Vairogdziedzera autoimunitāte: ar Th17-asociēto citokīnu un patomorfoloģisko mehānismu izpēte Hašimoto tireoidīta un Greivsa slimības patoģenēzē. Promocijas darba kopsavilkums

Pēdējo dekāžu laikā ievērojami pieaugusi tādu autoimūno vairogdziedzera slimību (AIVS) kā Greivsa slimība (GS) un Hašimoto tireoidīts (HT) saslimstība un izplatība. Ir būtiski identificēt imunoloģiskos un patomorfoloģiskos faktorus, kas iesaistīti vairogdziedzera autoimunitātes izcelsmē. Lai gan 1. un 2. tipa T līdzētājšūnu (Th) izmainīta aktivitāte AIVS patoģenēzē uzskatāma par pierādītu, nesen veiktie pētījumi liecina arī par iespējamu Th17 šūnu lomu AIVS attīstībā. Turklāt Th šūnu diferenciāciju var ietekmēt selēna deficīts, veicinot šūnu un humorālās imūnatbildes disfunkciju. Šī promocijas darba mērķis bija izpētīt Th17 šūnu nozīmi HT un GS patoģenēzē, lietojot dažādas morfoloģiskās diagnostikas metodes un xMAP tehnoloģiju, kā arī korelēt iegūtos datus ar selēna statusu. Sākotnēji pētījumā tika iekļauti 29 pieauguši pacienti ar AIVS, kuriem veikta tireoīdektomija, savukārt pētījuma klīniskā daļa ietvēra 52 pacientus ar pirmreizēji diagnosticētu, iepriekš neārstētu AIVS, kā arī 26 klīniski veselas pētāmās personas, kuras iekļautas kontroles grupā. Lai izvērtētu ar Th17 saistītu imūnatbildi AIVS ietvaros, tika mērīts ar Th17 saistīto citokīnu – interleikīna (IL)-17, IL-22, IL-23, IL-6 un IL-10 – līmenis plazmā, kā arī IL-17, IL-23 un IL-1β imūnekspresijas izplatība un līmenis vairogdziedzera audos. Vairogdziedzera folikulu integritāte tika pētīta, analizējot blīvo šūnu savienojumu jeb slēgjūgļu proteīnu zonula occludens-1 un klaudīna-1 imūnekspresiju un korelējot iegūtos datus ar IL-17 un CD68 ekspresiju. Papildus tam tika noteikts selēna statuss plazmā. Starp AIVS un kontroles grupas pacientiem statistiski ticamas ar Th17 saistīto citokīnu plazmas līmeņa atšķirības netika konstatētas. Tomēr IL-17 ekspresijas līmenis tireocītos bija ievērojami augstāks pacientiem ar HT un GS nekā kontroles grupas pētāmajām personām, vienlaicīgi korelējot ar IL-23 un IL-1β imūnpozitivitāti HT grupā. Tika noteikta statistiski nozīmīga pozitīva korelācija starp ar Th17 saistīto citokīnu līmeni plazmā un hipertireozes smaguma pakāpi, kas bija neatkarīga no autoantivielu līmeņa, tādējādi norādot uz to iespējamo lomu GS patoģenēzē. HT pacientu vairogdziedzera audos tika novērotas tireocītu slēgjūgļu molekulārās pārmaiņas, uzsverot vairogdziedzera folikulu integritātes bojājumu, taču to saistība ar IL-17 netika konstatēta. Nozīmīgai vairogdziedzera enzīmu daļai, kura nodrošina gan antioksidatīvo un pretiekaisuma funkciju, gan aktivē vairogdziedzera hormonus mērķa audos, funkcionālai aktivitātei nepieciešams pietiekams daudzums selēna, tādēļ selēna deficīts tiek saistīts ar AIVS patoģenēzi un pat terapiju. Lai gan mūsu pētījumā starp AIVS pacientiem un kontroles grupu netika noteiktas statistiski nozīmīgas atšķirības plazmas selēna līmenī, rezultāti pierāda, ka selēna statuss plazmā pirmreizēji diagnosticētiem GS un HT pacientiem Latvijā ir suboptimāls. Analizējot selēna līmeni plazmā HT pacientiem, iegūta statistiska nozīmīga negatīva asociācija ar autoantivielu pret tireoperoksidāzi (TPO) titru, tādējādi liecinot par selēna imūnmodulējošo lomu AIVS patoģenēzē. Turklāt HT pacientiem ar augstāku autoantivielu pret TPO līmeni tika novērots zemāks selēna līmenis plazmā, kas liek domāt, ka selēna papildterapija šiem pacientiem var sniegt ieguvumus. Veicot šo pētījumu, tika iegūta nozīmīga informācija, kas padziļina mūsu zināšanas par vairogdziedzera autoimunitātes patoģenēzi, kā arī ar Th17 saistīto citokīnu lomu un regulatoro ietekmi, tomēr būtu nepieciešami turpmāki pētījumi, lai vēl precīzāk izpētītu Th17 šūnu lomu AIVS patoģenēzē. Pētījums apstiprina arī nepietiekamu selēna līmeni plazmā AVS pacientiem, tomēr, lai izprastu saistību starp selēna papildterapiju un imūnatbildes reakciju, nepieciešams iegūt vairāk klīnisko pētījumu datu.

Decreased ZO1 expression causes loss of time-dependent tight junction function in the liver of ob/ob mice

Diabetes patients are at a high risk of developing complications related to angiopathy and disruption of the signal transduction system. The liver is one of the multiple organs damaged during diabetes. Few studies have evaluated the morphological effects of adhesion factors in diabetic liver. The influence of diurnal variation has been observed in the expression and functioning of adhesion molecules to maintain tissue homeostasis associated with nutrient uptake. The present study demonstrated that the rhythm-influenced functioning of tight junction was impaired in the liver of ob/ob mice. The tight junctions of hepatocytes were loosened during the dark period in normal mice compared to those in ob/ob mice, where the hepatocyte gaps remained open throughout the day. The time-dependent expression of zonula occludens 1 (ZO1) in the liver plays a vital role in the functioning of the tight junction. The time-dependent expression of ZO1 was nullified and its expression was attenuated in the liver of ob/ob mice. ZO1 expression was inhibited at the mRNA and protein levels. The expression rhythm of ZO1 was found to be regulated by heat shock factor (HSF)1/2, the expression of which was reduced in the liver of ob/ob mice. The DNA-binding ability of HSF1/2 was decreased in the liver of ob/ob mice compared to that in normal mice. These findings suggest the involvement of impaired expression and functioning of adhesion factors in diabetic liver complications.

The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila

The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. Here, using a sensitized triple heterozygote-based genetic screening approach, we report the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. We demonstrate that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, We found that Pyd represses the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, our results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.

Beneficial Effects of Quercetin on Microcystin-LR Induced Tight Junction Defects

Quercetin has numerous functions including antioxidant and anti-inflammatory effects. The beneficial effect of quercetin against microcystin-LR (MC-LR)-induced testicular tight junctions (TJs) defects in vitro and in vivo were investigated. Significant reductions in transepithelial electrical resistance, occludin, and zonula occludens-1(ZO-1) levels were detected in the MC-LR-treated TM4 cells, and quercetin attenuated these effects. Interestingly, quercetin suppressed MC-LR-induced phosphorylation of protein kinase B (AKT). It effectively inhibited the accumulation of reactive oxygen species (ROS) in cells stimulated by MC-LR. In addition, ROS inhibitors blocked the TJ damage that is dependent on the AKT signaling pathway induced by MC-LR. In conclusion, our results suggest that alleviates MC-LR-impaired TJs by suppressing the ROS-regulated activation of the AKT pathway.

Sec6 modulates PP2A phosphatase activity through alteration of the binding affinity of PP2A in A and C subunits

Dephosphorylation of the activated the regulator of a-fetoprotein (Raf)-MAP/ERK kinase (MEK)-the extracellular signal-regulated protein kinase (ERK)-p90 ribosomal protein 6 kinase (RSK) cascade plays an essential role in regulating the magnitude and duration of kinase activation and the nature of the physiological response. The protein phosphatase 2A (PP2A), which is one of the major serine/threonine phosphatases, plays a pivotal role in various signaling pathways including cell cycle, metabolism, migration, and cell death. The impairment of PP2A activity is associated with various diseases including neurodegenerative disorders, autoimmune diseases, type II diabetes, and tumors. However, little is known about how cells control the assembly of PP2A subunits and stabilize PP2A phosphatase activity. In the present study, we demonstrate that Sec6 regulates PP2A phosphatase activity by modulating the binding affinity of the A and C subunits of PP2A, thereby modulating the phosphorylation of p90RSK at Thr359 and Ser380, glycogen synthase kinase 3b (GSK3b) at Ser9, and the expression of zinc finger E-box binding homeobox 1 (ZEB1), vimentin, and zonula occludens 3 (ZO-3).

The Cx43 Carboxyl-Terminal Mimetic Peptide αCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner

The Cx43 carboxyl-terminus (CT) mimetic peptide, αCT1, originally designed to bind to Zonula Occludens 1 (ZO1) and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing (ECIS), a TRITC-dextran Transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell–cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular chiral orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell–cell border localization of endothelial VE-cadherin, the tight junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1 binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

Morphological Features of the Testis among Autoimmune Mouse Model and Healthy Strains

Autoimmune diseases play a critical role in the progression of infertility in both sexes and their severity has been reported to increase with age. However, few reports have discussed their effect on the morphological features of the testis. Therefore, we compared the morphological alterations in the testes of autoimmune model mice (MRL/MpJ-Faslpr) and the control strain (MRL/MpJ) with those of their background strain (C57BL/6N) at 3 and 6 months. Furthermore, we analyzed the changes in spermatocytes, Sertoli cells, immune cells, and Zonula occludens-1 junctional protein by immunohistochemical staining. The MRL/MpJ-Faslpr mice showed a significant increase in the serum Anti-double stranded DNA antibody level, relative spleen weight, and seminiferous luminal area when compared with other studied two strains. In contrast, a significant decrease in the relative testis weight, and numbers of both Sertoli, meiotic spermatocyte was observed in MRL/MpJ-Faslpr and MRL/MpJ mice compared with C57BL/6N mice especially at 6 months. Similarly, Zonula occludens-1 junctional protein positive cells showed a significant decrease in the same strains at 6 months. However, no immune cell infiltration could be observed among the studied three strains. Our findings suggest that the increase in autoimmune severity especially with age could lead to infertility through loss of spermatogenic and Sertoli cells, rather than the disturbance of the blood–testis barrier.