| Background:Autoimmune diseases(AIDs)are inflammatory diseases that mistakenly attack normal tissues and organs due to the abnormal activation of the immune system.According to statistics,autoimmune diseases affect about 10%of the world’s population and have become a global health problem.Toll-like receptors(TLRs)are the first line of defense to construct the human immune system which have highly conserved pattern recognition receptors.Toll-like receptors activate a variety of immune cells such as Macrophages(Mφs)and Dendritic cells(DCs).Abnormally activated macrophages and dendritic cells release inflammatory cytokines,chemokines and co-stimulatory molecules,leading to inflammation and autoimmune diseases.Activated cdc42 bound protein kinase 1(ACK1)is a non-receptor tyrosine kinase involved in a variety of signaling pathways and physiological processes.However,the role of ACK1 in activation of TLR pathway,inflammation and pathogenesis of autoimmune diseases has not been reported.Therefore,further exploration of the role and mechanism of ACK1 in inflammation and autoimmune diseases is expected to provide a new direction for the treatment of inflammation and autoimmune diseases.Objective:To investigate the effect of ACK1 on TLR pathway mediated activation of macrophages and dendritic cells and the pathogenesis of inflammatory and autoimmune diseases.To explore whether targeting ACK1 can alleviate the pathogenesis of inflammatory and autoimmune diseases.It provides target reference and new direction for the research of inflammation and autoimmune diseases.Method:1.The effect of activation of TLR4/TLR7/TLR9 pathway on ACK1 expression in macrophages and dendritic cells was analyzed.(1)Culture and treatment of macrophages and dendritic cells in vitro:C57BL/6 mice were selected to take tibia and femur of both hind limbs and isolate bone marrow cells.BMDMs differentiation was stimulated by mouse recombinant Granulocyte-macrophage colony-stimulating factor(GM-CSF).Recombinant recombinant murine IL-4 and GM-CSF were used to stimulate BMDCs differentiation.All cell cultures were performed in an incubator at 37℃ and 5%CO2.(2)The expression of ACK1 was detected by flow cytometry and Western blot.2.Effects of ACK1 on TLR pathway-mediated macrophage activation:Lentivirus with high expression of ACK1(ACK1-LV)and lentivirus with interference expression of ACK1(RNAi-ACK1-LV)were constructed.The above lentiviruses were used to infect BMDMs according to the instructions and the effects of high and low expression on TLR pathway-mediated macrophage activation were analyzed after appropriate time.(1)The expressions of CD86 and CD40 on macrophage surface were detected by flow cytometry;(2)The content of inflammatory factors in cell supernatant was detected by ELISA;(3)Proteins were collected and the activation of the MAPKs and NF-κB pathways were investigated by Western blot analysis.3.At the cell research level,explore the effect of targeting ACK1 on TLRs pathwaymediated activation of macrophages and dendritic cells.(1)The effect of ACK1 inhibitor AIM-100 treatment on the vitality of macrophages and dendritic cells:macrophages and dendritic cells were treated with different concentrations for 24 h and 48 h respectively and the vitality of the two kinds of cells was detected by Cell TiterLumiTM luminescence cell viability detection kit;(2)To study the effect of ACK1 inhibitor AIM-100 treatment on TLR pathway induced activation of macrophages and dendritic cells:macrophages and dendritic cells were treated with different concentrations of AIM-100 respectively and TLR4/7/9 agonist was added to stimulate macrophages and dendritic cells 2 hours later.Flow cytometry was used to detect the expressions of CD40 and CD86 and ELISA was used to detect the expressions of inflammatory factors.The activation of MAPKs and NF-κB pathways was detected by Western blot 1 h later.4.To explore the effect of targeting ACK1 on endotoxin shock in mice at the animal level.Endotoxin shock model of mice:C57BL/6 8-week-old female mice were used in the following experiments.In the high-dose LPS model,AIM-100(5,10,20 μg/g)or control was intraperitoneally injected and LPS(37.5 μg/g)was intraperitoneally injected 2 hours later.The survival of each mouse was monitored for up to 3 days.In low-dose LPS models,mice were pretreated with AIM-100(5,10,20 μg/g)or a control for the first 2 hours and intraperitoneally injected with LPS(10 μg/g).(1)To observe the high dose of LPS in the model mice of vital signs,activity index and survival rate and analysis of records;(2)The vital signs of mice in the low-dose LPS model collected the eyeball blood and serum to detect the expression of inflammatory factors after the 3 hours later;(3)The vital signs of mice in the low-dose LPS model were observed.12 h later,the liver and lungs were collected for HE section staining to observe the damage;the apoptosis of lung and liver cells was observed by TUNEL;(4)To observe the low dose of LPS in mice model of vital signs,12 hours after collection lymph nodes and spleen and flow cytometry detected the activation of macrophages and dendritic cells.5.To explore the effect of AIM-100 on TLR7 ligand IMQ induced lupus mice at the animal level.Imiquimod induced lupus in mice:Female C57BL/6 mice were applied with imiquimod(IMQ)to the right ear three times a week.AIM-100(20 μg/g)was injected intraperitoneally and the control group was treated with placebo.After 10 weeks,mice were sacrificed,and corresponding experiments were collected from eyeball blood,spleen and kidney to observe the effect of AIM-100 on IMQ-induced lupus model.(1)The expression of dsDNA in peripheral blood was detected;(2)The degree of spleen enlargement and weight were observed;(3)HE sections of spleen and kidney were made to observe the pathological changes;(4)The expression of CD40 and CD86 in macrophages and dendritic cells was detected by flow cytometry;(5)IgG and IgM deposits in the kidneys were detected.RESULTS:1.TLRs up-regulated ACK1 expression in macrophages and dendritic cells.2.Overexpression of ACK1 significantly promoted TLR pathway-mediated macrophage activation while interference with ACK1 expression significantly inhibited TLR pathwaymediated macrophage activation.(1)Compared with the control BMDMs,the expressions of CD86 and CD40 on the cell surface of BMDMs with high ACK1 expression were significantly increased after stimulation by LPS,R848 or CpG-1826.The expressions of CD86 and CD40 on the cell surface of BMDMs with low ACK1 expression were significantly decreased after LPS,R848 or CpG-1826 stimulation.(2)Compared with the control BMDMs,the contents of IL-12 and TNF-α in cell culture supernatant of BMDMs with high ACK1 expression were significantly increased after stimulation by LPS,R848 or CpG-1826.The contents of IL-12 and TNF-α in cell culture supernatant of BMDMs with low ACK1 expression were significantly reduced after stimulation by LPS,R848 or CpG-1826.(3)Compared with control BMDMs,the phosphorylation of p38,Erk,JNK and p65 was significantly enhanced in BMDMs with high ACK1 expression after stimulation by LPS,R848 or CpG-1826.The phosphorylation of p38,Erk,JNK and p65 was significantly decreased in BMDMs with low ACK1 expression after stimulation by LPS,R848 or CpG1826.Overexpression of ACK1 significantly promoted TLR pathway-mediated macrophage activation,while interference with ACK1 expression significantly inhibited TLR pathwaymediated macrophage activation.3.ACK1 inhibitor AIM-100 significantly inhibits TLRs pathway-mediated activation of macrophages and dendritic cells.(1)AIM-100 had no significant effect on the cell viability of macrophages and dendritic cells when the concentration was lower than 20 μM.(2)AIM-100 significantly inhibited TLRs pathway-mediated macrophage and dendritic cell activation:The expressions of CD86 and CD40 on the surface of AIM-100 treated BMDMs;the contents of IL-6,IL-12 and TNF-α in the culture supernatant and the phosphorylation of p38,Erk,JNK and p65 were significantly decreased compared with the control agent treated BMDMs.The same phenomenon was observed in BMDCs.4.Drug ACK1 inhibitor AIM-100 alleviated the condition of endotoxic shock in mice.(1)It was found that AIM-100 could significantly reduce the mortality of mice with endotoxic shock and with the increase of drug concentration within a certain dose,the survival rate of mice was higher;(2)ELISA results showed that the serum levels of TNF-α,IL-6 and IL-12 in AIM-100-treated endotoxic shock mice were significantly lower than those in vehicle-treated endotoxic shock mice;(3)Hematoxylin and eosin(HE)staining showed that AIM-100 significantly alleviated liver and lung injury induced by LPS;TUNEL showed that the apoptosis of lung and liver cells of AIM-100 treated mice with endotoxin shock was relatively low;(4)The expression levels of CD86 and CD40 in macrophages and dendritic cells in lymph nodes and spleen of endotoxin-shock mice treated with AIM-100 were significantly lower than those of endotoxin-shock mice treated with control.5.Targeting ACK1 alleviates the disease of TLR7 ligand IMQ-induced lupus mice.(1)Imq-induced lupus mice treated with AIM-100 showed significantly lower levels of serum dsDNA expression than those treated with control;(2)The spleen enlargement degree and weight of IMQ-induced lupus mice treated with control agent were significantly higher than those of AIM-100 IMQ-induced lupus mice;(3)Hematoxylin and eosin(HE)staining showed that AIM-100 treated mice with IMQ-induced lupus exhibited significant inhibition on lymphoid cell infiltration and diffuse expansion of renal mesangial matrix.(4)The expression levels of CD86 and CD40 in spleen macrophages and dendritic cells of IMQ-induced lupus mice treated with AIM-100 were decreased compared with those treated with control agent;(5)The kidneys of IMQ-induced lupus mice treated with AIM-100 showed relatively less IgG and IgM deposition than the kidneys of IMQ mice treated with a control agent.Conclusion:1.TLRs up-regulate the expression of ACK1 in macrophages and dendritic cells;2.ACK1 promotes TLRs pathway-mediated activation of macrophages and dendritic cells;3.The targeted ACK1 significant inhibition of the TLRs pathway mediated activation of macrophages and dendritic cells;4.Targeting ACK1 can significantly relieve inflammation and autoimmune diseases. |
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