The neutrophil NLRC4 inflammasome selectively promotes IL-1β maturation without pyroptosis during acute Salmonella challenge.

The macrophage NLRC4 inflammasome drives potent innate immune responses against Salmonella by eliciting caspase-1-dependent proinflammatory cytokine production (e.g., interleukin-1β [IL-1β]) and pyroptotic cell death. However, the potential contribution of other cell types to inflammasome-mediated host defense against Salmonella was unclear. Here, we demonstrate that neutrophils, typically viewed as cellular targets of IL-1β, themselves activate the NLRC4 inflammasome during acute Salmonella infection and are a major cell compartment for IL-1β production during acute peritoneal challenge in vivo. Importantly, unlike macrophages, neutrophils do not undergo pyroptosis upon NLRC4 inflammasome activation. The resistance of neutrophils to pyroptotic death is unique among inflammasome-signaling cells so far described and allows neutrophils to sustain IL-1β production at a site of infection without compromising the crucial inflammasome-independent antimicrobial effector functions that would be lost if neutrophils rapidly lysed upon caspase-1 activation. Inflammasome pathway modification in neutrophils thus maximizes host proinflammatory and antimicrobial responses during pathogen challenge.

Characterization of NALP2 and NALP3 IL-1β processing inflammasomes

Résumé II y a cinq ans, la découverte d'un nouveau domaine, le PYD domaine, lié aux domaines de la mort, a permis la description de la nouvelle famille des NALP protéines. L'analyse structurelle de cette famille de protéines révéla la présence de deux autres domaines, impliqués dans l'oligomerisation, NACHT, et la détection des ligands, Leucine rich repeats ou LRR. Cette architecture protéique est homologue à celle qui est décrite pour les NODs, les Tol1 récepteurs et tes protéines de résistance chez les plantes. Cette homologie suggère une possible implication des NALPs dans la régulation de l'immunité innée. Premièrement, nous avons décrit les composants minimaux qui permettent à l'inflammasomeNALP3 d'activer la caspase pro-inflammatoire, caspase-1. En comparaison à NALP1, NALP3 ne contient pas de FIIND domaine, ni de CARD domaine en C-terminus et n'interagit pas avec caspase-5. Nous avons découvert une protéine très homologue au C-terminus de NALP1, Cardinal, qui se lie au NACHT domaine de NALP2 et NALP3 par l'intermédiaire de son FIIND domaine. Cardinal possède la capacité d'interagir avec caspase-l, mais seul ASC semble être nécessaire à la maturation de la prointerleukine-1β suite à la stimulation de NALP3. Deuxièmement, notre étude s'est concentrée sur la nature du stimulus capable d'induire la formation et l'activation de l'inflammasome-NALP3. Nous avons démontré que l'ajout de muramyl dipeptide (MDP), produit à partir de la digestion enzymatique de peptidoglycaris bactériens, induit à la fois l'expression de la proIL-1β par la voie NOD2 et sa maturation en IL-1β active par la voie NALP3. Bien que le MDP active l'inflammasome-NALP3, il est incapable d'induire la sécrétion de l'IL-1β mature dans la lignée cellulaire THP1, comparé aux monocytes primaires humains. Cette différence pourrait être liée à l'absence, dans les THP1, de la protéine Filamin, qui est proposée d'interagir avec Cardinal. L'implication de NALP3 dans la maturation de l'IL-lb est confirmée suite à la découverte de mutations sur le gène CIAS1/NALP3/cryopyrin associées à trois maladies auto-inflammatoires : le syndrome de Muckle-Wells (MWS), l'urticaire familial au froid (FCU) et le syndrome CINCA/NOMID. Une élévation constitutive de la maturation et de la sécrétion de la proIL-1β en absence de stimulation MDP est détectée dans les macrophages des patients Muckle-Wells. En conclusion, nos études ont démontré que l'inflammasome-NALP3 doit être finement régulé pour éviter une activité incontrôlée qui représente la base moléculaire des symptômes associés aux syndromes auto-inflammatoires liés à NALP3. Summary Five years ago, the description of the NALP family originated from the discovery of a new death-domain fold family, the PYD domain. NALP contains aprotein-protein interaction domain (PYD), an oligomerization domain (NACHT) and a ligand-sensing domain, leucine rich repeats or LRR. This protein architecture shares similarity with receptors involved in immunity, such as NODS, Toll receptors (TLRs) and related plant resistance proteins, and points to an important role of NALPs in defense mechanisms. We first described the minimal complex involved in the pro-inflammatory Interleukin-1beta (IL-1β) cytokine maturation, called the inflammasome, which contains NALP3. In contrast to NALP1, NALP3, like other members of the NALP family, is devoid of C-terminal FIIND and CARD domains and does not interact with the pro-inflammatory caspase-5. Interestingly, a homolog of the C-terminal portion of NALP1 was found in the human genome and was named Cardinal. We found that NALP2 and NALP3 interact with the CARD-containing proteins Cardinal. Cardinal is able to bind to caspase-1 but is not required for IL-1β maturation through NALP3 activation, as demonstrated for the adaptor ASC. Secondly, our study focused on the stimuli involved in the activation of the NALP3 inflammasome. MDP was shown to induce the expression of proIL1β through NOD2 and then the maturation into active IL-1β by activation of the NALP3 inflammasome. However, in the monocytic THP1 cell line, secretion of IL-1β upon MDP stimulation seems to be independent of the inflammasome activation compared to human primary monocytes. This difference might be linked to a Cardinal-interacting protein, filamin. Until now, the role of Cardinal and filamin is still unknown and remains to be elucidated. Finally, mutations in the NALP3/cryopyrin/CIAS1 gene are associated with three autoinflammatory diseases: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. Constitutive, elevated IL-1β maturation and secretion, even in the absence of MDP stimulation, was observed in macrophages from Muckle-Wells patients and confirmed a key role for the NALP3 inflammasome in innate immunity In conclusion, our studies describes the formation of the NALP3 inflammasome and suggests that this complex has to be tightly regulated to avoid an increased deregulated inflammasome activity that is the molecular basis for the symptoms associated with NALP3-dependent autoinflammatory disorders.

Cutting edge: cyclic polypeptide and aminoglycoside antibiotics trigger IL-1β secretion by activating the NLRP3 inflammasome.

Clinical use of antibiotics is based on their capacity to inhibit bacterial growth via bacteriostatic or bacteriocidal effects. In this article, we show that the aminoglycoside antibiotic neomycin, the cyclic lipopeptide antibiotic polymyxin B, and the cyclic peptide antibiotics gramicidin and tyrothricin can induce IL-1β secretion in bone marrow dendritic cells and macrophages. LPS priming was required to trigger the transcription and translation of pro-IL-1β but was independent of TNFR or IL-1R signaling. All four antibiotics required the NLRP3 inflammasome, the adaptor ASC, and caspase-1 activation to secrete IL-1β, a process that depended on potassium efflux but was independent of P2X7 receptor. All four antibiotics induced neutrophil influx into the peritoneal cavity of mice, which required NLRP3 only in the case of polymyxin B. Together, certain antibiotics have the potential to directly activate innate immunity of the host.

Characterization of a specific 20- to 25-kD interleukin-1 inhibitor from cultured human lung macrophages.

Alveolar macrophages have the ability to downregulate immune processes in vitro. We have recently suggested the presence of interleukin-1 (IL-1) inhibitors in the supernatants of human bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis or sarcoidosis. In the present study, we further analyze the cellular origin and the biologic properties of a 20- to 25-kD IL-1 inhibitor spontaneously produced by cultured human alveolar macrophages (AM). The inhibitor blocks IL-1-induced prostaglandin E2 production by human fibroblasts and the IL-1-related increase of phytohemagglutinin-induced murine thymocyte proliferation. After rigorous IL-1 alpha and IL-1 beta depletion, supernatants of lung macrophages specifically block the binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1 in a dose-dependent manner. These results indicate that AM from both normal donors and patients produce a specific IL-1 inhibitor that may be of importance in protecting the alveolar environment from the deleterious effects of excessive IL-1 production.

Type I IFN-mediated regulation of IL-1 production in inflammatory disorders.

Although contributing to inflammatory responses and to the development of certain autoimmune pathologies, type I interferons (IFNs) are used for the treatment of viral, malignant, and even inflammatory diseases. Interleukin-1 (IL-1) is a strongly pyrogenic cytokine and its importance in the development of several inflammatory diseases is clearly established. While the therapeutic use of IL-1 blocking agents is particularly successful in the treatment of innate-driven inflammatory disorders, IFN treatment has mostly been appreciated in the management of multiple sclerosis. Interestingly, type I IFNs exert multifaceted immunomodulatory effects, including the reduction of IL-1 production, an outcome that could contribute to its efficacy in the treatment of inflammatory diseases. In this review, we summarize the current knowledge on IL-1 and IFN effects in different inflammatory disorders, the influence of IFNs on IL-1 production, and discuss possible therapeutic avenues based on these observations.

Canakinumab vs triamcinolone acetonide for treatment of acute flares and prevention of recurrent flares in "difficult to treat" gouty arthritis

Monosodium urate crystal deposition seen in gout stimulates IL-1 beta OR IL-1_; release. Canakinumab, a long-acting, fully human anti- IL-1 beta OR IL-1_; monoclonal antibody, effectively neutralizes IL-1 beta OR IL-1_;. Methods: This was an 8-week, dose-ranging, multi-center, blinded, doubledummy, active-controlled trial. Patients (aged 18-80 years) with an acute gout flare, refractory to or contraindicated to NSAlDs and/or colchicine, were randomized to one dose of canakinumab 10, 25, 50, 90, 150 mg s.c. or triamcinolone acetonide (TA) 40 mg i.m. Primary variable was assessed as pain intensity at 72 h post-dose (0-100 mm VAS). Secondary variables included pain intensity 24 and 48 h post-dose, time to 50% reduction in pain intensity, time to recurrence of gout flares up to 8 weeks post-dose, and rescue medication use. Results: 191/200 enrolled patients (canakinumab, n_143; TA, n_57) completed the study. Canakinumab showed significant dose-dependent pain reduction at 72 h. Canakinumab 150 mg showed superior pain relief versus TA starting from 24 h: estimated mean difference in pain intensity on VAS was -11.5 (24 h), -18.2 (48 h), and -19.2 (72 h) (all p_0.05). Canakinumab 150 mg provided a rapid onset of pain relief: median time to 50% reduction in pain was reached at 1 day with canakinumab 150 mg versus 2 days with TA (p_0.0006). At Week 8, recurrent flares occurred in 1 patient (3.7%) on canakinumab 150 mg versus 25 (44.6%) patients on TA (relative risk reduction, 94%; p_0.006). During 7 days post-dose, 6 patients (22.2%) on canakinumab 150 mg, and 31 patients (55.4%) on TA, took rescue medication. Time to first rescue medication was significantly longer with canakinumab 150 mg versus TA (hazard ratio, 0.36; p_0.02). Serious adverse events (canakinumab _lsqb_n_4_rsqb_ and TA _lsqb_n_1_rsqb_) were considered not treatment-related by investigators and no patient discontinued due to adverse events. Conclusions: Canakinumab 150 mg was well-tolerated, provided rapid and sustained pain relief in patients with acute gout flares, and significantly reduced the recurrent flare risk by 94% at 8-weeks post-dose compared with triamcinolone acetonide.

Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome.

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.

Targeting IL-1β and IL-17A driven inflammation during influenza-induced exacerbations of chronic lung inflammation.

For patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. Although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. In this study, we report that interleukin-1β (IL-1β) and interleukin-17A (IL-17A) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. Using a mouse model of disease, our data shows a role for IL-1β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. We further report a role for IL-17A as a mediator of IL-1β induced neutrophilia at early time points during influenza-induced exacerbations. Blocking of IL-17A or IL-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. Therefore, IL-17A and IL-1β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation.

B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A.

Relationship of IL-1 and TNF-α polymorphisms with Helicobacter pylori in gastric diseases in a Brazilian population

It is well known that the risk of development of gastric cancer (GC) in Helicobacter pylori-infected patients depends on several factors. Thus, the aim of this study was to investigate the effect of proinflammatory cytokine gene polymorphisms for IL-1β, IL-1RN and TNF-α on the development of GC in a Brazilian population. A total of 202 biopsies obtained from Brazilian patients with chronic gastritis and GC were included in the study. Infection with H. pylori cagA+ was determined by the polymerase chain reaction (PCR) as previously described. IL-1β, IL-1RN and TNF-α polymorphism genotyping was performed by restriction fragment length polymorphism PCR. Associations between gene polymorphisms, clinical diseases and virulence markers were evaluated using either the χ² test or the Fisher exact test. Our results demonstrated that the IL-1β -511 C/C and IL-1β -511 C/T alleles were associated with chronic gastritis in H. pylori-positive patients (P = 0.04 and P = 0.05, respectively) and the IL-1β -511 C/C genotype was associated with GC (P = 0.03). The frequency of IL-1RN alleles from patients with chronic gastritis and GC indicated that there was no difference between the genotypes of the groups studied. Similar results were found for TNF-α -308 gene polymorphisms. Our results indicate that the IL-1β -511 C/C and C/T gene polymorphisms are associated with chronic gastritis and GC development in H. pylori-infected individuals.

Psychological stress alters the ultrastructure and increases IL-1β and TNF-α in mandibular condylar cartilage

Psychological factors can be correlated with temporomandibular disorders (TMDs), but the mechanisms are unknown. In the present study, we examined the microstructural changes and expression of proinflammatory cytokines in mandibular condylar cartilage of the temporomandibular joint (TMJ) in a psychological stress animal model. Male Sprague-Dawley rats (8 weeks old, 210 ± 10 g) were randomly divided into 3 groups: psychological stress (PS, N = 48), foot shock (FS, N = 24), and control (N = 48). After inducing psychological stress using a communication box with the FS rats for 1, 3, or 5 weeks, PS rats were sacrificed and compared to their matched control littermates, which received no stress and were killed at the same times as the PS rats. Body and adrenal gland weight were measured and corticosterone and adrenocorticotropic hormone levels were determined by radioimmunoassay. After hematoxylin-eosin staining for histological observation, the ultrastructure of the TMJ was examined by scanning electron microscopy. Transcription and protein levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evaluated by ELISA and semi-quantitative RT-PCR. The PS group showed a significantly higher adrenal gland weight after 3 weeks of stress and higher hormone levels at weeks 1, 3, and 5. Histopathological changes and thinning cartilage were apparent at weeks 3 and 5. In the PS group, TNF-α increased at 1, 3, and 5 weeks and IL-1β increased significantly after 1 and 3 weeks of stress, and then decreased to normal levels by 5 weeks. Psychological stress increased plasma hormone levels and RT-PCR indicated increased IL-1β and TNF-α expression in the TMJ in a time-dependent manner. These results suggest that cytokine up-regulation was accompanied by stress-induced cartilage degeneration in the mandibular condyle. The proinflammatory cytokines play a potential role in initiating the cartilage destruction that eventually leads to the TMDs.

Efecto in vitro de diferentes dosis de Mycobacterium tuberculosis en la expresión de IL-1 de membrana y moléculas HLA-DR en macrófagos humanos

Tesis (Maestría en Ciencias con Especialidad en Inmunología) UANL