F.L. van de Veerdonk

Inflammasome-Independent Regulation of IL-1-Family Cytokines

Induction, production, and release of proinflammatory cytokines are essential steps to establish an effective host defense. Cytokines of the interleukin-1 (IL-1) family induce inflammation and regulate T lymphocyte responses while also displaying homeostatic and metabolic activities. With the exception of the IL-1 receptor antagonist, all IL-1 family cytokines lack a signal peptide and require proteolytic processing into an active molecule. One such unique protease is caspase-1, which is activated by protein platforms called the inflammasomes. However, increasing evidence suggests that inflammasomes and caspase-1 are not the only mechanism for processing IL-1 cytokines. IL-1 cytokines are often released as precursors and require extracellular processing for activity. Here we review the inflammasome-independent enzymatic processes that are able to activate IL-1 cytokines, paying special attention to neutrophil-derived serine proteases, which subsequently induce inflammation and modulate host defense. The inflammasome-independent processing of IL-1 cytokines has important consequences for understanding inflammatory diseases, and it impacts the design of IL-1-based modulatory therapies.

Crohn's disease-associated ATG16L1 polymorphism modulates pro-inflammatory cytokine responses selectively upon activation of NOD2

Objective Autophagy has recently been shown to modulate the production of pro-inflammatory cytokine production and to contribute to antigen processing and presentation through the major histocompatibility complex. Genetic variation in the autophagy gene ATG16L1 has been recently implicated in Crohns disease pathogenesis. The mechanisms underlying this association are not yet known, although experimental models suggest an inhibitory effect of autophagy on interleukin 1β (IL-1β) responses. Here, the effect of ATG16L1 genetic variation on cytokine responses has been assessed in humans. Design and setting Peripheral blood mononuclear cells from healthy individuals and patients with Crohns disease with different ATG16L1 genotypes were stimulated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain 2 (NOD2), with or without the autophagy inhibitor 3-methyladenine. Induction of cytokine production and related factors were measured at the mRNA and protein level. Furthermore, protein levels of ATG16L1 were assessed by western blot. Results The present study demonstrates that cells isolated from individuals bearing the ATG16L1 Thr300Ala risk variant, which is shown to affect ATG16L1 protein expression upon NOD2 stimulation, display increased production of the pro-inflammatory cytokines IL-1β and IL-6, specifically after stimulation with NOD2 ligands. In contrast, no differences were found when cells were stimulated with TLR2 or TLR4 agonists. These findings were confirmed in two independent cohorts of volunteers and in a group of patients with Crohns disease. The increased production could be ascribed to increased mRNA expression, while processing of pro-IL-1β by caspase-1 activation was not affected. The effect of the ATG16L1 polymorphism was abrogated when autophagy was blocked. Conclusions The present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1β and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohns disease.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

The role of NLRs and TLRs in the activation of the inflammasome

Background: Interleukin-1β is one of the most important pro-inflammatory cytokines. In contrast to other cytokines, activation of IL-1β requires processing from an inactive precursor by the cysteine protease caspase-1. Caspase-1 forms a protein platform called the inflammasome, together with proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family. Objective/methods: A review of literature investigating the stimulation of IL-1β production by microbial pathogens and their components. Results/conclusions: To produce IL-1β, macrophages need a double stimulation with Toll like receptor (TLR) ligands that induce gene transcription, and NLR agonists (such as ATP or muramyl dipeptide (MDP)) that activate the inflammasome. Monocytes can release active IL-1β upon stimulation with TLR ligands alone. This probably represents an adaptation of each cell type to its environment.

Neutrophil-Mediated Inhibition of Proinflammatory Cytokine Responses

Neutrophils (polymorphonuclear neutrophils [PMNs]) play an elaborate role in the innate immune response against invading pathogens. Recent research provided evidence that PMNs can play a modulatory role in inflammation next to their primary role of phagocytosis. In the current study, we investigated whether neutrophils can modulate the innate immune response against Candida albicans. Production of the proinflammatory cytokines IL-1β and TNF-α by human PBMCs in response to C. albicans or LPS was decreased by coculture of PMNs; however, the anti-inflammatory cytokine IL-10 remained unaffected. Using Transwells and cells of patients with chronic granulomatous disease, we show that this downregulation of proinflammatory cytokine production was independent of phagocytosis and reactive oxygen species but was dependent on a soluble factor. We suggest that neutrophil-derived proteases are responsible for the downregulation of IL-1β and TNF-α, as cytokine production could be recovered by addition of α1-antitrypsin, an endogenous inhibitor of serine proteases. PMN lysates and neutrophil elastase could degrade recombinant human IL-1β and TNF-α but not IL-10, and this could be inhibited by addition of α1-antitrypsin. Moreover, we also provide evidence that the dampening effect of PMNs is present in vivo in a murine zymosan-induced arthritis model and a murine experimental endotoxemia model. Altogether, our data show that PMNs can dampen the proinflammatory response to C. albicans by protease-mediated degradation of cytokines. This observation suggest that PMNs might play a important regulatory role in the host defense against C. albicans and can be important for understanding the regulation of inflammation in general.

Differential effects of IL-17 pathway in disseminated candidiasis and zymosan-induced multiple organ failure.

The role of the IL-17 pathway in antifungal host defense is controversial. Several studies suggested that IL-17 is crucial for the protection against Candida infection, whereas other studies reported that IL-17 may contribute to inflammatory pathology and worsening of fungal disease. To address these discrepancies, we assessed the differential role of IL-17 pathway in two models of fungal sepsis: intravenous infection with live Candida albicans, in which fungal growth is the main cause of mortality, and zymosan-induced multiple organ failure, in which the inflammatory pathology drives the mortality. First, IL-17 receptor-deficient (IL-17RA−/−) mice showed increased mortality and higher fungal loads in the kidneys in the model of disseminated candidiasis, partly caused by lower neutrophil recruitment in the IL-17RA−/− mice. Second, IL-17RA−/− mice were not protected against the multiorgan failure induced by zymosan. These data demonstrate that IL-17 does not have a major contribution to the inflammatory pathology leading to organ failure in fungal sepsis and support the concept that the IL-17 pathway is protective in antifungal host defense.

Aspergillus fumigatus–Induced IL-22 Is Not Restricted to a Specific Th Cell Subset and Is Dependent on Complement Receptor 3

Th cell responses induced by Aspergillus fumigatus have been extensively investigated in mouse models. However, the requirements for differentiation and the characteristics of A. fumigatus–induced human Th cell subsets remain poorly defined. We demonstrate that A. fumigatus induces Th1 and Th17 subsets in human PBMCs. Moreover, we show that the cytokine IL-22 is not restricted to a specific Th subset, in contrast to IL-17A. The pattern recognition and cytokine pathways that skew these Aspergillus-induced Th cell responses are TLR4- and IL-1–, IL-23–, and TNF-α–dependent. These pathways are of specific importance for production of the cytokines IL-17A and IL-22. Additionally, our data reveal that the dectin-1/Syk pathway is redundant and that TLR2 has an inhibitory effect on Aspergillus-induced IL-17A and IL-22 production. Notably, blocking complement receptor (CR)3 significantly reduced Aspergillus-induced Th1 and Th17 responses, and this was independent on the activation of the complement system. CR3 is a known receptor for β-1,3-glucan; however, blocking CR3 had significant effects on Th cell responses induced by heat-killed Aspergillus conidia, which have minimal β-glucan expression on their cell surface. Collectively, these data characterize the human Th cell subsets induced by Aspergillus, demonstrate that the capability to produce IL-22 is not restricted to a specific T cell subset, and provide evidence that CR3 might play a significant role in the adaptive host defense against Aspergillus, although the ligand and its action remain to be elucidated.

Autophagy is redundant for the host defense against systemic Candida albicans infections

Autophagy has been demonstrated to play an important role in the immunity against intracellular pathogens, but very little is known about its role in the host defense against fungal pathogens such as Candida albicans. Therefore, the role of autophagy for the host defense against C. albicans was assessed by complementary approaches using mice defective in autophagy, as well as immunological and genetic studies in humans. Although C. albicans induced LC3-II formation in macrophages, myeloid cell-specific ATG7−/− mice with defects in autophagy did not display an increased susceptibility to disseminated candidiasis. In in vitro experiments in human blood mononuclear cells, blocking autophagy modulated cytokine production induced by lipopolysaccharide, but not by C. albicans. Furthermore, autophagy modulation in human monocytes did not influence the phagocytosis and killing of C. albicans. Finally, 18 single-nucleotide polymorphisms in 13 autophagy genes were not associated with susceptibility to candidemia or clinical outcome of disease in a large cohort of patients, and there was no correlation between these genetic variants and cytokine production in either candidemia patients or healthy controls. Based on these complementary in vitro and in vivo studies, it can be concluded that autophagy is redundant for the host response against systemic infections with C. albicans.

Pattern recognition pathways leading to a Th2 cytokine bias in allergic bronchopulmonary aspergillosis patients

Allergic bronchopulmonary aspergillosis (ABPA) is characterised by an exaggerated Th2 response to Aspergillus fumigatus, but the immunological pathways responsible for this effect are unknown.

Milder clinical hyperimmunoglobulin E syndrome phenotype is associated with partial interleukin-17 deficiency.

Mutations in the signal transducer and activator of transcription 3 (STAT3) were reported to cause hyperimmunoglobulin E syndrome (HIES). The present study investigates T helper type 17 (Th17) responses triggered by the relevant stimuli Staphylococcus aureus and Candidia albicans in five ‘classical’ HIES patients, and a family with three patients who all had a milder HIES phenotype. We demonstrate that patients with various forms of HIES have different defects in their Th17 response to S. aureus and C. albicans, and this is in line with the clinical features of the disease. Interestingly, a partial deficiency of interleukin (IL)‐17 production, even when associated with STAT3 mutations, leads to a milder clinical phenotype. We also observed defective Th17 responses in patients with the ‘classical’ presentation of the disease but without STAT3 mutations. These data demonstrate that defective IL‐17 production in response to specific pathogens can differ between patients with HIES and that the extent of the defective Th17 response determines their clinical phenotype.