Multi-regulatory potency of USP1 on inflammasome components promotes pyroptosis in thyroid follicular cells and contributes to the progression of Hashimoto's thyroiditis.

BACKGROUND: Inflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis. METHODS: Our study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation. RESULTS: Our findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram. CONCLUSIONS: Our study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT.

Effects and action mechanism of gonadotropins on ovarian follicular cells: A novel role of Sphingosine-1-Phosphate (S1P). A review.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective Gs protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting Gq proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells.

miRNA expression in PCOS: unveiling a paradigm shift toward biomarker discovery.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that affects a substantial percentage of women, estimated at around 9-21%. This condition can lead to anovulatory infertility in women of childbearing age and is often accompanied by various metabolic disturbances, including hyperandrogenism, insulin resistance, obesity, type-2 diabetes, and elevated cholesterol levels. The development of PCOS is influenced by a combination of epigenetic alterations, genetic mutations, and changes in the expression of non-coding RNAs, particularly microRNAs (miRNAs). MicroRNAs, commonly referred to as non-coding RNAs, are approximately 22 nucleotides in length and primarily function in post-transcriptional gene regulation, facilitating mRNA degradation and repressing translation. Their dynamic expression in different cells and tissues contributes to the regulation of various biological and cellular pathways. As a result, they have become pivotal biomarkers for various diseases, including PCOS, demonstrating intricate associations with diverse health conditions. The aberrant expression of miRNAs has been detected in the serum of women with PCOS, with overexpression and dysregulation of these miRNAs playing a central role in the atypical expression of endocrine hormones linked to PCOS. This review takes a comprehensive approach to explore the upregulation and downregulation of various miRNAs present in ovarian follicular cells, granulosa cells, and theca cells of women diagnosed with PCOS. Furthermore, it discusses the potential for a theragnostic approach using miRNAs to better understand and manage PCOS.

Changes of B cell subsets in different types of diabetes and its effect on the progression of latent autoimmune diabetes in adults.

PURPOSE: Developmental abnormalities in B cells is one of the key players in autoimmune diabetes, but little is known about its role in latent autoimmune diabetes in adults (LADA). This study aimed to investigate the distribution of B cell subsets in different types of diabetes and to analyze their correlations with other biochemical parameters. METHODS: A total of 140 participants were prospectively enrolled from January 2021 to December 2022. Diabetes-related autoantibodies and laboratory indicators were tested. Flow cytometry was used to analyze the percentage of circulating B cell subsets and T follicular cells. The correlation of B cell subsets with different indicators was assessed by Spearman's correlation method. RESULTS: We observed that the Naïve phenotype cells tended to be less frequent in patients with diabetes than in healthy controls. The frequency of plasmablasts (PB) and Breg cell-related phenotype (B10) were significantly higher in LADA. Notably, the percentage of PB was positively associated with levels of islet cell antibody (ICA) and insulin autoantibody (IAA), but inversely associated with fasting C-peptide (FCP), further indicating that PB may promote the destruction of ß-cell in patients with diabetes. CONCLUSIONS: This study showed that patients with LADA had significantly altered frequencies of B cell subsets, particularly in the naïve to memory B cell ratio. Our study provided valuable information on the distribution characteristics of B cell subsets in LADA and suggested the feasibility of B-cell targeted therapy in LADA patients.

Changes of allergic inflammation and immunological parameters after Alt a 1 and A. alternata immunotherapy in mice.

Background: The efficacy of allergen-specific subcutaneousimmunotherapy (SCIT) with Alt a 1 of the fungus A. alternata is still unknown. Yet, few studies compare the therapeutic effects and immunological mechanisms of Alt a 1 and A. alternata extracts. We aim to explore and compare the changes in allergic inflammation and immunological mechanisms of Alt a 1 and A. alternata in mice. Methods: Female BALB/c mice administrated recombinant Alt a 1 (rAlt a 1), native Alt a 1 (nAlt a 1), and A. alternata. Lung histology, airway hyper-reactivity (AHR), bronchoalveolar lavage fluid (BALF) cytokine levels, serum immunoglobulin responses, the expression of Bcl-6, the percentages of T follicular helper cells (Tfh), cytokine-related Tfh subtypes, regulatory B cells (Breg), and IL-10+ Breg cells were detected. Results: High-purity nAlt 1 protein was obtained. SCIT with Alt a 1 and Alternaria decreased airway and lung inflammation, including improvement of lung pathology, lower levels of AHR, reduction of total cell numbers, and IL-4 and IL-13 levels in BALF. Furthermore, Alt a 1-SCIT effectively suppressed the IgE responses, elevated IgG titers, and was superior in decreasing the expression of Bcl-6. Additionally, Alternaria-SCIT significantly decreased the expression of Tfh cells, L-4+ Tfh, and IL-5+ Tfh cells in the spleen, whereas Alt a 1 showed superior therapeutic effects in the lymph node. IL-13+ Tfh cells in these two treatment groups not being significant. IL-17A+ Tfh cells were alleviated most effectively after A. alternata-SCIT in both the spleen and lymph node. Intriguingly, IL-10+ Breg cells decreased remarkably in response to SCIT with rAlt a 1. Conclusions: Treatments with Alt a 1 and A. alternata extracts had beneficial effects on allergic inflammation. Alt a 1-SCIT resulted in prominent improvement in the immunoglobulin responses, Bcl-6, and IL-10+ Breg cells. Alternaria-SCIT was more likely to suppress the expression of Tfh and cytokine-related Tfh subtypes.

Perturbations in spike-specific peripheral T follicular helper cells in SARS-CoV2 breakthrough convalescent individuals immunized by BBV152 vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov2) infection has caused an increase in mortality and morbidity, but with vaccination, the disease severity has significantly reduced. With the emergence of various variants of concern (VOCs), the vaccine breakthrough infection has also increased. Here we studied circulating spike-specific T follicular response (cTfh) in infection-naïve vaccinees and convalescent vaccinees (individuals who got the Delta breakthrough infection after two doses of BBV152 vaccine) to understand their response as they are the most crucial cells that are involved in vaccine-mediated protection by helping in B-cell maturation. Our results indicated that cTfh cells in both the groups recognized the wild-type and Delta spike protein but memory response to the wild-type spike was superior in infection-naïve than in the convalescent group. The cytokine response, particularly interleukin-21 (IL-21) from cTfh, was also higher in infection-naïve than in convalescent vaccinees, indicating a dampened cTfh response in convalescent vaccinees after breakthrough infection. Also, there was a positive correlation between IL-21 from cTfh cells and neutralizing antibodies of infection-naïve vaccinees. Multiple cytokine analysis also revealed higher inflammation in convalescent vaccinees. Our data indicated that the necessity of a third booster dose may be individual-specific depending on the steady-state functional phenotype of immune cells.

Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control.

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin ß1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin ß1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

Amount of antigen, T follicular helper cells and affinity of founder cells shape the diversity of germinal center B cells: A computational study.

A variety of B cell clones seed the germinal centers, where a selection stringency expands the fitter clones to generate higher affinity antibodies. However, recent experiments suggest that germinal centers often retain a diverse set of B cell clones with a range of affinities and concurrently carry out affinity maturation. Amid a tendency to flourish germinal centers with fitter clones, how several B cell clones with differing affinities can be concurrently selected remains poorly understood. Such a permissive selection may allow non-immunodominant clones, which are often rare and of low-affinity, to somatically hypermutate and result in a broad and diverse B cell response. How the constituent elements of germinal centers, their quantity and kinetics may modulate diversity of B cells, has not been addressed well. By implementing a state-of-the-art agent-based model of germinal center, here, we study how these factors impact temporal evolution of B cell clonal diversity and its underlying balance with affinity maturation. While we find that the extent of selection stringency dictates clonal dominance, limited antigen availability on follicular dendritic cells is shown to expedite the loss of diversity of B cells as germinal centers mature. Intriguingly, the emergence of a diverse set of germinal center B cells depends on high affinity founder cells. Our analysis also reveals a substantial number of T follicular helper cells to be essential in balancing affinity maturation with clonal diversity, as a low number of T follicular helper cells impedes affinity maturation and also contracts the scope for a diverse B cell response. Our results have implications for eliciting antibody responses to non-immunodominant specificities of the pathogens by controlling the regulators of the germinal center reaction, thereby pivoting a way for vaccine development to generate broadly protective antibodies.

Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes.

The effects of methylene blue (MB) on cromakalim-induced K+ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3-300 µM, MB inhibited K+ currents (IC50: 22.4 µM) activated by cromakalim, which activates KATP channels. MB inhibited cromakalim-activated K+ currents in a noncompetitive and voltage-independent manner. The respective EC50 and slope values for cromakalim-activation of K+ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 µM MB. The inhibition of cromakalim-induced K+ currents by MB was not altered by pretreatment with the Ca2+ chelator BAPTA, which suggests that MB does not influence Ca2+-activated second messenger pathways. K+ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the KATP ligand [3H]glibenclamide are not altered by MB in a concentration range between 1 µM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K+ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K+ currents in follicular cells of Xenopus oocytes.

GLIS3 regulates transcription of thyroid hormone biosynthetic genes in coordination with other thyroid transcription factors.

BACKGROUND: Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several thyroid hormone (TH) biosynthetic genes in thyroid follicular cells. Whether and to what extent, GLIS3 regulates thyroid gene transcription in coordination with other thyroid transcriptional factors (TFs), such as PAX8, NKX2.1 and FOXE1, is poorly understood. METHODS: PAX8, NKX2.1, and FOXE1 ChIP-Seq analysis with mouse thyroid glands and rat thyrocyte PCCl3 cells was performed and compared to that of GLIS3 to analyze the co-regulation of gene transcription in thyroid follicular cells by these TFs. RESULTS: Analysis of the PAX8, NKX2.1, and FOXE1 cistromes identified extensive overlaps between these TF binding loci and those of GLIS3 indicating that GLIS3 shares many of the same regulatory regions with PAX8, NKX2.1, and FOXE1, particularly in genes associated with TH biosynthesis, induced by thyroid stimulating hormone (TSH), and suppressed in Glis3KO thyroid glands, including Slc5a5 (Nis), Slc26a4, Cdh16, and Adm2. ChIP-QPCR analysis showed that loss of GLIS3 did not significantly affect PAX8 or NKX2.1 binding and did not cause major alterations in H3K4me3 and H3K27me3 epigenetic signals. CONCLUSIONS: Our study indicates that GLIS3 regulates transcription of TH biosynthetic and TSH-inducible genes in thyroid follicular cells in coordination with PAX8, NKX2.1, and FOXE1 by binding within the same regulatory hub. GLIS3 does not cause major changes in chromatin structure at these common regulatory regions. GLIS3 may induce transcriptional activation by enhancing the interaction of these regulatory regions with other enhancers and/or RNA Polymerase II (Pol II) complexes.