Formononetin attenuates hepatic injury in diabetic mice by regulating macrophage polarization through the PTP1B/STAT6 axis.

BACKGROUND: Formononetin (FNT) is an isoflavone known for its anti-inflammatory properties and has been shown to reduce insulin resistance in Type 2 Diabetes Mellitus (T2DM). However, its effects and the underlying mechanisms in diabetic liver injury remain largely unexplored. METHODS: We established a T2DM-induced liver injury mouse model by feeding high-fat diet, followed by injecting streptozotocin. The mice were then treated with FNT and the liver function in these mice was assessed. Macrophage markers in FNT-treated T2DM mice or human THP-1 cells were evaluated using flow cytometry, RT-qPCR, and Western blotting. The expression of PTP1B and STAT6 in mouse liver tissues and THP-1 cells was analyzed. Molecular docking predicted the interaction between PTP1B and STAT6, which was validated via co-immunoprecipitation (Co-IP) and phos-tag analysis. Microscale thermophoresis (MST) assessed the binding affinity of FNT to PTP1B. RESULTS: FNT treatment significantly ameliorated blood glucose levels, hepatocyte apoptosis, inflammatory response, and liver dysfunction in T2DM mice. Moreover, FNT facilitated M2 macrophage polarization in both T2DM mice and high glucose (HG)-induced THP-1-derived macrophages. The PTP1B/STAT6 axis, deregulated in T2DM mice, was normalized by FNT treatment, which counteracted the T2DM-induced upregulation of PTP1B and downregulation of phosphorylated STAT6. Molecular docking and subsequent analyses revealed that PTP1B binds to and dephosphorylates STAT6 at the S325A site. In contrast, FNT strongly binds to PTP1B and influences its expression at the K116A site, promoting M2 polarization of THP-1 cells via downregulation of PTP1B. CONCLUSION: Formononetin mitigates diabetic hepatic injury by fostering M2 macrophage polarization via the PTP1B/STAT6 axis.

Solitary fibrous tumor of thyroid: Case report.

Solitary fibrous tumour is a relatively rare soft tissue fibroblastic tumour, accounting for approximately 2% of soft tissue tumours. It has been described primarily as a tumour of the pleural cavity; however, up to 70% of cases occur elsewhere, in any anatomical location, which can make diagnosis difficult. If this is the diagnosis being considered, the STAT6 antibody is currently available with high sensitivity and specificity. In this paper we describe the case of a 72-year-old female patient, followed up and treated by an outpatient endocrinologist for a multinodular euthyroid goitre for several years. Due to complete nodular remodelling of the left lobe of the thyroid gland and sonographic findings of several small nodules in the right lobe of the thyroid gland, total thyroidectomy was recommended to the patient. The operation was performed at the ENT department in Jindrichuv Hradec Hospital. Material from the operation was subsequently sent for histopathological examination. Several hyperplastic colloid nodules and a small oncocytic adenoma were detected microscopically in the right lobe of the thyroid gland. In the left lobe, an imprecisely delineated, greyish-white lesion measuring 2 x 1.8 x 1.5 cm was observed on the section. Microscopically, the tumour consisted of spindle-shaped cells in a focally hyalinised stroma. In the immunohistochemical examination, tumour cells reacted positively with the CD34 antibody, and negatively with antibodies against thyroglobulin, cytokeratins (CK AE1/AE3) and S100 protein. Further immunohistochemical examinations (Bcl2, CD99, STAT6) with positive results were supplemented upon consultation at a higher facility. Based on morphology and the results of the immunohistochemical examinations, the tumour was diagnosed as a solitary fibrous tumour of the thyroid gland. This is a relatively unusual finding in this location; according to literature, only a few dozen cases have been described.

Malvidin From Malva sylvestris L. Ameliorates Allergic Responses in Ovalbumin-Induced Allergic Rhinitis Mouse Model via the STAT6/GATA3 Pathway.

BACKGROUND: Malva sylvestris L. (commonly known as mallow) has been widely used in traditional Tibetan formulations to treat allergic rhinitis (AR), and malvidin is a key anti-inflammation constituent of this plant. OBJECTIVE: The present study aimed to evaluate the potential therapeutic effect and mechanism of malvidin in an AR mouse model. METHODS: Malvidin's efficacy was evaluated in an AR mouse model induced by ovalbumin (OVA) sensitization and challenge. The factors, such as nasal symptoms, serum OVA-specific immunoglobulin E (IgE) levels, histological changes in the nasal mucosa, and expressions of Th1, Th2, Th17, and Tregs and their cytokines, were assessed. Western blotting was used to analyze the effect of malvidin on signal transducer and activator of transcription 6 (STAT6) and GATA3 expression levels. RESULTS: Malvidin reduced the allergic symptoms and serum levels of OVA-specific IgE in the AR model. Histological analysis indicated that malvidin alleviates nasal mucosal edema, eosinophil infiltration, and goblet cell proliferation. In addition, it altered the expression of Th1/Th2/Th17-related cytokines, enhanced the Treg population, and reduced Th2-mediated immunity by suppressing the phosphorylation of STAT6 and expression of the GATA3 protein. CONCLUSIONS: Malvidin significantly improved allergic symptoms in an OVA-induced AR mouse model by modulating Th1/Th2 immune responses and suppressing the STAT6/GATA3 pathway, indicating its potential as a naturally sourced agent for AR management.

Involvement of M2 macrophages polarization in PM2.5-induced COPD by upregulating MMP12 via IL4/STAT6 pathway.

Biomass-related airborne fine particulate matter (PM2.5) is an important risk factor for chronic obstructive pulmonary disease (COPD). Macrophage polarization has been reported to be involved in PM2.5-induced COPD, but the dynamic characteristics and underlying mechanism of this process remain unclear. Our study established a PM2.5-induced COPD mouse model and revealed that M2 macrophages predominantly presented after 4 and 6 months of PM2.5 exposure, during which a notable increase in MMP12 was observed. Single cell analysis of lung tissues from COPD patients and mice further revealed that M2 macrophages were the dominant macrophage subpopulation in COPD, with MMP12 being involved as a hub gene. In vitro experiments further demonstrated that PM2.5 induced M2 polarization and increased MMP12 expression. Moreover, we found that PM2.5 increased IL-4 expression, STAT6 phosphorylation and nuclear translocation. Nuclear pSTAT6 then bound to the MMP12 promoter region. Furthermore, the inhibition of STAT6 phosphorylation effectively abrogated the PM2.5-induced increase in MMP12. Using a coculture system, we observed a significantly reduced level of E-cadherin in alveolar epithelial cells cocultured with PM2.5-exposed macrophages, while the decrease in E-cadherin was reversed by the addition of an MMP12 inhibitor to the co-culture system. Taken together, these findings indicated that PM2.5 induced M2 macrophage polarization and MMP12 upregulation via the IL-4/STAT6 pathway, which resulted in alveolar epithelial barrier dysfunction and excessive extracellular matrix (ECM) degradation, and ultimately led to COPD progression. These findings may help to elucidate the role of macrophages in COPD, and suggest promising directions for potential therapeutic strategies.

tsRNA-GlyGCC promotes colorectal cancer progression and 5-FU resistance by regulating SPIB.

BACKGROUND: tRNA-derived small RNAs (tsRNAs) are newly discovered non-coding RNA, which are generated from tRNAs and are reported to participate in several biological processes in diseases, especially cancer; however, the mechanism of tsRNA involvement in colorectal cancer (CRC) and 5-fluorouracil (5-FU) is still unclear. METHODS: RNA sequencing was performed to identify differential expression of tsRNAs in CRC tissues. CCK8, colony formation, transwell assays, and tumor sphere assays were used to investigate the role of tsRNA-GlyGCC in 5-FU resistance in CRC. TargetScan and miRanda were used to identify the target genes of tsRNA-GlyGCC. Biotin pull-down, RNA pull-down, luciferase assay, ChIP, and western blotting were used to explore the underlying molecular mechanisms of action of tsRNA-GlyGCC. The MeRIP assay was used to investigate the N(7)-methylguanosine RNA modification of tsRNA-GlyGCC. RESULTS: In this study, we uncovered the feature of tsRNAs in human CRC tissues and confirmed a specific 5' half tRNA, 5'tiRNA-Gly-GCC (tsRNA-GlyGCC), which is upregulated in CRC tissues and modulated by METTL1-mediated N(7)-methylguanosine tRNA modification. In vitro and in vivo experiments revealed the oncogenic role of tsRNA-GlyGCC in 5-FU drug resistance in CRC. Remarkably, our results showed that tsRNA-GlyGCC modulated the JAK1/STAT6 signaling pathway by targeting SPIB. Poly (ß-amino esters) were synthesized to assist the delivery of 5-FU and tsRNA-GlyGCC inhibitor, which effectively inhibited tumor growth and enhanced CRC sensitive to 5-FU without obvious adverse effects in subcutaneous tumor. CONCLUSIONS: Our study revealed a specific tsRNA-GlyGCC-engaged pathway in CRC progression. Targeting tsRNA-GlyGCC in combination with 5-FU may provide a promising nanotherapeutic strategy for the treatment of 5-FU-resistance CRC.

Advances in the molecular biology of the solitary fibrous tumor and potential impact on clinical applications.

Solitary fibrous tumor (SFT) is a rare fibroblastic mesenchymal neoplasm. The current classification has merged SFT and hemangiopericytoma (HPC) into the same tumor entity, while the risk stratification models have been developed to compensate for clinical prediction. Typically, slow-growing and asymptomatic, SFT can occur in various anatomical sites, most commonly in the pleura. Histologically, SFT consists of spindle to oval cells with minimal patterned growth, surrounded by stromal collagen and unique vascular patterns. Molecularly, SFT is defined by the fusion of NGFI-A-binding protein 2 (NAB2) and signal transducer and activator of transcription 6 (STAT6) genes as NAB2-STAT6. This fusion transforms NAB2 into a transcriptional activator, activating early growth response 1 (EGR1) and contributing to SFT pathogenesis and development. There are several fusion variants of NAB2-STAT6 in tumor tissues, with the most frequent ones being NAB2ex4-STAT6ex2 and NAB2ex6-STAT6ex16/ex17. Diagnostic methods play a crucial role in SFT clinical practice and basic research, including RT-PCR, next-generation sequencing (NGS), FISH, immunohistochemistry (IHC), and Western blot analysis, each with distinct capabilities and limitations. Traditional treatment strategies of SFT encompass surgical resection, radiation therapy, and chemotherapy, while emerging management regimes include antiangiogenic agents, immunotherapy, RNA-targeting technologies, and potential targeted drugs. This review provides an update on SFT's clinical and molecular aspects, diagnostic methods, and potential therapies.

Multiomics analysis identified IL-4-induced IL1RL1high eosinophils characterized by prominent cysteinyl leukotriene metabolism.

BACKGROUND: Clinical studies have demonstrated that IL-4, a type 2 cytokine, plays an important role in the pathogenesis of chronic rhinosinusitis and eosinophilic asthma. However, the direct effect of IL-4 on eosinophils remains unclear. OBJECTIVE: We aimed to elucidate the inflammatory effects of IL-4 on the functions of human eosinophils. METHODS: A multiomics analysis comprising transcriptomics, proteomics, lipidomics, quantitative RT-PCR, and flow cytometry was performed by using blood eosinophils from healthy subjects stimulated with IL-4, IL-5, or a combination thereof. RESULTS: Transcriptomic and proteomic analyses revealed that both IL-4 and IL-5 upregulate the expression of γ-gultamyl transferase 5, a fatty acid-metabolizing enzyme that converts leukotriene C4 into leukotriene D4. In addition, IL-4 specifically upregulates the expression of IL-1 receptor-like 1 (IL1RL1), a receptor for IL-33 and transglutaminase-2. Additional transcriptomic analysis of cells stimulated with IL-13 revealed altered gene expression profiles, characterized by the upregulation of γ-gultamyl transferase 5, transglutaminase-2, and IL1RL1. The IL-13-induced changes were not totally different from the IL-4-induced changes. Lipidomic analysis revealed that IL-5 and IL-4 additively increased the extracellular release of leukotriene D4. In vitro experiments revealed that STAT6 and IL-4 receptor-α control the expression of these molecules in the presence of IL-4 and IL-13. Analysis of eosinophils derived from patients with allergic disorders indicated the involvement of IL-4 and IL-13 at the inflamed sites. CONCLUSIONS: IL-4 induces the proallergic phenotype of IL1RL1high eosinophils, with prominent cysteinyl leukotriene metabolism via STAT6. These cellular changes represent potential therapeutic targets for chronic rhinosinusitis and eosinophilic asthma.

CD34-Negative Malignant Renal Solitary Fibrous Tumor: Case Report and Diagnostic Insights.

Solitary fibrous tumors (SFTs) are rare fibroblastic neoplasms with diverse biological behaviors and widespread distribution. Primary renal SFTs are uncommon, and their malignant variants, especially those that are CD34 negative, are even rarer. This study presents a case of malignant renal SFT in a 57-year-old female, focusing on its immunomorphological features. On gross examination, the tumor's large size (11.5 cm) was remarkable. Microscopic analysis showed high cellularity, diffuse sheets of moderately pleomorphic ovoid cells, prominent staghorn vessels, tumor cell necrosis, and a high mitotic count. Immunohistochemistry revealed strong positivity for STAT6, vimentin, and Bcl-2 and, notably, negativity for CD34. The presence of the NAB2::STAT6 gene fusion was confirmed through fluorescence in situ hybridization. This case emphasizes the need to consider SFT in the differential diagnosis of unusual renal tumors, even when CD34 is negative. The infrequency, morphological diversity, and resemblance to other tumors make diagnosing renal SFTs challenging. Accurate identification and classification as benign or malignant are crucial for proper clinical management and prognosis.

Malignant solitary fibrous tumor of the kidney with IGF2 secretion and without hypoglycemia.

BACKGROUND: Solitary fibrous tumor (SFT) is a rare fibroblastic mesenchymal tumor that mostly involves the pleura and infrequently involves extra-pleural sites. De novo SFT of the kidney is uncommon, and malignant SFT is extremely rare. CASE PRESENTATION: We report a case of a 51-year-old man with a large malignant SFT in the left kidney. Pathological examination confirmed the diagnosis of SFT based on typical morphology, nuclear STAT6 expression, and NAB2-STAT6 gene fusion. The malignant subtype was determined by a large tumor size (≥ 15 cm) and high mitotic counts (8/10 high-power fields). KRAS mutation was identified by DNA sequencing. Insulin-like growth factor 2 (IGF2) was diffusely and strongly expressed in tumor cells, however, hypoglycemia was not observed. Hyperglycemia and high adrenocorticotropic hormone (ACTH) concentration were observed one month after surgery. Hormone measurements revealed normal blood cortisol and aldosterone levels, and increased urinary free cortisol level. A pituitary microadenoma was identified using brain magnetic resonance imaging, which may be responsible for the promotion of hyperglycemia. CONCLUSIONS: We report a case of renal malignant SFT with a KRAS mutation, which was previously unreported in SFT and may be associated with its malignant behavior. Additionally, we emphasize that malignant SFT commonly causes severe hypoglycemia due to the production of IGF2. However, this effect may be masked by the presence of other lesions that promote hyperglycemia. Therefore, when encountering a malignant SFT with diffuse and strong IGF2 expression and without hypoglycemia, other lesions promoting hyperglycemia need to be ruled out.

Daphnetin alleviates allergic airway inflammation by inhibiting T-cell activation and subsequent JAK/STAT6 signaling.

Allergic asthma is a major health burden on society as a chronic respiratory disease characterized by inflammation and muscle tightening around the airways in response to inhaled allergens. Daphne kiusiana Miquel is a medicinal plant that can suppress allergic airway inflammation; however, its specific molecular mechanisms of action are unclear. In this study, we aimed to elucidate the mechanisms by which D. kiusiana inhibits allergic airway inflammation. We evaluated the anti-inflammatory effects of the ethyl acetate (EA) fraction of D. kiusiana and its major compound, daphnetin, on murine T lymphocyte EL4 cells stimulated with phorbol 12-myristate 13-acetate and ionomycin in vitro and on asthmatic mice stimulated with ovalbumin in vivo. The EA fraction and daphnetin inhibited T-helper type 2 (Th2) cytokine secretion, serum immunoglobulin E production, mucus secretion, and inflammatory cell recruitment in vivo. In vitro, daphnetin suppressed intracellular Ca2+ mobilization (a critical regulator of nuclear factor of activated T cells) and functions of the activator protein 1 transcription factor to reduce interleukin (IL)-4 and IL-13 expression. Daphnetin effectively suppressed the IL-4/-13-induced activation of Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6) signaling in vitro and in vivo, thereby inhibiting the expression of GATA3 and PDEF, two STAT6-target genes responsible for producing Th2 cytokines and mucins. These findings indicate that daphnetin suppresses allergic airway inflammation by stabilizing intracellular Ca2+ levels and subsequently inactivating the JAK/STAT6/GATA3/PDEF pathway, suggesting that daphnetin is a promising alternative to existing asthma treatments.

The Secretome of Adult Murine Hookworms Is Shaped by Host Expression of STAT6.

Co-evolutionary adaptation of hookworms with their mammalian hosts has been selected for immunoregulatory excretory/secretory (E/S) products. However, it is not known whether, or if so, how host immunological status impacts the secreted profile of hematophagous adult worms. This study interrogated the impact of host Signal transducer and activator of transcription 6 (STAT6) expression during the experimental evolution of hookworms through the sequential passage of the life cycle in either STAT6 deficient or WT C57BL/6 mice. Proteomic analysis of E/S products by LC-MS showed increased abundance of 15 proteins, including myosin-3, related to muscle function, and aconitate hydratase, related to iron homeostasis. However, most E/S proteins (174 of 337 unique identities) were decreased, including those in the Ancylostoma-secreted protein (ASP) category, and metallopeptidases. Several identified proteins are established immune-modulators such as fatty acid-binding protein homologue, cystatin, and acetylcholinesterase. Enrichment analysis of InterPro functional categories showed down-regulation of Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP), Astacin-like metallopeptidase, Glycoside hydrolase, and Transthyretin-like protein groups in STAT6 KO-adapted worms. Taken together, these data indicate that in an environment lacking Type 2 immunity, hookworms alter their secretome by reducing immune evasion proteins- and increasing locomotor- and feeding-associated proteins.

Unveiling the Rarity: A Case Report on Solitary Fibrous Tumor of the Thyroid Gland.

Solitary Fibrous Tumor (SFT) rarely manifests within the thyroid gland, an organ predominantly associated with epithelial carcinomas. This case report explores the clinical narrative of a 70-year-old patient presenting with a sizable SFT localized in the left lobe of the thyroid, posing diagnostic challenges uncommon in thyroid nodules. The report delves into the clinical history, radiological findings, pathological assessments, and therapeutic interventions, contributing to the limited literature on thyroidal SFTs. The patient's ultrasound revealed a substantial thyroid mass causing tracheal and vascular displacement, categorized as TIRADS 3. Fine needle aspiration indicated mesenchymal origin, prompting further investigation. Contrast-enhanced computed tomography depicted a well-defined lesion with varied enhancement, compressing surrounding structures. Histopathology confirmed a spindle cell proliferation, prompting immunohistochemistry revealing CD34, STAT6, and Bcl-2 positivity, aligning with SFT characteristics. The rarity of thyroidal SFTs poses diagnostic challenges, necessitating reliance on immunohistochemistry for accurate differentiation from other spindle cell neoplasms. Radiological investigations, including ultrasound and magnetic resonance imaging, contribute to preoperative planning. The case underscores the importance of meticulous pathological examination, emphasizing the utility of immunohistochemistry in confirming SFT diagnosis. The report enhances understanding among clinicians, pathologists, and researchers, guiding improved diagnostic accuracy and tailored treatment strategies for future occurrences of thyroidal SFTs.

Nicotinamide phosphoribosyltransferase prompts bleomycin-induced pulmonary fibrosis by driving macrophage M2 polarization in mice.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible, fatal interstitial lung disease lacking specific therapeutics. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD) salvage biosynthesis pathway and a cytokine, has been previously reported as a biomarker for lung diseases; however, the role of NAMPT in pulmonary fibrosis has not been elucidated. Methods: We identified the NAMPT level changes in pulmonary fibrosis by analyzing public RNA-Seq databases, verified in collected clinical samples and mice pulmonary fibrosis model by Western blotting, qRT-PCR, ELISA and Immunohistochemical staining. We investigated the role and mechanism of NAMPT in lung fibrosis by using pharmacological inhibition on NAMPT and Nampt transgenic mice. In vivo macrophage depletion by clodronate liposomes and reinfusion of IL-4-induced M2 bone marrow-derived macrophages (BMDMs) from wild-type mice, combined with in vitro cell experiments, were performed to further validate the mechanism underlying NAMPT involving lung fibrosis. Results: We found that NAMPT increased in the lungs of patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis. NAMPT inhibitor FK866 alleviated BLM-induced pulmonary fibrosis in mice and significantly reduced NAMPT levels in bronchoalveolar lavage fluid (BALF). The lung single-cell RNA sequencing showed that NAMPT expression in monocytes/macrophages of IPF patients was much higher than in other lung cells. Knocking out NAMPT in mouse monocytes/macrophages (Namptfl/fl;Cx3cr1CreER) significantly alleviated BLM-induced pulmonary fibrosis in mice, decreased NAMPT levels in BALF, reduced the infiltration of M2 macrophages in the lungs and improved mice survival. Depleting monocytes/macrophages in Namptfl/fl;Cx3cr1CreER mice by clodronate liposomes and subsequent pulmonary reinfusion of IL-4-induced M2 BMDMs from wild-type mice, reversed the protective effect of monocyte/macrophage NAMPT-deletion on lung fibrosis. In vitro experiments confirmed that the mechanism of NAMPT engaged in pulmonary fibrosis is related to the released NAMPT by macrophages promoting M2 polarization in a non-enzyme-dependent manner by activating the STAT6 signal pathway. Conclusions: NAMPT prompts bleomycin-induced pulmonary fibrosis by driving macrophage M2 polarization in mice. Targeting the NAMPT of monocytes/macrophages is a promising strategy for treating pulmonary fibrosis.

Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids.

BACKGROUND & AIMS: Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells. METHODS: We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation. RESULTS: Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids. CONCLUSIONS: IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration.

A first-in-class inhibitor of HSP110 to potentiate XPO1-targeted therapy in primary mediastinal B-cell lymphoma and classical Hodgkin lymphoma.

BACKGROUND: Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) are distinct hematological malignancies of B-cell origin that share many biological, molecular, and clinical characteristics. In particular, the JAK/STAT signaling pathway is a driver of tumor development due to multiple recurrent mutations, particularly in STAT6. Furthermore, the XPO1 gene that encodes exportin 1 (XPO1) shows a frequent point mutation (E571K) resulting in an altered export of hundreds of cargo proteins, which may impact the success of future therapies in PMBL and cHL. Therefore, targeted therapies have been envisioned for these signaling pathways and mutations. METHODS: To identify novel molecular targets that could overcome the treatment resistance that occurs in PMBL and cHL patients, we have explored the efficacy of a first-in-class HSP110 inhibitor (iHSP110-33) alone and in combination with selinexor, a XPO1 specific inhibitor, both in vitro and in vivo. RESULTS: We show that iHSP110-33 decreased the survival of several PMBL and cHL cell lines and the size of tumor xenografts. We demonstrate that HSP110 is a cargo of XPO1wt as well as of XPO1E571K. Using immunoprecipitation, proximity ligation, thermophoresis and kinase assays, we showed that HSP110 directly interacts with STAT6 and favors its phosphorylation. The combination of iHSP110-33 and selinexor induces a synergistic reduction of STAT6 phosphorylation and of lymphoma cell growth in vitro and in vivo. In biopsies from PMBL patients, we show a correlation between HSP110 and STAT6 phosphorylation levels. CONCLUSIONS: These findings suggest that HSP110 could be proposed as a novel target in PMBL and cHL therapy.

Targeted Demethylation of FOXP3-TSDR Enhances the Suppressive Capacity of STAT6-deficient Inducible T Regulatory Cells.

In vitro induced T regulatory cells (iTregs) are promising for addressing inflammation-driven diseases. However, current protocols for the generation and expansion of iTregs fail to induce extensive demethylation of the Treg-specific demethylated region (TSDR) within the FOXP3 gene, recognized as the master regulator for regulatory T cells (Tregs). This deficiency results in the rapid loss of Foxp3 expression and an unstable regulatory phenotype. Nevertheless, inhibition of STAT6 signaling effectively stabilizes Foxp3 expression in iTregs. Thus, this study aimed to develop a protocol combining epigenetic editing with STAT6 deficiency to improve iTregs' ability to maintain stable suppressive function and a functional phenotype. Our findings demonstrate that the combination of STAT6 deficiency (STAT6-/-) with targeted demethylation of the TSDR using a CRISPR-TET1 tool leads to extensive demethylation of FOXP3-TSDR. Demethylation in STAT6-/- iTregs was associated with enhanced expression of Foxp3 and suppressive markers such as CTLA-4, PD-1, IL-10, and TGF-ß. Furthermore, the edited STAT6-/- iTregs exhibited an increased capacity to suppress CD8+ and CD4+ lymphocytes and could more efficiently impair Th1-signature gene expression compared to conventional iTregs. In conclusion, the deactivation of STAT6 and TSDR-targeted demethylation via CRISPR-TET1 is sufficient to induce iTregs with heightened stability and increased suppressive capacity, offering potential applications against inflammatory and autoimmune diseases.

Suppressive Activity of Boiogito, a Japanese Traditional Kampo Medicine, on Periostin Secretion in Human Fibroblast-Like Synoviocytes In Vitro.

Background Knee osteoarthritis (KOA) is a prevalent degenerative disease that affects the knee joints, particularly among individuals aged over 40 years. It leads to pain, stiffness, and reduced quality of life; affects approximately 300 million individuals worldwide; and is increasing, particularly in developed nations. Although treatments for KOA range from conservative measures to surgical interventions, such as total knee arthroplasty (TKA), the financial burden of TKA in many countries underscores the urgent need for effective conservative therapies. The pathophysiology of KOA involves articular cartilage degeneration, increased subchondral bone turnover, synovitis, and periarticular soft tissue contracture. Abnormal bone turnover, intensified by factors, such as weight gain and knee injury, precedes cartilage degeneration. Synovitis, characterized by inflammation in the synovial tissue, plays a crucial role in perpetuating the disease by triggering a cascade of catabolic and proinflammatory mediators, including cytokines, such as interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-13. Periostin, an extracellular matrix protein, is implicated in KOA progression, with its levels increasing with disease severity. Materials & methods In this study, the preventive effect of boiogito (BOT), a traditional herbal medicine, on periostin secretion in human fibroblast-like synoviocytes (hFLS) stimulated by IL-13 was investigated. Synoviocyte Growth Medium and recombinant human IL-13 were used for cell culture and stimulation. BOT was dissolved in phosphate-buffered saline and applied to cell cultures. Periostin secretion and mRNA expression were measured using enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction, respectively. Cell viability was assessed using an MTT assay, and signal transducer and activator of transcription factor 6 (STAT6) phosphorylation was examined using Western blotting. Results IL-13 stimulation of hFLS significantly increased periostin secretion, with levels rising above 20 ng/mL after 72 h of stimulation. Pretreatment with BOT dose-dependently suppressed periostin secretion, with doses of 1,000 µg/mL significantly reducing periostin levels. Furthermore, BOT inhibited periostin mRNA expression and STAT6 phosphorylation in IL-13-stimulated hFLS, suggesting its potential in modulating IL-13-mediated inflammatory pathways in KOA. Conclusion This study demonstrated the preventive effect of BOT on periostin secretion in IL-13-stimulated hFLS, highlighting its potential as a therapeutic agent for KOA. By inhibiting periostin production and downstream signaling pathways, BOT may offer a promising conservative treatment option for KOA, addressing the inflammatory cascade implicated in disease progression. Further research is warranted to elucidate the specific herbal components responsible for the therapeutic effects of BOT and to validate its efficacy in clinical settings.

Efficacy of Laurus nobilis L. for Tight Junction Protein Imbalance in Leaky Gut Syndrome.

Laurus nobilis L. (LNL) belongs to the evergreen Lauraceae family. It is native to the Mediterranean and widely distributed in the southern United States, Europe, and the Middle East. LNL is rich in active ingredients of the sesquiterpene lactone series and has been reported to have antioxidant, anti-inflammatory, and anticancer effects. And parthenolide, known as a sesquiterpene lactone-based compound, inhibits the activation of lipopolysaccharide-binding protein (LBP), which is a major trigger for leaky gut syndrome. However, the effectiveness of LNL in improving the state of increased intestinal permeability has not yet been reported. Therefore, we demonstrated the efficacy of LNL, which is known to be rich in parthenolide, in improving intestinal permeability induced by IL-13. We investigated the improvement in permeability and analyzed major tight junction proteins (TJs), permeability-related mechanisms, weight and disease activity indices, and corresponding cytokine mechanisms. LNL maintained TJs homeostasis and clinical improvement by reducing increased claudin-2 through the inhibition of IL-13/STAT6 activation in TJ-damaged conditions. These results are expected to be effective in preventing leaky gut syndrome through the TJ balance and to further improve intestinal-related diseases, such as inflammatory bowel disease.

SHP2 mediates STAT3/STAT6 signaling pathway in TAM to inhibit proliferation and metastasis of lung adenocarcinoma.

OBJECTIVE: This study examines SHP2's influence on the STAT3/STAT6 pathway in tumor-associated macrophages (TAMs) and its impact on lung adenocarcinoma proliferation and metastasis. METHODS: Lung cancer A549 and NCI-H1688 cell lines were subcutaneously injected into nude mice. Macrophages were isolated using flow cytometry and analyzed for CD163, CD206, and Arginase-1 levels via western blot. Similarly, the effect on THP1 cell-associated proteins was assessed. The impact on A549 and NCI-H1688 cell migration, invasion, and proliferation was evaluated through wound healing, Transwell assays, and CCK8. RESULTS: Compared to controls, the sh-RNA SHP2 group showed increased tumor volume and higher expression levels of CD163, CD206, Arginase-1, p-STAT3, p-STAT6, IL-4, IL-10, and various cathepsins in macrophages and THP1 cells. However, p-STAT1 and p-STAT5 levels remained unchanged. The sh-RNA SHP2 group also demonstrated enhanced migration, invasion, and proliferation in both cell lines. CONCLUSIONS: SHP2 negatively affects the STAT3/STAT6 pathway in TAMs, promoting M2 polarization and cathepsin secretion, which enhances lung adenocarcinoma cell proliferation and metastasis.