BACKGROUND: Increasing evidence indicates that oxidative stress and interferon γ (IFNγ)-driven cellular immune responses are responsible for the pathogenesis of vitiligo. However, the connection between oxidative stress and the local production of IFNγ in early vitiligo remains unexplored. The aim of this study was to identify the mechanism underlying the production of IFNγ by mast cells and its impact on vitiligo pathogenesis. METHODS: Skin specimens from the central, marginal, and perilesional skin areas of active vitiligo lesions were collected to characterize changes of mast cells, CD8 + T cells, and IFNγ-producing cells. Cell supernatants from hydrogen peroxide (H 2 O 2 )-treated keratinocytes (KCs) were harvested to measure levels of soluble stem cell factor (sSCF) and matrix metalloproteinase (MMP)-9. A murine vitiligo model was established using Mas-related G protein-coupled receptor-B2 (MrgB2, mouse ortholog of human MrgX2) conditional knockout (MrgB2 -/- ) mice to investigate IFNγ production and inflammatory cell infiltrations in tail skin following the challenge with tyrosinase-related protein (Tyrp)-2 180 peptide. Potential interactions between the Tyrp-2 180 peptide and MrgX2 were predicted using molecular docking. The siRNAs targeting MrgX2 and the calcineurin inhibitor FK506 were also used to examine the signaling pathways involved in mast cell activation. RESULTS: IFNγ-producing mast cells were closely aligned with the recruitment of CD8 + T cells in the early phase of vitiligo skin. sSCF released by KCs through stress-enhanced MMP9-dependent proteolytic cleavage recruited mast cells into sites of inflamed skin (Perilesion vs . lesion, 13.00 ± 4.00/high-power fields [HPF] vs . 26.60 ± 5.72/HPF, P <0.05). Moreover, IFNγ-producing mast cells were also observed in mouse tail skin following challenge with Tyrp-2 180 (0 h vs . 48 h post-recall, 0/HPF vs . 3.80 ± 1.92/HPF, P <0.05). The IFNγ + mast cell and CD8 + T cell counts were lower in the skin of MrgB2 -/- mice than in those of wild-type mice (WT vs . KO 48 h post-recall, 4.20 ± 0.84/HPF vs . 0.80 ± 0.84/HPF, P <0.05). CONCLUSION Mast cells activated by MrgX2 serve as a local IFNγ producer that bridges between innate and adaptive immune responses against MCs in early vitiligo. Targeting MrgX2-mediated mast cell activation may represent a new strategy for treating vitiligo.
Vitiligo/metabolism* ; Mast Cells/immunology* ; Animals ; Interferon-gamma/metabolism* ; Mice ; Humans ; Melanocytes/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Female ; Matrix Metalloproteinase 9/metabolism* ; Stem Cell Factor/metabolism*

OBJECTIVES: To investigate the therapeutic effects of inulin-type oligosaccharides of <i>Morinda officinalisi> (IOMO) in a murine model of <i>Streptococcus pneumoniaei> meningitis (SPM) and explore its possible mechanisms. METHODS: A total of 120 male C57BL/6J mice were randomly assigned into Sham, SPM+Saline, SPM+IOMO (25 mg/kg), and SPM+IOMO (50 mg/kg) groups. After modeling, the mice received daily gavage of saline or IOMO at the indicated doses for 7 consecutive days, and the changes in symptom scores and mortality of the mice were monitored. Brain pathology and neuronal injury of the mice were assessed using HE and Nissl staining, and qRT-PCR was performed to detect mRNA levels of the inflammatory mediators. Brain edema and blood-brain barrier (BBB) permeability of the mice were evaluated by measuring brain water content and Evans blue (EB) staining; Western blotting was used to analyze the expressions of BBB-associated proteins, and flow cytometry was employed to detect IFN‑γ expression level in the infiltrating lymphocytes. Open-field test (OFT) and novel object recognition test (NORT) were conducted to assess learning and memory ability of the mice on day 21 after modeling. RESULTS: IOMO treatment at 50 mg/kg significantly reduced the symptom scores and mortality rate of SPM mice, alleviated brain damage, and downregulated mRNA levels of IL-6, TNF‑α, IL-1β, IL-18, IFN‑γ, iNOS, NLRP3, ASC, caspase-1 and GSDMD in the brain tissue. IOMO treatment also decreased brain water content and EB leakage, upregulated VE-cadherin and occludin expressions, and suppressed AQP4, iNOS, and IFN‑γ levels of the mice. IOMO-treated mice exhibited improved learning and memory compared with the saline-treated mice on day 21 after SPM modeling. CONCLUSIONS IOMO alleviates SPM symptoms, reduces mortality, and mitigates cognitive deficits in mice possibly by suppressing cerebral inflammation and protecting BBB functions.
Animals ; Morinda/chemistry* ; Mice, Inbred C57BL ; Male ; Mice ; Meningitis, Pneumococcal/drug therapy* ; Blood-Brain Barrier/metabolism* ; Inulin/therapeutic use* ; Oligosaccharides/therapeutic use* ; Disease Models, Animal ; Interferon-gamma/metabolism* ; Brain Edema

OBJECTIVES: To investigate the mechanism by which transforming growth factor‑β (TGF‑β) regulates major histocompatibility complex class I (MHC-I) expression in hepatocellular carcinoma (HCC) cells and its role in immune evasion of HCC. METHODS: HCC cells treated with TGF‑β alone or in combination with SB-431542 (a TGF-β type I receptor inhibitor) were examined for changes in MHC-I expression using RT-qPCR and Western blotting. A RNA interference experiment was used to explore the role of miR-23a-3p/IRF1 signaling in TGF‑β‑mediated regulation of MHC-I. HCC cells with different treatments were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the changes in HCC cell proliferation was assessed using CCK-8 and colony formation assays. T-cell cytotoxicity in the co-culture systems was assessed with lactate dehydrogenase (LDH) release and JC-1 mitochondrial membrane potential assays, and T-cell activation was evaluated by flow cytometric analysis of CD69 cells and ELISA for TNF-α secretion. RESULTS: TGF‑β treatment significantly suppressed MHC-I expression in HCC cells and reduced T-cell activation, leading to increased tumor cell proliferation and decreased HCC cell death in the co-culture systems. Mechanistically, TGF-β upregulated miR-23a-3p, which directly targeted IRF1 to inhibit MHC-I transcription. Overexpression of miR-23a-3p phenocopied TGF‑β‑induced suppression of IRF1 and MHC-I. CONCLUSIONS We reveal a novel immune escape mechanism of HCC, in which TGF‑β attenuates T cell-mediated antitumor immunity by suppressing MHC-I expression through the miR-23a-3p/IRF1 signaling axis.
Humans ; MicroRNAs/genetics* ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Interferon Regulatory Factor-1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Histocompatibility Antigens Class I/metabolism* ; Cell Line, Tumor ; Tumor Escape ; Coculture Techniques

Tricellulin, a key tricellular tight junction (TJ) protein, is essential for maintaining the barrier integrity of acinar epithelia against macromolecular passage in salivary glands. This study aims to explore the role and regulatory mechanism of tricellulin in the development of salivary gland hypofunction in Sjögren's syndrome (SS). Employing a multifaceted approach involving patient biopsies, non-obese diabetic (NOD) mice as a SS model, salivary gland acinar cell-specific tricellulin conditional knockout (Tric^CKO) mice, and IFN-γ-stimulated salivary gland epithelial cells, we investigated the role of tricellulin in SS-related hyposalivation. Our data revealed diminished levels of tricellulin in salivary glands of SS patients. Similarly, NOD mice displayed a reduction in tricellulin expression from the onset of the disease, concomitant with hyposecretion and an increase in salivary albumin content. Consistent with these findings, Tric^CKO mice exhibited both hyposecretion and leakage of macromolecular tracers when compared to control animals. Mechanistically, the JAK/STAT1/miR-145 axis was identified as mediating the IFN-γ-induced downregulation of tricellulin. Treatment with AT1001, a TJ sealer, ameliorated epithelial barrier dysfunction, restored tricellulin expression, and consequently alleviated hyposalivation in NOD mice. Importantly, treatment with miR-145 antagomir to specifically recover the expression of tricellulin in NOD mice significantly alleviated hyposalivation and macromolecular leakage. Collectively, we identified that tricellulin deficiency in salivary glands contributed to hyposalivation in SS. Our findings highlight tricellulin as a potential therapeutic target for hyposecretion, particularly in the context of reinforcing epithelial barrier function through preventing leakage of macromolecules in salivary glands.
Sjogren's Syndrome/complications* ; Animals ; Xerostomia/etiology* ; Mice ; Mice, Inbred NOD ; MARVEL Domain Containing 2 Protein/metabolism* ; Humans ; Mice, Knockout ; Disease Models, Animal ; Interferon-gamma ; Salivary Glands/metabolism* ; Tight Junctions/metabolism* ; MicroRNAs/metabolism* ; Female

Objective To explore the regulatory effects of cytokines interleukin(IL)-1β,IL-6,tumor necrosis factor alpha(TNF-ɑ),gamma interferon(IFN-γ),granulocyte colony-stimulating factor(G-CSF),and granulocyte-macrophage colony-stimulating factor(GM-CSF)on spontaneous pyroptosis in neutrophils.Methods Neutrophils isolated from mouse bone marrow by density-gradient centrifugation were cultured <i>in vitroi> for 20 h with or without 10,50 or 100 ng/mL IL-1β,IL-6,IFN-γ,G-CSF or GM-CSF,or for 12 h with or without 1,10 or 50 ng/mL TNF-α.After incubation,cells were stained with annexin Ⅴ(AV)/propidium iodide(PI),and the proportions and absolute number of neutrophils undergoing different forms of cell death were determined by fluorescence microscopy combined with manual counting.Pyroptotic neutrophils were identified by cell morphology in conjunction with AV/PI staining.Flow cytometry with counting beads was employed to measure the proportions and number of AV/PI-stained Ly6g⁺neutrophils in different forms of cell death.Western blotting was employed to assess the cleavage and activation levels of cysteinyl aspartate-specific proteinase-3(caspase-3)and gasdermin E(GSDME).Results Treatment with IL-1β or IL-6 had no significant effect on the proportion or number of neutrophils undergoing spontaneous pyroptosis.After 12 h of treatment with TNF-α at 1,10,and 50 ng/mL,the proportions of pyroptotic neutrophils were(14.79±0.45)%,(19.99±3.02)%,and(20.66±1.99)%,respectively,higher than that[(10.22±1.12)%]in the untreated control(<i>Pi>=0.024,<i>Pi><0.001,and <i>Pi><0.001,respectively).Treatment with 10,50,and 100 ng/mL IFN-γ for 20 h reduced the proportion of pyroptotic neutrophils from(17.43±1.88)%to 12.00%(all <i>Pi><0.001).G-CSF at 10,50,and 100 ng/mL reduced the proportion of pyroptotic cells to around 6.00%and greatly inhibited the cleavage of both caspase-3 and GSDME.After 20 h of treatment with 10,50,and 100 ng/mL GM-CSF,the proportions of pyroptotic neutrophils decreased to(7.52±0.53)%,(5.27±2.30)%,and(0.64±1.11)%,respectively.Conclusions Neither IL-1β nor IL-6 affects GSDME-mediated spontaneous pyroptosis in neutrophils.TNF-ɑ induces spontaneous pyroptosis in neutrophils,whereas IFN-γ,G-CSF,and GM-CSF demonstrate inhibitory effects.
Pyroptosis/drug effects* ; Animals ; Neutrophils/cytology* ; Mice ; Cytokines/pharmacology* ; Interleukin-1beta/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Cells, Cultured ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Interferon-gamma/pharmacology* ; Interleukin-6/pharmacology*

The innate immune response is the first line of defense for the host against viral infections. Targeted degradation of pathogenic microorganisms through autophagy, in conjunction with pattern recognition receptors synergistically inducing the production of interferon (IFN), constitutes an important pathway for the body to resist viral infections. Rubicon, a Run domain Beclin 1-interacting and cysteine-rich domain protein, has an inhibitory effect on autophagy and IFN production. On the one hand, Rubicon, as a component of the phosphoinositide 3-kinase (PI3K) complex, interacts with different domains of vacuolar protein sorting 34 (Vps34), ultraviolet radiation resistance associated gene (UVRAG), guanosine triphosphate (GTP) kinase, and RAS oncogene family member 7 (Rab7) to mediate the inhibition of autophagy maturation; on the other hand, Rubicon inhibits the ubiquitination of nuclear factor κB essential modulator (NEMO) and the dimerization of interferon regulatory factor 3 (IRF3), thereby blocking the signal transduction related to IFN production. Research has revealed that various viruses, such as Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis B virus (HBV), Sendai virus (SeV), and hepatitis C virus (HCV), achieve innate immune evasion by regulating the expression or function of Rubicon. Rubicon is expected to be a new target for antiviral therapy.
Humans ; Autophagy/immunology* ; Immunity, Innate ; Interferons/immunology* ; Immune Evasion ; Animals ; Virus Diseases/virology* ; Signal Transduction ; Viruses/immunology* ; Intracellular Signaling Peptides and Proteins/immunology* ; Autophagy-Related Proteins

Objective To explore the effect of Evodiamine (Evo) on the immune function of allergic rhinitis (AR) rats and the regulatory mechanism on C-C motif chemokine ligand 2 (CCL2)/ C-C motif chemokine receptor 2 (CCR2) pathway. Methods The related targets of Evo-AR-immune function were screened by network pharmacology, and the protein interaction network diagram of intersecting targets was constructed. The AR rat model was established by ovalbumin (OVA) combined with aluminium hydroxide, and the rats were divided into six groups: a normal control (NC) group, a model group, a Loratadine (LOR) group, an Evodiamine low dose (Evo-L) group, a Evodiamine high dose (Evo-H) groups, and an Evo-H combined with CCL2 group. After the last administration, the symptoms of rats in each group were scored; ELISA was applied to detect the levels of histamine, immunoglobulin E (IgE), interleukin 4 (IL-4), IL-13 and interferon γ (IFN-γ); Diff-Quick staining solution was applied to detecte the number of cells in the nasal lavage fluid (NALF); hematoxylin eosin (HE) staining was applied to observe the pathological changes of nasal mucosa tissue; real-time quantitative PCR was applied to detect the levels of CCL2 and CCR2 mRNA in tissue; Western blot was applied to detect the expression levels of CCL2, CCR2 and CXC motif chemokine ligand 8 (CXCL8) proteins in nasal mucosa. Results There were eight intersection targets of EVo-AR-immune function, and protein interaction network diagram showed that CXCL8 was the core target. Compared with the NC group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in the model group were increased, while the level of IFN-γ was decreased. Compared with the model group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in LOR and Evo groups were decreased, while the level of IFN-γ was increased. Further use of CCL2 recombinant protein for compensatory experiments revealed that the improvement effect of Evo on immune function in AR rats was reversed by CCL2. Conclusion Evo can improve the immune function of AR rats, and its mechanism may be related to the inhibition of the CCL2/CCR2 pathway.
Animals ; Receptors, CCR2/immunology* ; Signal Transduction/drug effects* ; Chemokine CCL2/immunology* ; Rats ; Rhinitis, Allergic/metabolism* ; Immunoglobulin E/blood* ; Quinazolines/pharmacology* ; Male ; Interferon-gamma ; Rats, Sprague-Dawley ; Interleukin-13 ; Histamine ; Interleukin-4/immunology* ; Disease Models, Animal

Objective To explore the effect of miR-582-5p on Mycobacterium tuberculosis (Mtb)-infected macrophages by regulating dual specificity phosphatase 1 (DUSP1). Methods THP-1 macrophages were divided into six groups: control group, Mtb group, inhibitor-NC group, miR-582-5p inhibitor group, miR-582-5p inhibitor+si-NC group, and miR-582-5p inhibitor+si-DUSP1 group. QRT-PCR was applied to detect the gene expression of miR-582-5p and DUSP1 in cells. ELISA kit was used to detect the levels of interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). CCK-8 method was applied to detect cell proliferation. Flow cytometry was applied to detect cell apoptosis rate. Western blot analysis was used to measure the protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X (BAX), and cleaved-caspase 3 (c-caspase-3) in cells. In addition, the target relationship between miR-582-5p and DUSP1 was verified. Results Compared with the control group, the expression of miR-582-5p, levels of IFN-γ, IL-6, TNF-α, IL-1β, bacterial load and OD450 values (24 h, 48 h), and the protein expression of Bcl2 in macrophages were higher in the Mtb group, while the mRNA expression of DUSP1, apoptosis rate, and the protein expression levels of c-caspase-3, BAX and DUSP1 were lower. Compared with the Mtb group and the inhibitor-NC group, the above-mentioned indicators in the miR-582-5p inhibitor group were partially reversed. Down-regulation of DUSP1 expression partially reversed the inhibitory effect of down-regulation of miR-582-5p expression on Mtb-infected macrophages. Conclusion Inhibiting the expression of miR-582-5p can up-regulate DUSP1, thereby inhibiting the proliferation and inflammatory response of Mtb-infected macrophages and promoting cell apoptosis.
Humans ; Macrophages/metabolism* ; Dual Specificity Phosphatase 1/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Tuberculosis/microbiology* ; Apoptosis/genetics* ; THP-1 Cells ; Cell Proliferation/genetics* ; Interferon-gamma/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics*

Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

Objectives To investigate interleukin 36γ (IL-36γ) expression, and analyze the influence of IL-36γ to CD8⁺ T cell activity in chronic obstructive pulmonary diseases (COPD) patients with secondary fungal pneumonia. Methods Peripheral blood was collected from 47 COPD patients, 39 COPD patients with secondary fungal pneumonia, and 20 controls. Bronchial alveolar lavage fluid (BALF) was isolated from 27 COPD patients with secondary fungal pneumonia. CD8⁺ T cells were purified. The levels of four IL-36 isoforms in plasma and BALF were measured by enzyme linked immunosorbent assay (ELISA). CD8⁺ T cells were stimulated with recombinant human IL-36γ. The levels of interferon γ(IFN-γ), tumor necrosis factor α(TNF-α), perforin and granzyme B in the cultured supernatants were measured by ELISA. Recombinant human IL-36γ-stimulated CD8⁺ T cells were co-cultured with NCI-H1882 cells in either direct cell-to-cell contact or Transwell^TM manner. The levels of IFN-γ, TNF-α, and lactate dehydrogenase in the cultured supernatants were assessed. The percentage of target cell death was calculated. Results Plasma IL-36α, IL-36β, and IL-36γ levels were significantly elevated in both COPD group and COPD with secondary fungal pneumonia group compared with those in control group. However, only plasma IL-36γ level was higher in COPD with secondary fungal pneumonia group than that in COPD group [(200.11±99.95)pg/mL vs (53.03±87.18)pg/mL, P=0.023]. There was no remarkable difference in plasma IL-36 receptor antagonist level among three groups. IL-36γ level in BALF from infectious site was higher than that from non-infectious site in COPD with secondary fungal pneumonia group [(305.82±59.60)pg/mL vs (251.93±76.01)pg/mL, P=0.011]. IL-36γ stimulation enhanced IFN-γ, TNF-α, perforin and granzyme B secreted by CD8⁺ T cells. When IL-36γ-stimulated CD8⁺ T cells were directly mixed with NCI-H1882 cells for co-culture, the percentage of cell death was increased [(16.06±3.67)% vs (11.47±2.36)%, P=0.002]. When using Transwell^TM plate for non-contact co-culture, IL-36γ-stimulated CD8⁺ T cell-mediated death of NCI-H1882 cells showed no significant difference compared to that without stimulation [(4.77±0.78)% vs (4.99±0.92)%, P=0.554]. Conclusion IL-36γ level in plasma and infectious site is elevated in COPD patients with secondary fungal pneumonia, which enhances the cytotoxicity of CD8⁺ T cells in peripheral blood and infectious microenviroment.
Humans ; Pulmonary Disease, Chronic Obstructive/complications* ; CD8-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Aged ; Middle Aged ; Interferon-gamma/metabolism* ; Interleukin-1/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Lung Diseases, Fungal/complications* ; Bronchoalveolar Lavage Fluid/chemistry* ; Perforin/metabolism* ; Pneumonia/immunology* ; Granzymes/metabolism*