Cytokine Storm and Inflammation in Ricin-Induced Lung Injury

The gene expression profiles of pro‐inflammatory cytokines/chemokines in bronchoalveolar lavage fluids (BALFs) at 0, 4, 8, 12, 24, 48, and 72 h post‐ricin challenge were examined with multiplex cytokine quantification assays. A total of 12 major cytokines/chemokines were highly upregulated in BALFs from 4 to 72 h post‐ricin challenge (Figure 2A), suggesting the occurrence of an inflammatory cytokine storm during DAD progression. Multiple inflammatory features were also observed in parallel histological examinations (Figure S3A,B). In detail, the abundance of IL‐6 and CXCL1 was significantly upregulated at 8 h and sustained at high levels from 8 through 48 h. GM‐CSF and CXCL2 were robustly induced at 12 h and gradually decreased at 24 and 48 h, although they remained much higher than at 0 h. The two classic proinflammatory mediators Il‐1β and TNF‐α showed a moderate increase from 4 to 48 h, and a significant increase at 72 h. Overall, ricin‐induced DAD expression patterns were characteristic of a cytokine storm, with an excess release of multiple pro‐inflammatory cytokines/chemokines, and asynchronous changes in released cytokines/chemokines denoting diverse inflammatory responses in different lung cell populations during DAD progression.
To further investigate the production sources of these cytokines/chemokines, we analyzed their gene expression profiles using UMAP plots (Figure 2B) and discrete cell populations (Figure S4A). Fibroblasts and neutrophils were the main cell populations that had higher expressed levels of these cytokines/chemokines. After ricin exposure, fibroblasts served as the major source of CXCL1, which acted as the chemoattract for mobile neutrophils (Figure 2C). We next assessed the recruitment and trafficking mechanisms of immune cells by analyzing the expression of chemokines and their corresponding receptors based on scRNA‐seq data (Figure S4B). Several potential mediators, including Cxcl1 (to Cxcr1 and Cxcr2), Cxcl9 (to Cxcr3), and Cxcl10 (to Cxcr3) exhibited high‐levels of gene expression in fibroblasts and might be involved in neutrophil recruitment.
In summary, fibroblasts and neutrophils represent the major source of multiple pro‐inflammatory cytokines/chemokines and play an important role in promoting inflammation and a cytokine storm during DAD progression.