| Background:The exact pathophysiological mechanism of hyperuricemia and gout is not fully understood.Many studies had discovered a close relationship between gout and the immune system.Both NK cells and iNKT cells have the functions of anti-infection,anti-tumor and immunomod?lation.Many studies have implicated the roles of NK cells and iNKT cells in the pathogenesis of various metabolic diseases,such as diabetes and obesity.However,little is known about the alterations of these cells in patients with hyperuricemia and gout,and it is not clear whether they are involved in the occurrence and development of the gout and hyperuricemia.Aim:We aimed to investigate the relationship between hyperuricemia,gout and NK cells,through establish an animal model of hyperuricemia and gout.The relationship between hyperuricemia,gout and NK cells was further clarified in the patients.The changes of iNKT cells in gout and HUA patients were also detected in order to fully understand the effect of uric acid on the immune system.In addition,we investigated the relationship between uric acid and the above immune cell subsets and cytokines in hyperuricemia and gout patients,in order to further understand the relationship between HUA,gout and immune system.Methods:1.Animal model of hyperuricemia and gout(1)C57BL/6 mice were divided into 6 groups: healthy control group(CS),hyperuricemia group(HUA),12-hour gout group(12H),24-hour gout group(24H),48-hour gout group(48H),72-hour gout group(72H),10 mice in each group.(2)The hyperuricemia mouse model was established by intraabdominal injection of oxazinic acid 300mg/kg·bw once a day,7 days totally.On the basis of hyperuricemia model,Air-Pouch model was established by subcutaneous injection ofair into the back and 3mg MSU crystal was injected into the air-pouch cavities to establish gout model.(3)Separated the serum to measure the serum uric acid level.(4)The infiltration of inflammation cells was observed by HE stain of the Air-Pouch membrane.(5)The percentage,subsets and receptors of NK cells in peripheral blood was detected by flow cytometry.(6)The percentage,subsets and receptors of NK cells in the air-pouch lavage fluid was detected by flow cytometry.(7)The percentage,subsets and receptors of NK cells in spleen was detected by flow cytometry.(8)The levels of IFN-?,IL-6,IL-10,TNF-? in serum and lavage fluid were detected by CBA.2.Study in the patients with hyperuricemia and gout(1)A total of 20 untreated asymptomatic HUA patients,20 acute gouty arthritis(AG)patients,20 intercritical gout(IG)patients and 20 control subjects(CS)were recruited for this study.All patients were followed up at 4 and 24 weeks.(2)Serum uric acid,blood glucose,triglyceride and cholesterol were measured in CS and patients before and after follow-up.(3)The alterations of NK cells,iNKT cels,NKT cells,T cells and B cells were deteced by flow cytometry.(4)The levels of serum IFN-??IL-6?IL-10?IL-2?IL-4?TNF-? were measured using CBA.(5)The potential correlation between the indexes of NK cells,iNKT cells and cytokines,clinical indexes was analyzed.Results:1.Animal model of hyperuricemia and gout(1)The levels of uric acid in HUA and gout group were higher than those in CS.HE staining of air-pouch membrane in gouty group showed inflammatory cell infiltration,which proved that hyperuricemia and gout model were successfully established.(2)Alteration of NK cell percentage: the percentage of NK cells in peripheral blood of HUA mice decreased.After injection of MSU crystal,NK cells in spleen andlavage fluid of gout mice decreased at first and then increased,and NK cells in peripheral blood decreased after 72 hours.?(3)Changes of NK cell receptor: the expression of NKG2 D in peripheral blood and lavage fluid of HUA mice decreased,while the percentage of NKG2D+NK cells in lavage fluid of gout mice decreased slightly.(4)The alterations of NK cell subsets: In HUA mice,the percentage of CD11b+CD27+NK cells in peripheral blood decreased and CD11b-CD27+NK cells increased in spleen.CD11b+CD27+NK cells in lavage fluid of gout mice first decreased and then increased,CD11b+CD27-NK cells increased first and then decreased,the percentage of CD11b+CD27-NK cells in peripheral blood increased,the proportion of CD11b+CD27+NK cells decreased,and the changes of spleen NK cell subsets were unobvious.(5)The change of activated NK cells: the proportion of CD69 NK cells in HUA and gout mice lavage fluid increased.(6)The change of cytokines: the concentration of IL-6,TNF-?,IL-10 in peripheral blood of HUA and gout group was increased;the level of cytokines in lavage fluid wasn't alter in HUA group,but the level of TNF-?,IFN-? and IL-6 in gout group was higher.2.Study in the patients with hyperuricemia and gout(1)There was no obvious difference about the uric acid concentration in patients with HUA,AG and IG.After follow-up,the rate of uric acid level which reached the target in each group was low.(2)The absolute numbers of NK cells,NKG2D+NK cells in the peripheral blood were lower in the HUA patients when compared with the CS,but there was no change after follow-up.There were a negative correlation between the NK cells ?NKG2D+NK cells with BMI before and after follow-up.The number of CD4+iNKT cells in HUA group was higher than that in CS group,and positively correlated with SUA,BMI and IL-10.24 weeks later,the number of CD4+iNKT cells was still positively correlated with IL-10.The number of CD4-CD8-iNKT cells,CD8+iNKT cells was lower than that in CS group,but there was no change after follow-up.(3)The absolute number of NK cells and NKG2D+NK cells in AG patients were decreased at the time of acute gout attack,and were negatively correlated with IL-6 and 24-hour urine uric acid(UUA).The absolute number of NK cells and theexpression of NKG2 D returned to normal after remission of joint symptoms.The absolute number of CD4+iNKT cells in AG patients was higher than that in CS group,and positive correlated with serum uric acid;the absolute number of CD8+iNKT cells,CD4-CD8-iNKT cells were lower than CS group.There was no dynamic change in each subgroup during the follow-up.(4)There was no change in absolute number of NK cells and their subsets in patients with IG,but the absolute number of inhibitory receptor NKG2 A was increased,and there was a positive correlation between the NKG2 A and UUA.The number of CD8+iNKT cells and CD4-CD8-iNKT cells in IG group was lower than that in CS group before and after follow-up.(5)The absolute number of CD107a+NK cells in HUA,AG and IG patients increased and decreased during follow-up,but there was no difference among the three groups.There was no difference of the absolute number of IFN-?+NK cells,IFN-?+iNKT cells in the three groups when compared with CS.(6)The concentration of IL-10 in peripheral blood of patients with HUA,AG and IG increased before and after follow-up,while the concentration of IL-6 in patients with AG increased when recruitment,but decreased at the time of follow-up.Conclusions:1.The levels of IL-10 in hyperuricemia,gout model mice and patients increased,and the level of IL-6 in the gout model mice and the patients increased,which proved the hyperuricemic state can caused the abnormal secretion of the inflammatory factors.2.There were danymic alterations of subsets and receptors of NK cells from the peripheral blood,spleen and lavage fluid of HUA and gout mice,which evidenced the elevated levels of serum uric acid and the stimulation of MSU crystals,can cause the alterations and redistribution of NK cells in the circulation,tissue and organ.3.The number of NK cells and NKG2D+NK cells decreased in the peripheral blood of patients with hyperuricemia and AG,and the number of NKG2A+NK cells increased in IG patients,further suggested that NK cells had different alterations in HUA and different stages of gout.4.The number of CD4+iNKT cells increased in the peripheral blood of patients with hyperuricemia,and the number of CD4-CD8-iNKT cells,CD8+iNKT cells in the patients with hyperuricemia and gout decreased,indicating the imbalance of the iNKTcell subsets in the patients with hyperuricemia and gout.5.The cytotoxicity of NK cells in HUA and gout patients increased,but reached normal with the prolongation of the course of disease.The ability of NK cells and iNKT cells to secrete IFN-? in HUA and gout patients was not affected by uric acid level. |
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