Dennd1A Suppresses Inflammatory Response In Macrophages During Sepsis

Background:Sepsis is a life-threatening organ dysfunction caused by dysregulated inflammatory response to infection.At present,there are no effective treatment for sepsis.Macrophages express multiple pattern recognition receptors and play an important role in sepsis.It is of great significance to explore new regulatory mechanisms of macrophages during sepsis.The Rho family of small GTP-binding proteins(Rho-GTPases),as an important molecular switch in cell signal transduction,switch between active and inactive states by binding to GTP or GDP,and play a key regulatory role in a variety of cellular processes.Members of the guanine nucleotide exchange factor(GEF)family promote the conversion between GTP and GDP,and ensure that Rho-GTPases can be activated in a specific space and time,which is an important regulator of Rho-GTPase activity.Studies have shown that a variety of GEF are involved in regulating the endocytosis of macrophages and vesicular transport of early endosomes.DENN domain protein can play the role of GEF.DenndlA(DENN domain containing 1A)is a protein containing DENN(differentially expressed in Neoplastic Versus Normal cells)domain,which is widely expressed in various tissues.Dennd1A deletion affects the development of liver,primordial germ cells and blood cells,but its effect on macrophages and related inflammation has not been reported so far.In this paper,we used conditional knockout mice to study the regulatory effect of DenndlA on macrophage function and sepsis.Methods and results1.Dennd1A is highly expressed in macrophagesTo determine the possibility of DenndlA regulating macrophages,we first detected the expression of DenndlA in various immune cells by qPCR,and found that DenndlA was highly expressed in macrophages.2.Macrophage-specific DenndlA knockout significantly promotes the expression of inflammatory factors in macrophagesTo investigate the role of DenndlA in macrophage immune regulation,we constructed a DenndlA specific knockout in myeloid cells(Lyz2-iCre+/-DenndlAflox/flox,MKO)mice and control(DenndlA flox/flox,WT)mice,and the knockout efficiency of macrophages was measured.The peritoneal macrophages were isolated and stimulated with LPS,IL-4 and Poly I:C.The expression of cytokines was detected by fluorescence quantification and ELISA.The results showed that under the stimulation of LPS and Poly I:C,the expression of macrophage inflammatory factors in Denndl A knockout mice increased.3.Macrophage-specific Dennd1A knockout significantly promotes the inflammatory response of sepsis in miceIn order to further explore the role of macrophage DenndlA in the sepsis of mice,we established LPS Shock and cecal puncture ligation models with the above-mentioned macrophage knockout DenndlA mice and control mice.Further analysis showed significantly shorter survival in LPS and CLP treated MKO mice than WT mice.Results of HE and RT-PCR showed that MKO mice had more necrotic areas and higher levels of IL-6 and other inflammatory factors in the lung.This was further confirmed by ELISA.4.Dennd1A deletion does not affect macrophage phagocytosisIn order to study the effect of DenndlA on the phagocytosis of macrophages,we used the peritoneal macrophages of DenndlA knockout mice for the fluorescent microsphere phagocytosis test.Flow cytometry and fluorescence microscope observation showed that the phagocytic capacity of macrophages did not affect after knockout.5.Dennd1A inhibits phagocytosis and TLR4 signaling in macrophagesWe next performed flow cytometry,fluorescence staining and western blot to test whether DenndlA affects the endocytosis of macrophage surface receptors and whether it affects the downstream signal molecules of TLR4,and found that DenndlA inhibited the downstream signal molecules of TLR4.Conclusion and significanceThis study has proved for the first time that DenndlA can inhibit the inflammatory reaction of macrophages and protect mice from sepsis induced inflammation.However,the detail mechanisms need to be further explored.