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.

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.

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.

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.

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.

Comprehensive Analysis of the Immunosuppressive Function of Regulatory T Cells in Human Hepatocellular Carcinoma Tissues.

BACKGROUND: Immune-based therapies are commonly employed to combat hepatocellular carcinoma (HCC). However, the presence of immune-regulating elements, especially regulatory T cells (Tregs), can dramatically impact the treatment efficacy. A deeper examination of the immune-regulation mechanisms linked to these inhibitory factors and their impact on HCC patient outcomes is warranted. METHODS: We employed multicolor fluorescence immunohistochemistry (mIHC) to stain Foxp3, cytokeratin, and nuclei on an HCC tissue microarray (TMA). Leveraging liver cancer transcriptome data from TCGA, we built a prognostic model focused on Treg-associated gene sets and represented it with a nomogram. We then sourced liver cancer single-cell RNA sequencing data (GSE140228) from the GEO database, selectively focusing on Treg subsets, and conducted further analyses, including cell-to-cell communication and pseudo-time trajectory examination. RESULTS: Our mIHC results revealed a more substantial presence of Foxp3+Tregs in HCC samples than in adjacent normal tissue samples (P < .001). An increased presence of Foxp3+Tregs in HCC samples correlated with unfavorable patient outcomes (HR = 1.722, 95% CI:1.023-2.899, P = .041). The multi-factorial prognosis model we built from TCGA liver cancer data highlighted Tregs as a standalone risk determinant for predicting outcomes (HR = 3.84, 95% CI:2.52-5.83, P < .001). Re-analyzing the scRNA-seq dataset (GSE140228) showcased distinctive gene expression patterns in Tregs from varying tissues. Interactions between Tregs and other CD4+T cell types were predominantly governed by the CXCL13/CXCR3 signaling pathway. Communication pathways between Tregs and macrophages primarily involved MIF-CD74/CXCR4, LGALS9/CD45, and PTPRC/MRC1. Additionally, macrophages could influence Tregs via HLA-class II and CD4 interactions. CONCLUSION: An elevated presence of Tregs in HCC samples correlated with negative patient outcomes. Elucidating the interplay between Tregs and other immune cells in HCC could provide insights into the modulatory role of Tregs within HCC tissues.

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.

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.

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.

FOXF1 reverses lung fibroblasts transdifferentiation via inhibiting TGF-ß/SMAD2/3 pathway in silica-induced pulmonary fibrosis.

Silicosis is one of the most common and severe types of pneumoconiosis and is characterized by lung dysfunction, persistent lung inflammation, pulmonary nodule formation, and irreversible pulmonary fibrosis. The transdifferentiation of fibroblasts into myofibroblasts is one of the main reasons for the exacerbation of silicosis. However, the underlying mechanism of transcription factors regulating silicosis fibrosis has not been clarified. The aim of this study was to investigate the potential mechanism of transcription factor FOXF1 in fibroblast transdifferentiation in silica-induced pulmonary fibrosis. Therefore, a silicosis mouse model was established, and we found that FOXF1 expression level was significantly down-regulated in the silicosis group, and after overexpression of FOXF1 by adeno-associated virus (AAV), FOXF1 expression level was up-regulated, and silicosis fibrosis was alleviated. In order to further explore the specific regulatory mechanism of FOXF1 in silicosis, we established a fibroblasts transdifferentiation model induced by TGF-ß in vitro. In the model, the expression levels of SMAD2/3 and P-SMAD2/3 were up-regulated, but the expression levels of SMAD2/3 and P-SMAD2/3 were down-regulated, inhibiting transdifferentiation and accumulation of extracellular matrix after the overexpressed FOXF1 plasmid was constructed. However, after silencing FOXF1, the expression levels of SMAD2/3 and P-SMAD2/3 were further up-regulated, aggravating transdifferentiation and accumulation of extracellular matrix. These results indicate that the activation of FOXF1 in fibroblasts can slow down the progression of silicosis fibrosis by inhibiting TGF-ß/SMAD2/3 classical pathway, which provides a new idea for further exploration of silicosis treatment.

An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma.

Forkhead box protein 1 (FOXP1) serves as a tumour promoter or suppressor depending on different cancers, but its effect in oesophageal squamous cell carcinoma has not been fully elucidated. This study investigated the role of FOXP1 in oesophageal squamous cell carcinoma through bioinformatics analysis and experimental verification. We determined through public databases that FOXP1 expresses low in oesophageal squamous cell carcinoma compared with normal tissues, while high expression of FOXP1 indicates a better prognosis. We identified potential target genes regulated by FOXP1, and explored the potential biological processes and signalling pathways involved in FOXP1 in oesophageal squamous cell carcinoma through GO and KEGG enrichment, gene co-expression analysis, and protein interaction network construction. We also analysed the correlation between FOXP1 and tumour immune infiltration levels. We further validated the inhibitory effect of FOXP1 on the proliferation of oesophageal squamous cell carcinoma cells through CCK-8, colony formation and subcutaneous tumour formation assays. This study revealed the anticarcinogenic effect of FOXP1 in oesophageal squamous cell carcinoma, which may serve as a novel biological target for the treatment of tumour.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance.

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

Deciphering the developmental trajectory of tissue-resident Foxp3+ regulatory T cells.

Foxp3+ TREG cells have been at the focus of intense investigation for their recognized roles in preventing autoimmunity, facilitating tissue recuperation following injury, and orchestrating a tolerance to innocuous non-self-antigens. To perform these critical tasks, TREG cells undergo deep epigenetic, transcriptional, and post-transcriptional changes that allow them to adapt to conditions found in tissues both at steady-state and during inflammation. The path leading TREG cells to express these tissue-specialized phenotypes begins during thymic development, and is further driven by epigenetic and transcriptional modifications following TCR engagement and polarizing signals in the periphery. However, this process is highly regulated and requires TREG cells to adopt strategies to avoid losing their regulatory program altogether. Here, we review the origins of tissue-resident TREG cells, from their thymic and peripheral development to the transcriptional regulators involved in their tissue residency program. In addition, we discuss the distinct signalling pathways that engage the inflammatory adaptation of tissue-resident TREG cells, and how they relate to their ability to recognize tissue and pathogen-derived danger signals.

Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

[Xixin Decoction regulates Th17/Treg balance and transcription factor expression in senescence-accelerated mouse-prone].

This study aims to investigate the effect of Xixin Decoction on the T helper 17 cell(Th17)/regulatory T cell(Treg) ba-lance of intestinal mucosa and the expression of related transcription factors in the senescence-accelerated mouse-prone 8(SAMP8) model. Fifty 14-week male mice of SAMP8 were randomized by the random number table method into model group, probiotics group, and high-, medium-, and low-dose Xixin Decoction groups, with 10 mice in each group. Ten 14-week male mice of senescence-acce-lerated mouse-resistant 1(SAMR1) served as control group. After 10 weeks of feeding, the mice were administrated with correspon-ding drugs for 10 weeks. Morris water maze test was carried out to examine the learning and memory abilities of mice. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of secretory immunoglobulin A(SIgA) in the intestinal mucosa, and flow cytometry to detect the percentage content of Th17 and Treg in the intestinal mucosa. Western blot was performed to determine the protein levels of retinoid-related orphan receptor gamma t(RORγt) and forkhead box p3(Foxp3) in the mouse colon tissue. Compared with control group, the escape latency of mice in model group was significantly prolonged(P<0.01), and the number of times of crossing the platform and the residence time in the target quadrant were significantly reduced within 60 s(P<0.01), intestinal mucosal SIgA content was significantly decreased(P<0.01), Th17 content was increased(P<0.05), Treg content was decreased(P<0.01), the expression of RORγt protein was increased and Foxp3 protein was decreased in colon(P<0.01). Compared with the model group, high-dose Xixin Decoction group improved the learning and memory ability(P<0.05 or P<0.01). Probiotics group and high-and medium-dose Xixin Decoction group increased the content of SIgA in intestinal mucosa(P<0.05 or P<0.01), decreased percentage content of Th17 and increased the percentage content of Treg in intestinal mucosa(P<0.05 or P<0.01). Furthermore, they down-regulated the protein level of RORγt and up-regulated the protein level of Foxp3 in the intestinal mucosa(P<0.01). In conclusion, Xixin Decoction may act on intestinal mucosal immune barrier, affect gut-brain information exchange, and improve the learning and memory ability of SAMP8 by promoting SIgA secretion and regulating the Th17/Treg balance and the expression of RORγt and Foxp3.

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.

Experience-Dependent Behavioral Plasticity in Avoiding Epigallocatechin Gallate (EGCG) Requires DAF-16/FOXO in the AIY Interneurons of Caenorhabditis elegans.

Bitterness and astringency are the aversive tastes in mammals. In humans, aversion to bitterness and astringency may be reduced depending on the eating experience. However, the cellular and molecular mechanisms underlying plasticity in preference to bitter and astringent tastants remain unknown. This study aimed to investigate the preference plasticity to bitter and astringent tea polyphenols, including catechins and tannic acids, in the model animal Caenorhabditis elegans. C. elegans showed avoidance behavior against epigallocatechin gallate (EGCG), tannic acid, and theaflavin. However, they displayed diminishing avoidance against EGCG depending on their EGCG-feeding regime at larval stages. Additionally, the behavioral plasticity in avoiding EGCG required the transcription factor DAF-16/FOXO. Isoform-specific deletion mutant analysis and cell-specific rescue analysis revealed that the function of daf-16 isoform b in AIY interneurons is necessary for experience-dependent behavioral plasticity to EGCG.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.