Integrated Visualization of Neutrophil Recruitment and Inflammatory Cytokine Profiling in Bronchoalveolar Lavage...

Bronchoalveolar lavage fluid (BALF) is the most directly accessible sampling matrix for studying lower airway inflammation, yet conventional analyses rely on single-marker flow cytometry combined with analyte-by-analyte enzyme-linked immunosorbent assay (ELISA), an approach that limits information density and obscures cell-factor coupling. This study presents an integrated visualization workflow that couples standardized collection and preprocessing with a seven-marker neutrophil and monocyte flow cytometry panel, a 13-analyte multiplex cytokine assay, and a unified analysis pipeline based on uniform manifold approximation and projection (UMAP), FlowSOM metaclustering, hierarchical clustering, Spearman correlation networks, and principal component analysis (PCA). The workflow was applied to a single-center prospective cohort of 112 adults undergoing bronchoscopy, comprising 64 patients with microbiologically confirmed pulmonary infection and 48 non-infection controls. Cytological and flow cytometric readouts revealed a pronounced shift from a macrophage-dominant to a neutrophil-dominant alveolar profile during infection, with the neutrophil percentage rising from a median of 3.2% to 68.4% and the absolute neutrophil concentration increasing approximately 50-fold. Multiplex profiling resolved a coordinated three-module cytokine response, with chemokine, proinflammatory, and regulatory modules all upregulated and CXCL8/IL-8, IL-1β, IL-6, CXCL1/GRO-α, and G-CSF showing the strongest single-marker discrimination within the study cohort (area under the curve [AUC] 0.89 to 0.94). Integrative visualization using PCA, a correlation matrix, and a network graph revealed a tightly coupled neutrophil-chemokine axis at the participant level, with BALF neutrophil count emerging as the central network hub. The workflow uses only material routinely obtained from clinically indicated BAL, requires no additional sample volume, and provides an information-dense framework for resolving airway inflammation in respiratory infection that may be feasible in tertiary-care laboratories equipped with multiparameter flow cytometry and multiplex bead-array platforms, extending into longitudinal, single-cell, and therapeutic-evaluation studies. Broader clinical adoption of individual analytes as biomarkers will require prospective validation in independent multicenter cohorts.