Persistently active interferon-γ pathway and expansion of T-bet+ B cells in a subset of patients with childhood-onset systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease causing significant morbidity and mortality, despite important improvements in its management in the last decades. The objective of this work is to investigate the role of IFN-γ in the pathogenesis of childhood-onset systemic lupus erythematosus (cSLE), evaluating the crosstalk between IFN-α and IFN-γ and the expression of T-bet, a transcription factor induced by IFN-γ, in B cells of patients with cSLE. Expression levels of both IFN-α and IFN-γ-induced genes were upregulated in patients with cSLE. We found increased serum levels of CXCL9 and CXCL10 in patients with cSLE. Type I IFN score decreased with initiation of immunosuppressive treatment; conversely, type II IFN score and levels of CXCL9 were not significantly affected by immunosuppressive treatment. Type II IFN score and CXCL9 were significantly higher in patients with lupus nephritis. We observed the expansion of a population of naïve B cells expressing T-bet in a cluster of patients with cSLE. IFN-γ, but not IFN-α, induced the expression of T-bet in B cells. Our data suggest that IFN-γ is hyperactive in cSLE, especially in patients with lupus nephritis, and it is not modulated by therapy. Our data reinforce the potential of IFN-γ as a therapeutic target in SLE.

Monocytes From Patients With Macrophage Activation Syndrome and Secondary Hemophagocytic Lymphohistiocytosis Are Hyperresponsive to Interferon Gamma.

Objective: To investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease. Methods: Fresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry. Results: Monocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001]. Statistically significant correlations between IFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found. Conclusion: Our data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.

IFNAR2 Deficiency Causing Dysregulation of NK Cell Functions and Presenting With Hemophagocytic Lymphohistiocytosis.

We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/ß receptor 2 (IFNAR2) gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient's cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon ex vivo stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient's natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.

Movement disorders in ADAR1 disease: Insights from a comprehensive cohort.

ADAR1 variants are associated to rare and heterogenous neurological conditions, including Aicardi-Goutières syndrome type 6, bilateral striatal necrosis, and dyschromatosis symmetrica hereditaria. Movement disorders (MDs) commonly occur in ADAR1-related diseases although a complete overview on the phenomenology has not been provided yet. Here, a cohort of 57 patients with ADAR1-related diseases, including 3 unpublished patients and 54 previously reported cases, was reviewed. Data on demographics, clinical features of MDs, genetics and biomarkers were collected and descriptive statistics, group analysis for genotype and logistic regression were run. Manifestations of MD characterized the onset of ADAR1-related disease in 60% of patients. Specifically, dystonia occurred in 39% of cases, even as severe status dystonicus, while prevalence of other MDs was lower. Patients often presented brain lesions (>90%) and progressive disease course (43%), fatal in some cases. Clinical presentation and outcome differed among patients with distinct genotype. This review shows that phenomenology of MDs in ADAR1-related diseases is wide and heterogeneous, although a severe motor syndrome (often characterized by dystonia) secondary to brain lesions represents the most common manifestation. Waiting for future development of disease-modifying treatments, an appropriate symptomatic intervention is crucial for ADAR1 patients. Accordingly, a deeper knowledge of phenomenology is fundamental.

An unusual presentation of purine nucleoside phosphorylase deficiency mimicking systemic juvenile idiopathic arthritis complicated by macrophage activation syndrome.

BACKGROUND: Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition that presents with fever, rash and arthritis. At onset systemic features are predominant and the diagnosis may be a challenge. Secondary hemophagocytic lymphohistiocytosis (sHLH) forms may be associated with different disorders, including rheumatic diseases, and this form is called macrophage activation syndrome (MAS). CXCL9 levels, a chemokine induced by IFNγ, are significantly elevated in patients with sHLH or MAS and are correlated with laboratory features of disease activity. High levels of IL-18 have been reported in patients with MAS during sJIA, as well as in some patients with sHLH and IL-18 is indeed known to induce IFNγ production. FINDINGS: We report a patient with a clinical presentation highly suggestive for systemic juvenile idiopathic arthritis (sJIA) onset complicated by MAS, and was later diagnosed with purine nucleoside phosphorylase (PNP)-deficiency with HLH. Some unusual features appeared when HLH was controlled and further investigations provided the correct diagnosis. Serum CXCL9 and IL-18 levels were found markedly elevated at disease onset, during the active phase of MAS and decreased progressively during the course. CONCLUSION: The reported case underlines the potential difficulties in discriminating sJIA from other causes of systemic inflammation. Furthermore, this supports the notion that especially in young children with a sJIA-like disease other mimicking conditions should be actively sought for. CXCL9 and IL-18 levels suggested that patients with PNP-deficiency may have a subclinical activation of the IFNγ pathway and indeed they are predisposed to develop sHLH.

Variable Clinical Phenotypes and Relation of Interferon Signature with Disease Activity in ADA2 Deficiency.

OBJECTIVE: An upregulation of type I interferon (IFN) stimulated genes [IFN score (IS)] was described in patients with adenosine deaminase 2 deficiency (DADA2). We describe the clinical course of 5 such patients and the role of IS as a marker of disease activity and severity. METHODS: Expression levels of IS were determined by quantitative real-time PCR. RESULTS: Five white patients were identified as carrying CECR1 mutations. The IS before treatment was elevated in 4 out of 5 patients and decreased after treatment. CONCLUSION: Our data confirm the high variability of DADA2 and suggest type I IS as a biomarker of disease activity.

Muscle Expression of Type I and Type II Interferons Is Increased in Juvenile Dermatomyositis and Related to Clinical and Histologic Features.

OBJECTIVE: To evaluate the expression of type I interferon (IFNα/ß)- and type II IFN (IFNγ)-inducible genes in muscle biopsy specimens from patients with juvenile dermatomyositis (DM) and to correlate their expression levels with histologic and clinical features. METHODS: Expression levels of IFN-inducible genes and proinflammatory cytokines were assessed by quantitative polymerase chain reaction in muscle biopsy specimens from patients with juvenile DM (n = 39), patients with Duchenne's muscular dystrophy (DMD), and healthy controls. Muscle biopsy sections were stained and scored for severity of histopathologic features. The charts of patients with juvenile DM were reviewed for clinical features at the time of sampling and long-term outcomes. RESULTS: Muscle expression levels of IFNα/ß-inducible genes (type I IFN score), IFNγ, IFNγ-inducible genes (type II IFN score), and tumor necrosis factor (TNF) were significantly higher in juvenile DM patients not receiving glucocorticoid therapy before muscle biopsy (n = 27) compared to DMD patients (n = 24) (type I IFN score, P < 0.0001; type II IFN score, P < 0.001; TNF, P < 0.05) and healthy controls (n = 4) (type I IFN score, P < 0.01; type II IFN score, P < 0.01; TNF, P < 0.05). Immunofluorescence staining of muscle biopsy sections from untreated juvenile DM patients showed increased immunoreactivity for IFNγ and HLA class II molecules compared to controls. Type I and type II IFN scores were correlated with typical histopathologic features of juvenile DM muscle biopsy samples, such as infiltration of endomysial CD3+ cells (type I IFN score, r = 0.68; type II IFN score, r = 0.63), perimysial CD3+ cells (type I IFN score, r = 0.59; type II IFN score, r = 0.66), CD68+ cells (type II IFN score, r = 0.46), and perifascicular atrophy (type I IFN score, r = 0.61; type II IFN score, r = 0.77). Juvenile DM patients with a high type I IFN score, a high type II IFN score, and high TNF expression levels showed more severe disease activity at biopsy (P < 0.05). In addition, juvenile DM patients with a high type II IFN score at biopsy reached clinically inactive disease significantly later than patients with low type II IFN score (log rank chi-square value 13.53, P < 0.001). CONCLUSION: The increased expression of IFN-inducible genes in the muscle in juvenile DM patients and their association with histologic and clinical features further support a pathogenic role for both type I and type II IFNs in juvenile DM.

Switched Memory B Cells Are Increased in Oligoarticular and Polyarticular Juvenile Idiopathic Arthritis and Their Change Over Time Is Related to Response to Tumor Necrosis Factor Inhibitors.

OBJECTIVE: To investigate whether abnormalities in B cell subsets in patients with juvenile idiopathic arthritis (JIA) correlate with clinical features and response to treatment. METHODS: A total of 109 patients diagnosed as having oligoarticular JIA or polyarticular JIA were enrolled in the study. B cell subsets in peripheral blood and synovial fluid were analyzed by flow cytometry. RESULTS: Switched memory B cells were significantly increased in patients compared to age-matched healthy controls (P < 0.0001). When patients were divided according to age at onset of JIA, in patients with early-onset disease (presenting before age 6 years) the expansion in switched memory B cells was more pronounced than that in patients with late-onset disease and persisted throughout the disease course. In longitudinal studies, during methotrexate (MTX) treatment, regardless of the presence or absence of active disease, the number of switched memory B cells increased significantly (median change from baseline 36% [interquartile range {IQR} 15, 66]). During treatment with MTX plus tumor necrosis factor inhibitors (TNFi), in patients maintaining disease remission, the increase in switched memory B cells was significantly lower than that in patients who experienced active disease (median change from baseline 4% [IQR -6, 32] versus 41% [IQR 11, 73]; P = 0.004). The yearly rate of increases in switched memory B cells was 1.5% in healthy controls, 1.2% in patients who maintained remission during treatment with MTX plus TNFi, 4.7% in patients who experienced active disease during treatment with MTX plus TNFi, and ~4% in patients treated with MTX alone. CONCLUSION: Switched memory B cells expand during the disease course at a faster rate in JIA patients than in healthy children. This increase is more evident in patients with early-onset JIA. TNFi treatment inhibits this increase in patients who achieve and maintain remission, but not in those with active disease.

Functional and Morphological Improvement of Dystrophic Muscle by Interleukin 6 Receptor Blockade.

The anti-inflammatory agents glucocorticoids (GC) are the only available treatment for Duchenne muscular dystrophy (DMD). However, long-term GC treatment causes muscle atrophy and wasting. Thus, targeting specific mediator of inflammatory response may be more specific, more efficacious, and with fewer side effects. The pro-inflammatory cytokine interleukin (IL) 6 is overproduced in patients with DMD and in the muscle of mdx, the animal model for human DMD. We tested the ability of inhibition of IL6 activity, using an interleukin-6 receptor (Il6r) neutralizing antibody, to ameliorate the dystrophic phenotype. Blockade of endogenous Il6r conferred on dystrophic muscle resistance to degeneration and alleviated both morphological and functional consequences of the primary genetic defect. Pharmacological inhibition of IL6 activity leaded to changes in the dystrophic muscle environment, favoring anti-inflammatory responses and improvement in muscle repair. This resulted in a functional homeostatic maintenance of dystrophic muscle. These data provide an alternative pharmacological strategy for treatment of DMD and circumvent the major problems associated with conventional therapy.