Part 5: Allogeneic HSCT in refractory SJIA with lung disease; recent cases from centers in North America & Europe.

It has been increasingly recognized that there is a subset of patients with refractory systemic JIA, who have failed all available medications and may benefit from HSCT. The increasing experience with HSCT in SJIA, suggests that despite the complicated post-HSCT course, short-term, the transplanted patients either achieved SJIA remission or reduced burden of disease. Longer follow-up, however, is needed to better define the long-term outcomes. The discussion at the NextGen 2022 conference was focused on the optimal timing for the procedure, the need for a good control of inflammatory SJIA activity prior to HSCT, and the role of the reduced intensity conditioning regimens as there was a remote concern that such regimens might increase the risk of SJIA relapse after the transplantation. There was unanimous agreement about the importance of long-term registries to address these questions.

Vertebral Body Reshaping after Fractures: An Important Index of Recovery in Glucocorticoid-Treated Children.

PURPOSE: In this 6-year study we identified factors associated with spontaneous vertebral body reshaping in glucocorticoid (GC)-treated children with leukemia, rheumatic disorders, and nephrotic syndrome. METHODS: Subjects were 79 children (mean age 7.4 years) who had vertebral fracture (VF) evaluation on lateral spine radiographs at least 1 year after VF detection. VF were graded using the modified Genant semiquantitative method and fracture burden for individuals was quantified using the spinal deformity index (SDI; sum of grades from T4 to L4). RESULTS: Sixty-five children (82.3%) underwent complete vertebral body reshaping (median time from VF detection to complete reshaping 1.3 years by Cox proportional hazard modeling). Of 237 VF, the majority (83.1%) ultimately reshaped, with 87.2% reshaping in the thoracic region vs 70.7% in the lumbar region (P = .004). Cox models showed that (1) every g/m2 increase in GC exposure in the first year after VF detection was associated with a 19% decline in the probability of reshaping; (2) each unit increase in the SDI at the time of VF detection was associated with a 19% decline in the probability of reshaping [hazard ratio (HR) = 0.81; 95% confidence interval (CI) = 0.71, 0.92; P = .001]; (3) each additional VF present at the time of VF detection reduced reshaping by 25% (HR = 0.75; 95% CI = 0.62, 0.90; P = .002); and (4) each higher grade of VF severity decreased reshaping by 65% (HR = 0.35; 95% CI = 0.21, 0.57; P < .001). CONCLUSION: After experiencing a VF, children with higher GC exposure, higher SDI, more severe fractures, or lumbar VF were at increased risk for persistent vertebral deformity.

E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

S100A8/A9-alarmin promotes local myeloid-derived suppressor cell activation restricting severe autoimmune arthritis.

Immune-suppressive effects of myeloid-derived suppressor cells (MDSCs) are well characterized during anti-tumor immunity. The complex mechanisms promoting MDSC development and their regulatory effects during autoimmune diseases are less understood. We demonstrate that the endogenous alarmin S100A8/A9 reprograms myeloid cells to a T cell suppressing phenotype during autoimmune arthritis. Treatment of myeloid precursors with S100-alarmins during differentiation induces MDSCs in a Toll-like receptor 4-dependent manner. Consequently, knockout of S100A8/A9 aggravates disease activity in collagen-induced arthritis due to a deficit of MDSCs in local lymph nodes, which could be corrected by adoptive transfer of S100-induced MDSCs. Blockade of MDSC function in vivo aggravates disease severity in arthritis. Therapeutic application of S100A8 induces MDSCs in vivo and suppresses the inflammatory phenotype of S100A9ko mice. Accordingly, the interplay of T cell-mediated autoimmunity with a defective innate immune regulation is crucial for autoimmune arthritis, which should be considered for future innovative therapeutic options.

Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy.

BACKGROUND: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. METHODS: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. RESULTS: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. CONCLUSION: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

The roles of toll-like receptor 4, CD33, CD68, CD69, or CD147/EMMPRIN for monocyte activation by the DAMP S100A8/S100A9.

The S100A8/A9 heterocomplex is an abundant damage-associated molecular pattern and mainly expressed by monocytes, inflammatory activated keratinocytes and neutrophilic granulocytes. The heterocomplex as well as the heterotetramer are involved in a variety of diseases and tumorous processes. However, their detailed mode of action and especially which receptors are involved hereby remains to be fully revealed. Several cell surface receptors are reported to interact with S100A8 and/or S100A9, the best studied being the pattern recognition receptor TLR4. RAGE, CD33, CD68, CD69, and CD147, all of them are involved as receptors in various inflammatory processes, are also among these putative binding partners for S100A8 and S100A9. Interactions between S100 proteins and these receptors described so far come from a wide variety of cell culture systems but their biological relevance in vivo for the inflammatory response of myeloid immune cells is not yet clear. In this study, we compared the effect of CRISPR/Cas9 mediated targeted deletion of CD33, CD68, CD69, and CD147 in ER-Hoxb8 monocytes on S100A8 or S100A9 induced cytokine release with TLR4 knockout monocytes. Whereas deletion of TLR4 abolished the S100-induced inflammatory response in monocyte stimulation experiments with both S100A8 and S100A9, knockouts of CD33, CD68, CD69, or CD147 revealed no effect on the cytokine response in monocytes. Thus, TLR4 is the dominant receptor for S100-triggered inflammatory activation of monocytes.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

UVA1 radiation attenuates pro-inflammatory functions in human monocytes.

UVA1 therapy is effective in the treatment of inflammatory and autoimmune skin diseases. The mode of action of UVA1 therapy is not completely understood and especially data on cells of the innate immune system like monocytes, which are critically involved in many inflammatory processes, are sparse. We wanted to answer the question whether UVA1 irradiation alters functional properties of human monocytes. We treated human peripheral blood monocytes in vitro with 2 J/cm2 UVA1 light, incubated the cells for 48 h and examined both functional properties and alterations in the gene and protein expression profile. While UVA1 did not alter cell viability or susceptibility to apoptosis inducing agents, it decreased the capacity of monocytes for phagocytosis and to eliminate infectious agents like Leishmania major. Moreover, we measured a significantly reduced production of interleukin (IL)-1ß mRNA in lipopolysaccharide activated monocytes after UVA1 treatment. Importantly, UVA1-treated monocytes not only produce less IL-1ß, but also upregulate expression of the anti-inflammatory IL-1ß decoy receptor. Our data provide evidence that UVA1 radiation not only interferes with fundamental monocyte properties like phagocytosis, pathogen killing and activation, but could also specifically attenuate pro-inflammatory IL-1 effects. This might constitute a hitherto unknown anti-inflammatory mechanism of UVA1 in human monocytes.

Role of S100A8/A9 in Platelet-Neutrophil Complex Formation during Acute Inflammation.

Acute respiratory distress syndrome (ARDS) due to pulmonary infections is associated with high morbidity and mortality. Upon inflammation, the alarmin S100A8/A9 is released and stimulates neutrophil recruitment mainly via binding to Toll-like receptor 4 (TLR4). TLR4 is also expressed on platelets, which modulate the immune response through direct interaction with leukocytes. In a murine model of Klebsiella pneumoniae-induced pulmonary inflammation, global S100A9 deficiency resulted in diminished neutrophil recruitment into the lung alveoli and neutrophil accumulation in the intravascular space, indicating an impaired neutrophil migration. A lack of TLR4 on platelets resulted in reduced neutrophil counts in the whole lung, emphasising the impact of TLR4-mediated platelet activity on neutrophil behaviour. Flow cytometry-based analysis indicated elevated numbers of platelet-neutrophil complexes in the blood of S100A9-/- mice. Intravital microscopy of the murine cremaster muscle confirmed these findings and further indicated a significant increase in neutrophil-platelet complex formation in S100A9-/- mice, which was reversed by administration of the S100A8/A9 tetramer. An in vitro bilayer assay simulated the murine alveolar capillary barrier during inflammation and validated significant differences in transmigration behaviour between wild-type and S100A9-/- neutrophils. This study demonstrates the role of S100A8/A9 during platelet-neutrophil interactions and neutrophil recruitment during pulmonary inflammation.

Associations of Betatrophin/ANGPTL8 with Septic Dyslipidemia in Human Peritonitis: An Explorative Analysis.

Sepsis is defined by life-threatening organ dysfunction mediated by the host's response to infection. This can result in septic dyslipidemia, which is involved in the neutralization of pathogen-related lipids. Knowledge of the regulatory mechanisms of septic dyslipidemia is incomplete. The cytokine betatrophin/Angiopoietin-like protein 8 (ANGPTL8) plays a role in the regulation of triacylglyceride metabolism, though its function in septic dyslipidemia remains unknown. Sixty-six patients were enrolled in a cross-sectional study. Circulating concentrations and adipose tissue (AT) mRNA expression of betatrophin/ANGPTL8 were studied in patients suffering from peritoneal sepsis. Insulin-resistant individuals and subjects without metabolic derangement/systemic inflammation were enrolled as controls. All underwent open abdominal surgery. Circulating betatrophin/ANGPTL8 was analyzed by an enzyme-linked immunosorbent assay and AT mRNA expression levels were assessed by real-time PCR. Standard laboratory analyses including lipid electrophoresis were evaluated. Sepsis patients showed pronounced septic dyslipidemia (p < 0.05 for all major lipid classes). Despite comparable betatrophin/ANGPTL8 mRNA expression in AT (p = 0.24), we found significantly increased circulating betatrophin/ANGPTL8 with septic dyslipidemia (p = 0.009). Expression levels of betatrophin/ANGPTL8 in AT correlated with circulating concentrations in both control groups (r = 0.61; p = 0.008 and r = 0.43; p = 0.034), while this association was undetectable in sepsis. After stratification, betatrophin/ANGPTL8 remained associated with hypertriacylglyceridemia (p < 0.05).

S100A8-mediated metabolic adaptation controls HIV-1 persistence in macrophages in vivo.

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in "sterile" inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

Alarming and Calming: Opposing Roles of S100A8/S100A9 Dimers and Tetramers on Monocytes.

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

The Good and the Bad: Monocytes' and Macrophages' Diverse Functions in Inflammation.

Monocytes and macrophages are central players of the innate immune response and play a pivotal role in the regulation of inflammation. Thereby, they actively participate in all phases of the immune response, from initiating inflammation and triggering the adaptive immune response, through to the clearance of cell debris and resolution of inflammation. In this review, we described the mechanisms of monocyte and macrophage adaptation to rapidly changing microenvironmental conditions and discussed different forms of macrophage polarization depending on the environmental cues or pathophysiological condition. Therefore, special focus was placed on the tight regulation of the pro- and anti-inflammatory immune response, and the diverse functions of S100A8/S100A9 proteins and the scavenger receptor CD163 were highlighted, respectively. We paid special attention to the function of pro- and anti-inflammatory macrophages under pathological conditions.

C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9.

The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but are completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of S100a8 and S100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout (KO)-based screening approach in immortalized murine monocytes, we identified the transcription factor C/EBPδ as a central regulator of S100a8 and S100a9 expression. We showed that S100A8/A9 expression and thereby neutrophil recruitment and cytokine release were decreased in C/EBPδ KO mice in a mouse model of acute lung inflammation. S100a8 and S100a9 expression was further controlled by the C/EBPδ antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within S100a8 and S100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100a8 and S100a9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterized by S100a8 and S100a9 overexpression.

In vivo imaging of microenvironmental and anti-PD-L1-mediated dynamics in cancer using S100A8/S100A9 as an imaging biomarker.

PURPOSE: As a promotor of tumor invasion and tumor microenvironment (TME) formation, the protein complex S100A8/S100A9 is associated with poor prognosis. Our aim was to further evaluate its origin and regulatory effects, and to establish an imaging biomarker for TME activity. METHODS: S100A9-/-cells (ko) were created from syngeneic murine breast cancer 4T1 (high malignancy) and 67NR (low malignancy) wildtype (wt) cell lines and implanted into either female BALB/c wildtype or S100A9-/- mice (n = 10 each). Anti-S100A9-Cy5.5-targeted fluorescence reflectance imaging was performed at 0 h and 24 h after injection. Potential early changes of S100A9-presence under immune checkpoint inhibition (anti-PD-L1, n = 7 vs. rat IgG2b as isotype control, n = 3) were evaluated. RESULTS: In S100A9-/-mice contrast-to-noise-ratios were significantly reduced for wt and S100A9-/-tumors. No significant differences were detected for 4T1 ko and 67NR ko cells as compared to wildtype cells. Under anti-PD-L1 treatment S100A9 presence significantly decreased compared with the control group. CONCLUSION: Our results confirm a secretion of S100A8/S100A9 by the TME, while tumor cells do not apparently release the protein. Under immune checkpoint inhibition S100A9-imaging reports an early decrease of TME activity. Therefore, S100A9-specific imaging may serve as an imaging biomarker for TME formation and activity.

Fast and global reorganization of the chloroplast protein biogenesis network during heat acclimation.

Photosynthesis is a central determinant of plant biomass production, but its homeostasis is increasingly challenged by heat. Little is known about the sensitive regulatory principles involved in heat acclimation that underly the biogenesis and repair of chloroplast-encoded core subunits of photosynthetic complexes. Employing time-resolved ribosome and transcript profiling together with selective ribosome proteomics, we systematically deciphered these processes in chloroplasts of Chlamydomonas reinhardtii. We revealed protein biosynthesis and altered translation elongation as central processes for heat acclimation and showed that these principles are conserved between the alga and the flowering plant Nicotiana tabacum. Short-term heat exposure resulted in specific translational repression of chlorophyll a-containing core antenna proteins of photosystems I and II. Furthermore, translocation of ribosome nascent chain complexes to thylakoid membranes was affected, as reflected by the increased accumulation of stromal cpSRP54-bound ribosomes. The successful recovery of synthesizing these proteins under prolonged acclimation of nonlethal heat conditions was associated with specific changes of the co-translational protein interaction network, including increased ribosome association of chlorophyll biogenesis enzymes and acclimation factors responsible for complex assembly. We hypothesize that co-translational cofactor binding and targeting might be bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.

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.

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.