| Liver diseases are one of major threats to human health. Investigation of pathogenesis and prophylaxis has been drawn great attention. Immune-mediated liver injury plays an important role in the pathogenesis of various liver diseases. However, the pathophysiological mechanisms have not been fully elucidated. The Con A-induced hepatitis in mice is one of the typical models for studying the pathogenesis of immune-mediated liver injury and drug screening. The Con A-induced immune liver injury in mice is similar to human autoimmune hepatitis and viral hepatitis. It is characterized by the activation of T cells (especially CD4+), and markedly increases the production of inflammatory cytokines which cause liver cell injuries.G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein and comprises one subfamily of the ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs). GIT2, regulator of GPCR internalization and resensitization, participates in actin cytoskeleton assembly, focal adhesin dynamics, cell migration and the spatial localization of signaling molecules. GIT2 is highly conserved across mammalian and avian species. The GIT2 homology between human and mouse is 86% and that between human and chicken is 89%. GIT2 is widely distributed, highly expressed in heart, brain, spleen, lung, kidney, thymus, liver, muscle, testis, and is more highly expressed with aging. GIT2-/- mice displayed anxiety-like behavior in the absence of depressive-like responses, bone mineral density and bone volume decreased, as well as osteoblastic activity diminished with the consistent number of osteoblasts.GIT2 is closely associated with the immune system. Lack of C terminal domain in the GIT2 isoform is mainly observed in the immune cells. GIT2-/- mice display multiple symptoms of immunodeficiency because the deficiency of GIT2 inhibite the chemotaxis and superoxide production in neutrophils. GIT2 is a component of the Gβγ-signaling complex and forms a linear complex of PAK1-αPIX-GIT2, which is necessary for immunological synapse function. The immunological synapse is essential for antigen recognition, proliferation and activation of T cell, and plays a key role in cellular immune response and humoral immune response. In addition, the deficiency of GIT2 was found to inhibit the development of double-positive T cells to single-positive CD4+ T cells. GIT2 plays a critical role in the development and activation of T cells by participating in the single-positive selection of CD4+ T cells and the formation of immunological synapse. Therefore, GIT2 may regulate the natural and acquired immunity.GIT2 may have immune function in viral hepatitis and autoimmune hepatitis based on its major role in the development and activation of T cells, while the activation of T cells causes liver injury induced by viral hepatitis and autoimmune hepatitis. However, the function of GIT2 in the immune-mediated liver injury is still not clarified.A Con A-induced immune-mediated hepatitis model in mice was set up in this study to investigate the role and mechanism of GIT2 in immune-mediated hepatitis. It may provide a new strategy and target for clinical therapy of viral hepatitis and autoimmune hepatitis. In addition, CCl4 and D-GalN/LPS induced liver injury model and partial hepatectomy were used to preliminarily explore the role of GIT2 in liver injury caused by other factors because of the complex etiology of liver injury. This study would contribute to the comprehensive understanding of the role of GIT2 in liver injury.1. The phenotypic analysis of GIT2-/- micePhenotypic analysis of GIT2-/- mice provides us with the experimental basis of animal level for understanding the role of GIT2 in liver injuries.GIT2-/- mice were identified by PCR and Western-blot on the DNA and protein level individually. GIT2-/- mice were born at the expected mendelian ratio and were fertile. The obvious phenotyic defects of adult GIT2-/- mice were not detected. The organ index indicated that the spleen index of GIT2-/- mice is about 2 times higher than that of wild type (GIT2+/+) mice (t=5.650, P=0.001), while as for the liver index, thymus index, kidney index and heart index no obvious difference was observed. In addition, the spleen volume of GIT2-/- mice was significantly larger than that of GIT2+/+ mice. The pathological analysis showed that the liver tissues of GIT2-/- mice were normal, marrow hyperplasia was red, local myeloid metaplasia changed, immature hematopoietic cells increased, megakaryocytes were visible, white pulp lymphoid follicle mild atrophy, and tissue structure was basically normal. The complete blood count and liver function indexes of GIT2-/- mice were normal. Differential genes between GIT2-/- and GIT2+/+ enriched in metabolism, redox, phosphorylation, apoptosis, ion transport, transferase activity, oxygen activity, ATP binding, hydrolase activity was observed with Agilent 60K gene chip of mice. Differential genes were also associated with activation of T and B cells, regulation and inflammatory factor production of macrophage. These results suggested GIT2 may have an effect on liver metabolic detoxication, glycogen synthesis, protein synthesis, energy supply, antioxidant function, ion metabolism and immune system.As for the proportion of CD4-CD8- T cell, CD4+CD8+T cell, CD4+CD8- T cell and CD4-CD8+T cell in thymus, and the proportion of CD4-T cell and CD8+T cell in spleen and lymph node no difference was observed by the flow cytometry. The proportion of CD4+T in liver of GIT2-/- mice was lower than that of GIT2+/+ mice (t= 9.210, P=0.000), while the proportion of CD8+T in liver of GIT2-/- mice was higher than that of GIT2+/+ mice (t=5.707, P=0.001). And the numbers of CD4+ T cells, CD8+T cells and B cells in spleen of GIT2-/- mice were greater than those of GIT2+/+ mice (t=3.880, P=0.018; t=5.804, P=0.004;t=6.631, P=0.003); the number of CD4+ T cells in liver of GIT2-/- mice was smaller than that of GIT2+/+ mice (t= 14.208, P=0.000), while the number of CD8+ T cells in liver of GIT2-/- mice was greater than that of GIT2+/+ mice (t=3.833, P=0.003). The results indicated that GIT2-/- mice developed splenomegaly and splenic extramedullary hematopoiesis, and GIT2 may have an effect on liver immune system.2. Deficiency of GIT2 prevented Con A-induced immune hepatitis in miceIn order to demonstrate the role of GIT2 in the occurrence and development of human liver diseases,10 viral hepatitis liver tissues,11 active cirrhosis liver tissues and 29 cirrhosis liver tissues were examined with immunohistochemistry. The results indicated that the expression of GIT2 in pathological human liver tissues was upregulated. In addition, the expression of GIT2 was tested in the mice with Con A-induced immune liver injury by Real-time PCR and Western-blot. The results showed that mRNA expression of GIT2 increased after injection of Con A, and reached the peak after ConA treatment for 1 hour. The upregulation of GIT2 was also observed and the upregulation was kept for 24 hours after injection of Con A. The results demonstrated that GIT2 was closely associated with the development of human liver diseases and Con A-induced immune liver injuries in mice.On the basis of the demonstrated role of GIT2 in liver injury, the function of GIT2 was further investigated in Con A-induced immune hepatitis. Compared with the GIT2+/+ mice, GIT2-/- mice had higher survival rate after being given a lethal dose of Con A (25 mg/kg) via tail vein injection. The upregulated levels of serum ALT and AST of GIT2-/- mice were significantly reduced as expected after the injection of 15mg/kg Con A (ALT t3h=25.866, P3h=0.000; t6h=18.331, P6h=0.000; t12h=36.621, P12h=0.000; t24h=32.017, P24h=0.000; AST t3h=6.328, P3h=0.003; t6h=7.377,6h P=0.002; t12h=20.522, P12h=0.000; t24h=67.625, P24h=0.000), and the massive necrosis of liver was also attenuated in GIT2-/- mice (t=4.362, P=0.045). It was also detected that the TUNEL apoptosis assay significantly reduced hepacyte apoptosis in GIT2-/- mice (t=6.621, P=0.003). These results demonstrated that deficiency of GIT2 protected the liver from the Con A-induced immune liver injury.The GIT2 gene was delivered into GIT2-/- mice by a hydrodynamics-based gene transfection in vivo using a naked pCMV-myc-GIT2 plasmid to understand the reverse effect on GIT2-/- mice injected with Con A. The increased serum ALT & AST (t=6.804, P=0.002;t=4.677, P=0.001) and more hepatocyte necrosis (t=4.023, P=0.016) in GIT2-/- mice were observed with delivered pCMV-myc-GIT2 after injection of Con A for 24 hours. This suggested GIT2 turnover can reverse the resistance of GIT2-/- mice to Con A-induced immune liver injury. The results further demonstrated deficiency of GIT2 protected the liver from the Con A-induced immune liver injury.3. The protective mechanism of GIT2 deletion on Con A-induced immune hepatitis in miceOn the basis of the specific protection of deficiency of GIT2 on the Con A-induced immune liver injury the protective mechanism of GIT2 deletion was further investigated in Con A-induced immune hepatitis.The absolute numbers of various immunocytes were determined after injection of Con A for 6 hours and 12 hours in the GIT2+/+ and GIT2-/- mice. The total mononuclear cells (MNC) number and the absolute numbers of CD3+T cells, CD4+ T cells, CD8+ T cells, NK cells, Kupffer cells, DC cells and B cells in GIT2+/+ and GIT2-/- mice increased respectively in Con A-induced immune liver injury model compared with the control group, while the numbers of these immunocytes in GIT2-/-mice injected with Con A were smaller than those of GIT2+/+ mice injected with Con A. In addition, compared with the control group, the proportions of CD4+CD25+Foxp3+ Tregs cells in GIT2+/+ and GIT2-/- mice also increased after Con A treatment for 12 hours, while the proportions of CD4+CD25+Foxp3+Tregs cells in GIT2-/- mice injected with Con A were bigger than those of GIT2+/+ mice injected with Con A. The results suggested that the protective mechanisms of GIT2 deficiency on Con A-induced immune liver injuries may be the deficiency of GIT2 inhibited most of immunocytes infiltration and promoted CD4+D25+Foxp3+Tregs cells infiltration.The liver infiltrating lymphocytes cause hepatocyte damage by releasing various inflammatory cytokines. The intracellsular stain, Cytometric Bead Array (CBA) assay and Real-time PCR were used to detect the inflammatory cytokines, which were produced by CD3+ T cells, secreted to serum, and their mRNA level presented in the homogenized liver. Compared with the GIT2+/+ mice, obviously lower levels of inflammatory cytokines TNF-α IFN-γ d IL-4 produced by CD3+ T cells were observed in GIT2-/- mice after injection of Con A for 1 hour. The factors of TNF-α、 IFN-T. IL-2, IL-4, IL-6 and IL-17 largely decreased in serum of GIT2-/- mice after injection of Con A for 3 & 6 hours (TNF-α 3a = 3.707, P3a =0.014; t6a = 2.874, P6a =0.035; IFN-T t3h = 7.132, P3h =0.002; t6a = 8.480, P6h =0.009; IL-2 t3a = 8.096, P3a =0.000; t6a = 2.989, P6h =0.017; IL-4 t3h = 16.817, P3a =0.002; IL-6 t3a = 4.430, P3a=0.003; t6a = 6.103, P6a =0.004; IL-17A t3a = 5.967, P3h =0.004), while IL-10, a protection inflammatory cytokine, was much higher than that of GIT2+/+ mice (t3h = 3.474, P3a =0.018; t61a = 3.699, P61a =0.021). The mR),IA levels of inflammatory cytokinesofTNF-a, IFN-T. IL-2, IL-4, IL-6, MIP-1, MCP-1, iNOSandlP-10 in GIT2-/- mice were much lower than those of GIT2+/+ mice after injection of CortA for 3 & 6 hours, while mRNA level of IL-10 in GIT2-/- mice was much higher than that of the GIT2+/+ mice. The results suggested that GIT2 deficiency may down-regulate proinflammatory cytokines and up-regulate inhibiting flammatory cytokines to exert Con A-induced immune liver injuryFinally, given the important role of CD4+ T cells in Con A-induced immune liver injuries, the CD4+T ceils were separated from splenocytes and incubated for 24-----72 hours with Con A in vitro. The proportions of CD4+ CD25~ cells and CD4~ CD69~cells were lower in CD4+ T cells with GIT2 deficiency than that of GIT2+/+ CD4+ T cells after 24 hours incubation. The levels of inflammatory cytokines produced by CD4+ T cells with GIT2 deficiency were lower than those of GIT2+/+ CD4+ T cells after 48 hours incubation (TNF-α t1μg/ml = 48.050, P1μg/ml=0.000; t2.5μg/ml= 26.201, P2.5μg/ml =0.000; t5μg/ml= 16.986, P5μg/ml =0.000; IFN-γ t2.5μg/ml = 3.064, P2.5μtg/ml =0.038; t5μg/ml = 5.470, P5μg/ml =0.005; IL-2 t5μg/ml = 5.301, P5μg/m= =0.006; IL-4 t25μg/m~ = 5.783, P2.5μg/ml =0.004; t5μg/ml = 7.619, P5μg/ml =0.015; IL-6 t1μg/ml = 19.756, P1μg/ml =0.000; IL-10 t1μg/ml = 7.080, P1μg/ml =0,002; t2.5μg/ml = 19.086, P2.5μg/ml =0.000; t5μg/ml = 7.645, P5μg/ml =0.002; IL-17A t1μg/ml = 9.880, P1μg/m =0.001 ; t2.5μg/ml = 11.963, P2.5μg/ml =0.000; t5μg/ml = 4.482, P5μg/ml =0.008). The proliferation of CD4+ T cells with GIT2 deficiency was decreased compared with GIT2+/+ CD4+T cells after 72 hours incubation (t= 3.838, P=0.012; t 6.215, P=0.001). The results indicated that GIT2 deficiency inhibited the activation, proliferation of CD4+ T cells, which were stimulated by Con A in vitro, and their inflammatory cytokines production. Therefore, it was confirmed GIT2 deficiency inhibited CD4+ T cells function of Con A-induced immune liver injury.In order to further understand the protective molecular mechanism of GIT2 deletion on Con A-induced liver injury by inhibiting the function of T cells, CD3+ and CD4+ T cells were stimulated by anti-CD3 plus anti-CD28 and PMA/Ionomycin in vitro, respectively. It was found that the stimulation of anti-CD3 plus anti-CD28 revealed a consistent decrease in the activation and proliferation of GIT2 deficient CD3+ and CD4+ T cells, while the activation and proliferation induced by PMA plus Ionomycin was normal. The results suggested the TCR signaling of GIT2 deficient T cells was inhibited, but its cell cycle was normal. From the results above, it can be concluded that the protective molecular mechanism may be that GIT2 deficiency inhibited TCR signaling.4. Effect of GIT2 deficiency on other liver injuriesGiven the complex etiology of liver injury, the CCl4 model, D-GalN/LPS model and the partial hepatectomy model were used to investigate the effects of GIT2 in the chemical liver injury, endotoxin acute liver injury and regeneration capacity. It was found that the survival rate of GIT2-/- mice was lower than that of GIT2+/+ mice and the liver pathological damage of GIT2-/- mice was more severe than that of GIT2+/+ mice in the CCl4 model and the D-GalN/LPS model. The levels of ALT and AST of both mice showed no significant difference in the CCl4 model, while, the levels of ALT and AST were lower than those of GIT2+/+ mice in the D-GalN/LPS model (t= 4.125, P=0.015; t=5.149,P=0.007). The levels of ALT, AST of GIT2+/+ and GIT2-/-mice increased and peaked respectively at 6h after partial hepatectomy, and those of the GIT2-/- mice were slightly lower than those of the GIT2+/+ mice. The liver pathological damage of both mice showed no significant difference. It was found that the GIT2-/- mice hepatocyte proliferation ability was stronger than that of GIT2+/+ mice, especially at 48h and 72h with Ki-67 immunohistochemical detection. The results suggested that GIT2 deficiency aggravated CCl4 and D-GalN/LPS induced liver injury but contributed to the liver cells regeneration. The study on the liver diseases needs comprehensive considerations.The following points may be concluded based on the above findings:1. GIT2 may have an effect on liver metabolic detoxication, glycogen synthesis, protein synthesis, energy supply, antioxidant function, ion metabolism and immune system.2. GIT2 is closely associated with human liver diseases and Con A-induced immune liver injury in mice. GIT2 deficiency can prevent Con A-induced immune liver injuries.3. The protective mechanisms of GIT2 deficiency on Con A-induced immune liver injury may exert by inhibiting most of immunocytes (CD3+ T cells, CD4+ T cells, CD8+T cells, NK cells, Kupffer cells, DC cells and B cells) infiltration and promoting CD4+D25+Foxp3+Tregs cells infiltration, as well as decreasing the production of proinflammatory cytokines (TNF-a, IFN-y, IL-2, IL-4, IL-6, MIP-1, MCP-1, iNOS and IP-10) and increasing IL-10 level. Inhibiting CD4+ T cells function is most important among those protective mechanisms.4. GIT2 deficiency aggravates CCl4 and D-GalN/LPS induced liver injury but it contributes to the regeneration of liver cells. |
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