Systemic autoinflammatory diseases (SAIDs) are defined by dysregulation of the innate immune system and episodes of sterile inflammation (1). Because of overlapping clinical manifestations, diagnosis usually depends on recognizing specific phenotypic patterns, often supported by genetic testing (2). Nevertheless, a notable proportion of individuals presenting with autoinflammatory features do not fulfill the criteria for any established SAID or for conditions such as periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA), nor do they carry pathogenic mutations in genes associated with hereditary SAIDs. These patients are categorized as having syndrome of undifferentiated recurrent fever (SURF) (3).

This creates particular challenges in interpreting genetic findings in atypical cases with prominent inflammatory features, especially when common variants are identified in pleiotropic genes like nucleotide-binding oligomerization domain-containing protein 2 (NOD2). NOD2 encodes a cytosolic pattern recognition receptor essential for innate immune defense, as it mediates proinflammatory signaling pathways (4). Mutations in NOD2 have been associated with granulomatous autoinflammatory conditions, including Blau syndrome (BS), inflammatory bowel disease (IBD), and NOD2-associated autoinflammatory disease (NAID) (4, 5). Recently, non-pathogenic NOD2 variants have been increasingly reported in patients diagnosed with SURF, although the number of cases remains limited (6–9). Across these studies, all patients exhibited recurrent fever, with abdominal pain, rash, and musculoskeletal symptoms as the most frequently reported manifestations (6, 8, 9). However, given the limited evidence to date and the observation that some patients also carried co-existing variants in other genes, the role of non-pathogenic NOD2 variants in shaping clinical features or influencing disease course remains uncertain.

In light of the limited knowledge regarding the inflammatory pathways driving SURF and its diverse clinical presentation, this study aims to characterize the clinical features, treatment outcomes, and functional impact of NOD2 variants in patients with SURF.

Although SURF is classified as an autoinflammatory disorder without consistent associations to pathogenic gene mutations, recent studies have increasingly identified benign or VUS alterations across several genes, including NOD2, supporting the concept of a multifactorial etiology (6–9). Similarly, NOD2-associated diseases are regarded as genetically transitional diseases (GTDs), situated between monogenic and polygenic conditions, where mutations are necessary but not solely sufficient for disease onset (16). Moreover, even in monogenic Blau syndrome, reduced-penetrance variants have been documented, underscoring the complexity of NOD2-driven inflammation (17). Nevertheless, the functional impact of non-pathogenic NOD2 variants in SURF remains uncertain. Moreover, targeted NGS panels may aid diagnosis in a subset of patients with suspected AIDs, but their diagnostic yield remains limited (18), requiring clinical and functional correlation. Against this background, our study is, to the best of our knowledge, the first to investigate their functional consequences by specifically examining NF-κB activation and cytokine production. Our results demonstrate aberrant NOD2-mediated signaling, with distinct NF-κB activity and cytokine patterns that appear to be linked to specific genotypes. Notably, elevated levels of IL-2, TNF-α, IL-6, and IL-8 in SURF patients compared to HCs suggest that these cytokines may contribute to the underlying disease mechanisms.

Despite carrying the same NOD2 variant, patients exhibited variable clinical features, with recurrent fever in all cases, most frequently accompanied by abdominal and limb pain, while half of the cohort also developed cutaneous involvement in the form of various rashes. Additional manifestations such as rash, myalgia, and arthritis were in line with previous SURF cohorts (6–8, 19, 20). While some reports emphasize phenotypic variability (7, 8, 19), others suggest a more uniform presentation influenced by genetic factors (20). A chronic inflammatory course affecting the gastrointestinal tract or musculoskeletal system occurred in 33.3% of our patients—higher than the 12.3% previously reported (7)—possibly reflecting our smaller sample size or the universal presence of NOD2 variants. When compared with the limited number of published SURF cases carrying NOD2 variants (6, 8, 9), our findings were consistent in showing recurrent fever frequently accompanied by abdominal pain, musculoskeletal symptoms, and rash. Unlike the existing literature, however, rare features such as oral ulcers, pharyngitis, lymphadenopathy, hepatosplenomegaly, pericarditis, conjunctivitis, and periorbital edema were not detected. Notably, the granulomatous inflammation of the skin and eyes observed in our cohort has not been previously reported in SURF patients with NOD2 variants. Given the established link between NOD2 and granulomatous inflammation in Blau syndrome and Crohn disease (4, 5), our findings highlight the need for further studies to determine whether granulomatous inflammation constitutes a distinct phenotypic subset of SURF or merely reflects a coincidental association.

In the absence of standardized treatment protocols, SURF management typically involves colchicine, corticosteroids, and IL-21 inhibitors (21). Although prior studies have described good colchicine response rates in over half of patients (6, 7, 20), with colchicine-sensitive cases proposed to represent a more homogeneous subgroup (20), we observed fewer complete responders, possibly reflecting broader clinical variability. Recent surveys indicate that 70.8% of clinicians prescribe corticosteroids during flares based on severity rather than as routine therapy (22). Similarly, corticosteroids in our series were used on demand for disease flares or chronic organ inflammation, with all treated patients improving. Moreover, Papa et al. (6) reported that SURF patients carrying NOD2 variants demonstrated partial response to colchicine but achieved complete remission with corticosteroids, a finding consistent with our results and suggesting a potential association between NOD2 variants and limited colchicine responsiveness but favorable corticosteroid outcomes. Additionally, one-third of patients required long-term immunomodulatory therapy beyond intermittent steroids, highlighting the need for personalized treatment approaches in SURF, particularly for those with chronic or organ-specific inflammatory involvement. Larger multicenter studies will be essential to confirm these observations and refine treatment strategies in SURF.

Although cytokine patterns differ across SAIDs, with elevated cytokines often contributing to pathogenesis (23), the underlying mechanisms of SURF remain poorly defined. Moreover, current functional data are largely limited to studies comparing SURF with PFAPA or FMF (8, 11, 19). Previous studies identified a persistent IL-1 signature in SURF tonsils and noted trends toward higher IL-21β, IL-6, IL-8, and IL-17A levels in some SURF cases compared to PFAPA (8, 19). Pyrin inflammasome involvement has also been proposed in SURF pathophysiology (11). In contrast to these findings, another study demonstrated that anti–IL-1 blockade with anakinra was ineffective in three out of four colchicine-resistant patients, suggesting an IL-1β–independent inflammatory pattern in this subgroup (6). In our cohort, we observed significantly elevated IL-2 levels, a cytokine primarily implicated in Treg homeostasis in autoimmune conditions (24). In FMF, Treg dysfunction has been proposed; for instance, Rimar et al. (25) described Treg expansion following attacks, while another study noted elevated IL-10 and TGF-β, both known Treg inducers (26). However, contrasting data indicate reduced Treg numbers in FMF, suggesting impaired regulatory capacity may exacerbate disease severity (27). Elevated sIL-2R levels in FMF patients, even during remission, correlated with erythrocyte sedimentation rate and activated CD4+CD69+ T cells, implicating IL-2 signaling in persistent low-grade inflammation (28). These findings raise the possibility that IL-2-driven immune activation in SURF may reflect altered Treg function, contributing to immune dysregulation.

Variants like L1007fs, G908R, and R702W are associated with both CD and NAID. While functional studies have classified these variants as loss-of-function in CD due to reduced NF-κB activation (29–31), IBD models and CD patients without NOD2 mutations also display impaired proinflammatory cytokine production (32, 33). This diminished response appears independent of NOD2 genotype and distinct from healthy controls (34), suggesting additional contributors to defective immunity in CD.

Conversely, G908R has been linked to enhanced IL-8 and TNF-α production in familial sarcoidosis, despite reduced NF-κB signaling, pointing to macrophage-driven chronic inflammation (35). Although some NAID-associated NOD2 variants act as gain-of-function mutations, compound heterozygous IVS8 + 158 and R702W variants have shown loss-of-function features in NAID, yet R702W may have different effects in NAID versus CD, consistent with distinct clinical presentations (36, 37). Our findings suggest that R702W, G908R, P268S/V955I, and R702W/P268S may be associated with robust proinflammatory cytokine responses despite varied NF-κB activation, differing from their reported loss-of-function classification in CD (29, 30). This may indicate that NOD2 variants act differently depending on disease context, possibly through compensatory signaling or interactions with other innate immune pathways (38). Tissue-specific NOD2 expression and local immune environments may also shape these responses.

Notably, while the R702W variant was present in both healthy control and SURF patients, only the latter displayed markedly elevated cytokine levels, indicating this variant may contribute to inflammation under disease conditions but not in healthy states. Furthermore, patients carrying P268S/V955I (P8) and G908R (P9) showed high cytokine production at baseline and after MDP stimulation, even in the absence of detectable NF-κB activation, implying alternative inflammatory pathways may be involved. Interestingly, G908R appeared to exhibit divergent NF-κB activation between two patients: one (P2) had baseline and MDP-induced p-IκBα expression, while the other (P9) lacked p-IκBα under both conditions, highlighting the influence of additional regulatory mechanisms on NF-κB signaling. Despite these differences, both patients produced significantly elevated cytokine levels compared to HCs.

Another important finding was that the L682F and L1007fs variants in SURF patients, along with R587C in BS, were suggestive of an association with a hypoinflammatory profile, characterized by absent NF-κB activation and diminished cytokine responses compared to HCs and other NOD2 variants. The SURF patient carrying L1007fsinsC (P4) presented with chronic gastrointestinal symptoms (nonspecific focal colitis) but lacked endoscopic, radiologic, or histologic evidence of IBD, consistent with the expected loss-of-function phenotype seen in CD (29, 30). Although R587C is classified as pathogenic in BS, its functional role remains debated (39–42). Matsuda et al. (40) reported weak spontaneous NF-κB activation with R587C compared to R334W, while our data—aligned with Parkhouse et al. (41)— seem to support a hypoinflammatory effect, reinforcing emerging evidence of loss-of-function features in BS-related NOD2 variants (42).

The R753Q variant showed cytokine levels comparable to HCs both at baseline and after MDP stimulation, despite constitutive NF-κB activation, which may suggest minimal impact on NOD2-driven immune responses and seems to support its classification as likely benign. In contrast, A1000T and P268S variants appeared to be associated with NF-κB activation and mild inflammatory activity—greater than HCs but lower than more strongly proinflammatory variants. Although P268S has not been shown to significantly alter NOD2 function (29, 30), its potential role in autoinflammation remains uncertain. Given the limited functional data on A1000T and P268S, further investigation is needed to clarify their contributions in SURF.

Detection of p-p38 across all samples, including HCs and regardless of MDP stimulation, suggests that p38 MAPK activation may occur independently of NOD2-mediated signaling. Possible explanations may include activation via alternative pathways or basal cellular stress in PBMCs, with handling and culture conditions potentially contributing (43). This may underscores the complexity of p38 MAPK regulation and the need for further study into its broader role in immune modulation and inflammation in SURF.

Our study’s primary limitation was the small cohort, attributable to the rarity of SURF and funding constraints that limited control recruitment. The lack of whole exome sequencing (WES) and segregation analyses may have led to undetected rare pathogenic variants in other genes. Nonetheless, it should be noted that many patients with undefined autoinflammatory syndromes remain without a genetic diagnosis even after comprehensive genomic testing (44). Furthermore, while samples were obtained during disease flares, studying treatment-naïve patients in larger cohorts will be essential to better elucidate disease mechanisms. Despite these challenges, a major strength of our work is the integration of long-term clinical follow-up with functional analysis of NOD2 variants, which may provide valuable insights into potential disease pathways.

Overall, our findings suggest that NOD2 variants may act as disease modulators rather than primary drivers of SURF. This may highlight the importance of further functional studies to evaluate NOD2 signaling within disease-relevant immune contexts, as identical mutations might trigger inflammation through distinct molecular routes depending on the condition. Future research should explore additional genetic, epigenetic, or environmental contributors that may influence inflammatory responses in SURF, helping to refine our understanding of NOD2-linked autoinflammatory disorders.