We read with great interest the recent article by Martin et al on the role of interleukin-1β (IL-1β) secretion in Blau syndrome (1). To evaluate synergistic effects, the authors measured IL-1β secretion levels in peripheral blood mononuclear cells (PBMCs) isolated from 5 patients with Blau syndrome, compared with levels in the PBMCs of 5 controls, after cells were stimulated with muramyldipeptide, Pam3Cys (a Toll-like receptor-2 [TLR-2] agonist), lipopolysaccharide (LPS), or with combinations of muramyldipeptide and either Pam3Cys or LPS. In addition, Martin and colleagues presented 2 case reports in which recombinant human IL-1 receptor antagonist (anakinra) was not effective in treating Blau syndrome. Finally, the authors stated that Blau syndrome is not mediated by excess IL-1 activity.
We would like to present evidence that is consistent with the findings of Martin et al and to offer our own hypotheses. We also have data on IL-1β secretion levels in the PBMCs of 2 patients with Blau syndrome with an arginine-to-tryptophan mutation at position 334 of NOD-2 (R334W) (Figure 1A). These patients (whose cases have been reported previously ) had both been receiving prednisolone (15 mg/day [0.3 mg/kg]). The methods of isolation of PBMCs and the analysis of cytokine concentrations in culture supernatants were described by us in a previous report (3), and the analysis of patient materials was approved by the Human Research Ethical Committee of Shinshu University. We evaluated secretion levels of IL-1β, tumor necrosis factor α (TNFα), IL-6, and IL-8 in the culture supernatants of PBMCs that were left untreated for 8 hours or incubated with muramyldipeptide (10 ng/ml or 1 μg/ml) (a NOD-2 stimulatory ligand), LPS (0.1 ng/ml or 10 ng/ml) (a TLR-4 stimulatory ligand), or muramyldipeptide (10 ng/ml or 1 μg/ml) combined with a low amount of LPS (0.1 ng/ml). The data showed that IL-1β secretion from PBMCs isolated from patients with Blau syndrome remained undetectable without stimulation and was low after treatment with muramyldipeptide alone. These findings were the same as in healthy controls. PBMCs isolated from patients exhibited a lower response to LPS stimulation than cells from healthy controls. Notably, the synergistic stimulatory effect of the combination of muramyldipeptide and LPS on IL-1β secretion, which was observed in healthy controls, was not observed in patients with Blau syndrome, while the secretion levels of TNFα, IL-6, and IL-8 in the PBMCs of patients exhibited normal responses compared with healthy volunteers (Figures 1B–D). These observations are consistent with those reported by Martin et al (1).
IL-1β synthesis is known to be regulated by a 2-step process: transcriptional and translational regulation, and posttranslational regulation. In the first step, activation of the transcription factor NF-κB leads to transcription of the proIL-1β gene, which can be translated to proIL-1β. The next step is maturation of proIL-1β by inflammasome, which is known to be an IL-1β–processing platform composed of Nod-like receptors, ASC, and caspase 1 (4). The secretion of IL-1β in PBMCs is known to be synergistically induced by muramyldipeptide and LPS. It has also been reported that this synergistic effect of NOD-2 and TLR on IL-1β maturation is caspase 1 dependent and that the activation of caspase 1 and the release of mature IL-1β by muramyldipeptide is NOD-2 dependent (5).
Mutated NOD2 in patients with Blau syndrome is thought to be a constitutive active form of NOD-2, which has been found to lead to constitutive NF-κB activation in studies performed in vitro (6, 7). The results of those studies do not contradict the data presented by Martin et al (1) or our own data (Figure 1), because the in vitro observation in HEK 293T cells transfected with the mutated form of NOD2 (R334W) reflects primary initiated high-level NF-κB activation. Activation of NF-κB by constitutive activated mutated NOD-2 occurs via induction of a signaling complex, including RICK/RIP2 and IKK complex. Constitutive NF-κB activation then induces a negative feedback regulator, such as A20, a downstream regulator of RICK/RIP2 (8); therefore, we hypothesize that such a negative feedback regulator may affect inflammasome for IL-1β secretion either directly or indirectly. IL-1β secretion due to a synergistic effect of muramyldipeptide and LPS was not observed in PBMCs isolated from patients with Blau syndrome. Therefore, it is possible that the pathogenesis of Blau syndrome may be related to suppression of IL-1β synthesis.