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*

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 screen anti-tumor drugs that improve antigen processing and presentation in acute myeloid leukemia (AML) cells. METHODS: A TCR-like or TCR mimic antibody that can specifically recognize HLA-A*0201:WT1_126-134 ( RMFPNAPYL) complex (hereafter referred to as HLA-A2:WT1) was synthesized to evaluate the function of antigen processing and presentation machinery (APM) in AML cells. AML cell line THP1 was incubated with increasing concentrations of IFN-γ, hypomethylating agents (HMA), immunomodulatory drugs (IMiD), proteasome inhibitors (PI) and γ-secretase inhibitors (GSI), followed by measuring of HLA-ABC, HLA-A2 and HLA-A2:WT1 levels by flow cytometry at consecutive time points. RESULTS: The TCR-like antibody we generated only binds to HLA-A*0201⁺WT1⁺ cells, indicating the specificity of the antibody. HLA-A2:WT1 level of THP-1 cells detected with the TCR-like antibody was increased significantly after co-incubation with IFN-γ, showing that the HLA-A2:WT1 TCR like antibody could evaluate the function of APM. Among the anti-tumor agents screened in this study, GSI (LY-411575) and HMA (decitabine and azacitidine) could significantly increase the HLA-A2:WT1 level. The IMiD lenalidomide and pomalidomide could aslo upregulate the expression of HLA-A2:WT1 complex under certain concentrations of the drugs and incubation time. As proteasome inhibitors, carfilzomib could significantly decreased the expression of HLA-A2:WT1, while bortezomib had no significant effect on HLA-A2:WT1 expression. CONCLUSION HLA-A2:WT1 TCR-like antibody can effectively reflect the APM function. Some of the anti-tumor drugs can affect the APM function and immunogenicity of tumor cells.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Antineoplastic Agents/pharmacology* ; Antigen Presentation/drug effects* ; HLA-A2 Antigen/immunology* ; Receptors, Antigen, T-Cell/immunology* ; Cell Line, Tumor ; Interferon-gamma

Ossification of the posterior longitudinal ligament (OPLL) is a condition comprising ectopic bone formation from spinal ligaments. This disease is a leading cause of myelopathy in the Asian population. However, the molecular mechanism underlying OPLL and efficient preventive interventions remain unclear. Here, we performed single-cell RNA sequencing and revealed that type I interferon (IFN) signaling was activated in the ossified ligament of patients with OPLL. We also observed that IFN-β stimulation promoted the osteogenic differentiation of preosteoblasts in vitro and activated the ossification-related gene SPP1, thereby confirming the single-cell RNA sequencing findings. Further, blocking the IFN-α/β subunit 1 receptor (IFNAR1) using an anti-IFNAR1 neutralizing antibody markedly suppressed osteogenic differentiation. Together, these results demonstrated that the type I IFN signaling pathway facilitated ligament ossification, and the blockade of this signaling might provide a foundation for the prevention of OPLL.
Humans ; Signal Transduction ; Interferon Type I/metabolism* ; Ossification of Posterior Longitudinal Ligament/genetics* ; Gene Expression Profiling ; Single-Cell Analysis ; Osteogenesis/genetics* ; Receptor, Interferon alpha-beta/metabolism* ; Male ; Female ; Cell Differentiation ; Middle Aged

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.
Humans ; HEK293 Cells ; CRISPR-Cas Systems ; DEAD Box Protein 58/metabolism* ; Signal Transduction ; Receptors, Immunologic/metabolism* ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/metabolism* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Interferon-Induced Helicase, IFIH1/metabolism* ; Transcription Factor RelA/metabolism* ; Interferon-beta/metabolism*

OBJECTIVE: KRAS gene is one of the most common mutations of proto-oncogenes in human tumors, G12V is one of the most common mutation types for KRAS. It's challenging to chemically acquire the targeted drug for this mutation. Recent studies reported that this mutation peptides can form a neoepitope for T cell recognition. Our study aims to clone the T cell receptor (TCR) which specifically recognizes the neoepitope for KRAS G12V mutation and constructs TCR engineered T cells (TCR-T), and to investigate if TCR-Ts have strong antitumor response ability. METHODS: In this study, tumor infiltrating lymphocytes were obtained from one colorectal cancer patient carrying KRAS G12V mutation. Tumor-reactive TCR was obtained by single-cell RT-5' rapid-amplification of cDNA ends PCR analysis and introduced into peripheral blood lymphocytes to generate TCR-Ts. RESULTS: We obtained a high-affinity TCR sequence that specifically recognized the HLA-A^*11:01-restricted KRAS G12V_8-16 epitope: KVA11-01. KVA11-01 TCR-T could significantly kill various tumor cells such as PANC-1, SW480 and HeLa (overexpressing HLA-A^*11:01 and KRAS G12V), and secreting high levels of interferon-γ (IFN-γ). Non-specific killing experiments suggested KVA11-01 specifically recognized tumor cells expressing both mutant KRAS G12V and HLA-A^*11:01. In vivo assay, tumor inhibition experiments demonstrated that infusion of approximately 1E7 KVA11-01 TCR-T could significantly inhibit the growth of subcuta-neously transplanted tumors of PANC-1 and HeLa (overexpressing HLA-A^*11:01 and KRAS G12V) cells in nude mice. No destruction of the morphologies of the liver, spleen and brain were observed. We also found that KVA11-01 TCR-T could significantly infiltrate into tumor tissue and had a better homing ability. CONCLUSION KVA11-01 TCR-T cells can effectively target a variety of malignant tumor cells carrying KRAS G12V mutation through in vitro and in vivo assay. KVA11-01 TCR-T cells have excellent biological activity, high specificity of target antigen and homing ability into solid tumor tissue. KVA11-01 TCR-T is expected to be an effective treatment for patients with KRAS G12V mutant solid malignancies.
Animals ; DNA, Complementary ; Epitopes ; HLA-A Antigens ; Humans ; Interferon-gamma ; Mice ; Mice, Nude ; Mutation ; Neoplasms ; Proto-Oncogene Proteins p21(ras)/genetics* ; Receptors, Antigen, T-Cell/genetics*

The aim of this paper was to study the influence of triptolide in the immune response pathways of acquired immune deficiency syndrome( AIDS). Target proteins of triptolide and related genes of AIDS were searched in PubChem and Gene databases on line. Molecular networks and canonical pathways comparison analyses were performed by bioinformatics software( IPA). There were 15 targets proteins of triptolide and 258 related genes of AIDS. Close biological relationships of molecules of triptolide and AIDS were established by networks analysis. There were 21 common immune response pathways of triptolide and AIDS,including neuroinflammation signaling pathway,Th1 and Th2 activation pathway and role of pattern recognition receptors in recognition of bacteria and viruses. Triptolide stimulated immune response pathways by the main molecules of IFNγ,JAK2,NOD1,PTGS2,RORC. IFNγ is the focus nodes of triptolide and AIDS,and regulates genes of AIDS directly or indirectly. Triptolide may against AIDS by regulating molecules IFNγ in immune response pathways.
Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; Computational Biology ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Gene Regulatory Networks ; Humans ; Interferon-gamma ; genetics ; Phenanthrenes ; pharmacology ; Receptors, Pattern Recognition ; immunology ; Signal Transduction ; T-Lymphocytes ; immunology

Imiquimod, a toll-like receptor agonist, is a topical immunomodulator that induces the production of several cytokines including interferon-alpha, which shows antifibrotic properties. We hypothesized that the antifibrotic effect of imiquimod would soften fibrotic skin lesions. Therefore, we applied topical imiquimod with topical tacrolimus or systemic acitretin in patients with refractory lesions of myxedema, angiolymphoid hyperplasia, and generalized discoid lupus erythematosus and confirmed improvement in fibrotic lesions in these patients. Thus, we conclude that use of imiquimod improves fibrotic skin lesions. This report describes our experience with the treatment of this condition along with a review of the related literature.
Acitretin ; Angiolymphoid Hyperplasia with Eosinophilia ; Cytokines ; Fibrosis ; Humans ; Hyperplasia ; Interferon-alpha ; Lupus Erythematosus, Discoid ; Myxedema ; Skin ; Tacrolimus ; Toll-Like Receptors

Objective To analyze the levels of cytokines (IL-2,IFN-γ,IL-6,IL-10) associated with Th1 and Th2 cells in HLA-DQ8 transgenic mice model of ocular experimental autoimmune myasthenia gravis (oEAMG) induced by recombinant H-AChR γ subunit immunization.Methods DQ8 mice were immunized with 20 μg of AChR γ subunit,20 μg of crude E. coli extract (E. coli group),or complete Freund's adjuvant (CFA) only (CFA group). All mice were immunized on days 0,30,and 60. Mice were euthanized 28 days after the third immunization,and draining lymph node cells (LNC) and spleen lymphocytes were cultured in vitro. The supernatant was collected to observe the interleukin(IL)-2,interferon(IFN)-γ,IL-6,IL-10 production by ELISA.Results LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice exhibited significantly enhanced IFN-γ (F=76.332,P<0.001;F=34.865,P<0.001) and IL-2 (F=42.835,P<0.001;F=38.030,P<0.001),which associated with Th1 cells,as compared to E. coli group and CFA group. There were no significant differences in IL-6 (F=1.325,P=0.284;F=1.935,P=0.166) and IL-10 (F=0.908,P=0.417;F=1.189,P=0.322) levels,which secreted by Th2 cells,among these three groups.Conclusion Th1 cytokines play key roles in the pathogenesis of oEAMG,while the mechanism of Th2 cytokines for oEAMG remains unclear.
Animals ; Cytokines ; Escherichia coli ; HLA-DQ Antigens ; Interferon-gamma ; Mice ; Mice, Transgenic ; Receptors, Cholinergic ; Th1 Cells ; Th2 Cells

Aryl hydrocarbon receptor (AhR) regulates both innate and adaptive immune responses by sensing a variety of small synthetic and natural chemicals, which act as its ligands. AhR, which is expressed in dendritic cells (DCs), regulates the differentiation of DCs. However, effects of AhR on the differentiation of DCs are variable due to the heterogeneity of DCs in cell surface marker expression, anatomical location, and functional responses. The plasmacytoid DCs (pDCs), one of DC subsets, not only induce innate as well as adaptive immune responses by secreting type I interferons and pro-inflammatory cytokines, but also induce IL-10 producing regulatory T cell or anergy or deletion of antigen-specific T cells. We showed here that AhR ligands indoxyl 3-sulfate (I3S) and indole-3-carbinol (I3C) inhibited the development of pDCs derived from bone marrow (BM) precursors induced by FMS-like tyrosine kinase 3 ligand (Flt3L). I3S and I3C downregulated the expression of signal transducer and activator of transcription 3 (STAT3) and E2-2 (Tcf4). In mice orally treated with I3S and I3C, oral tolerance to dinitrofluorobenzene was impaired and the proportion of CD11c⁺B220⁺ cells in mesenteric lymph nodes was reduced. These data demonstrate that AhR negatively regulates the development of pDCs from BM precursors induced by Flt3L, probably via repressing the expression of STAT3.
Animals ; Bone Marrow ; Cell Differentiation ; Cytokines ; Dendritic Cells* ; Dinitrofluorobenzene ; fms-Like Tyrosine Kinase 3* ; Immune Tolerance ; Interferon Type I ; Interleukin-10 ; Ligands* ; Lymph Nodes ; Mice ; Population Characteristics ; Receptors, Aryl Hydrocarbon* ; STAT3 Transcription Factor ; T-Lymphocytes ; Vascular Endothelial Growth Factor Receptor-1*