Blau syndrome (MIM #186580) is a rare, systemic granulomatous disease caused by mutations in the nucleotide-binding oligomerization domain 2 (NOD2) gene and is inherited in an autosomal dominant manner. It is classified as an autoinflammatory syndrome, a concept that has received increasing attention in recent years.

In 1985, Blau (1) reported a family that caused granulomas in the skin, eyes, and joints over four generations. In 1990, Pastores et al. (2) reported a mother and daughter with similar symptoms and considered them to be from the same category as the diseases reported by Blau; thus, they named the disease Blau syndrome. Subsequently, a linkage analyses of a large pedigree were presumed, and discovered that Blau syndrome and Crohn’s disease have mutations in the same gene (3). In 2001, when mutations in the NOD2 gene were identified in Crohn’s disease (4, 5), Miceli-Richard et al. (6) examined the NOD2 gene in four families with Blau syndrome, and identified three gene mutations.

Idiopathic sarcoidosis pediatric cases are rare, yet it is known that in a small subset of patients, peak onset occurs before the age of four years (less than 0.5% of all sarcoidosis cases) (7). The clinical features in these cases were the absence of bilateral hilar lymphadenopathy, and the presence of joint symptoms. Hence, these cases were occasionally referred to as early-onset sarcoidosis (EOS), and the difference from idiopathic sarcoidosis debated (8). Initially no gene abnormalities were found in two solitary cases of EOS (6). We collected ten EOS cases in Japan and identified the same NOD2 mutations in these patients as those reported for Blau syndrome (9), and now Blau syndrome and EOS are considered to be the same disease (10, 11).

NOD2 consists of 1,040 amino acids and has a three domain structure. The N-terminus contains two caspase activation and recruitment domain s (CARD), which are important as in signal transduction; the centrally located NOD region is involved in polymerization; and the C-terminus contains a leucine-rich repeat (LRR). The LRR recognizes muramyl dipeptide (MDP), a component of bacterial cell walls. In Blau syndrome, most patients have mutations in exon 4, the NOD region. Most of these mutations are missense, with single amino acid substitutions. The 334th amino acid of NOD2 is a hot spot for mutations. Typically, arginine (R) is mutated to glutamine (Q) or tryptophan (W) in this region (p.R334Q and p.R334W). Among the 50 patients with NOD2 mutations currently identified in Japan, the p.R334W mutation was most common (fifteen cases), followed by the p.R587C (nine cases), and then the p.R334Q (five cases) (12).

It is still unclear how the mutations identified in Blau syndrome are involved in the formation of granulomas. When NOD2 was shown to recognize MDP, an experimental system to overexpress the NOD2 genes into HEK293 cells was used, in which the mutant with a frameshift in the LRR frequently identified in Crohn’s disease was shown to be hyporesponsive to MDP (13). In contrast, mutants identified in Blau syndrome spontaneously promoted NF-κB transcription in the same experimental system, even without the addition of MDP (14). This system of assessing NF-κB transcriptional capacity using luciferase as an indicator is still today a very useful system for confirming whether identified mutations are those associated with Blau syndrome, and indeed all 15 mutants we identified showed spontaneous NF-κB transcriptional enhancement in the absence of MDP (9, 15). This ligand-independent NF-κB activation induced by mutations has been observed in another group of hereditary autoinflammatory syndromes known as cryopyrin-associated periodic syndromes (CAPS). CAPS is caused by mutations in the NLRP3 gene, another NOD family receptor (16). Interestingly, the p.R334W mutation in the NOD2 gene frequently found in Blau syndrome, corresponds to a missense mutation in an analogous position of the NLRP3 gene, the p.R260W mutation in CAPS. These mutations suggest a common molecular mechanism, inducing the autoactivation of NOD family receptors, between these two inflammatory diseases (17).

Nevertheless, we observe an overproduction of IL-1β in peripheral blood monocytes extracted from CAPS patients and the macrophages established from CAPS patients-derived induced pluripotent stem (iPS) cells compared to healthy controls (18, 19), whereas a reduced response to MDP to produce inflammatory cytokines in the peripheral blood of patients with Blau syndrome were reported (20, 21). This lower responsiveness to MDP at the cellular level was also observed in studies using iPS cells differentiated into macrophages established from our patients (22), or knock-in mice with a corresponding Nod2 mutation identified in Blau syndrome (23). Since granulomas may arise from the inability to eliminate foreign substances and pathogens, it is possible that a reduced response to MDP at the cellular level may induce chronic inflammation, leading to granuloma formation in Blau syndrome.

The clinical phenotype of Blau syndrome is characterized by a distinct triad of skin, joint and eye disorders.

Blau syndrome is currently treated with various therapies, with multiple reports on their effectiveness ( Table 1 ). However, there is no specific cure for the disease. Currently, the relationship between the variant of the genetic mutation and the response to treatment is not clear.

An investigation into the cellular phenotypes of Blau syndrome may be necessary to evaluate the efficacy of anti-TNF therapy since the pharmacological mechanism behind their effectiveness in Blau syndrome is unknown.

The IFNγ signal has been reported to be increased in localized skin lesions of idiopathic sarcoidosis and Blau syndrome (71) and IFNγ upregulates the expression of NOD2, acting as a priming signal, as previously reported (22). This was established through iPS cells from patients with Blau syndrome that were differentiated into macrophages and confirmed that IFNγ treatment enhanced the expression of NOD2. The enhanced expression of NOD2 through IFNγ priming was observed regardless of the presence or absence of the NOD2 mutations. However, the spontaneous transcriptional enhancement of NF-κB and the production of proinflammatory cytokines were observed only in cells that contained the NOD2 mutation associated with Blau syndrome. These effects were not found in cells where the NOD2 mutation was corrected to the wild-type amino acid using the CRISPR-Cas9 system. Of interest, skin lesions have appeared following BCG vaccination in some patients with Blau syndrome. Upon review of fifty patients in the cohort from Japan, the attending physician provided information indicating that BCG vaccination may have induced symptoms in nine patients (12). Since IFNγ is a cytokine that is highly associated with BCG-mediated immune responses, BCG vaccination may be involved in NOD2-mediated inflammatory responses through the induction of NOD2 expression.

While verifying the studies using Blau syndrome patient-derived iPS cells, additional, surprising results were obtained (72). Macrophages, differentiated from the peripheral blood of Blau syndrome who did not receive anti-TNF treatment, released inflammatory cytokines after being primed with IFNγ, as described above. In contrast, macrophages differentiated from patients treated with anti-TNF, did not produce proinflammatory cytokines, similar to macrophages differentiated from healthy subjects. This was despite anti-TNF patient macrophages being primed with IFNγ and being differentiated ex vivo for one week with unchanged culture conditions. A comprehensive gene expression analysis of all three groups indicated that the cells from the anti-TNF treatment patient group had a nearly identical gene expression pattern as the cells differentiated from healthy individuals, without the NOD2 mutation. Furthermore, when primed with IFNγ, cells from these two groups (healthy individuals and anti-TNF treated patients) behaved in the same manner and had gene expression patterns that differed from macrophages differentiated from patients not receiving an anti-TNF agent. This study suggests that prior exposure to a proinflammatory cytokine, such as TNFα, at the monocytic or earlier progenitor cell stage is an important determinant for the macrophage response to IFNγ. Thus, the use of anti-TNF antibodies for Blau syndrome treatment is appropriate in the sense that long-term administration of anti-TNF antibodies may correct the abnormalities that occur in the early progenitor stage by blocking the autoinflammatory loop and restoring the threshold for which IFNγ stimulation triggers an inflammatory response in macrophages ( Figure 1 ).

At present, no specific treatment for Blau syndrome exists, based on its etiology, and the disease is treated empirically. However, early diagnosis will allow for the prediction of future symptoms that are likely to appear, and prompt treatment will prevent or delay the onset of severe symptoms (joint contractures and blindness), which significantly impair the patient’s quality of life. Future accumulation of a large number of patients, with detailed information on disease progression, treatment, and prognosis, would be advantageous to analyze disease pathogenesis and establish a specific treatment based on disease etiology.

TM, NaK, RT-I, YU, SN, MS, ST, and NoK contributed to conception and design of the study. TM and NaK wrote the first draft of the manuscript. NaK and MS created the first draft of the figure, while RT-I created the final figure. All authors have read, contributed to revisions of the manuscript, and approved the submitted version.

This work was supported in part by the Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine (JP21bm0104001); the Acceleration Program for Intractable Diseases Research Utilizing Disease-Specific iPS Cells (17935423); the Practical Research Project for Rare/Intractable Diseases (17929899) from AMED (MS); a research grant from MHLW (NaK and NoK); a JSPS KAKENHI Grant Number 19K08784, 22K08380; and a Lexi’s Legacy Research Grant from Cure Blau syndrome Foundation (NaK).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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