Faculty Bibliography

Background/Purpose: Using whole exome sequencing (WES), we identified a de novo mutation in DYSF encoding dysferlin in 2 unrelated patients with systemic inflammation and sterile pulmonary abscesses. Unlike dysferlin mutations that cause muscular dystrophies, the patients have no muscle disease, but a robust clinical response to IL-1 blockade suggested inflammasome activation and the presentation with sterile pulmonary abscess formation raised questions about an efferocytosis defect Methods: We characterized monocytes and neutrophil activation and function to assess the mechanism of the IL-1 dependent inflammation by flowcytometry, immunofluoroscence, ELISA, cytokine array, survival assay and we developed an efferocytosis assay. U937 cells expressing mutant cell line was used to further understand the mutation.
Result(s): Monocyte and monocyte-derived M1 and M2 macrophages (MDM) stimulated with LPS and ATP-released high IL-1 serum levels, that was higher in the DYSF patients' monocytes and M1 and M2 MDM compared to healthy controls (HC), and was comparable to the augmented IL-1 production in NOMID. Dysferlin colocalizes with NLRP3 in LPS activated monocyte and in M1 and M2 MDMs. Expression levels of ASC, and Caspase-1 were increased in the DYSF patients monocytes. M2-MDMs from both patients expressed proinflammatory mediators, high CXCL1(P< 0.05), CCL2, IL-6 and IL-8. We observed LPS and ATP activation-induced altered nucleur integrity in M2-MDMs from both patients (70% and 50% respectively) compared to healthy controls. In neutrophils, LPS and ATP activation did not increase IL-1b was not upregulated although MIF, IL-16 and IL-8 levels were observed. We hypothesized that either delayed neutrophil apoptosis or clearence contribute to the lung abscess formation. While neutrophil apoptosis was normal, patients' M2 MDM ability to clear healthy control neutrophils by efferocytosis was significantly impaired. Coculturing patients' neutrophils with macrophages from healthy donors didn't result in abnormal effereocytosis, thus suggesting an efferocytosis defect caused by mutant monocyte derived macrophages. The abnormal efferocyutosis was reproduced in dysferlin nutant U937 cells which showed a defect in phagosome maturation.
Conclusion(s): The de novo GOF mutation in dysferlin in two patients with systemic inflammation and sterile lung abscesses reveal a novel role of dysferlin in regulating inflammasome activation in monocytes and macrophage maturation and in neutriophil efferocytosis. Our data expand on a previously unidentified role of dysferlin in regulating M2-macrophage efferocytosis of neutrophils as a mechanisms for lung abscess formation

OBJECTIVE:To identify novel heterozygous LPIN2 mutations in a patient with Majeed syndrome and characterize the pathomechanisms that lead to the development of sterile osteomyelitis. METHODS:Targeted genetic analysis and functional studies assessing monocyte responses, macrophage differentiation, and osteoclastogenesis were conducted to compare the pathogenesis of Majeed syndrome to interleukin-1 (IL-1)-mediated diseases including neonatal-onset multisystem inflammatory disease (NOMID) and deficiency of the IL-1 receptor antagonist (DIRA). RESULTS:A 4-year-old girl of mixed ethnic background presented with sterile osteomyelitis and elevated acute-phase reactants. She had a 17.8-kb deletion on the maternal LPIN2 allele and a splice site mutation, p.R517H, that variably spliced out exons 10 and 11 on the paternal LPIN2 allele. The patient achieved long-lasting remission receiving IL-1 blockade with canakinumab. Compared to controls, monocytes and monocyte-derived M1-like macrophages from the patient with Majeed syndrome and those with NOMID or DIRA had elevated caspase 1 activity and IL-1β secretion. In contrast, lipopolysaccharide-stimulated, monocyte-derived, M2-like macrophages from the patient with Majeed syndrome released higher levels of osteoclastogenic mediators (IL-8, IL-6, tumor necrosis factor, CCL2, macrophage inflammatory protein 1α/β, CXCL8, and CXCL1) compared to NOMID patients and healthy controls. Accelerated osteoclastogenesis in the patient with Majeed syndrome was associated with higher NFATc1 levels, enhanced JNK/MAPK, and reduced Src kinase activation, and partially responded to JNK inhibition and IL-1 (but not IL-6) blockade. CONCLUSION/CONCLUSIONS:We report 2 novel compound heterozygous disease-causing mutations in LPIN2 in an American patient with Majeed syndrome. LPIN2 deficiency drives differentiation of proinflammatory M2-like macrophages and enhances intrinsic osteoclastogenesis. This provides a model for the pathogenesis of sterile osteomyelitis which differentiates Majeed syndrome from other IL-1-mediated autoinflammatory diseases.

BACKGROUNDUndifferentiated systemic autoinflammatory diseases (USAIDs) present diagnostic and therapeutic challenges. Chronic interferon (IFN) signaling and cytokine dysregulation may identify diseases with available targeted treatments.METHODSSixty-six consecutively referred USAID patients underwent underwent screening for the presence of an interferon signature using a standardized type-I IFN-response-gene score (IRG-S), cytokine profiling, and genetic evaluation by next-generation sequencing.RESULTSThirty-six USAID patients (55%) had elevated IRG-S. Neutrophilic panniculitis (40% vs. 0%), basal ganglia calcifications (46% vs. 0%), interstitial lung disease (47% vs. 5%), and myositis (60% vs. 10%) were more prevalent in patients with elevated IRG-S. Moderate IRG-S elevation and highly elevated serum IL-18 distinguished 8 patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, 2 patients were compound heterozygous for potentially novel LRBA mutations, 4 patients harbored potentially novel splice variants in IKBKG (which encodes NF-κB essential modulator [NEMO]), and 6 patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutières syndrome-like (AGS-like) phenotypes, 5 patients carried mutations in either SAMHD1, TREX1, PSMB8, or PSMG2. Two patients had anti-MDA5 autoantibody-positive juvenile dermatomyositis, and 7 could not be classified. Patients with LRBA, IKBKG, and SAMD9L mutations showed a pattern of IRG elevation that suggests prominent NF-κB activation different from the canonical interferonopathies CANDLE, SAVI, and AGS.CONCLUSIONSIn patients with elevated IRG-S, we identified characteristic clinical features and 3 additional autoinflammatory diseases: IL-18-mediated PAP and recurrent MAS (IL-18PAP-MAS), NEMO deleted exon 5-autoinflammatory syndrome (NEMO-NDAS), and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD). The IRG-S expands the diagnostic armamentarium in evaluating USAIDs and points to different pathways regulating IRG expression.TRIAL REGISTRATIONClinicalTrials.gov NCT02974595.FUNDINGThe Intramural Research Program of the NIH, NIAID, NIAMS, and the Clinical Center.

BACKGROUND: Deficiency of IL-1 receptor antagonist (DIRA) is a rare autoinflammatory disease that presents with life-threatening systemic inflammation, aseptic multifocal osteomyelitis, and pustulosis responsive to IL-1-blocking treatment. This study was performed (a) to investigate rilonacept, a long-acting IL-1 inhibitor, in maintaining anakinra-induced inflammatory remission in DIRA patients, (b) to determine doses needed to maintain remission, and (c) to evaluate the safety and pharmacokinetics of rilonacept in young children (<12 years). METHODS: Six mutation-positive DIRA patients (children, ages 3-6 years), treated with daily anakinra, were enrolled into an open-label pilot study of subcutaneous rilonacept for 24 months. Clinical symptoms and inflammatory blood parameters were measured at all visits. A loading dose (4.4 mg/kg) was administered, followed by once weekly injections (2.2 mg/kg) for 12 months. Dose escalation (4.4 mg/kg) was allowed if inflammatory remission was not maintained. Subjects in remission at 12 months continued rilonacept for an additional 12 months. RESULTS: Five of six patients required dose escalation for findings of micropustules. Following dose escalation, all patients were in remission on weekly rilonacept administration, with stable laboratory parameters for the entire study period of 24 months. All children are growing at normal rates and have normal heights and weights. Quality of life improved while on rilonacept. No serious adverse events were reported. CONCLUSION: Rilonacept was found to maintain inflammatory remission in DIRA patients. The once weekly injection was well tolerated and correlated with increased quality of life, most likely related to the lack of daily injections. TRIAL REGISTRATION: ClinicalTrials.gov NCT01801449. FUNDING: NIH, NIAMS, and NIAID.