Flow Cytometric Expression of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Protein: An Effective Screening Tool for Blau Syndrome

Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disorder presenting in early childhood and characterized by a triad of granulomatous arthritis, dermatitis, and uveitis. It is caused by variants in the nucleotide oligomerization domain 2 (NOD2) gene that encode for the NOD2 protein, having two caspase recruitment (CARD) domains, a centrally located NACHT domain and six leucine-rich repeats (LRRs) [1]. This cytosolic pattern recognition receptor protein is expressed in various immune cells and recognizes muramyl dipeptide (MDP) derived from intracellular bacterial lipopolysaccharides and triggers an immune response by activating the NFKB pathway. Mutations in the NOD2 gene decrease the capability of spontaneous oligomerization of the protein. Over the years, the number of NOD2 mutations associated with BS has expanded, and most variants are found at or near the nucleotide-binding NOD/NACHT domain or extending into the C-terminal region of the LRR structure, with the R334W variant being the most common. Frequent variants of uncertain significance, incomplete penetrance, as well as recognition of asymptomatic carriers have made genotype–phenotype correlation difficult to understand. Clinically, patients with BS are often diagnosed late, and as a result, patients develop significant ocular morbidity and/or end-organ damage. So, early identification is crucial for timely intervention and management. Genetic testing is expensive and time-consuming, so a need was felt to have a flow cytometry-based rapid screening test to identify the subset of patients who would benefit from further evaluation by expensive and time-consuming genetic tests. Flow cytometry enables rapid and cost-effective assessment of NOD2 protein levels in different immune cell subsets, with same-day turnaround time, making it particularly useful in acute or resource-constrained clinical settings.

Patients with clinical suspicion of BS were screened for NOD2 variants and enrolled in the study after informed written consent. The patients were registered at the Pediatric Immune Deficiency Clinic and Pediatric Rheumatology Clinic, Advanced Pediatric Centre, PGIMER, Chandigarh, India. The clinical profile of 11 BS patients (10 children, 1 adult) from our cohort was published (PMID: 36189202) highlighting that 54.5% of patients had a classic triad, while the frequency of arthritis, dermatitis, and uveitis was 100%, 81.8%, and 72.7%, respectively. Among these 11 patients, flow cytometry and gene expression studies could be carried out on only 9 patients, as 1 patient died and one was lost to follow up. Whole blood (5 mL) was obtained from patients with BS and healthy controls (HC). NOD2 protein expression was assessed in BS (n = 9) and HC (n = 7) by intracellular staining of different immune cells by flow cytometry. Isolation of peripheral blood mononuclear cells (PBMCs) was carried out from patients with BS and HC using the density gradient centrifugation method. Cells were stained with a titrated volume of mouse anti-human CD3-FITC (561802- Becton Dickinson, USA), mouse anti-human CD19-APC (555415- Becton Dickinson, USA) mouse anti-human CD16 BV421 (562878-Becton Dickinson, USA), mouse anti-human CD14 PE-Cy7 (560919-Becton Dickinson, USA), mouse anti-human CD11c PerCP-Cy5.5 (565227-Becton Dickinson, USA), mouse anti-human CD4 BV510 (563094-Becton Dickinson, USA), and mouse anti-human CD8 BUV395 (563796- Becton Dickinson, USA). The cells were then washed, lysed, and fixed using lyse and fix (558049, Becton Dickinson, USA), permeabilized using Triton X-100, and incubated for 10 min. Finally, cells were stained with mouse anti-human NOD2 labeled with PE (NB100-524PE- Novus Biosciences, USA) and acquired on the BD LSRFortessa X-20 flow cytometer. The proportion of T cells and subsets, B cells, NK cells, and monocyte subsets was assessed. The gating strategy for assessing NOD2 protein expression in different immune cells is highlighted in Figure 1. Statistical comparison among patients and healthy control was performed by non-parametric t test (not normally distributed data) followed by Mann–Whitney U test and value of p < 0.05 was considered significant. Results were analyzed using GraphPad Prism 9 Software (8.3.0.538). Graphs were obtained from GraphPad. The data variability was presented using median ± interquartile range (IQR).

We corroborated flow cytometry results with NOD2 gene expression on whole blood using real time polymerase chain reaction in patients with BS (n = 9) and HC (n = 8). For gene expression, total RNA was isolated using QIAamp RNA Blood Mini Kit (Qiagen) as per standard protocol and quantified. cDNA was synthesized from messenger RNA (mRNA) using cDNA synthesis Kit. Gene specific primers were designed, and Ct method was used to calculate quantitative gene expression (Kenneth and Thomas, 2001).

The patients with BS had lower NOD2 protein expression expressed as delta median fluorescence intensity (ΔMFI) across all immune cells: T cells, CD4 + T cells, CD8 + T cells, B cells, NK cells, classical monocytes, intermediate monocytes and non-classical monocytes as compared to healthy control. However, a significant difference in ∆MFI was obtained for B cells (p = 0.03) as well as for CD8 + T cells (p = 0.05). The ∆MFI, median ± IQR of NOD2 expression on different immune subsets in patients and healthy controls has been highlighted in Table 1.

Gene expression analysis revealed reduced NOD2 gene expression in patients with BS (p = 0.04) as compared to healthy controls. Expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-18) was reduced in BS patients (ns) while comparable values were noted for TNF-ɑ and transcription regulators of inflammation (NF-κB1 and NF-κB2) (Figure 2). It is difficult to correlate NOD2 protein expression with clinical outcomes in this small cohort. However, we tried to correlate NOD2 protein expression with clinical parameters like age at disease onset, presence of family history, uveitis and treatment response. Two patients with reduced NOD2 protein expression in all immune cell subsets had early onset of disease with classical Blau syndrome, positive family history and required multiple immunosuppressive therapies.

Early identification is crucial for timely intervention and management of BS. Genetic sequencing of the NOD2 gene remains the gold standard for diagnosis. However, genetic sequencing can be expensive and time-consuming. Flow cytometric assessment of NOD2 protein expression presents a promising alternative for screening due to its cost-effectiveness and rapid turnaround. While it does not replace traditional genetic testing, which will remain the definitive diagnostic tool, flow cytometry serves as a valuable pre-screening method to identify high-risk individuals who should undergo confirmatory genetic sequencing. Flow cytometry will also enable the identification of individuals who are otherwise detected negative by most common hot spot genetic screenings but are clinically symptomatic. Flow cytometry has been successfully used in other conditions, such as Wiskott Aldrich syndrome and Bruton's Tyrosine Kinase deficiencies. In BS, reduced NOD2 protein expression has been observed in mutation-positive patients, suggesting it could be a useful marker for the disease.

Despite advancements in understanding BS, the exact mechanism by which NOD2 variants cause the syndrome remains unclear. It is particularly unclear whether these variants result in gain-of-function (GOF) or loss-of-function (LOF) mutations. The hypothesis of GOF mutations has been widely accepted due to the autosomal dominant inheritance pattern of BS [2]. Supporting this hypothesis, in vitro studies using overexpression systems have shown that BS-associated NOD2 mutations lead to hyperactivation of the NF-κB and MAPK pathways, resulting in increased inflammatory cytokine production [3]. Contrarily, clinical studies challenge the GOF hypothesis [4]. For example, peripheral blood cells from BS patients do not exhibit spontaneous cytokine release and show reduced IL-1β response compared to controls, which is not consistent with the hyperactive immune response seen in GOF conditions [4]. There are few in vivo studies in the literature so far. Moreover, these observations are more aligned with the LOF mutations seen in Crohn's disease (CD), where NOD2 mutations fail to activate the NF-κB pathway [3, 5].

The conflicting evidence from in vitro and clinical studies suggests that the pathogenesis of BS may involve a more complex mechanism than a straightforward GOF or LOF mutation. It underscores the necessity for further research to elucidate the precise role of NOD2 in BS. A comprehensive understanding of NOD2 signaling and its impact on immune regulation in BS is vital. This requires collaborative efforts across basic research, translational studies, and clinical investigations to unravel the intricacies of NOD2-related immune dysregulation and improve patient outcomes in Blau syndrome.

The present study compares NOD2 protein expression by flow cytometry. Patients with the R334W variant in the NOD2 gene have lower expression of NOD2 protein in B cells and CD8 + T cells by flow cytometry. Real-time PCR analysis also revealed reduced expression of the NOD2 gene and pro-inflammatory cytokines in patients with BS. Flow-based assessment may serve as a rapid screening test for patients with uveitis and a diagnostic aid for identifying patients with BS. Our results are consistent with in vivo studies and showed reduced NOD2 protein expression by flow cytometry as well as by qPCR in R334W variant-positive BS patients and reduced inflammatory cytokine production, hence supporting/suggesting loss-of-function (LOF) in Blau syndrome. There is a need to develop animal models of NOD2 mutated mice and perform functional validation of our findings.

The present study includes a small number of patients from a single center, which is a limitation that can indeed lead to sample variability and may limit the robustness of statistical power and generalizability of our findings. BS being a rare condition and recruiting a larger cohort presents significant logistical and clinical challenges. Despite the limited sample size, consistent trends were observed across multiple outcome measures and appropriate statistical analyses to account for the sample limitations. Furthermore, the study provides valuable preliminary insights that open many new avenues and provide groundwork for future multi-center studies with larger cohorts for functional validation.

Conceptualization: D.S.; Writing-original draft preparation: R.K., J.S.; Writing-review & editing: D.S., A.R., V.G., S.S.; Formal analysis: R.K., J.S., K.A., A.R. Clinical care: V.G., D.S., S.S. R.K. & J.S. have equally contributed and share the first authorship. All authors have approved the final version of the manuscript.

The authors declare no conflicts of interest.