B. pertussis tracheal cytotoxin biases NOD signaling to suppress IL-1 mediated inflammation and evade adaptive immunity

Bordetella pertussis, the causative agent of whooping cough, is a Gram-negative bacterium, which colonizes the respiratory tract and evades clearance through multiple virulence factors. One such factor, tracheal cytotoxin (TCT), is a monomeric peptidoglycan (PGN) fragment released extracellularly due to inefficient recycling by the permease AmpG. Although TCT is known to be a potent NOD1 agonist in vitro, its role in vivo remains poorly defined. Here, we demonstrate that TCT release during infection suppresses pulmonary inflammation. Mice infected with a TCT over-releasing strain, TCT(+), exhibited reduced lung immunopathology compared to wild-type or TCT-under releasing, TCT(−), strains. Reporter assays revealed that TCT(+) preferentially enhanced NOD1 activation, while TCT(−) strains skewed signaling towards NOD2. In vivo, NOD2 knockout mice displayed reduced lung inflammation, implicating NOD2-driven responses in pulmonary pathology. Transcriptomic analysis identified IL-1β as closely associated with expression of NOD2 but not NOD1. In support, TCT(+) infection led to reduced IL-1β protein levels than WT or TCT(−). CellChat analysis of single-cell RNA-sequencing data identified alveolar macrophages as the primary source of IL-1β and airway fibroblasts as key responders. Infected airway fibroblasts upregulated inflammatory chemokines such as CXCL13 and CCL19, promoting lymphocyte recruitment. Proteomic profiling and flow cytometry confirmed reduced chemokine levels and B cell recruitment in TCT(+) infections. Importantly, IL-1 receptor knockout mice showed significantly impaired recruitment of B and CD4+ T cells and higher bacterial burdens, despite unchanged innate cell infiltration. This underscores the role of IL-1 in coordinating adaptive immunity. Together, these findings support a model in which TCT release skews host signaling towards NOD1, suppresses NOD2 and IL-1 mediated fibroblast activation, blunts chemokine-driven lymphocyte recruitment, and impairs adaptive immune clearance. These results suggest that TCT release is an immune evasion strategy to dampen inflammation, prolong persistence and prevent the development of protective adaptive responses and highlights divergent outcomes following engagement of NOD1 or NOD2 during infection.