Mindy S. Lo

Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Mediate Inhibition of Stat2 Expression and Alpha/Beta Interferon Responsiveness

ABSTRACT Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-α/β signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-α/β response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.

New insights into the immunopathogenesis of systemic lupus erythematosus

The aetiology of systemic lupus erythematosus (SLE) is multifactorial, and includes contributions from the environment, stochastic factors, and genetic susceptibility. Great gains have been made in understanding SLE through the use of genetic variant identification, mouse models, gene expression studies, and epigenetic analyses. Collectively, these studies support the concept that defective clearance of immune complexes and biological waste (such as apoptotic cells), neutrophil extracellular traps, nucleic acid sensing, lymphocyte signalling, and interferon production pathways are all central to loss of tolerance and tissue damage. Increased understanding of the pathogenesis of SLE is driving a renewed interest in targeted therapy, and researchers are now on the verge of developing targeted immunotherapy directed at treating either specific organ system involvement or specific subsets of patients with SLE. Accordingly, this Review places these insights within the context of our current understanding of the pathogenesis of SLE and highlights pathways that are ripe for therapeutic targeting.

cAMP response element modulator α controls IL2 and IL17A expression during CD4 lineage commitment and subset distribution in lupus

Appropriate expression of IL-2 plays a central role during the priming and differentiation of T cells. A tight balance between IL-2 and the effector cytokine IL-17A is essential for immune homeostasis. Epigenetic mechanisms have been documented as a key component of cytokine regulation during lineage commitment. The molecular mechanisms that induce chromatin remodeling are less well understood. We investigated epigenetic regulators that mediate the diametric expression of IL-2 and IL-17A in naive, central memory, and effector memory CD4+ T cells. We demonstrate that cAMP response modulator (CREM)α contributes to epigenetic remodeling of IL2 in effector memory T cells through the recruitment of DNMT3a. CREMα also reduces CpG-DNA methylation of the IL17A promoter. CREMα expression is regulated at the epigenetic level by CpG-DNA methylation, which allows increased CREMα expression in effector memory CD4+ T cells. T cells from patients with systemic lupus erythematosus (SLE) express increased levels of CREMα and exhibit a phenotype that is similar to effector memory CD4+ T cells with epigenetically predetermined expression patterns of IL-2 and IL-17A. We conclude that CREMα mediates epigenetic remodeling of the IL2 and IL17A gene during T-cell differentiation in favor of effector memory T cells in health and disease.

Treatment of systemic lupus erythematosus: new advances in targeted therapy

Treatment for systemic lupus erythematosus (SLE) has traditionally been restricted to broad‐based immunosuppression, with glucocorticoids being central to care. Recent insights into lupus pathogenesis promise new, selective therapies with more favorable side effect profiles. The best example of this is belimumab, which targets the B cell cytokine BLyS and has now received Food and Drug Administration (FDA) approval for its use in SLE. Strategies targeting other cytokines, such as interleukin 6 (IL‐6) and interferon (IFN)‐α, are also on the horizon. Blockade of costimulatory interactions between immune cells offers another opportunity for therapeutic intervention, as do small molecule inhibitors that interfere with cell signaling pathways. We review here the current strategies for SLE treatment, with particular focus on therapies now in active pharmaceutical development. We will also discuss new understandings in lupus pathogenesis that may lead to future advances in therapy.

Modification of the Stat1 SH2 Domain Broadly Improves Interferon Efficacy in Proportion to p300/CREB-binding Protein Coactivator Recruitment

A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference inspontaneousdimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-β (that depends on Stat1/Stat2 heterodimerization) as well as IFN-γ (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-γ. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy.

The impact of RSV, adenovirus, influenza, and parainfluenza infection in pediatric patients receiving stem cell transplant, solid organ transplant, or cancer chemotherapy.

RVIs are a significant cause of morbidity and mortality in immunocompromised children. We analyzed the characteristics and outcomes of infection by four respiratory viruses (RSV, adenovirus, influenza, and parainfluenza) treated at a pediatric tertiary care hospital in a retrospective cohort of patients who had received cancer chemotherapy, hematopoietic stem cell, or SOT. A total of 208 infections were studied among 166 unique patients over a time period of 1993–2006 for transplant recipients, and 2000–2005 for patients with cancer. RSV was the most common respiratory virus identified. There were 17 (10% of all patients) deaths overall, of which 12 were at least partly attributed to the presence of a RVI. In multivariate models, LRT symptoms in the absence of upper respiratory symptoms on presentation (OR 10.2 [2.3, 45.7], p = 0.002) and adenoviral infection (OR 3.7 [1.1, 12.6], p = 0.034) were significantly associated with poor outcome, defined as death or disability related to RVI. All of the deaths occurred in patients who had received either solid organ or HSCT. There were no infections resulting in death or disability in the cancer chemotherapy group.

Outcomes in Hospitalized Pediatric Patients With Systemic Lupus Erythematosus

OBJECTIVE: Disparities in outcomes among adults with systemic lupus erythematosus (SLE) have been documented. We investigated associations between sociodemographic factors and volume of annual inpatient hospital admissions with hospitalization characteristics and poor outcomes among patients with childhood-onset SLE. METHODS: By using the Pediatric Health Information System, we analyzed admissions for patients aged 3 to <18 years at index admission with ≥1 International Classification of Diseases, Ninth Revision code for SLE from January 2006 to September 2011. Summary statistics and univariable analyses were used to examine demographic characteristics of hospital admissions, readmissions, and lengths of stay. We used multivariable logistic regression analyses, controlling for patient gender, age, race, ethnicity, insurance type, hospital volume, US census region, and severity of illness, to examine risk factors for poor outcomes. RESULTS: A total of 10 724 admissions occurred among 2775 patients over the study period. Hispanic patients had longer lengths of stay, more readmissions, and higher in-hospital mortality. In multivariable analysis, African American race was significantly associated with ICU admission. African American race and Hispanic ethnicity were associated with end-stage renal disease and death. Volume of patients with SLE per hospital and hospital location were not significantly associated with outcomes. CONCLUSIONS: In this cohort of hospitalized children with SLE, race and ethnicity were associated with outcomes. Further studies are needed to elucidate the relationship between sociodemographic factors and poor outcomes in patients with childhood-onset SLE.

Next-Generation Sequencing Reveals Restriction and Clonotypic Expansion of Treg Cells in Juvenile Idiopathic Arthritis.

Treg cell–mediated suppression of Teff cells is impaired in juvenile idiopathic arthritis (JIA); however, the basis for this dysfunction is incompletely understood. Animal models of autoimmunity and immunodeficiency demonstrate that a diverse Treg cell repertoire is essential to maintain Treg cell function. The present study was undertaken to investigate the Treg and Teff cell repertoires in JIA.

Predicting Coronary Artery Aneurysms in Kawasaki Disease at a North American Center: An Assessment of Baseline z Scores

Background Accurate risk prediction of coronary artery aneurysms (CAAs) in North American children with Kawasaki disease remains a clinical challenge. We sought to determine the predictive utility of baseline coronary dimensions adjusted for body surface area (z scores) for future CAAs in Kawasaki disease and explored the extent to which addition of established Japanese risk scores to baseline coronary artery z scores improved discrimination for CAA development. Methods and Results We explored the relationships of CAA with baseline z scores; with Kobayashi, Sano, Egami, and Harada risk scores; and with the combination of baseline z scores and risk scores. We defined CAA as a maximum z score (zMax) ≥2.5 of the left anterior descending or right coronary artery at 4 to 8 weeks of illness. Of 261 patients, 77 patients (29%) had a baseline zMax ≥2.0. CAAs occurred in 15 patients (6%). CAAs were strongly associated with baseline zMax ≥2.0 versus <2.0 (12 [16%] versus 3 [2%], respectively, P<0.001). Baseline zMax ≥2.0 had a C statistic of 0.77, good sensitivity (80%), and excellent negative predictive value (98%). None of the risk scores alone had adequate discrimination. When high‐risk status per the Japanese risk scores was added to models containing baseline zMax ≥2.0, none were significantly better than baseline zMax ≥2.0 alone. Conclusions In a North American center, baseline zMax ≥2.0 in children with Kawasaki disease demonstrated high predictive utility for later development of CAA. Future studies should validate the utility of our findings.

Methods and Protocols to Study T Cell Signaling Abnormalities in Human Systemic Lupus Erythematosus

Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). T cell receptor (TCR/CD3)-mediated stimulation of SLE T cells show increased protein tyrosine phosphorylation of cellular proteins with faster kinetics, heightened calcium flux response, and decreased IL-2 production. The molecular mechanisms of T cell signaling abnormalities in SLE T cells are complex. Current research has been directed towards investigating various factors that contribute to abnormal tyrosine phosphorylation, intracellular calcium response, and cytokine production. Central to this dysfunction is the aberrant expression and function of the TCR/CD3ζ chain. Latest developments suggest multiple explanations are involved, including altered receptor structure, supramolecular assembly, modulation of membrane clustering, aberrant cellular distribution, and pre-compartmentalization with lipid-rafts. The methods and protocols described here pertaining to T cell signaling abnormalities in SLE T cells are optimized in many ways and are derived by the combined task and continuous efforts of many researchers in the lab over a long period of time. These simplified protocols can be readily applied to study T cell signaling abnormalities in SLE to identify the genetic, molecular, and biochemical factors contributing to aberrant immune cell function and unravel the pathophysiology of SLE.