasomal degradation through a mechanism dependent on the SOCS3 SOCS box thus preventing further activation of STAT3.
SOCS3; JAK/STAT; LIF; SOCS box; embryonic stem cells
SOCS1 can regulate TLR-mediated signal transduction, yet mechanistic studies in murine macrophages have been confusing and contradictory. This study has used an adenoviral transfection system to determine the role of SOCS1 in the regulation of TNFα production by activated human monocytes. Monocytes were infected with AdV-SOCS1 or with an empty vector control, AdV-GFP, for 24 h prior to activation with the TLR4 ligand, LPS. SOCS1 did not regulate TNFα mRNA or protein production within the first two hours of TLR4 activation. However, SOCS1 suppressed the sustained production of TNFα by primary human monocytes and synovial fluid macrophages ex vivo. In addition, SOCS1 regulated the production of IL-6, but not IL-10, by monocytes. Analysis of the early signaling pathway downstream of TLR4 demonstrated that SOCS1 had no regulatory effect on the activation or on the DNA binding capacity of NFκB. The late effects of LPS are mediated in part through the MyD88-independent pathway activating IRF3 and initiating the production of IFNβ. In response to adenoviral infection and prior to LPS exposure, monocytes expressed enhanced levels of IFNβ and Myxovirus A (MxA) mRNA, an anti-viral molecule characterizing IFNβ activity. These two genes were reduced in AdV-SOCS1-infected cells. Further, SOCS1 regulated IFN-dependent pathways in LPS-activated cells as evidenced by reduced IFNβ production and STAT1 phosphorylation.
Using AdV-infection to dissect SOCS1 control of IFN-dependent pathways, this study suggests that SOCS1-regulation of the IFN-dependent component of the LPS-induced TLR4 signaling pathway may contribute to the down-regulation of inflammatory cytokine production by AdV-SOCS1-infected human monocytes.
Human; Inflammation; Signal Transduction; TNF; Toll-like receptor
Macrophages lacking SPSB2 have increased NO production and enhanced pathogen-killing capabilities due to decreased ubiquitin-mediated destruction of iNOS.
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.
The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4(59–77) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.
HSQC, heteronuclear single quantum coherence; SPSB, SPRY domain- and SOCS box-containing protein; hPar-4, human prostate apoptosis response protein-4; hSPSB, human SPSB; ITC, isothermal titration calorimetry; mSPSB, murine SPSB; PDB, Protein Data Bank; GST, glutathione S-transferase; PEG, polyethylene glycol; X-ray crystallography; NMR; ITC; protein structure; protein–peptide interaction
Objectives To determine whether the UK Clinical Aptitude Test (UKCAT) adds value to the selection process for school leaver applicants to medical and dental school, and in particular whether UKCAT can reduce the socioeconomic bias known to affect A levels.
Design Cohort study
Setting Applicants to 23 UK medical and dental schools in 2006.
Participants 9884 applicants who took the UKCAT in the UK and who achieved at least three passes at A level in their school leaving examinations (53% of all applicants).
Main outcome measures Independent predictors of obtaining at least AAB at A level and
UKCAT scores at or above the 30th centile for the cohort, for the subsections and the entire test.
Results Independent predictors of obtaining at least AAB at A level were white ethnicity (odds ratio 1.58, 95% confidence interval 1.41 to 1.77), professional or managerial background (1.39, 1.22 to 1.59), and independent or grammar schooling (2.26, 2.02 to 2.52) (all P<0.001). Independent predictors of achieving UKCAT scores at or above the 30th centile for the whole test were male sex (odd ratio 1.48, 1.32 to 1.66), white ethnicity (2.17, 1.94 to 2.43), professional or managerial background (1.34, 1.17 to 1.54), and independent or grammar schooling (1.91, 1.70 to 2.14) (all P<0.001). One major limitation of the study was that socioeconomic status was not volunteered by approximately 30% of the applicants. Those who withheld socioeconomic status data were significantly different from those who provided that information, which may have caused bias in the analysis.
Conclusions UKCAT was introduced with a high expectation of increasing the diversity and fairness in selection for UK medical and dental schools. This study of a major subgroup of applicants in the first year of operation suggests that it has an inherent favourable bias to men and students from a higher socioeconomic class or independent or grammar schools. However, it does provide a reasonable proxy for A levels in the selection process.
The Suppressor Of Cytokine Signalling (SOCS) proteins were, as their name suggests, first described as inhibitors of cytokine signalling. While their actions clearly now extend to other intracellular pathways, they remain key negative regulators of cytokine and growth factor signalling. In this review we focus on the mechanics of SOCS action and the complexities of the mouse models that have underpinned our current understanding of SOCS biology.
cytokine signalling; SOCS; SH2 domain; SOCS box; inflammation
Suppressors of cytokine signaling (SOCSs) are key regulators of cytokine-induced responses in hematopoietic as well as nonhematopoietic cells. SOCS1 and SOCS3 have been shown to modulate T-cell responses, whereas the roles of other SOCS family members in the regulation of lymphocyte function are less clear. Here, we report the generation of mice with a targeted disruption of the Socs5 gene. Socs5−/− mice were born in a normal Mendelian ratio and were healthy and fertile. We found that SOCS5 is expressed in primary B and T cells in wild-type mice. However, no abnormalities in the lymphocyte compartment were seen in SOCS5-deficient mice. We examined antigen- and cytokine-induced proliferative responses in B and T cells in the absence of SOCS5 and found no deviations from the responses seen in wild-type cells. Because SOCS5 has been implicated in Th1 differentiation, we also investigated the importance of SOCS5 in T helper cell responses. Unexpectedly, SOCS5-deficient CD4 T cells showed no abnormalities in Th1/Th2 differentiation and Socs5−/− mice showed normal resistance to infection with Leishmania major. Therefore, although SOCS5 is expressed in primary B and T cells, it appears to be dispensable for the regulation of lymphocyte function.
SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/− mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/− mice weighed approximately 10% less than wild-type littermates.