Study On Host Response Factor GM-CSF After Inhalation Of Ricin

The lethal dose of ricin toxin is extremely low at 0.0001 mg/kg for intravenous injection and 0.20 mg/kg for oral administration.At present,no effective treatment is known,and it is listed as a class B priority biological threat agent by the National Institutes of Health（NIAID）in the Biodefense Strategic Plan research.In this study,we first screened and evaluated cytokines that play an important role in the toxicity induced by ricin inhalation using antibody blocking and gene knockout mice.A series of key factors,including IL-6,IL-18,GM-CSF,IL-1β,MCP-1,TNFαand GM-CSF R alpha,were selected based on preliminary toxicological experiments of ricin inhalation in the laboratory.After inhalation of ricin,Csf2ra^[-/-]gene knockout mice showed a good rescue effect,suggesting that GM-CSF is a key host response factor affecting the toxicity of ricin inhalation.Compared with wild mice,lung inflammation in Csf2ra^[-/-]gene knockout mice was significantly reduced after inhalation of castor oil.Flow cytometry showed that the Csf2ra^[-/-]gene knockout mice had a developmental deficiency in macrophages and significantly reduced neutrophils.Cytokines were detected in lung lavage fluid at12,24 and 72 h after ricin poisoning.Compared with wild-type mice（C57BL/6J）,the secretion levels of various inflammatory cytokines in lung lavage fluid of Csf2ra^[-/-]gene knockout mice increased,suggesting negative feedback regulation in the body;this result needs to be further explored.Routine transcriptomic analysis of mouse lung tissue and pathological biopsy results showed that the increased survival rate of Csf2ra^[-/-]mice after ricin inhalation is likely due to reduced inflammation compared with wild-type mice.We hypothesize that alveolar macrophages and GM-CSF are crucial inducers of inflammatory death.We next explored the effect of GM-CSF on macrophages.At the cell level,the effects of GM-CSF on mouse bone marrow-induced macrophages（BMDM）were investigated by measuring cytokine levels in cell culture supernatant and analyzing the changes of conventional transcriptomics.At 4 and 12 h after ricin attack,GM-CSF induced BMDM to produce a severe inflammatory response,including secretion of MCP-3,MIP-1β,MIP-1α,RANTES and other chemokines,which can chemotact macrophages,neutrophils,T cells,B cells and other inflammatory cells.Transcriptome results also indicated that GM-CSF stimulation of monocyte macrophages is a key factor in the development of inflammation.In addition to up-regulating genes that promote cell proliferation and differentiation,such as KIF11,DLGAP5,ZFR,TACC2,COX6C,and EMG1,chemokines of chemotactic inflammatory cells,such as CXCL3,CCL5,RANTES,CCL6,and CCL2,were present.In a gene function clustering analysis,we found that the main function differences between gene clusters observed were in the inflammatory response and neutrophil chemotactic cytokines,including the cell’s response to lipopolysaccharide activation,the activation of chemokines,lymphocyte chemotaxis,immune response,monocyte chemotaxis,and cytokine-mediated inflammatory processes.These responses is a pro-inflammatory effect.This is in contrast to established GM-CSF actions on macrophages or blood-derived monocytes in a concentration-dependent manner that promotes survival and/or polarization/differentiation and release of inflammatory cytokines such as IL-1,IL-6,etc.We found that GM-CSF attracts through chemotaxis a variety of inflammatory cells and stimulates more intense inflammation after acting on macrophages.In conclusion,GM-CSF is a key factor in the lethal process of ricin inflammation and induces macrophages to secrete other chemokines,that lead to a cascading amplification of inflammation.knockout Knockout of the GM-CSF receptor has a clear rescue effect.This provides a theoretical basis for the rescue of ricin poisoning in the future.