The Relationship Between Toll-like Receptor7,9and Human Parvovirus B19in Hashimoto’s Thyroiditis

[Background]Hashimoto’s thyroiditis (HT) is the most common organ-specific autoimmune diseases inold women. Clinically, there were no special symptoms in its early stage, but in its latestage it can cause hypothyroidism, or even induce thyroid cancers. A variety ofautoantibodies in the patient’s serum can be detected. Its main pathological changes wereas follows: a large number of lymphocytes infiltration, secondary lymphoid follicle withobvious germinal centers formation, as well as thyroid follicular epithelial cellseosinophilic degeneration and progressive distruction until fibrosis in the end. AlthoughHT has been focused for more than a century after the first reported in1912, the etiologyand pathogenesis remain unclear. Currently, HT is closely related to both genetic andenvironmental factors, and it is believed that virus infection might play an important rolein it. But the mechanism is not clear.Human parvovirus B19(B19) is a non-enveloped and single-stranded DNA virus. Humans are the only host. B19infection is common and associated with a variety ofdiseases, especially autoimmune diseases. Our laboratories and other foreign groupspointe out that the incidence of HT is closely related to B19infection, but its pathogenesisis not clear.Toll-like receptors (TLRs) are one kind of cell transmembrane receptors andpathogen pattern recognition receptors which were found in recent years. It can causecellular immune response by activating intracellular transduction signal, and play a role asa bridge between the innate and adaptive immune. TLRs are able to recognize viruses andother pathogenic microorganisms, activate the innate immune response causing theproduction of large amounts of co-stimulatory molecules and proinflammatory cytokines,and then induce adaptive immune responses. As important members of the TLR family,TLR7and TLR9are known to identify virus. TLR7mainly expresses in plasmacytoiddendritic cells, recognizes single-stranded (ssRNA) virus and activates MyD88and IRF7to mediate anti-viral immunity. TLR9presents not only in memory B lymphocytes andplasmacytoid dendritic cells, but also in some normal epithelial cells, including intestinalepithelial cells, lung epithelial cells and so on. It recognizes CpG DNA and also activatesMyD88and IRF7to mediate anti-viral immunity. According to the researches, TLR7andTLR9are both associated with anti-viral immunity, but because of the negative regulationof the virus, TLR is in the sustained activation state. As a result, a large number ofinflammatory cytokines are produced, which causes chronic inflammation, autoimmunedisorders and other diseases. TLR7and TLR9are associated with various autoimmunediseases, but not involving HT.Based on the above information, we want to know whether TLR7and TLR9andB19work together in the pathogenesis of HT. The answer to this question will not onlyhave important significance to further elucidate the pathogenesis of HT, but also providenew ideas for the treatment of HT. To do this, we have revealed the relationship betweenB19and TLR7,9in HT thyroid tissue by using immunohistochemical staining of theserial sections, confocal laser microscope with immunofluorescence double labeling andco-immunoprecipitation, and want to provide viral evidence to the mechanism of B19 infection.[Objects]To explore the relevance of TLR7,9and B19in HT pathogenesis.[Methods]1. To detect the expression of TLR7,9and B19in41cases HT thyroid specimens byEnVision immunohistochemical staining and to analyze their expression correlation.2. To analyze the co-localization of TLR7and B19, TLR9and B19in HT thyroidtissues by immunofluorescence double labeling confocal laser microscope.3. To analyze the interaction of B19and TLR9by co-immunoprecipitation.[Results]1. Both B19and TLR9were distributed mainly in thyroid follicular epithelial cellswhich showed eosinophilic degeneration. We found that34cases showedTLR9-positive and33cases were B19-positive. But both B19and TLR9werenegative in20cases of normal thyroid tissue. Statistical analysis showed that TLR9and B19-positive expression rates were82.93%(34/41) and80.49%(33/41)respectively. And the expressions of TLR9and B19in HT have a very significantdifference, compared with normal thyroid.(p＜0.001）.2. The serial section staining showed that B19was positive while TLR9was alsopositive, but TLR7was negative in the same area. Spearman rank correlationanalysis showed a positive correlation between B19and TLR9(r=0.594, P=0.001),but no relationship between B19and TLR7.3. The double-labeling immunofluorescence suggested that co-localization of TLR9and B19antigen was in the cytoplasm of the degenerative thyroid follicularepithelial cells. The negative control was negative. And TLR7was also negative in the degenerative follicular epithelial cells.4. Co-immunoprecipitation test showed that B19and TLR9worked together in freshHT thyroid tissues.[Conclusions]We found for the first time that TLR9may be a receptor for recognizing B19antigen in the pathogenesis of HT, but not TLR7.