Interaction And Mechanism Between Neutrophil Extracellular Traps And Different Antigen-presenting Cells In Hashimoto’s Thyroiditis

Objectives Hashimoto’s thyroiditis(HT)is the most common autoimmune thyroid disease(AITD).The incidence rate of HT is increasing.It is closely related to immune disorders.Neutrophil extracellular traps(NETs)is a new death mode of neutrophils,forming net-shape extracellular fibers composed of DNA,histones and other neutrophil specific microbicidal granular proteins.Recent studies have suggested that NETs are related to a variety of autoimmune diseases while few research exists between NETs and HT.The purpose of this study is to explore the immune effect and mechanism of NETs on professional and non-professional antigen-presenting cells of HT,and the following effect on T lymphocyte differentiation.The study in-depth will help to broaden new vision of HT pathogenesis,and provide a theoretical basis for the design and development of drugs targeting NETs to prevent and treat HT.Methods Firstly,we focused on the experimental autoimmune thyroiditis(EAT)constructed in BALB/c mice,a classical animal model of HT.The differences of neutrophil forming NETs,immune cell subsets,cytokines,thyroid inflammation,vitamin D and DNasel levels were detected,and we explored the above effect in calcitriol intervention group.Further,we verify the difference in the formation of NETs in the peripheral blood of clinical HT patients.Then,professional antigen-presenting cells in EAT mice,i.e.plasmacytoid dendritic cells(pDC),and one of the non-professional antigen-presenting cells,thyroid epithelial cells,were taken as the research objects in vitro.Through primary cell culture,cell coculture,immunomagnetic bead sorting,flow cytometry,cellular immunofluorescence,flow cytometry,Western blot,RT-PCR and other key technologies,we explored how NETs affect pDC and the following T lymphocyte differentiation,and explored the immunomodulatory effects of NETs on the antigen presentation ability and cell adhesion ability of thyroid epithelial cells.Results EAT mice and HT patients were more prone to NETs forming and EAT mice was with decrease eliminating ability of NETs.EAT mice were with Th17 differentiation advantage and decreased Treg cells.NETs was positively correlated with Th17 cells and negatively with Treg cells.Calcitriol can partially reverse the above immune abnormalities.There was abnormal maturation and activation of pDC in EAT mice.NETs can further promote the maturation and activation of pDC,induce naive CD4+T cells into Th17 differentiation advantage,reduce the differentiation of Treg cells which could inhibit immunity,and ultimate may break the immune tolerance.TLR9 inhibitor can inhibit the above NETs’ effect,indicating that TLR9 pathway is involved in the maturation and activation of pDC by NETs.Human thyroid epithelial cell line Nthy on 3-1 could absorb NETs and transport them to the cytoplasm through endocytosis.Then by activating TLR9 pathway,NETs promoted the expression of MHC-II and key transcription factor CIITA,increased the expression of adhesion factor ICAM-1 and apoptosis related pathway FAS.It is suggested that NETs could regulate the immune function of thyroid epithelial cells through TLR9 pathway.Conclusions This study revealed the phenomena of excessive formation and decreased degradation of NETs in EAT mice and HT patients.NETs as endogenous DNA and protein complexes,can activate professional and nonprofessional antigen-presenting cells through TLR9 pathway,improve antigenpresenting ability and regulate immune phenotype,and may participate in systemic inflammatory immune disorder and local inflammatory destruction of thyroid in HT disease.Part Ⅰ Formation of Neutrophil Extracellular Traps in EAT Mice and Intervention of Vitamin DObjective To investigate the formation of Neutrophil Extracellular Traps(NETs)in experimental autoimmune thyroiditis(EAT)mice and the intervention of calcitriol.Methods Six-week-old female BALB/c mice were randomly divided into EAT model group(EAT group,n=8),calcitriol intervention group(VD group,n=8)and control group(control group,n=8).The mice in EAT and VD groups were immunized subcutaneously with porcine thyroglobulin and fed with high iodine.The mice in VD group were injected intraperitoneally with calcitriol.The mice in control group were injected subcutaneously with adjuvant,injected intraperitoneally with PBS,and drank drinking water from normal animals.After 8 weeks,the neutrophils in peripheral blood of mice were isolated and purified.The formation of NETs was observed by scanning electron microscope.The Th17 and Treg cell subsets in spleen were identified and quantified by cellular immunofluorescence and flow cytometry.The thyroglobulin antibody(TGAb),thyroid peroxidase antibody(TPOAb),type Ⅰ deoxyribonuclease(DNase-I),1,25-dihydroxyvitamin D3[1,25(OH)2D3]and the levels of interleukin-17(IL-17)and interleukin-10(IL-10)were measured.Paraffin sections,HE staining and MHC-II immunohistochemistry were performed on the thyroid gland.At the same time,5 patients with Hashimoto’s thyroiditis and 5 healthy volunteers were selected to isolate and purify peripheral blood neutrophils.The amount of NETs formation under the same PMA stimulation was compared by immunofluorescence.Results(1)The thyroiditis score,TGAb,TPOAb and MHC-II expression in thyroid epithelial cells in EAT group were significantly higher than those in control group.The VD intervention group significantly lowered the thyroiditis score,TPOAb and MHC-II expression compared with EAT group.(2)Compared with control group,the spleen Th17 cell subsets,IL-17 levels and Th17/Treg ratio in the EAT group increased significantly,the Treg cell subsets and IL-10 levels decreased significantly,the formation of NETs increased significantly,and the levels of DNase-I and 1,25(OH)2D3 decreased significantly.Compared with EAT group,VD group significantly decreased the proportion of Th17 cells,the level of IL-17 and the ratio of Th17/Treg,and significantly increased the proportion of Treg cells.After VD intervention,the levels of DNase-I and 1,25(OH)2D3 increased,but there was no significant difference compared with EAT group.(3)NETs formation was positively correlated with Th17 cell subsets and negatively correlated with Treg cell subsets.(4)Under the same conditions,the peripheral blood neutrophils of HT patients were more likely to form NETs than those of normal people.Conclusion There is an imbalance of CD4+T lymphocyte differentiation in EAT mice,which is manifested by the increase of Th17 cells and the decrease of Treg cells.Both EAT mice and HT patients were more prone to NETosis.At the same time,the degradation ability of NETs decreased in EAT mice.The formation of NETs was positively correlated with Th17 cells and negatively correlated with Treg cells.The vitamin D level of EAT mice decreased,and calcitriol supplementation could alleviate thyroid inflammation and immunophenotype,autoantibodies,excessive formation of NETs with degradation disorder,imbalance of Th17/Treg ratio.Part Ⅱ Neutrophil Extracellular Traps Activates Plasmacytoid Dendritic Cells Through TLR9 Pathway and Induces Imbalance of CD4+T Lymphocyte DifferentiationObjective To investigate the stimulation and activation of neutrophil extracellular traps(NETs)on professional antigen-presenting cells:plasmacytoid dendritic cells(pDCs)and the subsequent differentiation of CD4+T lymphocytes,and to explore the role of TLR9 pathway.Methods Six-week-old female BALB/c mice were randomly divided into EAT model group(EAT group)and control group(control group)for 8 weeks.The EAT group was immunized subcutaneously with porcine thyroglobulin and fed with iodine water,while the control group was injected subcutaneously with adjuvant and drank normal drinking water at the same time.After 8 weeks,the spleen single cell suspension was aseptically extracted,and the spleen pDC of the two groups were purified by immunomagnetic beads sorting method.The purity and maturity of pDCs were detected by flow cytometry.In addition,neutrophils from peripheral blood of normal mice were isolated and purified,and stimulated by PMA in vitro for formation and purification of NETs.In vitro,experiment was set up as sole pDC group,pDC+NETs group,and pDC+NETs+TLR9 inhibitor group.After 20 hours of culture,parts were subjected to flow cytometry to detect the changes of pDC maturation and activation.The remaining cells were co-cultured with naive CD4+T cells from normal mice sorted by immunomagnetic beads.In addition,sole CD4+T cell culture group was set up.T cell differentiation was detected in the co-culture system by flow cytometry after 7 days.Results(1)The maturity of spleen pDCs in EAT mice was higher than that of normal mice,which was characterized by the significant increase of CD40 and CD86;(2)NETs can promote the expression of antigen presenting molecule MHC-II and costimulatory molecule CD40,and promote the activation and maturation of pDC in vitro,especially those derived from EAT.TLR9 inhibitor could partially reverse the above results;(3)The pDC stimulated by NETs induced naive CD4+T lymphocytes differentiating into Th17,with the trend of decreasing Treg cells.TLR9 inhibitors could inhibit the above differentiation directions,especially those derived from EAT mice,suggesting that NETs can promote the antigen presenting ability of pDC through TLR9 pathway and induce T lymphocytes to differentiate into Th17 and reduce Treg differentiation.Conclusion EAT mice have abnormal increase of mature pDC.NETs can promote the maturation and activation of pDC through TLR9 signaling pathway,and then start the differentiation advantage of Th17 cells and reducing Treg,which may break the immune tolerance,and lead to or aggravate the expansion and persistence of systemic chronic inflammation in EAT.Part Ⅲ The Immune Regulation of Neutrophil Extracellular Traps on Thyroid Epithelial Cells and the Mechanism of TLR9 PathwayObjective To investigate the immunoregulation of neutrophil extracellular traps(NETs)on non-professional antigen-presenting cells:human thyroid epithelial cell line Nthy-ori 3-1 and the involvement of TLR9 pathway.Methods Neutrophils from peripheral blood of healthy volunteers were extracted and purified to prepare NETs,stimulated by PMA in vitro.The expression of MHC-II,EEA1,MPO and TLR9 in Nthy-ori 3-1 were detected by western blot under different NETs concentrations.The expression of MHC-II,CIITA,TLR9,MyD88,Fas and ICAM1 were detected by cellular immunofluorescence,flow cytometry and real-time fluorescent quantitative PCR in Nthy-ori 3-1 under NETs 50μg,with or without TLR9 inhibitor E6446 dihydrochloride.Results(1)Human thyroid epithelial cell Nthy-ori 3-1 could absorb NETs and transport them into the cytoplasm through endocytosis;(2)NETs stimulated the expression of MHC-II and TLR9 proteins in Nthy-ori 3-1 cells in a concentration dependent manner,and increased the transcription of MHC-II,CIITA,TLR9,Fas,ICAM1 and MyD88 at the mRNA level;(3)TLR9 inhibitor can inhibit the protein expression and mRNA transcription of MHC-II and CIITA in Nthy-ori 3-1 cells induced by NETs.Conclusion Human thyroid epithelial cell Nthy-ori 3-1 can absorb NETs and transport them to the cytoplasm through endocytosis,then promote the expression of MHC-II,CIITA,ICAM1 and Fas through TLR9 pathway,increase the antigen presentation ability,adhesion ability and promote apoptosis pathway,which may be related to local immune regulation and thyroid tissue destruction in HT disease.