Metabolism And Polarization Abnormalities Of Macrophages Are Involved In The Pathogenesis Of Autoimmune Thyroiditis In NOD.H-2^h4 Mice

Objective:Autoimmune thyroiditis（AIT）is a typical organ-specific autoimmune disease.The pathological feature of AIT is the infiltration of a large number of immune cells among thyroid follicular cells.The activated immune cells cause damage,apoptosis and necrosis of thyroid follicular cells through the release of inflammation factors,and eventually progress to hypothyroidism.The infiltrating immune cells are mainly CD4⁺T cells and macrophages.Macrophages can be polarized into M1 or M2 macrophages in different microenvironments due to their high degree of plasticity.Among them,M1phenotype secretes inflammatory factors and participates in the inflammatory response.The polarization direction of macrophages is directly related to metabolic reprogramming.Immunometabolism studies have found that resting macrophages mainly metabolize energy production through oxidative phosphorylation（OXPHOS）,while aerobic glycolysis after stimulation by lipopolysaccharide（LPS）becomes the important energy metabolism pathway.This metabolic change is called metabolic reprogramming.M1 macrophage infiltration has been found in a variety of autoimmune diseases,including rheumatoid arthritis,atherosclerosis,systemic lupus erythematosus and inflammatory bowel disease,but there is a lack of research on immune metabolism related to AIT.Previous studies have shown that the treatment of 2-Deoxy-D-glucose（2-DG）and metformin effectively inhibits the polarization of macrophages to M1phenotype and reduces the secretion of proinflammatory cytokines.Alleviated the progression of rheumatoid arthritis（RA）.Its mechanism may be related to metabolic regulatory proteins,such as adenosine monophosphate-activated protein kinase（AMPK）and Hypoxia-inducible factor 1-alpha（HIF-1α）.The purpose of this study was to investigate the important metabolic pathway and polarization subtypes of macrophages in AIT mice.To investigate whether 2-DG and metformin inhibit the aerobic glycolysis of M1 type macrophages through AMPK/HIF-1αpathway can correct the polarization imbalance of macrophages,and ultimately reduce the inflammatory infiltration of AIT thyroid tissue.Methods:NOD.H-2^h4female mice at 4 weeks of age were randomly divided into four groups,each with 15 mice.The control（CON）group mice were fed with sterile water for 12weeks;the mice in the AIT group were fed with sterile water containing 0.05%sodium iodide for 8 weeks;The 2-DG and Metformin（MET）groups were fed with sterile water containing 0.05%sodium iodide for 8 weeks,and then fed with 5 mg/ml 2-DG and 3mg/ml metformin in sterile water for 4 weeks.Animals in each group were sacrificed at the 16 weeks of age,and apical blood,thyroid tissue,and spleen tissue were collected.ELISA measures the serum anti-thyroglobulin antibodies（Tg Ab）level;hematoxylin-eosin（HE）staining to observe the lymphatic infiltration of thyroid tissue;immunofluorescence method was used to locate and detect the expression of M1 and M2macrophages and AMPK and HIF-1αin thyroid tissue;Extract spleen-derived macrophages to measure extracellular acidification rate（ECAR）and oxygen consumption rate（OCR）,and analyze macrophage polarization by flow cytometry;Western Blot was used to detect the expression of AMPK/HIF-1αpathway and glycolysis enzymes,such as:hexokinase 2（HK2）,phosphofructokinase（PFKP）,pyruvate kinase M2（PKM2）expression.Results:1.The occurrence of autoimmune thyroiditis in NOD.H-2^h4miceCompared with the AIT group,the CON group,2-DG and MET groups had significantly lower thyroiditis scores and serum Tg Ab levels（P<0.05）.The thyroiditis score and serum Tg Ab levels of the CON group were not significantly different from those of the 2-DG and MET groups（P>0.05）.2.Analysis of the proportion of subtypes of macrophages polarization（1）The results of flow cytometry experiments showed that the ratio of M1/M2 in the AIT group was significantly higher than that in the CON,2-DG and MET groups（P<0.05）.The ratio of M1/M2 in the 2-DG and MET groups was significantly There was no significant difference in the CON group（P>0.05）.（2）The results of immunofluorescence staining showed that there were significantly more M1 macrophages in the thyroid tissue of mice in the AIT group than in the CON,2-DG and MET groups（P<0.05）.Compared with the AIT group and the CON group,the M2 macrophages in the 2-DG and MET groups were significantly increased（P<0.05）.3.Metabolism of mouse spleen-derived macrophagesCompared with the CON group,the ECAR and OCR values of the AIT group were significantly higher（P<0.05）.Compared with the AIT group,the ECAR and OCR of the2-DG and MET groups were significantly lower（P<0.05）.4.The expression of mouse AMPK/HIF-1αpathway and glycolytic pathway enzymesWestern Blot results showed that compared with the CON group,the expressions of HIF-1αand key glycolytic enzymes HK2,PFKP,and PKM2 in the AIT group were significantly increased（P<0.05）.HIF-1αand HK2,PFKP,and PFKP in the 2-DG and MET groups The expression of PKM2 was significantly lower than that of AIT（P<0.05）,but the expression of AMPK was higher than that of AIT group and CON group（P<0.05）.Compared with the CON group,the expression of HIF-1αin the AIT and 2-DG groups was significantly higher（P<0.05）,but there was no significant difference in the MET group（P>0.05）.Conclusion:Compared with the AIT group,2-DG and metformin inhibited the glycolysis pathway by inhibiting the AMPK/HIF-1αpathway,reducing the polarization of macrophages to M1 and increasing M2 phenotype,finally reducing the progression of thyroid inflammation in mice.It is further clarified that the metabolic reprogramming of macrophages can affect the imbalance of their polarization and affect the development of autoimmune thyroiditis.