High-Dimensional Immunophenotyping Identifies Circulating Immune Signatures Associated with FeNO-Defined Asthma Phenotypes

Background: Asthma is a heterogeneous inflammatory airway disease traditionally classified into T2 high (eosinophilic) and T2 low inflammatory phenotypes. Fractional exhaled nitric oxide (FeNO) is commonly used as a biomarker of T2 inflammation, although nitric oxide also regulates monocyte maturation and T cell polarization. We hypothesized that FeNO identifies distinct systemic immune phenotypes in asthma. Methods: Peripheral immune cell subsets were characterized using OMIP - 69, a 40 - color high dimensional immunophenotyping panel encompassing innate and adaptive immune populations. Adults with asthma and healthy controls underwent peripheral blood immunophenotyping. FeNO was dichotomized at 25 ppb (FeNO - high [≥] 25 vs FeNO - low <25). Immune-cell subset proportions were summarized as medians (IQRs) and compared using the Wilcoxon rank-sum test. Logistic regression was used to evaluate associations between immune subsets and FeNO status. Results: Eighteen participants with asthma and seven healthy controls were enrolled. Asthmatic participants had higher BMI than healthy controls (median [IQR] asthma: 35.1 [28.9 - 37.7] vs healthy: 25.4 [22.6 - 26.7] kg/m2; p=0.003), although demographics were otherwise similar. Among asthmatics, 10 participants had FeNO - low asthma, and eight had FeNO - high asthma. FeNO - high asthma demonstrated increased classical monocytes (CD14++CD16 -) and compared with healthy controls (p=0.027), whereas FeNO - low asthma more closely resembled healthy controls. Intermediate monocytes (CD14++CD16+) were increased in FeNO - high versus FeNO - low (p= 0.08); a similar association was noted for classical monocytes (p = 0.13). In contrast, CCR7+ TCR; T - cells were significantly enriched in FeNO - low asthma (p=0.002). Logistic regression demonstrated positive associations between high - FeNO and classical monocytes (OR 16.06, 95% CI 1.56 - 1304.69; p=0.007) and intermediate monocytes (OR 4.19, 1.22 - 132.30; p=0.017), whereas CCR7+ {gamma} {delta} T - cells were negatively associated with high FeNO (OR 0.06, 0.00 - 0.47; p=0.001). Conclusions: Distinct systemic immune signatures identified by high-dimensional cytometry support the existence of biologically divergent FeNO - defined asthma endotypes and may inform future biomarker-driven approaches to patient stratification and precision therapy.