Monique Stoffels

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL‐1β in macrophages. Inflammasome activation in macrophages results from differences in cell‐wall architecture between yeasts and hyphae and is partly mediated by the dectin‐1/Syk pathway. These results define the dectin‐1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.

Inflammasome-independent modulation of cytokine response by autophagy in human cells

Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohns disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1β and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs) significantly enhances IL-1β after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFα. In line with this, induction of autophagy by starvation inhibited IL-1β production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases.

Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin-17.

OBJECTIVE Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of antinuclear autoantibodies. Increased apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE, but the underlying mechanisms remain elusive. During apoptosis apoptotic blebs are formed in which autoantigens are clustered. The cellular remnants after blebbing are referred to as apoptotic cell bodies. We undertook this study to compare the effects of apoptotic blebs and apoptotic cell bodies on maturation of dendritic cells (DCs) and their T cell stimulatory capacity in a murine setting. METHODS The uptake by DCs of apoptotic blebs and apoptotic cell bodies was analyzed by flow cytometry and confocal microscopy. DC maturation and DC-induced T cell activation were determined by measuring expression of costimulatory molecules using flow cytometry and by measuring production of cytokines using enzyme-linked immunosorbent assay. RESULTS DCs internalized apoptotic blebs more efficiently than apoptotic cell bodies. Incubation of DCs with apoptotic blebs resulted in increased CD40 and CD86 expression and increased interleukin-6 (IL-6) and tumor necrosis factor alpha production, while apoptotic cell bodies had no stimulatory effects. Using chloroquine, apoptotic bleb-induced DC maturation was shown to be independent of Toll-like receptors 3, 7, and 9. Interestingly, in cocultures with allogeneic T cells, bleb-matured DCs induced production of IL-2, interferon-gamma, and, in particular, IL-17, suggesting a Th1/Th17 response. CONCLUSION Apoptotic blebs, in contrast to apoptotic cell bodies, induce DC maturation, thereby providing DCs with increased Th17 cell stimulatory capacity. These data imply that apoptotic bleb-induced DC maturation represents an important driving force in the autoimmune response in SLE.

Sustained efficacy of the monoclonal anti-interleukin-1 beta antibody canakinumab in a 9-month trial in Schnitzler's syndrome

Objectives Schnitzlers syndrome is a chronic disabling autoinflammatory disorder, characterised by chronic urticaria, paraproteinemia and systemic inflammation. The interleukin (IL) 1 receptor antagonist anakinra is a very effective treatment, but requires daily injection and blocks both IL-1α and IL-1β. Canakinumab is a selective human monoclonal anti-IL-1β antibody with a long half-life. We investigated the long-term efficacy and safety of canakinumab in Schnitzlers syndrome. Methods In an open-label, single-treatment arm trial, eight patients with Schnitzlers syndrome received monthly injections with 150 mg canakinumab subcutaneously for 6 months, followed by a 3-month observation period. Primary outcome was complete or clinical remission at day 14. Secondary outcome measures included inflammatory markers, quality of life, time to relapse, safety and tolerability. Results After stopping anakinra, patients developed moderate to severe clinical symptoms. Canakinumab induced complete or clinical remission at day 14 in all eight patients. Median C-reactive protein concentrations decreased from 169 mg/l at baseline to less than 10 mg/l on day 14 and remained low or undetectable. One patient discontinued participation on day 39 because of return of symptoms while all others remained in complete or clinical remission during the 6-month treatment period. Relapse after last canakinumab dose occurred within 3 months in four patients. For two patients, remission continued several months post-study. Five patients reported at least one adverse event, predominantly mild upper respiratory tract infections. One patient died in a traffic accident. Conclusions In this 9-month study, monthly 150 mg canakinumab injection was an effective and well-tolerated treatment for Schnitzlers syndrome. Our data demonstrate that IL-1β plays a pivotal role in this disease. ClinicalTrials.gov NCT01276522.

MEFV mutations affecting pyrin amino acid 577 cause autosomal dominant autoinflammatory disease

Objectives Autoinflammatory disorders are disorders of the innate immune system. Standard genetic testing provided no correct diagnosis in a female patient from a non-consanguineous family of British descent with a colchicine-responsive autosomal dominant periodic fever syndrome. We aimed to unravel the genetic cause of the symptoms. Methods Whole exome sequencing was used to screen for novel sequence variants, which were validated by direct Sanger sequencing. Ex vivo stimulation with peripheral blood mononuclear cells was performed to study the functional consequences of the mutation. mRNA and cytokine levels were measured by quantitative PCR and ELISA, respectively. Results Whole exome sequencing revealed a novel missense sequence variant, not seen in around 6800 controls, mapping to exon 8 of the MEFV gene (c.1730C>A; p.T577N), co-segregating perfectly with disease in this family. Other mutations at the same amino acid (c.1730C>G; p.T577S and c.1729A>T; p.T577S) were found in a family of Turkish descent, with autosomal dominant inheritance of familial Mediterranean fever (FMF)-like phenotype, and a Dutch patient, respectively. Moreover, a mutation (c.1729A>G; p.T577A) was detected in two Dutch siblings, who had episodes of inflammation of varying severity not resembling FMF. Peripheral blood mononuclear cells from one patient of the index family showed increased basal interleukin 1β mRNA levels and cytokine responses after lipopolysaccharide stimulation. Responses normalised with colchicine treatment. Conclusions Heterozygous mutations at amino acid position 577 of pyrin can induce an autosomal dominant autoinflammatory syndrome. This suggests that T577, located in front of the C-terminal B30.2/SPRY domain, is crucial for pyrin function.

Mutations in the Mevalonate Kinase (MVK) Gene Cause Nonsyndromic Retinitis Pigmentosa

OBJECTIVE Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder characterized by night blindness and peripheral vision loss, and in many cases leads to blindness. Despite extensive knowledge about genes involved in the pathogenesis of RP, the genetic cause remains elusive in many patients. In this study, we aimed to identify novel genes that are involved in the cause of RP. DESIGN We present a case series with mutations in the mevalonate kinase (MVK) gene. PARTICIPANTS A total of 769 patients with nonsyndromic RP and 174 Dutch control individuals participated in this study. METHODS Exome sequencing analysis was performed in a proband of Dutch origin who was initially diagnosed with nonsyndromic autosomal recessive RP. Mutations in MVK were identified and subsequently tested for segregation within the patients family and screened in a large cohort of patients with genetically unsolved RP. Patients with mutations underwent extensive clinical reexamination. MAIN OUTCOME MEASURES Digital fundus photography, spectral-domain optical coherence tomography (OCT), and fundus autofluorescence analysis were performed in patients with MVK mutations. Mevalonate kinase (MK) enzyme activity was analyzed in cultured lymphoblastoid cells, and mevalonic acid levels were measured in urine samples. RESULTS Exome variant filtering and prioritization led to the identification of compound heterozygous mutations in MVK (p.I268T and p.A334T) in the proband and her affected brother. Screening of our nonsyndromic RP patient cohort revealed an additional individual who was homozygous for the p.A334T alteration. Clinical reevaluation of all 3 patients showed a classic form of RP with variable extraocular symptoms, such as history of recurrent childhood febrile crises in 2 patients, mild ataxia in 1, and renal failure in 1. All 3 affected individuals showed a significantly decreased MK activity and highly elevated levels of urinary mevalonic acid. CONCLUSIONS Although the MK activity in cells and mevalonic acid concentrations in urine are strongly aberrant and comparable to that in patients with systemic mevalonate kinase deficiency (MKD), only mild clinical symptoms related to this syndrome were observed in our patients. In the current article, we add another phenotype to the spectrum of diverging disorders associated with mutations in MVK.

Variable expression and treatment of PAPA syndrome

Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is an autosomal dominant autoinflammatory syndrome caused by a missense mutation in the PSTPIP1 gene on the long arm of chromosome 15. So far only three families have been described.1,–,7 In this letter we would like to describe three members of a new family with PAPA syndrome with variable symptoms and the effects of different treatments, including successful treatment during pregnancy with the interleukin 1 (IL-1) receptor antagonist anakinra. Case 1: A girl born in 1999. In 2002 she presented with an acute arthritis of the left ankle, accompanied by fever and an acute phase response (table 1). Septic arthritis was suspected, but blood and synovial fluid cultures remained negative. The symptoms persisted despite antibiotic treatment and diminished only gradually. After another episode of sterile arthritis, PAPA syndrome was suspected and genetically confirmed (c 688G>A (p.Ala 230 Thr) mutation in exon …

Hyper-IgD syndrome or mevalonate kinase deficiency.

Purpose of reviewThe hyper-IgD and periodic fever syndrome (HIDS) is one of the classical monogenetic hereditary autoinflammatory disorders, and together with the more severe mevalonic aciduria it is also known as ‘mevalonate kinase deficiency’ (MKD). In this study, we will give an overview of the primary research on mevalonate kinase deficiency published in the past 2 years. Recent findingsBesides an inventory of a number of recent case reports, literature review shows there are several interesting developments in the basic field of research. First, a group of articles was recently published on chemically instead of genetically induced MKD mouse and cell models, investigating the effects of several isoprenoid pathway intermediates. Second, another study confirms a role for small GTPases and their isoprenylation in the inflammatory response in mevalonate kinase deficiency. Lastly, there are now, finally, modest new indications about the role of IgD. SummaryBoth pathophysiological studies and clinical observations in the last 2 years have supported the central role of IL-1 in HIDS. There are some intriguing results and hypotheses about the link between isoprenoid metabolism and the IL-1 pathway through geranylgeranylation that deserve to be further examined.

TLR2/TLR4-dependent exaggerated cytokine production in hyperimmunoglobulinaemia D and periodic fever syndrome

OBJECTIVE The autoinflammatory hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is characterized by recurrent episodes of fever and inflammation. As part of the mevalonate kinase deficiency spectrum, it is caused by MVK mutations, resulting in decreased mevalonate kinase activity in the isoprenoid pathway. Although IL-1β is considered a major cytokine in its pathogenesis, IL-1 blockade is not successful in a proportion of patients. We aimed to further characterize the pro-inflammatory cytokine profile of HIDS. METHODS Peripheral blood mononuclear cells from HIDS patients and healthy donors were incubated with several stimuli. Cytokine concentrations were detected by ELISA. To analyse mRNA and protein expression, we performed quantitative RT-PCR and western blot, respectively. RESULTS We observed significant differences in cytokine production when cells were incubated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerization domain-containing 2 (NOD2). The increased ratio between active and inactive caspase-1 protein in HIDS patients could explain why these cells are more easily triggered to secrete IL-1β. This is apparently not regulated at the transcriptional level, since expression levels of caspase-1 and IL-1β mRNA were similar in patients and controls. Both anakinra and tocilizumab treatment resulted in decreased inflammation, both ex vivo as well as in vivo. CONCLUSION The increased cytokine secretion in HIDS is specific for TLR2, TLR4 and NOD2 ligation. Although IL-1β is important in the HIDS pathology, our data suggest it is a multicytokine disease. A more rigorous clinical trial is required to determine whether IL-6 receptor blockade may be considered in patients not responding to anakinra treatment.

The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases.

BackgroundThe extracellular domains of cytokine receptors are released during inflammation, but little is known about the shedding of Toll-like receptors (TLR) and whether they can be used as diagnostic biomarkers.MethodsThe release of sTLR2 and sTLR4 was studied in in-vitro stimulations, as well as in-vivo during experimental human endotoxemia (n = 11, 2 ng/kg LPS), and in plasma of 394 patients with infections (infectious mononucleosis, measles, respiratory tract infections, bacterial sepsis and candidemia) or non-infectious inflammation (Crohn’s disease, gout, rheumatoid arthritis, autoinflammatory syndromes and pancreatitis). Using C-statistics, the value of sTLR2 and sTLR4 levels for discrimination between infections and non-infectious inflammatory diseases, as well as between viral and bacterial infections was analyzed.ResultsIn-vitro, peripheral blood mononuclear cells released sTLR2 and sTLR4 by exposure to microbial ligands. During experimental human endotoxemia, plasma concentrations peaked after 2 hours (sTLR4) and 4 hours (sTLR2). sTLR4 did not correlate with cytokines, but sTLR2 correlated positively with TNFα (rs = 0.80, P < 0.05), IL-6 (rs = 0.65, P < 0.05), and IL-1Ra (rs = 0.57, P = 0.06), and negatively with IL-10 (rs = -0.58, P = 0.06), respectively. sTLR4 had a similar area under the ROC curve [AUC] for differentiating infectious and non-infectious inflammation compared to CRP: 0.72 (95% CI 0.66-0.79) versus 0.74 (95% CI 0.69-0.80) [P = 0.80], while sTLR2 had a lower AUC: 0.60 (95% CI 0.54-0.66) [P = 0.0004]. CRP differentiated bacterial infections better from viral infections than sTLR2 and sTLR4: AUC 0.94 (95% CI 0.90-0.96) versus 0.58 (95% CI 0.51-0.64) and 0.75 (95% CI 0.70-0.80), respectively [P < 0.0001 for both].ConclusionssTLRs are released into the circulation, and suggest the possibility to use sTLRs as diagnostic tool in inflammatory conditions.