Objective To explore the effects of S100 calcium-binding protein A9 (S100A9) gene knockout on the phenotype of systemic lupus erythematosus (SLE) in mice and to clarify the role of S100A9 in the pathogenesis of SLE. Methods Ten female C57BL/6 wild-type and S100A9 knockout (S100A9-KO ) mice were selected, with five wild-type and five S100A9-KO B6 mice receiving imiquimod (IMQ) cream to establish SLE mouse model. The other five wild-type and five S100A9-KO B6 mice were treated as control groups by wiping the skin of the right ear with a cotton swab. After 8 weeks, the mice were sacrificed. The serum was collected from each mouse to detect the levels of anti-double-stranded DNA (dsDNA) antibodies, immunoglobulin G (IgG), B cell activating factor (BAFF), and interleukin 6 (IL-6) using ELISA. The levels of serum creatinine were determined using a sarcosine oxidase method. Urine was collected to measure urinary protein concentration. Kidneys were collected and stained with hematoxylin and eosin (H&E) for evaluating histological changes. Results After IMQ treatment, the length and weight of spleen, levels of serum creatinine, anti-dsDNA antibodies, IgG, BAFF, IL-6, and urinary protein in the IMQ B6 group and IMQ S100A9-KO B6 group were significantly higher than those of the control groups. Lupus-like changes including increased glomerular volume and tubular epithelial swelling were observed in kidneys from the IMQ and IMQ S100A9-KO groups. However, compared with the IMQ B6 group, the IMQ S100A9-KO B6 group exhibited milder levels of serum and urine indicators as well as the lupus-like symptoms. Conclusion IMQ could induce lupus-like symptoms in both wild-type B6 mice and S100A9-KO B6 mice, but the lesions in S100A9 knockout mice are milder. Theses results suggested that S100A9 is involved in and promotes the pathogenesis of SLE.
Animals ; Lupus Erythematosus, Systemic/chemically induced* ; Female ; Calgranulin B/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Phenotype ; Mice ; Interleukin-6/blood* ; Disease Models, Animal ; Antibodies, Antinuclear/blood* ; B-Cell Activating Factor/blood* ; Immunoglobulin G/blood* ; Kidney/pathology*

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335^fl/fl FOXP3^creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4⁺ T cells, CD8⁺ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335^CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4⁺ and CD8⁺ T cells, along with their respective effector cells, was significantly higher in the Zfp335^CKO group than in the WT group. The proportions of CD4⁺ and CD8⁺ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335^CKO group compared to that of the WT group. In addition, the percentage of CD8⁺ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335^CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)⁺ Treg in the Zfp335^CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335^CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335^CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.
Animals ; T-Lymphocytes, Regulatory/metabolism* ; Mice ; CD8-Positive T-Lymphocytes/immunology* ; Neoplasms/genetics* ; Cell Line, Tumor ; Mice, Inbred C57BL ; Mice, Knockout ; DNA-Binding Proteins/genetics* ; Female

Objective To generate and characterize natural killer cell (NK cell)-conditional Cd226 gene knockout mice using Cre-loxP technology, and to explore the role of CD226 on NK cells in alleviating intestinal inflammation in a murine model of ulcerative colitis (UC). Methods NK cell-conditional Cd226 gene knockout mice were generated by crossing loxP-flanked Cd226 mice with Ncr1-Cre mice via the Cre-loxP system. Polymerase chain reaction (PCR) and agarose gel electrophoresis were used for genotyping. A UC model was established by dextran sulfate sodium (DSS) induction. Flow cytometry was performed to analyze CD226 expression levels on NK cells and the infiltration of related immune cells in colon tissues. Hematoxylin-eosin (HE) staining was performed to assess the degree of colonic inflammation. Results DNA gel electrophoresis and flow cytometry confirmed the successful generation of NK cell-specific Cd226 knockout mice. After conditional knockout of Cd226 in NK cells, inflammation in the UC mouse model was alleviated. Flow cytometry results showed a reduced proportion of NK cells in peripheral blood and the colon lamina propria, while HE staining demonstrated attenuated inflammatory responses. Conclusion Specific knockout of Cd226 in NK cells mitigates intestinal inflammation in UC mice by reducing NK cell numbers and inhibiting their pro-inflammatory functions.
Animals ; Colitis, Ulcerative/pathology* ; Killer Cells, Natural/metabolism* ; Mice, Knockout ; T Lineage-Specific Activation Antigen 1 ; Antigens, Differentiation, T-Lymphocyte/genetics* ; Mice ; Disease Models, Animal ; Mice, Inbred C57BL ; Male

Objective To investigate the role and mechanism of the zinc finger protein Kruppel-like transcription factor 9 (KLF9) in the stimulation of type I interferon expression induced by herpes simplex virus type 1 (HSV-1) in macrophages. Methods Agarose Gel electrophoresis, quantitative real-time PCR (qRT-PCR) and western blot analyses were employed to detect the KLF9 relative expression in bone marrow-derived macrophages (BMDMs) from Klf9^-/- (gKO) mice and wild-type (WT) mice. RNA-seq analysis was utilized to identify the potential targeted genes upon HSV-1 stimulation in BMDMs. ELISA was used to measure the potent of IFN-β in the supernatant of BMDMs derived from gKO and WT mice after HSV-1 stimulation. qRT-PCR analysis was employed to further confirm the changes of Ifnb1 and interferon-stimulated gene (ISG) such as interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), interferon-stimulated exonuclease gene 20 (Isg20), cholesterol 25-hydroxylase (Ch25h) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1). Western blot was used to detect the expression of phosphorylated interferon regulatory factor-3 (p-IRF3), IRF3, phosphorylated interferon regulatory factor-7 (p-IRF7), IRF7, phosphorylated nuclear factor-kappa B p65 (p-NF-κB p65) and NF-κB p65. CUT-Tag and ChIP-qPCR assay were utilized to confirm the binding region of KLF9 in Ifnb1. Results The KLF9 expression was significantly decreased in BMDMs from gKO mice compared with that from WT mice. The RNA-seq analysis showed that Klf9 deletion in BMDMs resulted in an impaired type I interferon signaling pathway. The qRT-PCR analysis revealed that Klf9 deletion in BMDMs led to a significant decrease of Ifnb1 and ISG such as Ifit1, Ch25h and Oasl1 except Isg20. Moreover, ELISA revealed that Klf9 knockout in BMDMs resulted in a significant decrease of IFN-β secreted from BMDMs. Mechanistically, KLF9 directly binds to the promoter of Ifnb1. Conclusion KLF9 is essential for macrophages to resist HSV-1 infection.
Animals ; Kruppel-Like Transcription Factors/physiology* ; Interferon-beta/metabolism* ; Macrophages/virology* ; Mice ; Herpesvirus 1, Human/physiology* ; Mice, Knockout ; Signal Transduction ; Mice, Inbred C57BL ; Interferon Regulatory Factor-3/genetics* ; Interferon Regulatory Factor-7/genetics* ; Gene Expression Regulation

Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout ( Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 ( Smoc1 ) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 ( Pax7 ) and myogenic factor 5 ( Myf5 ). After short-interfering RNA-mediated knocking down of Smoc1 , the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.
Animals ; Male ; Mice ; Testis/growth & development* ; Mice, Knockout ; Androgens/pharmacology* ; Testosterone/pharmacology* ; Receptors, LH/metabolism* ; Calcium-Binding Proteins/metabolism*

Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro , we reveal that BDNF -deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger ( Plzf ), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.
Animals ; Male ; Brain-Derived Neurotrophic Factor/genetics* ; Spermatogonia/cytology* ; Mice ; Spermatogenesis/genetics* ; Mice, Knockout ; Cell Differentiation ; Glial Cell Line-Derived Neurotrophic Factor/genetics* ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Cell Survival/physiology* ; Signal Transduction/physiology* ; Cell Proliferation/physiology*

Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.
Spermatogenesis/physiology* ; Animals ; Male ; Mice ; Epigenesis, Genetic ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Histones/metabolism* ; RNA Interference ; Testis/metabolism* ; Methylation ; Mice, Knockout ; Histone Demethylases

This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

OBJECTIVES: To investigate the role of a neural pathway from oxytocin (OXT) neurons in the paraventricular hypothalamic nucleus (PVN) to γ-aminobutyric acid (GABA) neurons (GABAergic neurons) in the trigeminal nucleus caudalis (TNC) in regulating pain sensitization in a mouse model of chronic migraine and to explore the underlying mechanisms. METHODS: A chronic migraine mouse model was established by intraperitoneal injection of nitroglycerin (NTG, 1 mg/mL, 10 mg/kg) on days 1, 3, 5, 7, and 9. The study consisted of four parts: PartⅠ: 24 male wild-type C57BL/6J mice were divided into four groups (n=6 in each), receiving single or repeated injection of NTG or saline, respectively. Immunofluorescence was used to detect c-Fos and OXT expression in the PVN. Part Ⅱ: 6 male OXT-Cre transgenic C57BL/6J mice were used for anterograde monosynaptic tracing combined with RNAscope and immunofluorescence to identify neural projections from PVN OXT neurons to TNC GABAergic neurons. Part Ⅲ: 30 male OXT-Cre transgenic C57BL/6J mice were bilaterally injected Cre-dependent chemogenetic activation virus into the PVN. These mice were randomly divided into five groups, with six mice in each group. Mice in the clozapine N-oxide (CNO) group and the control group were intra-peritoneally injected with 0.1 mg/mL of CNO solution (1 mg/kg) and the same volume of isotonic normal saline, respectively. 3 hours after the injection, the brain tissues were harvest and c-Fos immunofluorescence staining was performed to verify the efficiency of chemogenetic activation virus. Mice in the model control group and the CNO activated model group were subjected to chronic migraine modeling, with bilateral TNC injection of isotonic normal saline and CNO, respectively, on day 10. The mice in the negative control group were bilaterally intra-TNC injected with isotonic normal saline. After 30 minutes, the Von-Frey filament and acetone tests were used to assess the mechanical pain threshold and cold pain response time in the periorbital region of the mice in these three groups. Part Ⅳ: 24 male OXT-Cre transgenic C57BL/6J mice were bilaterally injected with the Cre-dependent chemogenetic activation virus into the PVN. These mice were randomly divided into four groups, with six mice in each group. Mice in the model control group, the CNO activated model group and the atosiban group were subjected to chronic migraine modeling. On day 10, mice in the negative control group and the model control group were intraperitoneally injected with isotonic normal saline, while mice in the CNO activated model group and the atosiban group were intraperitoneally injected with CNO. After 15 minutes, mice in the atosiban group were bilaterally intra-TNC injected with atosiban, while mice in other three groups were bilaterally intra-TNC injected with isotonic normal saline containing 1% dimethyl sulfoxide. After 15 minutes, the Von-Frey filament and acetone tests were used to assess the mechanical pain threshold and cold pain response time in the periorbital region of the mice. The GABA content in the bilateral TNC was detected by high-performance liquid chromatography (HPLC). RESULTS: Mice with chronic migraine models exhibited reduced periorbital mechanical pain thresholds and increased periorbital cold pain reaction time, accompanied by an increase in both the number of c-Fos⁺ neurons and the percentage of c-Fos⁺ OXT neurons in the PVN (all P<0.05). The anterograde tracing virus and RNAscope combined with immunofluorescence staining showed that PVN OXT neurons projected to TNC GABAergic neurons. Immuno-fluorescence staining demonstrated that compared with the control group, the percentage of c-Fos⁺ OXT neurons in the PVN of CNO group increased (P<0.05). In bilateral intra-TNC drug administration experiments, compared with the model control group, the periorbital mechanical pain threshold increased, and the periorbital cold pain reaction time decreased in the CNO activated model group (both P<0.05). In intraperitoneal drug administration experiments, compared with the CNO activate model group, the periorbital mechanical pain threshold decreased, and the periorbital cold pain reaction time increased in the atosiban group (both P<0.05). HPLC analysis showed that, compared with the negative control group, the model control group and the atosiban group, GABA level of TNC in the CNO activated model group increased (all P<0.05). CONCLUSIONS PVN OXT neurons exert a descending facilitatory effect on GABAergic neurons in the TNC via OXT release, thereby ameliorating pain sensitization in chronic migraine.
Animals ; Paraventricular Hypothalamic Nucleus/physiopathology* ; Male ; Mice, Inbred C57BL ; Migraine Disorders/physiopathology* ; Mice ; GABAergic Neurons/physiology* ; Oxytocin/physiology* ; Disease Models, Animal ; Neurons/physiology* ; Mice, Transgenic ; Neural Pathways ; Chronic Disease

OBJECTIVE: To explore the effect and mechanism of DNA methyltransferase 1 (DNMT1) knockout on the inhibition of acute myeloid leukemia (AML) by natural killer (NK) cells. METHODS: The peripheral blood NK cells of AML patients and controls were collected, and the mRNA and protein level of DNMT1 were measured by PCR and Western blot, respectively. The DNMT1 knockout mice were constructed to obtain NK^DNMT1-/- cells. The NK cells were stimulated with interleukin (IL)-12, IL-15, and IL-18 to construct memory NK cells, and then the interferon-γ (IFN-γ) levels were measured by ELISA. After co-culturing with memory NK cells and HL60 cells, the killing effect of NK^DNMT1-/- cells on HL60 cells was detected by LDH assay. Then, the HL60 cell apoptosis and NK cell NKG2D level were measured by flow cytometry. The perforin and granzyme B protein levels of NK cells were measured by Western blot. The AML model mice were constructed by injecting HL60 cells into the tail vein, meanwhile, memory NK cells were also injected, and then the mouse weights, CD33 positive rates, and survival time were detected. RESULTS: The mRNA and protein levels of DNMT1 in NK cells of AML patients were significantly higher than those in the control group (both P < 0.01), while the IFN-γ level induced by interleukin was significantly lower than that in the control group (P < 0.05). Compared with NK^DNMT1+/+ cells, the ability of NK^DNMT1-/- cells to secrete IFN-γ after interleukin stimulation was significantly increased (P < 0.05). The killing and apoptosis-inducing effects of NK^DNMT1-/- cells on HL60 cells were significantly stronger than those of NK^DNMT1+/+ cells (both P < 0.05). The NKG2D level and expression of perforin and granzyme B of NK^DNMT1-/- cells were significantly increased compared with NK^DNMT1+/+ cells (all P < 0.05). Compared with AML mice injected with NK^DNMT1+/+ cells, AML mice injected with NK^DNMT1-/- cells showed significantly increased body weight, decreased CD33 positive rate, and prolonged survival time (all P < 0.05). CONCLUSION Knocking out DNMT1 can enhance the inhibitory effect of NK cells on AML, which may be related to enhancing NK cell memory function.
Killer Cells, Natural/metabolism* ; Animals ; Leukemia, Myeloid, Acute ; Humans ; DNA (Cytosine-5-)-Methyltransferase 1 ; Mice ; Mice, Knockout ; HL-60 Cells ; Apoptosis ; Interferon-gamma/metabolism* ; Granzymes/metabolism* ; Perforin/metabolism* ; NK Cell Lectin-Like Receptor Subfamily K/metabolism*