Proteomic mapping identifies serum marker signatures associated with MIS-C specific hyperinflammation and cardiovascular manifestation.

Multisystem inflammatory syndrome in children (MIS-C) shares several clinical and immunological features with Kawasaki Disease (KD) and pediatric hyperinflammation, but the immuno-phenotypic overlap among these clinical mimics is still incompletely understood. Here we analyzed serum samples from treatment-naïve patients with MIS-C (n = 31) and KD (n = 11), pediatric hyperinflammation (n = 13) and healthy controls (HC, n = 10) by proximity extension assay (PEA) to profile 184 blood biomarkers. Collectively, immunophenotypic overlap between MIS-C and hyperinflammation exceeds overlap with KD. Overexpression of IL-17A in MIS-C and KD could best separate these conditions from hyperinflammatory conditions, while those were hallmarked by overabundance of adenosin deaminase and IL-18. Depletion in serum TNF-related subfamily member 9 (TNFRSF9) and apoptosis inducing ligand (TRAIL) linked with cardiovascular manifestations and myocarditis in MIS-C. Altogether, our analysis highlights important differences in molecular marker signatures also across different MIS-C and KD cohorts and suggests several previously unidentified molecular associations in context of cardiovascular inflammation.

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

A novel serum calprotectin (MRP8/14) particle-enhanced immuno-turbidimetric assay (sCAL turbo) helps to differentiate systemic juvenile idiopathic arthritis from other diseases in routine clinical laboratory settings.

BACKGROUND: Differential diagnosis in children with signs of unprovoked inflammation can be challenging. In particular, differentiating systemic juvenile idiopathic arthritis (SJIA) from other diagnoses is difficult. We have recently validated the complex of myeloid-related proteins 8/14 (MRP8/14, also known as S100A8/A9 complex or serum calprotectin) as a helpful biomarker supporting the diagnosis of SJIA. The results were subsequently confirmed with a commercial ELISA. However, further optimization of the analytical technology is important to ensure its feasibility for large-scale use in routine laboratory settings. METHODS: To evaluate the accuracy in identifying children with SJIA, the performance of a particle-enhanced immuno-turbidimetric assay for serum calprotectin (sCAL turbo) on an automated laboratory instrument was analyzed. Samples from 615 children were available with the diagnoses SJIA (n = 99), non-systemic JIA (n = 169), infections (n = 51), other inflammatory diseases (n = 126), and acute lymphoblastic leukemia (ALL, n = 147). In addition, samples from 23 healthy controls were included. RESULTS: The sCAL turbo assay correlated well with the MRP8/14 ELISA used in previous validation studies (r = 0.99, p < 0.001). It could reliably differentiate SJIA from all other diagnoses with significant accuracy (cutoff at 10,500 ng/ml, sensitivity 84%, specificity 94%, ROC area under curve 0.960, p < 0.001). CONCLUSIONS: Serum calprotectin analyses are a helpful tool supporting the diagnosis of SJIA in children with prolonged fever or inflammatory disease. Here, we show that an immuno-turbidimetric assay for detection of serum calprotectin on an automated laboratory instrument can be implemented in clinical laboratory settings to facilitate its use as a diagnostic routine test in clinical practice.

Inflammatory Biomarkers Can Differentiate Acute Lymphoblastic Leukemia with Arthropathy from Juvenile Idiopathic Arthritis Better Than Standard Blood Tests.

OBJECTIVE: To evaluate the predictive value of biomarkers of inflammation like phagocyte-related S100 proteins and a panel of inflammatory cytokines in order to differentiate the child with acute lymphoblastic leukemia (ALL) from juvenile idiopathic arthritis (JIA). STUDY DESIGN: In this cross-sectional study, we measured S100A9, S100A12, and 14 cytokines in serum from children with ALL (n = 150, including 27 with arthropathy) and JIA (n = 236). We constructed predictive models computing areas under the curve (AUC) as well as predicted probabilities in order to differentiate ALL from JIA. Logistic regression was used for predictions of ALL risk, considering the markers as the respective exposures. We performed internal validation using repeated 10-fold cross-validation and recalibration, adjusted for age. RESULTS: In ALL, the levels of S100A9, S100A12, interleukin (IL)-1 beta, IL-4, IL-13, IL-17, matrix metalloproteinase-3, and myeloperoxidase were low compared with JIA (P < .001). IL-13 had an AUC of 100% (95% CI 100%-100%) due to no overlap between the serum levels in the 2 groups. Further, IL-4 and S100A9 had high predictive performance with AUCs of 99% (95% CI 97%-100%) and 98% (95% CI 94%-99%), respectively, exceeding both hemoglobin, platelets, C-reactive protein, and erythrocyte sedimentation rate. CONCLUSIONS: The biomarkers S100A9, IL-4, and IL-13 might be valuable markers to differentiate ALL from JIA.

Case report: Idiopathic subglottic stenosis in a girl; successful treatment with macrolides.

An 8-year-old girl presented with treatment-refractory cough and inspiratory stridor. Bronchoscopies showed progressive scarring leading to narrowing of the proximal trachea (Myer-Cotton Grade 2) and epithelial metaplasia of the tracheal and bronchial mucosa. After excluding other causes of congenital and acquired tracheal stenosis, an idiopathic subglottic tracheal stenosis (iSGS) was diagnosed. Because of the patient's young age, a judicious therapeutic approach seemed appropriate. Therapy with azithromycin, followed by roxithromycin, was started. Symptoms almost completely subsided, spirometry normalized, and endoscopic and histologic findings improved considerably. Therapy has been continued for more than 3 years with normal lung function values, and no compromise on physical activities and development. In instances of iSGS, therapy with macrolides is worth considering before more invasive procedures such as dilatation, laser, intralesional injections, or surgical resection are performed.

An Immunological Axis Involving Interleukin 1ß and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial.

PURPOSE: A recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population. METHODS: Patients' and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1ß were analyzed for single or multiple marker (n = 22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation, and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics. RESULTS: Inflammation in IVIG-resistant KD (n = 16) is hallmarked by over-expression of innate immune mediators (particularly IL-6 > CXCL10 > S100A12 > IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n = 13) and MIS-C (n = 4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1ß signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1ß resulted in concentration-dependent LRG1 release. CONCLUSION: Our study identifies LRG1 as known trigger of endothelial activation and cardiac re-modeling to associate with IL-1ß signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

MRP8/14 serum levels as diagnostic markers for systemic juvenile idiopathic arthritis in children with prolonged fever.

OBJECTIVES: Differential diagnosis in children with prolonged fever is challenging. In particular, differentiating systemic-onset JIA (SJIA) from infectious diseases is difficult. Biomarkers are needed that support the diagnostic work-up. The aim of this study was to validate the usefulness of Myeloid-related protein 8/14 (MRP8/14) measurements in the diagnostic work-up of febrile children and to transfer it to clinical practice. METHODS: Data for 1110 paediatric patients were included and divided into two cohorts: (cohort A) for validation of MRP8/14 test performance with three different testing systems: the experimental ELISA, commercial ELISA and an innovative (point-of-care test) lateral flow immunoassay (LFIA); (cohort B) to validate the diagnostic accuracy with the two latter assays. RESULTS: In cohort A (n = 940), MRP8/14 was elevated in SJIA (12 110 ± 2650 ng/ml mean ± 95% CI) compared with other diagnoses (including infections and autoinflammatory diseases; 2980 ± 510 ng/ml) irrespective of fever and anti-inflammatory treatment (P < 0.001). In untreated patients with fever (n = 195) MRP8/14 levels in SJIA (19 740 ± 5080 ng/ml) were even higher compared with other diagnoses (4590 ± 1160 ng/ml) (P < 0.001, sensitivity 73%, specificity 90%). In group B1, the performance of the tests was confirmed in untreated patients with fever (n = 170): commercial ELISA (sensitivity 79%, specificity 89%) and LFIA (sensitivity 84%, specificity 81%). Compared with ferritin, IL-18, ESR, soluble IL-2 receptor and procalcitonin, MRP8/14 showed the best accuracy. CONCLUSION: MRP8/14 serum analyses have been validated as a helpful tool supporting the diagnosis of SJIA in febrile children. The results could be confirmed with commercial ELISA and LFIA enabling a rapid diagnostic point-of-care screening test.

Patient parameters and response after administration of rituximab in pediatric mature B-cell non-Hodgkin lymphoma.

BACKGROUND: Mature aggressive B-cell lymphomas are heterogenous malignancies that make up more than half of all diagnosed non-Hodgkin lymphoma in children and adolescents. The overall survival rate increased over the last decades to 80%-90% due to fine tuning of polychemotherapy. However, new therapeutic implications are needed to further increase the overall survival. Current clinical trials analyze the therapeutic effect of rituximab in pediatric patients, while the mechanism of action in vivo is still not fully understood. METHODS: Effector molecules important for tumor defense were analyzed before and at day 5 after rituximab treatment via flow cytometry. Serum rituximab levels were measured with an ELISA. RESULTS: We evaluated patient parameters that may affect treatment response in relation to rituximab administration and serum rituximab levels. We indeed found a reduction of Fcγ receptor (FcγR) II levels after rituximab treatment in monocyte subtypes, whereas FcγRI expression was significantly increased. Serum levels of proinflammatory marker proteins S100A8/A9 and S100A12 significantly decreased after treatment to normal levels from an overall proinflammatory state before treatment. CD57, perforin, and granzyme B expression decreased after treatment, comprising a less cytolytic natural killer (NK) cell population. CONCLUSION: The highlighted effects of rituximab treatment on patient's immune response help in understanding the biology behind tumor defense mechanisms and effector function. After subsequent studies, these novel insights might be translated into patient care and could contribute to improve treatment of pediatric patients with mature aggressive B-cell lymphoma.

Discrimination of COVID-19 From Inflammation-Induced Cytokine Storm Syndromes Using Disease-Related Blood Biomarkers.

OBJECTIVE: Infection with the novel coronavirus SARS-CoV-2 triggers severe illness with high mortality in a subgroup of patients. Such a critical course of COVID-19 is thought to be associated with the development of cytokine storm, a condition seen in macrophage activation syndrome (MAS) and secondary hemophagocytic lymphohistiocytosis (HLH). However, specific data demonstrating a clear association of cytokine storm with severe COVID-19 are still lacking. The aim of this study was to directly address whether immune activation in COVID-19 does indeed mimic the conditions found in these classic cytokine storm syndromes. METHODS: Levels of 22 biomarkers were quantified in serum samples from patients with COVID-19 (n = 30 patients, n = 83 longitudinal samples in total), patients with secondary HLH/MAS (n = 50), and healthy controls (n = 9). Measurements were performed using bead array assays and single-marker enzyme-linked immunosorbent assay. Serum biomarker levels were assessed for correlations with disease outcome. RESULTS: In patients with secondary HLH/MAS, we observed pronounced activation of the interleukin-18 (IL-18)-interferon-γ axis, increased serum levels of IL-1 receptor antagonist, intercellular adhesion molecule 1, and IL-8, and strongly reduced levels of soluble Fas ligand in the course of SARS-CoV-2 infection. These observations appeared to discriminate immune dysregulation in critical COVID-19 from the well-recognized characteristics of other cytokine storm syndromes. CONCLUSION: Serum biomarker profiles clearly separate COVID-19 from MAS or secondary HLH in terms of distinguishing the severe systemic hyperinflammation that occurs following SARS-CoV-2 infection. These findings could be useful in determining the efficacy of drugs targeting key molecules and pathways specifically associated with systemic cytokine storm conditions in the treatment of COVID-19.

Definition and validation of serum biomarkers for optimal differentiation of hyperferritinaemic cytokine storm conditions in children: a retrospective cohort study.

BACKGROUND: Cytokine storm syndromes are life-threatening complications that can occur in children with rheumatic conditions (macrophage activation syndrome [MAS]), inherited cytotoxicity defects (ie, primary haemophagocytic lymphohistiocytosis [HLH]), or as a result of infection or malignancies (ie, secondary HLH). To adequately steer treatment, an early and clear discrimination of these entities is essential. We aimed to define and validate serum biomarker profiles that can differentiate between primary HLH, secondary HLH (predominantly infection-associated), and MAS associated with systemic juvenile idiopathic arthritis (systemic JIA-MAS). METHODS: In this multicentre, retrospective, cohort study, serum samples from patients (0-18 years) with a clinical diagnosis of primary HLH, secondary HLH, or systemic JIA-MAS were analysed by immunoassays for 55 cytokines and chemokines. Serum samples were collected from patients treated at seven clinical centres in Europe and North America. 15 serum biomarkers were validated using an independent commercial assay, and the diagnostic accuracy of the best performing biomarkers was tested in an independent validation cohort. FINDINGS: Serum samples were collected between Dec 7, 2010, and Jan 26, 2018. In the discovery cohort of 43 patients (24 girls and 19 boys) multi-marker analyses revealed distinct serum biomarker profiles associated with primary or secondary HLH versus systemic JIA-MAS. Ten biomarkers were identified that were differentially elevated in either HLH or systemic JIA-MAS and distinguished between these clinical entities, six of which were tested in an independent validation cohort of 79 patients (34 girls and 45 boys). Serum concentrations of S100A12 and interleukin-18, as well as ratios of both S100A12 and IL-18 with chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 were identified as the most promising candidates for differential diagnostics. INTERPRETATION: At initial presentation, when it is unclear whether a patient with excessive hyperferritinaemic inflammation has primary HLH, infection-associated secondary HLH, or MAS, high serum concentrations of S100A12 indicate an initial differential diagnosis of systemic JIA-MAS, thus helping to guide subsequent treatment decisions. We therefore suggest the inclusion of serum S100A12 and IL-18 in the diagnostic investigations for hyperferritinaemic syndromes; however, the definition and introduction of universially applicable cutoff values are still required. FUNDING: German Research Foundation, the Center for Interdisciplinary Clinical Research at University Hospital Muenster, the EU's Horizon 2020 research and innovation programme, and the Deutsche Kinderkrebsstiftung.

Gene-Dose Effect of MEFV Gain-of-Function Mutations Determines ex vivo Neutrophil Activation in Familial Mediterranean Fever.

Familial Mediterranean fever (FMF) is caused by mutations within the Mediterranean fever (MEFV) gene. Disease severity depends on genotype and gene dose with most serious clinical courses observed in patients with M694V homozygosity. Neutrophils are thought to play an important role in the initiation and perpetuation of inflammatory processes in FMF, but little is known about the specific characteristics of these cells in FMF patients. To further characterize neutrophilic inflammatory responses in FMF and to delineate gene-dose effects on a cellular level, we analyzed cytokine production and activation levels of isolated neutrophils derived from patients and subjects with distinct MEFV genotypes, as well as healthy and disease controls. Serum levels of interleukin-18 (IL-18) (median 11,485 pg/ml), S100A12 (median 9,726 ng/ml), and caspase-1 (median 394 pg/ml) were significantly increased in patients with homozygous M694V mutations. Spontaneous release of S100A12, caspase-1, proteinase 3, and myeloperoxidase (MPO) was restricted to ex vivo cultured neutrophils derived from patients with two pathogenic MEFV mutations. IL-18 secretion was highest in patients with two mutations but also increased in neutrophils from healthy heterozygous MEFV mutation carriers, exhibiting an ex vivo gene-dose effect, which was formerly described by us in patients' serum. CD62L (l-selectin) was spontaneously shed from the surface of ex vivo cultured neutrophils [median of geometric mean fluorescence intensity (gMFI) after 5 h: 28.8% of the initial level]. While neutrophils derived from healthy heterozygous mutation carriers again showed a gene-dose effect (median gMFI: 67.1%), healthy and disease controls had significant lower shedding rates (median gMFI: 83.6 and 82.9%, respectively). Co-culture with colchicine and/or stimulation with adenosine triphosphate (ATP) and lipopolysaccharide (LPS) led to a significant increase in receptor shedding. Neutrophils were not prevented from spontaneous shedding by blocking IL-1 or the NLRP3 inflammasome. In summary, the data demonstrate that ex vivo cultured neutrophils derived from FMF patients display a unique phenotype with spontaneous release of high amounts of IL-18, S100A12, MPO, caspase-1, and proteinase 3 and spontaneous activation as demonstrated by the loss of CD62L. Neutrophilic activation seems to be independent from IL-1 activation and displays a gene-dose effect that may be responsible for genotype-dependent phenotypes.

The Receptor for Advanced Glycation Endproducts (RAGE) Contributes to Severe Inflammatory Liver Injury in Mice.

Background: The receptor for advanced glycation end products (RAGE) is a multiligand receptor involved in a number of processes and disorders. While it is known that RAGE-signaling can contribute to toxic liver damage and fibrosis, its role in acute inflammatory liver injury and septic multiorgan failure is yet undefined. We examined RAGE in lipopolysaccharide (LPS) induced acute liver injury of D-galN sensitized mice as a classical model for tumor necrosis factor alpha (TNF-α) dependent inflammatory organ damage. Methods: Mice (Rage-/- and C57BL/6) were intraperitoneally injected with D-galN (300 mg/kg) and LPS (10 µg/kg). Animals were monitored clinically, and cytokines, damage associated molecular pattern molecules (DAMPs) as well as liver enzymes were determined in serum. Liver histology, hepatic cytokines as well as RAGE mRNA expression were analyzed. Cellular activation and functionality were evaluated by flow cytometry both in bone marrow- and liver-derived cells. Results: Genetic deficiency of RAGE significantly reduced the mortality of mice exposed to LPS/D-galN. Hepatocyte damage markers were reduced in Rage-/- mice, and liver histopathology was less severe. Rage-/- mice produced less pro-inflammatory cytokines and DAMPs in serum and liver. While immune cell functions appeared normal, TNF-α production by hepatocytes was reduced in Rage-/- mice. Conclusions: We found that RAGE deletion attenuated the expression of pro-inflammatory cytokines and DAMPs in hepatocytes without affecting cellular immune functions in the LPS/D-galN model of murine liver injury. Our data highlight the importance of tissue-specific RAGE-signaling also in acute inflammatory liver stress contributing to sepsis and multiorgan failure.

Alpha-1 Antitrypsin Deficiency and Pulmonary Morbidity in Patients with Primary Immunodeficiency Disease: A Single-Center Experience.

Background: Alpha-1 antitrypsin deficiency (AATD) is of importance in the pathogenesis of pulmonary emphysema, chronic obstructive pulmonary diseases (COPD), and bronchiectasis. Various pulmonary disorders are a typical feature of primary immunodeficiency disease (PID). This includes recurrent pulmonary infections, immunodysregulation, and autoinflammatory diseases. As a result, incidence of acute and chronic pulmonary diseases is higher. Interestingly, pulmonary morbidity in PID and AATD share similar features. To study the coexistence of AATD in patients suffering from PID, we performed the underlying investigation. Methods: We evaluated a study group of 149 patients (n = 149) with PID. In total, serum AAT concentrations were available for 110 patients (n = 110). For the identified patients, we analyzed both clinical associations and interactions. Results: Among the investigated patients, reduced serum AAT levels were detected in 7 patients. With regard to the genotype, PI∗ZZ was found in 2 patients, whereas PI∗MZ was observed in 5 patients. Independent of the underlying phenotype, obstructive lung diseases were found in 2 patients with PI∗ZZ and 2 patients with PI∗MZ. Conclusions: In Germany, the estimated percentage for PI∗ZZ and PI∗MZ is 0.01% and 1.9%, respectively. As demonstrated, the ratio in our study group was even higher. We identified seven patients with AATD. Since AATD contributes to pulmonary morbidity in PID patients, systematic underdiagnosis of the coexistence might yield a strong clinical impact. Hence, AAT analysis should be offered to all patients with confirmed PID diagnoses. To strengthen this finding, we suggest the investigation of larger databases.

Increased Prevalence of NLRP3 Q703K Variant Among Patients With Autoinflammatory Diseases: An International Multicentric Study.

Background: The NLRP3 inflammasome has been recognized as one of the key components of innate immunity. Gain-of-function mutations in the exon 3 of NLRP3 gene have been implicated in inflammatory diseases suggesting the presence of functionally important sites in this region. Q703K (c.2107C>A, p.Gln703Lys, also known in the literature as Q705K) is a common variant of NLRP3, that has been considered to be both clinically unremarkable or disease-causing with a reduced penetrance. Objectives: We aimed to investigate the potential genetic impact of the NLRP3 variant Q703K in patients with recurrent fever presenting with two autoinflammatory diseases: PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis) and CAPS (cryopyrin-associated periodic syndrome), as well as with undefined autoinflammatory disease (uAID). Methods: This is an international multicentric observational retrospective study characterizing the clinical phenotype of patients presenting with recurrent fever suspected to be of auto-inflammatory origin and where the Q703K NLRP3 variant was found. Monocytes of parents of 6 Q703K+ PFAPA patients were studied and levels of pro-inflammatory cytokines produced by monocytes of Q703K+ and Q703K- parents have been compared by ELISA. Results: We report 42 patients with the Q703K NLRP3 genetic variant: 21 were PFAPA patients, 6 had a CAPS phenotype, and 15 had an uAID. The phenotypes of PFAPA, CAPS and uAID were quite similar between Q703K positive and negative patients with the exception of increased prevalence of pharyngitis in the Q703K positive CAPS population compared to the negative one. The in vitro production of IL-1ß was not significantly different between Q703K+ and Q703K- monocytes from asymptomatic parents. Conclusion: The evidence we report in our study shows an increased prevalence of NLRP3 Q703K in patients with autoinflammatory diseases, suggesting an association between the Q703K variant and the risk of PFAPA, CAPS and uAID syndromes. However, we did not show a functional effect of this mutation on the inflammasome basal activity.

Synergistic Signaling of TLR and IFNα/ß Facilitates Escape of IL-18 Expression from Endotoxin Tolerance.

Rationale: IL-18 is a member of the IL-1 cytokine family, and elevated blood IL-18 concentrations associate with disease activity in macrophage activation syndrome (MAS) and poor clinical outcomes in severe inflammatory and septic conditions.Objectives: Although recent investigations provide mechanistic evidence for a contribution of IL-18 to inflammation and hyperinflammation in sepsis and MAS, we sought to study regulatory mechanisms underlying human IL-18 expression.Methods: Samples from in vivo and in vitro endotoxin rechallenge experiments, patients with inflammatory disease, and isolated human monocytes treated with various stimulants and drugs were tested for cytokine gene and protein expression. Serum IL-18 expression with or without JAK/STAT inhibition was analyzed in two MAS mouse models and in a patient with recurrent MAS.Measurements and Main Results: Peripheral blood and monocytic IL-18 expression escaped LPS-induced immunoparalysis. LPS-stimulated primary human monocytes revealed specific IL-18 expression kinetics controlled by IFNα/ß signaling. JAK/STAT inhibition or IFNß neutralization during LPS stimulation blunted cytokine expression. Similarly, microtubule-destabilizing drugs abrogated LPS-induced IL18 expression, but this effect could be fully reversed by addition of IFNα/ß. Ex vivo analysis of inflammatory disease patients' whole blood revealed strong correlation of type I IFN score and IL18 expression, whereas JAK/STAT inhibition strongly reduced IL-18 serum levels in two MAS mouse models and in a patient with recurrent MAS.Conclusions: Our data indicate that IL-18 (but not IL-1ß) production from human monocytes requires cooperative Toll-like receptor and IFNα/ß signaling. Interference with IFNα/ß expression or signaling following JAK/STAT inhibition may control catastrophic hyperinflammation in MAS.

Practice and consensus-based strategies in diagnosing and managing systemic juvenile idiopathic arthritis in Germany.

BACKGROUND: Systemic juvenile idiopathic arthritis (SJIA) is an autoinflammatory disease associated with chronic arthritis. Early diagnosis and effective therapy of SJIA is desirable, so that complications are avoided. The PRO-KIND initiative of the German Society for Pediatric Rheumatology (GKJR) aims to define consensus-based strategies to harmonize diagnostic and therapeutic approaches in Germany. METHODS: We analyzed data on patients diagnosed with SJIA from 3 national registries in Germany. Subsequently, via online surveys and teleconferences among pediatric rheumatologists with a special expertise in the treatment of SJIA, we identified current diagnostic and treatment approaches in Germany. Those were harmonized via the formulation of statements and, supported by findings from a literature search. Finally, an in-person consensus conference using nominal group technique was held to further modify and consent the statements. RESULTS: Up to 50% of patients diagnosed with SJIA in Germany do not fulfill the International League of Associations for Rheumatology (ILAR) classification criteria, mostly due to the absence of chronic arthritis. Our findings suggest that chronic arthritis is not obligatory for the diagnosis and treatment of SJIA, allowing a diagnosis of probable SJIA. Malignant, infectious and hereditary autoinflammatory diseases should be considered before rendering a diagnosis of probable SJIA. There is substantial variability in the initial treatment of SJIA. Based on registry data, most patients initially receive systemic glucocorticoids, however, increasingly substituted or accompanied by biological agents, i.e. interleukin (IL)-1 and IL-6 blockade (up to 27.2% of patients). We identified preferred initial therapies for probable and definitive SJIA, including step-up patterns and treatment targets for the short-term (resolution of fever, decrease in C-reactive protein by 50% within 7 days), the mid-term (improvement in physician global and active joint count by at least 50% or a JADAS-10 score of maximally 5.4 within 4 weeks) and the long-term (glucocorticoid-free clinically inactive disease within 6 to 12 months), and an explicit treat-to-target strategy. CONCLUSIONS: We developed consensus-based strategies regarding the diagnosis and treatment of probable or definitive SJIA in Germany.

Proinflammatory Cytokine Environments Can Drive Interleukin-17 Overexpression by γ/δ T Cells in Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Systemic-onset juvenile idiopathic arthritis (JIA) is speculated to follow a biphasic course, with an initial systemic disease phase driven by innate immune mechanisms and interleukin-1ß (IL-1ß) as a key cytokine and a second chronic arthritic phase that may be dominated by adaptive immunity and cytokines such as IL-17A. Although a recent mouse model points to a critical role of IL-17-expressing γ/δ T cells in disease pathology, in humans, both the prevalence of IL-17 and the role of IL-17-producing cells are still unclear. METHODS: Serum samples from systemic JIA patients and healthy pediatric controls were analyzed for the levels of IL-17A and related cytokines. Whole blood samples were studied for cellular expression of IL-17 and interferon-γ (IFNγ). CD4+ and γ/δ T cells isolated from the patients and controls were assayed for cytokine secretion in different culture systems. RESULTS: IL-17A was prevalent in sera from patients with active systemic JIA, while both ex vivo and in vitro experiments revealed that γ/δ T cells overexpressed this cytokine. This was not seen with CD4+ T cells, which expressed strikingly low levels of IFNγ. Therapeutic IL-1 blockade was associated with partial normalization of both cytokine expression phenotypes. Furthermore, culturing healthy donor γ/δ T cells in serum from systemic JIA patients or in medium spiked with IL-1ß, IL-18, and S100A12 induced IL-17 overexpression at levels similar to those observed in the patients' cells. CONCLUSION: A systemic JIA cytokine environment may prime γ/δ T cells in particular for IL-17A overexpression. Thus, our observations in systemic JIA patients strongly support a pathophysiologic role of these cells, as proposed by the recent murine model.

Monocyte-induced development of Th17 cells and the release of S100 proteins are involved in the pathogenesis of graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. However, the pathophysiology of GvHD remains poorly understood. In this study, we analyzed the induction of Th17 cells by monocytes of patients with GvHD in vitro, demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I-IV and chronic GvHD induce significantly increased levels of Th17 cells compared with patients without GvHD. S100 proteins are known to act as innate amplifier of inflammation. We therefore investigated the presence of S100 proteins in the stool, serum, and bowel tissue of patients with GvHD and the influence of S100 proteins on the induction of Th17 cells. Elevated levels of S100 proteins could be detected in patients with acute GvHD, demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, stimulation of monocytes with S100 proteins was found to promote Th17 development, emphasizing the role of S100 proteins in Th17-triggered inflammation. Altogether, our results indicate that induction of Th17 cells by activated monocytes and the stimulatory effects of proinflammatory S100 proteins might play a relevant role in the pathogenesis of acute GvHD. Regarding our data, S100 proteins might be novel markers for the diagnosis and follow-up of GvHD.

Validation of relapse risk biomarkers for routine use in patients with juvenile idiopathic arthritis.

OBJECTIVE: The myeloid-related proteins 8 and 14 (MRP-8/MRP-14) and neutrophil-derived S100A12 are biomarkers of inflammation. They can be used to determine the relapse risk in patients with juvenile idiopathic arthritis (JIA) after stopping antiinflammatory treatment. In this study, we tested the performance of different enzyme-linked immunosorbent assays (ELISAs) in order to validate systems available for routine use. METHODS: MRP-8/MRP-14 and S100A12 serum concentrations of 188 JIA patients in remission were analyzed. Commercially available test systems were compared to experimental ELISAs established in house. The ability of the assays to identify JIA patients at risk for relapse was analyzed. RESULTS: For MRP-8/MRP-14, the PhiCal Calprotectin and Buhlmann MRP8/14 Calprotectin ELISAs revealed hazard ratios of 2.3 and 2.1, respectively. For S100A12, the CircuLex S100A12/EN-RAGE ELISA revealed a hazard ratio of 3.1. The commercial assays allowed a JIA relapse prediction that was at least comparable to the experimental ELISAs. CONCLUSION: For the prediction of JIA relapse after stopping medication, the biomarkers MRP-8/MRP-14 and S100A12 can be determined by using assays that are available for routine use. The tested commercial MRP-8/MRP-14 and S100A12 ELISAs showed a performance comparable to well-established experimental ELISA protocols when assay-specific cutoffs for the indication of relapse prediction are thoroughly applied.