Thibaut De Smedt

A pilot study of IL-1 inhibition by anakinra in acute gout

Monosodium urate crystals stimulate monocytes and macrophages to release IL-1β through the NALP3 component of the inflammasome. The effectiveness of IL-1 inhibition in hereditary autoinflammatory syndromes with mutations in the NALP3 protein suggested that IL-1 inhibition might also be effective in relieving the inflammatory manifestations of acute gout. The effectiveness of IL-1 inhibition was first evaluated in a mouse model of monosodium urate crystal-induced inflammation. IL-1 inhibition prevented peritoneal neutrophil accumulation but TNF blockade had no effect. Based on these findings, we performed a pilot, open-labeled study (trial registration number ISRCTN10862635) in 10 patients with gout who could not tolerate or had failed standard antiinflammatory therapies. All patients received 100 mg anakinra daily for 3 days. All 10 patients with acute gout responded rapidly to anakinra. No adverse effects were observed. IL-1 blockade appears to be an effective therapy for acute gouty arthritis. The clinical findings need to be confirmed in a controlled study.

Cutting Edge: Alum Adjuvant Stimulates Inflammatory Dendritic Cells through Activation of the NALP3 Inflammasome

Adjuvants are vaccine additives that stimulate the immune system without having any specific antigenic effect of itself. In this study we show that alum adjuvant induces the release of IL-1β from macrophages and dendritic cells and that this is abrogated in cells lacking various NALP3 inflammasome components. The NALP3 inflammasome is also required in vivo for the innate immune response to OVA in alum. The early production of IL-1β and the influx of inflammatory cells into the peritoneal cavity is strongly reduced in NALP3-deficient mice. The activation of adaptive cellular immunity to OVA-alum is initiated by monocytic dendritic cell precursors that induce the expansion of Ag-specific T cells in a NALP3-dependent way. We propose that, in addition to TLR stimulators, agonists of the NALP3 inflammasome should also be considered as vaccine adjuvants.

Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.

Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohns disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Role of CD8alpha+ and CD8alpha- dendritic cells in the induction of primary immune responses in vivo.

Data from adoptive transfer of mature dendritic cells (DC) indicate that they are responsible for the induction of primary immunity. Two subclasses of DC have been recently identified in spleen that differ in their phenotype and in certain regulatory features. In vitro, both subsets have the capacity to activate naive T cells, although CD8α+ DC have been shown to induce T cell apoptosis and to stimulate lower levels of cytokines compared with CD8α− DC. The objective of this study was to analyze the function of these distinct DC types in vivo. Our results show that both subsets, pulsed extracorporeally with antigen and injected in the footpads of syngeneic mice, sensitize an antigen‐specific T cell primary response. However, CD8α+ cells trigger the development of Th1‐type cells, whereas CD8α− DC induce a Th2‐type response. These observations suggest that the Th1/Th2 balance in vivo is regulated by the antigen‐presenting‐cells of the primary immune responses. J. Leukoc. Biol. 66: 242–246; 1999.

Ox40 Costimulation Enhances the Development of T Cell Responses Induced by Dendritic Cells In Vivo

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4+ and CD8+ T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-γ−/− mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-γ during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4+ T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-γ secretion, and cytotoxic activity of Ag-specific CD8+ T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.

CD8α− and CD8α+ subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen‐presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse‐DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8α expression. We have studied the phenotype and maturation state of mouse splenic CD8α− and CD8α+ DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM‐CSF‐containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T‐cell priming capacity, transforming CD8α− and CD8α+ DCs into potent and equally efficient activators of naïve CD4+ and CD8+ T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels. Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.

Positive and Negative Regulation of Dendritic Cell Function by Lipopolysaccharide in Vivo

There is evidence that induction of primary immune responses in vitro and in vivo relies on the presentation of antigen by dendritic cells1. The unique property of DC to sensitize naive T lymphocytes may depend on a specialization of function over time that results in a reciprocal regulation of the capacity to process antigens and to activate T cells. Fresh DC process antigens and generate high levels of peptides/MHC complexes, whereas cultured DC have lost this capacity but have gained the property to prime T lymphocytes. This process of maturation occurs spontaneously in culture and correlates with increased expression of class II, B7-1 and B7-2 costimulatory molecules2–6.