Csf2ra deletion attenuates acute lung injuries induced by intratracheal inoculation of aerosolized ricin in mice

Abstract

Specific therapeutics are not available for acute lung injury (ALI) induced by ricin toxin (RT). Inhibiting the host immune response in the course of pulmonary ricinosis is hypothesized to be of benefit and can be achieved by impairing granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, thereby reducing the pro-inflammatory response to exogenous foreign body invasion. However, it is unknown whether mice with impaired GM-CSF signaling can survive after RT inhalation. To test this, colony stimulating factor 2 receptor alpha (Csf2ra) knockout (KO) mice that lack GM-CSF signaling and wild-type (WT) mice models of intratracheal exposure to a lethal dose (2× LD₅₀) of RT were established. Survival was greater in Csf2ra KO mice 21 days after RT inhalation compared with WT mice. Highly co-expressed genes that probably attenuated the pro-inflammatory response in the lung of Csf2ra KO mice were identified. Bioinformatics analysis revealed that transcriptome changes involved mostly inflammation-related genes after RT exposure in both Csf2ra KO mice and WT mice. However, the activity levels of pro-inflammatory pathways, such as the TNF signaling pathway and NF-κB signaling pathway, in Csf2ra KO mice were significantly decreased and the degree of neutrophil chemotaxis and recruitment inhibited after RT-exposure relative to WT mice. RT-qPCR and flow cytometry validated results of RNA-Seq analysis. This work provides potential avenues for host-directed therapeutic applications that can mitigate the severity of ALI-induced by RT.

Keywords: ALI; Csf2ra; GM-CSF receptor; RNA-Seq; RT; inflammation response.

Figure 1

Csf2ra KO mice acquire protection from RT-induced mortality and morbidity. (A) Survival curves for Csf2ra KO mice (n = 10) and WT mice (n = 10); survival rate of the two groups is significantly different (P = 0.0038). (B) H&E stained lung tissue from each treatment group at three time points. Scale bar, 250 μm. (C) Pathological score for each group. *P <0.05.

Figure 2

WGCNA identified gene co-expression networks in Csf2ra KO mice and WT mice. (A) Topological overlap matrix plot showing pairwise gene correlations within each module. Genes within different modules are labeled with different colors according to WGCNA’s conventions. (B) The top 15 KEGG entries for genes found in the “black” module. The x-axis represents gene count and the y-axis is KEGG terms. (C) The top 20 entries in GO analysis for biological processes in the “red” module.

Figure 3

DEGs analysis of RNA-Seq in lung tissue of Csf2a KO mice and WT mice after RT-exposure compared to control. (A) Bar chart showing the number of DEGs under different experimental conditions. (B) The top 25 entries in the KEGG pathway with significant accumulation of up- and downregulated genes in each group under different experimental conditions.

Figure 4

DEGs analysis of RNA-Seq in lung tissue of Csf2ra KO mice compared to WT mice at 4, 12 and 72 h post RT-inhalation. (A) The top 17 entries in the KEGG pathway of upregulated genes in Csf2ra KO mice or WT mice under different experimental conditions. (B) The TNF signaling pathway was significantly downregulated in Csf2ra KO mice relative to WT mice at 4 h post-RT inhalation. (C) The NF-κB pathway was significantly downregulated in Csf2ra KO mice relative to WT mice at 4 h post-inhalation. (D) RT-qPCR verification of genes identified in transcriptome analysis. Two-way ANOVAs followed by Sidak’s multiple comparison tests were used; *P <0.05. (E) Western blot analysis for p-p65, p65, p-IκB, IκB and TNF protein expression levels.

Figure 5

Temporal transcriptome analysis of lung tissues in Csf2ra KO mice and WT mice. (A) The nine clusters identified by the maSigPro algorithm for the selected genes. (B) Analysis of GO biological processes in Cluster 5. (C) Analysis of GO biological processes in Cluster 8. (D) Heat maps showing expression levels of genes in Cluster 8 in each group. (E) RT-qPCR verification of genes with consistently low expression in Csf2ra KO mice. Welch’s t test; **P <0.01, ***P <0.0001.

Figure 6

The degree of neutrophil chemotaxis and recruitment in Csf2ra ^-/- mice was decreased relative to WT mice. (A) Immune cell abundance analyzed by ImmuCellAI at different timepoints for Csf2ra KO mice and WT mice. (B) Bar graph showing the percentage of neutrophils measured by flow cytometry. Two-way ANOVAs followed by Sidak’s multiple comparison tests; *P <0.05.