FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement.

Long-range glutamatergic inputs originating from the cortex and thalamus are indispensable for striatal development, providing the foundation for motor and cognitive functions. Despite their significance, transcriptional regulation governing these inputs remains largely unknown. We investigated the role of a transcription factor encoded by a high-risk autism-associated gene, FOXP1, in sculpting glutamatergic inputs onto spiny projection neurons (SPNs) within the striatum. We find a neuron subtype-specific role of FOXP1 in strengthening and maturing glutamatergic inputs onto dopamine receptor 2-expressing SPNs (D2 SPNs). We also find that FOXP1 promotes synaptically driven excitability in these neurons. Using single-nuclei RNA sequencing, we identify candidate genes that mediate these cell-autonomous processes through postnatal FOXP1 function at the post-synapse. Last, we demonstrate that postnatal FOXP1 reinstatement rescues electrophysiological deficits, cell type-specific gene expression changes, and behavioral phenotypes. Together, this study enhances our understanding of striatal circuit development and provides proof of concept for a therapeutic approach for FOXP1 syndrome and other neurodevelopmental disorders.

Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system.

Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.

Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects.

Adaptive plasticity of natural interleukin-35-induced regulatory T cells (Tr35) that are required for T-cell immune regulation.

Background: IL-35 potently inhibits immune responses both in vivo and in vitro. However, the specific characteristics of IL-35-producing cells, including their developmental origin, cellular phenotype, and function, are unknown. Methods: By using a novel IL-35 reporter mouse (Ebi3-Dre-Thy1.1) and double transgenic fate-mapping reporter mice (35EbiT-Rosa26-rox-tdTomato reporter mice or Foxp3 fate-mapping system), we tracked and analyzed the differentiation and developmental trajectories of Tr35 cells in vivo. And then we investigated the therapeutic effects of OVA-specific Tr35 cells in an OVA-induced allergic airway disease model. Results: We identified a subset of cells, denoted Tr35 cells, that secrete IL-35 but do not express Foxp3. These cells have high expression of molecules associated with T-cell activation and can inhibit T-cell proliferation in vitro. Our analyses showed that Tr35 cells are a distinct subpopulation of cells that are independent of Tr1 cells. Tr35 cells exhibit a unique gene expression profile and tissue distribution. The presence of Thy1.1 (Ebi3) expression in Tr35 cells indicates their active secretion of IL-35. However, the proportion of ex-Tr35 cells (Thy1.1-) is significantly higher compared to Tr35 cells (Thy1.1+). This suggests that Tr35 cells possess the ability to regulate IL-35 expression rapidly in vivo. Tr35 cells downregulated the expression of the inflammatory cytokines IL-4, IFN-γ and IL-17A. However, once Tr35 cells lost IL-35 expression and became exTr35 cells, the expression of inflammatory cytokines was upregulated. Importantly, our findings indicate that Tr35 cells have therapeutic potential. In an OVA-induced allergic airway disease mouse model, Tr35 cell reinfusion significantly reduced airway hyperresponsiveness and histopathological airway and lung inflammation. Conclusions: We have identified a subset of Tregs, Tr35 cells, that are distinct from Tr1 cells. Tr35 cells can dynamically regulate the secretion of inflammatory cytokines by controlling IL-35 expression to regulate inflammatory immune responses.

CD4+ T cell heterogeneity in gestational age and preeclampsia using single-cell RNA sequencing.

A balance between pro-inflammatory decidual CD4+ T cells and FOXP3+ regulatory T cells (FOXP3+ Tregs) is important for maintaining fetomaternal tolerance. Using single-cell RNA-sequencing and T cell receptor repertoire analysis, we determined that diversity and clonality of decidual CD4+ T cell subsets depend on gestational age. Th1/Th2 intermediate and Th1 subsets of CD4+ T cells were clonally expanded in both early and late gestation, whereas FOXP3+ Tregs were clonally expanded in late gestation. Th1/Th2 intermediate and FOXP3+ Treg subsets showed altered gene expression in preeclampsia (PE) compared to healthy late gestation. The Th1/Th2 intermediate subset exhibited elevated levels of cytotoxicity-related gene expression in PE. Moreover, increased Treg exhaustion was observed in the PE group, and FOXP3+ Treg subcluster analysis revealed that the effector Treg like subset drove the Treg exhaustion signatures in PE. The Th1/Th2 intermediate and effector Treg like subsets are possible inflammation-driving subsets in PE.

ALKBH5-mediated m6A modification of circFOXP1 promotes gastric cancer progression by regulating SOX4 expression and sponging miR-338-3p.

Circular RNAs (circRNAs) have recently been suggested as potential functional modulators of cellular physiology processes in gastric cancer (GC). In this study, we demonstrated that circFOXP1 was more highly expressed in GC tissues. High circFOXP1 expression was positively associated with tumor size, lymph node metastasis, TNM stage, and poor prognosis in patients with GC. Cox multivariate analysis revealed that higher circFOXP1 expression was an independent risk factor for disease-free survival (DFS) and overall survival (OS) in GC patients. Functional studies showed that increased circFOXP1 expression promoted cell proliferation, cell invasion, and cell cycle progression in GC in vitro. In vivo, the knockdown of circFOXP1 inhibited tumor growth. Mechanistically, we observed ALKBH5-mediated m6A modification of circFOXP1 and circFOXP1 promoted GC progression by regulating SOX4 expression and sponging miR-338-3p in GC cells. Thus, our findings highlight that circFOXP1 could serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for GC.

CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Lactobacillus paracasei subsp. paracasei 2004 improves health and lifespan in Caenorhabditis elegans.

Recent research has highlighted the importance of the gut microbiome in regulating aging, and probiotics are interventions that can promote gut health. In this study, we surveyed several novel lactic acid bacteria to examine their beneficial effect on organismal health and lifespan in C. elegans. We found that animals fed some lactic acid bacteria, including L. acidophilus 1244 and L. paracasei subsp. paracasei 2004, grew healthy. Supplementation with the lactic acid bacterial strains L. acidophilus 1244 or L. paracasei subsp. paracasei 2004 significantly improved health, including food consumption, motility, and resistance to oxidative stressor, hydrogen peroxide. Our RNA-seq analysis showed that supplementation with L. paracasei subsp. paracasei 2004 significantly increased the expression of daf-16, a C. elegans FoxO homolog, as well as genes related to the stress response. Furthermore, daf-16 deletion inhibited the longevity effect of L. paracasei subsp. paracasei 2004 supplementation. Our results suggest that L. paracasei subsp. paracasei 2004 improves health and lifespan in a DAF-16-dependent manner.

FOXP4-mediated induction of PTK7 activates the Wnt/ß-catenin pathway and promotes ovarian cancer development.

Ovarian cancer (OV) poses a significant challenge in clinical settings due to its difficulty in early diagnosis and treatment resistance. FOXP4, belonging to the FOXP subfamily, plays a pivotal role in various biological processes including cancer, cell cycle regulation, and embryonic development. However, the specific role and importance of FOXP4 in OV have remained unclear. Our research showed that FOXP4 is highly expressed in OV tissues, with its elevated levels correlating with poor prognosis. We further explored FOXP4's function through RNA sequencing and functional analysis in FOXP4-deficient cells, revealing its critical role in activating the Wnt signaling pathway. This activation exacerbates the malignant phenotype in OV. Mechanistically, FOXP4 directly induces the expression of protein tyrosine kinase 7 (PTK7), a Wnt-binding receptor tyrosine pseudokinase, which causes abnormal activation of the Wnt signaling pathway. Disrupting the FOXP4-Wnt feedback loop by inactivating the Wnt signaling pathway or reducing FOXP4 expression resulted in the reduction of the malignant phenotype of OV cells, while restoring PTK7 expression reversed this effect. In conclusion, our findings underscore the significance of the FOXP4-induced Wnt pathway activation in OV, suggesting the therapeutic potential of targeting this pathway in OV treatment.

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

The role of microRNA-142a in Toxoplasma gondii infection-induced downregulation of Foxp3: implications for adverse pregnancy outcomes.

BACKGROUND: Toxoplasma gondii (T. gondii) is capable of infecting nearly all warm-blooded animals and approximately 30% of the global population. Though most infections are subclinical in immunocompetent individuals, congenital contraction can lead to severe consequences such as spontaneous abortion, stillbirth, and a range of cranio-cerebral and/or ocular abnormalities. Previous studies reported that T. gondii-infected pregnancy mice unveiled a deficit in both the amount and suppressive functions of regulatory T (Treg) cells, accompanied with reduced levels of forkhead box p3 (Foxp3). Recently, accumulative studies have demonstrated that microRNAs (miRNAs) are, to some extent, relevant to T. gondii infection. However, the link between alterations in miRNAs and downregulation of Foxp3 triggered by T. gondii has been only sporadically studied. METHODS: Quantitative reverse transcription polymerase chain reaction (RT-qPCR), protein blotting and immunofluorescence were employed to evaluate the impact of T. gondii infection and antigens on miRNA transcription and Foxp3 expression. Dual-luciferase reporter gene assays were performed to examine the fluorescence activity in EL4 cells, which were transfected with recombinant plasmids containing full-length/truncated/mutant microRNA-142a-3p (miR-142a) promoter sequence or wild type/mutant of Foxp3 3' untranslated region (3' UTR). RESULTS: We found a pronounced increase in miR-142a transcription, concurrent with a decrease in Foxp3 expression in T. gondii-infected mouse placental tissue. Similarly, comparable findings have been experimentally confirmed through the treatment of EL4 cells with T. gondii antigens (TgAg) in vitro. Simultaneously, miR-142a mimics attenuated Foxp3 expression, whereas its inhibitors markedly augmented Foxp3 expression. miR-142a promoter activity was elevated upon the stimulation of T. gondii antigens, which mitigated co-transfection of mutant miR-142a promoter lacking P53 target sites. miR-142a mimics deceased the fluorescence activity of Foxp3 3' untranslated region (3' UTR), but it did not affect the fluorescence activity upon the co-transfection of mutant Foxp3 3' UTR lacking miR-142a target site. CONCLUSION: In both in vivo and in vitro studies, a negative correlation was discovered between Foxp3 expression and miR-142a transcription. TgAg enhanced miR-142a promoter activity to facilitate miR-142a transcription through a P53-dependent mechanism. Furthermore, miR-142a directly targeted Foxp3 3' UTR, resulting in the downregulation of Foxp3 expression. Therefore, harnessing miR-142a may be a possible therapeutic approach for adverse pregnancy caused by immune imbalances, particularly those induced by T. gondii infection.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Regulatory role of PI16 in autoimmune arthritis and intestinal inflammation: implications for Treg cell differentiation and function.

BACKGROUND: Regulatory T cells (Tregs) are crucial in maintaining immune homeostasis and preventing autoimmunity and inflammation. A proportion of Treg cells can lose Foxp3 expression and become unstable under inflammation conditions. The precise mechanisms underlying this phenomenon remain unclear. METHODS: The PI16 gene knockout mice (PI16fl/flFoxp3Cre) in Treg were constructed, and the genotypes were identified. The proportion and phenotypic differences of immune cells in 8-week-old mice were detected by cell counter and flow cytometry. Two groups of mouse Naïve CD4+T cells were induced to differentiate into iTreg cells to observe the effect of PI16 on the differentiation and proliferation of iTreg cells, CD4+CD25+Treg and CD4+CD25- effector T cells (Teff) were selected and co-cultured with antigen presenting cells (APC) to observe the effect of PI16 on the inhibitory ability of Treg cells in vitro. The effects of directed knockout of PI16 in Treg cells on inflammatory symptoms, histopathological changes and immune cell expression in mice with enteritis and autoimmune arthritis were observed by constructing the model of antigen-induced arthritis (AIA) and colitis induced by dextran sulfate sodium salt (DSS). RESULTS: We identified peptidase inhibitor 16 (PI16) as a negative regulator of Treg cells. Our findings demonstrate that conditional knock-out of PI16 in Tregs significantly enhances their differentiation and suppressive functions. The conditional knockout of the PI16 gene resulted in a significantly higher abundance of Foxp3 expression (35.12 ± 5.71% vs. 20.00 ± 1.61%, p = 0.034) in iTreg cells induced in vitro compared to wild-type mice. Mice with Treg cell-specific PI16 ablation are protected from autoimmune arthritis (AIA) and dextran sulfate sodium (DSS)-induced colitis development. The AIA model of PI16CKO is characterized by the reduction of joint structure and the attenuation of synovial inflammation and in DSS-induced colitis model, conditional knockout of the PI16 reduce intestinal structural damage. Additionally, we found that the deletion of the PI16 gene in Treg can increase the proportion of Treg (1.46 ± 0.14% vs. 0.64 ± 0.07%, p < 0.0001) and decrease the proportion of Th17 (1.00 ± 0.12% vs. 3.84 ± 0.64%, p = 0.001). This change will enhance the shift of Th17/Treg toward Treg cells in AIA arthritis model (0.71 ± 0.06% vs. 8.07 ± 1.98%, p = 0.003). In DSS-induced colitis model of PI16CKO, the proportion of Treg in spleen was significantly increased (1.40 ± 0.15% vs. 0.50 ± 0.11%, p = 0.003), Th17 (2.18 ± 0.55% vs. 6.42 ± 1.47%, p = 0.017), Th1 (3.42 ± 0.19% vs. 6.59 ± 1.28%, p = 0.028) and Th2 (1.52 ± 0.27% vs. 2.76 ± 0.38%, p = 0.018) in spleen was significantly decreased and the Th17/Treg balance swift toward Treg cells (1.44 ± 0.50% vs. 24.09 ± 7.18%, p = 0.012). CONCLUSION: PI16 plays an essential role in inhibiting Treg cell differentiation and function. Conditional knock out PI16 gene in Treg can promote the Treg/Th17 balance towards Treg dominance, thereby alleviating the condition. Targeting PI16 may facilitate Treg cell-based therapies for preventing autoimmune diseases and inflammatory diseases. The research provides us with novel insights and future research avenues for the treatment of autoimmune diseases, particularly arthritis and colitis.

FOXP3 promote the progression of glioblastoma via inhibiting ferroptosis mediated by linc00857/miR-1290/GPX4 axis.

The oncogenic properties of members belonging to the forkhead box (FOX) family have been extensively documented in different types of cancers. In this study, our objective was to investigate the impact of FOXP3 on glioblastoma multiforme (GBM) cells. By conducting a screen using a small hairpin RNA (shRNA) library, we discovered a significant association between FOXP3 and ferroptosis in GBM cells. Furthermore, we observed elevated levels of FOXP3 in both GBM tissues and cell lines, which correlated with a poorer prognosis. FOXP3 was found to promote the proliferation of GBM cells by inhibiting cell ferroptosis in vitro and in vivo. Mechanistically, FOXP3 not only directly upregulated the transcription of GPX4, but also attenuated the degradation of GPX4 mRNA through the linc00857/miR-1290 axis, thereby suppressing ferroptosis and promoting proliferation. Additionally, the FOXP3 inhibitor epirubicin exhibited the ability to impede proliferation and induce ferroptosis in GBM cells both in vitro and in vivo. In summary, our study provided evidences that FOXP3 facilitates the progression of glioblastoma by inhibiting ferroptosis via the linc00857/miR-1290/GPX4 axis, highlighting FOXP3 as a potential therapeutic target for GBM.

The role of transcription factor FOXA1/C2/M1/O3/P1/Q1 in breast cancer.

Breast cancer is a common malignancy with the highest mortality rate among women worldwide. Its incidence is on the rise year after year, accounting for more than one-tenth of new cancers worldwide. Increasing evidence suggests that forkhead box (FOX) transcription factors play an important role in the occurrence and development of breast cancer. However, little is known about the relationship between the expression, prognostic value, function, and immune infiltration of FOX transcription factors in tumor microenvironment. We used bioinformatics to investigate expression and function of FOX factor in breast cancer. Our results revealed the expression levels of FOXA1 and FOXM1 were significantly higher in breast cancer tissues than in normal tissues. The high expression of mRNA in FOXA1 (P < .05), FOXM1 (P < .01), and FOXP1 (P < .05) groups was related to tumor stage. Survival analysis results showed that increased FOXP1 mRNA levels were significantly associated with overall survival (OS), recurrence-free survival (RFS), and distant metastasis-free survival (DMFS) in all patients with breast cancer (P < .05). Patients with the FOXA1 high-expression group had better RFS and DMFS than the low-expression group (P < .05), while patients with FOXM1 high-expression group had worse RFS, OS, and DMFS than the low-expression group (P < .05). Meanwhile, mutation analysis showed that genetic alterations in FOX transcription factors were significantly associated with shorter OS and progression-free survival (P < .05), but not with disease-free survival (P = .710) in patients with breast cancer. FOXP1, FOXA1, and FOXM1 may be used as potential biomarkers to predict the prognosis of patients with breast cancer. Functional enrichment indicated that FOX was mainly involved in cell division, cell senescence, cell cycle, and prolactin signaling pathway. In patients with breast cancer, FOXC2 expression was negatively correlated with the infiltration of B cells and positively correlated with the infiltration of neutrophils and dendritic cells. However, FOXM1 was negatively correlated with the infiltration of CD8 + T cells and macrophages and positively correlated with the infiltration of neutrophils and dendritic cells. These findings provided novel insights into the screening of prognostic biomarkers of the FOX family in breast cancer and laid a foundation for further research on the immune infiltration of the FOX transcription factor family members in tumors.

Umbilical cord mesenchymal stem cell-derived exosomes inhibits fibrosis in human endometrial stromal cells via miR-140-3p/FOXP1/Smad axis.

Endometrial fibrosis is the histologic appearance of intrauterine adhesion (IUA). Emerging evidences demonstrated umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-exo) could alleviate endometrial fibrosis. But the specific mechanism is not clear. In this study, we explored the effect of UCMSC-exo on endometrial fibrosis, and investigated the possible role of miR-140-3p/FOXP1/Smad axis in anti-fibrotic properties of UCMSC-exo. UCMSC-exo were isolated and identified. Transforming growth factor-ß (TGF-ß) was used to induce human endometrial stromal cell (HESC) fibrosis. Dual luciferase assay was performed to verify the relationship between miR-140-3p and FOXP1. The expressions of fibrotic markers, SIP1, and p-Smad2/p-Smad3 in HESCs stimulated with UCMSC-exo were detected by western blot. In addition, the effects of miR-140-3p mimic, miR-140-3p inhibitor and FOXP1 over-expression on endometrial fibrosis were assessed. The isolated UCMSC-exo had a typical cup-shaped morphology and could be internalized into HESCs. The expressions of fibrotic markers were significantly increased by TGF-ß, which was reversed by UCMSC-exo. MiR-140-3p in UCMSC-exo ameliorated TGf-ß-induced HESCs fibrosis. FOXP1 was identified as the direct target of miR-140-3p, which could inversely regulate miR-140-3p's function on HESCs fibrosis. Furthermore, we demonstrated that miR-140-3p in UCMSC-exo regulated Smad signal pathway to exert the anti-fibrotic effect in HESCs. The anti-fibrotic effect of UCMSC-derived exosomes against HESC fibrosis was at least partially achieved by miR-140-3p/FOXP1/Smad axis.

Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model. Injured IVDs treated with eEVs loaded with FOXF1 demonstrated robust sex-specific reductions in pain behaviors compared to control groups. Furthermore, significant restoration of IVD structure and function in animals treated with FOXF1 eEVs were observed, with significant increases in disc height, tissue hydration, proteoglycan content, and mechanical properties. This is the first study to successfully restore tissue function while modulating pain behaviors in an animal model of DBP using eEV-based non-viral delivery of transcription factor genes. Such a strategy can be readily translated to other painful musculoskeletal disorders.

Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function.

Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

Decreased Treg cells induced by bisphenol A is associated with up-regulation of PI3K/Akt/mTOR signaling pathway and Foxp3 DNA methylation in spleen of adolescent mice.

The current study aimed to explore whether bisphenol A (BPA) exposure aggravated the decrease in Tregs induced by ovalbumin (OVA) in adolescent female mouse models of asthma, and whether the process was associated with mTOR-mediated signaling pathways and DNA methylation levels. A total of 40 female C57BL/6 mice at the age of four weeks were used and divided into five groups after 1 week of domestication. Each group consisted of eight mice: the control group, OVA group, OVA + BPA (0.1 µg mL-1) group, OVA + BPA (0.2 µg mL-1) group, and OVA + BPA (0.4 µg mL-1) group. Results revealed that Foxp3 protein levels decreased in the spleens of mice exposed to BPA compared to those in the OVA group. After an elevation in BPA dose, the mRNAs of methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) were gradually upregulated. The mechanism was related to the activity of TLR4/NF-κB and PI3K/Akt/mTOR signaling pathways and the enhancement of Foxp3 DNA methylation. Our results, collectively, provided a new view for studying the mechanisms underlying BPA exposure-induced immune dysfunction. Investigation of the regulatory mechanisms of DNA methylation in the abnormal Th immune response caused by BPA exposure could help reveal the causes and molecular mechanisms underlying the high incidence of allergic diseases in children in recent years.