Pluripotent stem cell models of Blau syndrome reveal an IFN-γ-dependent inflammatory response in macrophages.

BACKGROUND: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. OBJECTIVES: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. METHODS: Blau syndrome-specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. RESULTS: IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ-dependent inflammatory response. CONCLUSIONS: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells.

Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.

[Changes in Japanese cedar sensitization rates of Japanese allergic children during the last 15 years].

In order to evaluate the changes in Japanese Cedar (JC) sensitization rates of allergic children, serum samples from 88 patients about 15 years ago (past group) and those from 91 current patients (present group) were randomly selected, and their JC specific IgE were measured with the CAP-RAST system. Sensitivity rate (class 2 or more) for JC of the present group was 65.9%, which was significantly higher than that of the past group, which was 46.6%. However, there was no significant difference between these two groups for children aged 6 or younger. For children aged 7 or older, the sensitivity rate of the present group was significantly higher than that of the past group. Thus, protection against JC sensitization, especially during early childhood, should be given serious attention.