Low Molecular Weight Hyaluronic Acid Promotes The Development Of Hashimoto’s Thyroiditis By Influencing Th17 Cell Differentiation

Background: Hashimoto’s thyroiditis(HT)is the most common autoimmune disease and one of the most common thyroid disorders,with an incidence of 0.3-1.5 per 1000 people.It is typically characterised by a lymphocytic infiltration of the thyroid tissue and replacement of the thyroid parenchyma by interstitial tissue,which is rich in collagen fibres and Hyaluronic Acid(HA).It has been shown that HA is involved in the regulation of the development of many autoimmune diseases,and we have previously found that low molecular weight hyaluronic acid(LMW HA)is highly expressed in the thyroid tissues of HT patients.However,the role and mechanism of LMW HA in HT is still unclear.Objective: To clarify the expression of LMW HA in the thyroid tissue of HT patients,further explore the role and mechanism of LMW HA in promoting the development of HT,and provide new ideas for the in-depth understanding of the pathogenesis of HT and the development of targeted drugs.Methods: Firstly,the differences in HA levels in thyroid tissue and serum between HT patients and control patients were measured by Elisa,and the differences in HA,LMW HA,hyaluronic acid synthase(HAS)and hyaluronidase(HYAL)expression in the paracancerous tissues of papillary thyroid cancer(PTC)patients with and without HT were detected by qRT-PCR,Western blot and immunohistochemistry,and the clinical correlation between HA concentration and HT was analyzed.Secondly,an experimental autoimmune thyroiditis(EAT)mouse model was constructed to mimic the development of HT,and the role of LMW HA in the development of EAT in mice was investigated by exogenous supplementation of LMW HA.The role of LMW HA in the development of EAT in mice was further investigated by inhibiting HA synthesis through oral administration of 4-Methylumbelliferone(4MU)prior to EAT induction.The severity score of inflammation in mouse thyroid tissue was scored by H&E staining.Then,the role of LMW HA in the pathogenesis of HT was investigated by measuring thyroid function,and thyroiditis-specific antibodies and immune-related inflammatory factors in EAT mice by Elisa.In addition,we also studying the relationship between serum HA concentrations and serum immune-related inflammatory factor concentrations in HT patients.Finally,the effects of exogenous increase the concentration of LMW HA and endogenous decrease the concentration of LMW HA on immune cells in the spleen of EAT mice were analyzed by flow cytometry to explore the possible mechanisms by which LMW HA promotes the development of EAT through its effects on the immune system.The mechanism of HA synthesis and secretion was also verified by extracting human thyroid-derived fibroblasts.Results: The HA levels in thyroid tissues and serum were significantly higher in HT patients than in control patients.LMW HA with a molecular weight of 20 k Da was also significantly increased,and hyaluronate synthase 2(HAS2)and hyaluronidase 2(HYAL2)were highly expressed in the thyroid tissues of HT patients.HA levels were positively correlated with serum FT3 and FT4 and HT-related antibodies(TPO-Ab and Tg-Ab),and negatively correlated with TSH.The EAT model could be successfully constructed by subcutaneous injection of PTg and CFA mixture,and could well mimic the development of HT.LMW HA increased the success rate of EAT induction and the severity of inflammation in mice,and was most obvious at the fourth week after induction.4MU inhibition of HA synthesis significantly reduced the incidence of EAT and the severity of inflammation.Serum inflammatory factors measured by Elisa revealed that LMW HA significantly increased serum IL-17 A,IL-1β and TNF-α concentrations in EAT mice,while 4MU significantly decreased serum IL-17 A,IL-1β and TNF-α concentrations in EAT mice.Additionally,serum IFN-γ,IL-17 A,IL-1β and TNF-αconcentrations were significantly higher in HT patients than in control patients,and that HA serum concentrations were positively correlated with IFN-γ,IL-17 A,IL-1β and TNF-α serum concentrations and negatively correlated with IL-4 and TGF-β serum concentrations.The flow cytometric analysis of mouse spleens at the fourth week after induction showed that exogenous increase in LMW HA promoted Dendritic cells(DCs)cell activation and Th17 cell differentiation in EAT mice,while 4 MU of endogenous decrease in LMW HA concentration inhibited DC cell activation and Th17 cell differentiation in EAT mice.In vitro,co-culture experiments of normal thyroid cell lines and fibroblasts with inflammatory factors revealed that fibroblasts synthesized and secreted more HA than normal thyroid cell lines.Among those inflammatory factors,IL-17 A has the most significant role in promoting HA synthesis and secretion by fibroblasts.IL-17 A promoted HA synthesis,secretion and upregulated the expression of HAS2,HYAL2,CD44 and TLR2 in a concentration-dependent manner in fibroblasts.Conclusion: LMW HA is increased in thyroid tissues of patients with HT.LMW HA contributes to the development of HT by promoting DC cell activation and Th17 cell differentiation.IL-17 A promotes HA synthesis and secretion by fibroblasts and upregulates HYAL2,which contributes to LMW HA production.4MU has potential therapeutic value for patients with HT by reducing LMW HA concentrations.