Abstract
Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define "stimulation score" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions.IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity in vitro This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by "stimulation score," agrees with in vivo cytokine response trends reported in the literature.