IL-21/IL-21R signaling is crucial in various immune diseases and cellular development, however, its role in bacterial pneumonia remains unclear. Here, IL-21R knockout (IL-21R^−/−) mice were more susceptible to Actinobacillus pleuropneumoniae (APP) than wild-type (WT) mice. High-dimensional mass cytometry analysis revealed that IL-21R deficiency inhibited neutrophil activation, decreased the numbers of monocytes and proinflammatory macrophages, and augmented the defective CD3^low T cells in the lungs. Intracellular cytokine staining showed decreased IFN-γ/TNF-α/IL-6 production in IL-21R^−/− mice, particularly in CD8⁺ T cells. Furthermore, a previously unrecognized Ly6C⁺Ly6G⁺CD4⁺ T cell subset emerged only in the lungs of WT mice post-APP infection, which was in an activated status with stronger secretion capacities of IL-10, IL-21, granzyme B, and perforin by flow cytometry. These cells polarized macrophages into M2- or M1- phenotype without/with infection, respectively, and enhanced proliferation, phagocytosis, and macrophage extracellular traps/ROS-mediated bactericidal activity of macrophages against-APP, Klebsiella pneumoniae, or Escherichia coli infection. Thus, our study demonstrated that IL-21 drives the differentiation of neutrophils, monocytes, and macrophages into pro-inflammatory subsets. IL-21-induced Ly6C⁺Ly6G⁺CD4⁺ T cells cooperate with macrophages to enhance bacterial clearance, providing a promising target for preventing bacterial pneumonia. (Figure presented.)

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) is a fatal respiratory disease that threatens the worldwide farming industry’s health. The immune responses of extrapulmonary tissues play an important role in developing porcine contagious pleuropneumonia; however, the immune responses of extrapulmonary tissues induced by APP are rarely uncovered. Here, we used high-dimensional mass cytometry to investigate the immune cell response in the spleen and peripheral blood during APP infection in mice. We found that the immune response triggered by APP was highly tissue-specific. Numerous infection time- or tissue-specific immune cell clusters, including previously unrecognized ones, were also identified in the spleen and peripheral blood. Integrative analysis of splenic lymphoid and myeloid cell clusters maps the dynamic immune response cellular network during APP infection. Surprisingly, during the early stages of APP infection, the majority of the top 6 cell clusters contributing to the infection time-specificity in the spleen were adaptive immune cell clusters rather than innate immune cell clusters, among which CD24hiMHCII+CD8+TEM cells exhibited a stronger expression of IFN-γ, IL-17A, and IL-10 compared to the CD24lo compartment. In peripheral blood, there was unprecedented heterogeneity in the immune cell composition. Also, peripheral immune cell clusters closely related to the severity of APP infection were identified. In summary, our data provide a systemic and comprehensive overview of the immune responses to APP infection in the spleen and peripheral blood. This provides a foundation for understanding the immune pathogenesis of APP and identifying potential diagnostic biomarkers and therapeutic targets.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.