| Purpose:To investigate the intestinal-associated retinal neuron degeneration and the role of Microglia/Macrophage in gut-retina axis,an intestinal mucosal immunity associated retina dysfunction mode was established,and further study the function of Steaps on Microglia/Macrophage.These provide a reference for underlying mechanism of immunoregulation on retinal neurodegeneration in gut-retina axis,and shed a new light on understanding the“gut-retina axis”related diseases.Method:Intestinal mucosal immunity associated retina dysfunction mice model were established by induction of dextran sodium sulfate?DSS?-induced chronic colitis.Electroretinography?ERG?was performed to evaluate retinal function.Retinal neuron degeneration was analyzed by immunohistochemistry.Flow Cytometer was used to detect microglia/macrophage activation and proliferation,as well as lymphocyte infiltration.In vivo depletion of CD4+T cells or blockade of MAdCAM-1 was performed to determine the role of“gut-homing”CD4+T cells recruited by microglia in retinal neurodegeneration.The potential role of Steaps in mouse retinal microglia was explored using RNA interference.The expressions of cytokines and chemokines during retinal inflammation were measured by qRTPCR.Retinal functions of Steap4-/-mice were evaluated to explicit the role of Steap4.Results:1)We successfully constructed a mouse model of intestinal mucosal immune disorder induced by DSS.ERG test showed that the amplitudes of ERG a-,b-wave in the retinas of mice with intestinal mucosal immune disorder were down-regulated to different degrees,and the oscillating potential was also significantly reduced,and the b-wave showed obvious delayed implicit times.2)HE results showed that the model mice had significantly thinner retinal inner nuclear layer and looser cell arrangement,but there was no significant change in the outer nuclear layer compared with the control group.3)Immunofluorescence staining results showed that DSS-induced intestinal mucosal immune disorder mice had severe damage of retinal bipolar cells,with a decrease in cell number and irregular dentritic fibers,and abnormal connections were also found in the synapses connecting RGC or photoreceptor cells.In addition,significant apoptosis and axon loss of RGC,as well as the demyelination of optic nerve were observed in DSS-treated mice.4)Intestinal mucosal immune disorder mice showed little change in the outer retinal nuclear layer,with only abnormal elongation of the outer segment by the 50th day.5)Fundus angiography showed no leakage and pathologic hyperplasia of retinal vessels after DSS feeding for 50 days.6)Flow cytometry and immunofluorescence staining analysis showed that intestinal mucosal immune disorders induced increased proliferation and activation of retinal microglia,accompanied with infiltration of peripheral macrophages.7)QRT-PCR showed that the expression levels of Tnfa,Il1b,Ifng and Il17a,and the chemokines Cxcl9,Cxcl10,Cxcl11,as well as the chemokine receptor Cxcr3 in the retina of mice with intestinal mucosal immune disorder were significantly increased.8)Flow cytometry and immunofluorescence staining revealed abnormal infiltration of CD4+T cells into retina of mice with intestinal mucosal immune disorder,mainly distributed in the inner layer of the retina.CD4 specific antibody intervention can significantly reduce the number of infiltrating CD4+T cells into retina,and improve the retinal immune microenvironment,and alleviate retinal dysfunction and neurodegeneration.9)Anti-CXCR3 antibody intervention can reduce infiltrating T cells regulated by microglia,alleviating the injury of retina in mice with intestinal mucosal immune disorder.10)According to the fluorescence staining of the blood vessels in the retinal whole mount,we found MAdCAM-1 were highly expressed on retinal microvessels of the intestinal mucosal immune disorder mice.11)QRT-PCR and immunofluorescence staining revealed that retinal infiltrating CD4+T cells were co-expressed?4?7,indicative of the“gut-homing”nature.12)Inhibitionn of MAdCAM-1 significantly reduced damage of retinal inner neurons in DSS-treated mice.13)The Steap family members stably expressed in colon and retina,but only Steap4 was found up-regulated in both tissues of DSS-treated mice,which was mainly expressed in Macrophages of colon and retinal inner neurons.14)in vitro experiments,expression of Steaps was up-regulated after stimulating by cytokines TNFa,IFNg,and IL17a in BV2 cells.The high expression of Steap4 gene was accompanied by the enhanced proliferation and activation of BV2 cells.15)when the Steap4 gene was knocked down,the expression level of genes related to proliferation and activation of BV2 cells was significantly reduced.16)At the age of 4.5 months,the retinal ERG of Steap4-/-mice was significantly changed,and the amplitude of a and b waves were significantly down-regulated.17)QRT-PCR showed that the retinal immune microenvironment of Steap4-/-mice was changed:the expression levels of cytokines Il1b and Ifng were increased,and the expression levels of genes related to the proliferation and activation of microglia/macrophage cells were also significantly increased.Conclusion:By constructing a DSS-induced gut-retina injury model,we identified that intestinal mucosal immune disorders can cause damage of the retinal inner neurons,resulting in changes in retinal visual function.Intestinal mucosal immune disorder could lead to the proliferation and activation of microglia in the retina,which induced changes in the retinal immune microenvironment,as well as the infiltration of peripheral macrophages and CD4+T cells,which damaged the inner retinal neurons and down-regulated retinal function.During this process,Steap4 may be involved in the regulation of microglia activation function and the injury of retinal neurons. |
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