CXCR3 deficiency decreases autoantibody production by inhibiting aberrant activated T follicular helper cells and B cells in lupus mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a high level of autoantibody production. T follicular helper (Tfh) cells and B cells participate in the development of SLE. Several studies have shown that CXCR3+ cells are increased in SLE patients. However, the mechanism through which CXCR3 influences lupus development remains unclear. In this study, we established lupus models to determine the role of CXCR3 in lupus pathogenesis. The concentration of autoantibodies was detected using the enzyme-linked immunosorbent assay (ELISA), and the percentages of Tfh cells and B cells were measured using flow cytometry. RNA sequencing (RNA-seq) was performed to detect the differentially expressed genes in CD4+ T cells from wild-type (WT) and CXCR3 knock-out (KO) lupus mice. Migration of CD4+ T cells in spleen section was assessed using immunofluorescence. CD4+ T cell function in helping B cells produce antibodies was determined using a co-culture experiment and supernatant IgG ELISA. Lupus mice were treated with a CXCR3 antagonist to confirm the therapeutic effects. We found that the expression of CXCR3 was increased in CD4+ T cells from lupus mice. CXCR3 deficiency reduced autoantibody production with decreased proportions of Tfh cells, germinal center (GC) B cells, and plasma cells. Expression of Tfh-related genes was downregulated in CD4+ T cells from CXCR3 KO lupus mice. Migration to B cell follicles and T-helper function of CD4+ T cells were reduced in CXCR3 KO lupus mice. CXCR3 antagonist AMG487 decreased the level of serum anti-dsDNA IgG in lupus mice. We clarify that CXCR3 may play an important role in autoantibody production by increasing the percentages of aberrant activated Tfh cells and B cells and promoting the migration and T-helper function of CD4+ T cells in lupus mice. Thus, CXCR3 may be a potential target for lupus therapy.

PRMT5 disruption drives antitumor immunity in cervical cancer by reprogramming T cell-mediated response and regulating PD-L1 expression.

Rationale: Protein arginine methyltransferase 5 (PRMT5) is an oncogene that promotes tumor cell proliferation, invasion and metastasis. However, the underlying mechanisms by which PRMT5 contributes to the progression of cervical cancer and especially the tumor microenvironment remain poorly understood. Methods: PRMT5 expression level was analyzed by Q-PCR, western blot, immunohistochemistry, and TCGA database. The role of PRMT5 in tumor growth was observed by transplanted tumor models, and the function of T cells in tumor microenvironment and in vitro co-culture system was investigated through flow cytometry. The transcriptional regulation of PRMT5 was analyzed using luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The therapeutic effect of PRMT5 inhibitor was evaluated in a cervical cancer cell line transplanted tumor model. Results: We observed that the mRNA and protein expression levels of PRMT5 were increased in cervical cancer tissues, and the high expression of PRMT5 was associated with poor outcomes in cervical cancer patients. The absence of PRMT5 significantly inhibited tumor growth in a cervical cancer transplanted tumor model, and importantly, PRMT5 absence in tumors led to increase the number and enhance the function of tumor infiltrating T cells. Mechanistically, PRMT5 enhanced the transcription of STAT1 through symmetric dimethylation of histone H3R2 and thus promoted PD-L1 expression in cervical cancer cells. Moreover, in an in vitro co-culture system, knockdown of PRMT5 in tumor cells could directly enhance the expression of IFN-γ, TNF-α and granzyme B in T cells. These results suggested that PRMT5 promoted the development of cervical cancer by the crosstalk between tumor cells and T cells. Furthermore, the PRMT5 inhibitor EPZ015666 treatment could suppress tumor growth in a cervical cancer transplanted tumor model. Conclusion: Our results clarify a new mechanism which PRMT5 knockdown in cervical cancer cells drives an antitumor function via reprogramming T cell-mediated response and regulating PD-L1 expression. Thus, our study highlights that PRMT5 may be a potential target for cervical cancer therapy.

Dual effect of IL-7/IL-7R signalling on the osteoimmunological system: a potential therapeutic target for rheumatoid arthritis.

The IL-7/IL-7R pathway plays a vital role in the immune system, especially in the inflammatory response. Monocytes/macrophages (osteoclast precursors) have been recently recognized as important participants in the osteoclastogenesis of rheumatoid arthritis (RA) patients. Here, we aimed to investigate the therapeutic potential of IL-7/IL-7R pathway in RA and to determine whether it could restrain osteoclastogenic functions and therefore ameliorate RA. Firstly, collagen-induced arthritis (CIA) mice were administered with IL-7Rα-target antibodies to assess their therapeutic effect on arthritis. We found that blockade of the IL-7/IL-7R pathway protected CIA mice from bone destruction in addition to inducing inflammatory remission, by altering the RANKL/RANK/OPG ratio and consequently decreasing osteoclast formation. To explore the effect and mechanism of this pathway, bone marrow cells were induced to osteoclasts and treated with IL-7, a STAT5 inhibitor or supernatants from T cells. The results showed that the IL-7/IL-7R pathway played a direct inhibitory role in osteoclast differentiation via STAT5 signalling pathway in a RANKL-induced manner. We applied flow cytometry to analyse the effect of IL-7 on T-cell RANKL expression and found that IL-7/IL-7R pathway had an indirect role in the osteoclast differentiation process by enhancing the RANKL expression on T cells. In conclusion, the IL-7/IL-7R pathway exhibited a dual effect on osteoclastogenesis of CIA mice by interacting with osteoimmunology processes and could be a novel therapeutic target for autoimmune diseases such as RA.

Salubrinal Alleviates Collagen-Induced Arthritis through Promoting P65 Degradation in Osteoclastogenesis.

Rheumatoid arthritis (RA) is a complex systemic autoimmune disorder that primarily involves joints, further affects the life quality of patients, and has increased mortality. The pathogenesis of RA involves multiple pathways, resulting in some patients showing resistance to the existing drugs. Salubrinal is a small molecule compound that has recently been shown to exert multiple beneficial effects on bone tissue. However, the effect of Salubrinal in RA has not been clearly confirmed. Hence, we induced collagen-induced arthritis (CIA) in DBA/1J mice and found that Salubrinal treatment decreased the clinical score of CIA mice, inhibiting joint damage and bone destruction. Furthermore, Salubrinal treatment downregulated osteoclast number in knee joint of CIA in mice, and suppressed bone marrow-derived osteoclast formation and function, downregulated osteoclast-related gene expression. Moreover, Salubrinal treatment inhibited RANKL-induced NF-κB signaling pathway, and promoted P65 degradation through the ubiquitin-proteasome system, further restrained RANKL-induced osteoclastogenesis. This study explains the mechanism by which Salubrinal ameliorates arthritis of CIA in mice, indicating that Salubrinal may be a potential drug for RA, and expands the potential uses of Salubrinal in the treatment of bone destruction-related diseases.

Protein arginine methyltransferase 5: a potential cancer therapeutic target.

BACKGROUND: PRMT5 is a type II protein arginine methyltransferase that methylates histone or non-histone proteins. Arginine methylation by PRMT5 has been implicated in gene transcription, ribosome biogenesis, RNA transport, pre-mRNA splicing and signal transduction. High expression of PRMT5 has been observed in various cancers and PRMT5 overexpression has been reported to improve cancer cell survival, proliferation, migration and metabolism and to inhibit cancer cell apoptosis. In addition, PRMT5 has been found to be required for cancer stem cell survival, self-renewal and differentiation. Several microRNAs have been shown to regulate PRMT5 expression. As PRMT5 has oncogene-like properties, several PRMT5 inhibitors have been used to explore their efficacy as potential drugs for different types of cancer, and three of them are now being tested in clinical trials. CONCLUSIONS: In this review, we summarize current knowledge on the role of PRMT5 in cancer development and progression, including its functions and underlying mechanisms. In addition, we highlight the rapid development of PRMT5 inhibitors and summarize ongoing clinical trials for cancer therapy. By affecting both tumor cells and the tumor microenvironment, PRMT5 inhibitors may serve as effective anti-cancer agents, especially when combined with immune therapies.

PD-1 and PD-L1 in cancer immunotherapy: clinical implications and future considerations.

Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling pathways and inhibits T cell activation. Moreover abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape, and the development of anti-PD-1/PD-L1 antibodies has recently become a hot topic in cancer immunotherapy. Here, we review the structure of PD-1 and PD-L1, the function of the PD-1/PD-L1 signaling pathway, the application of PD-1 or PD-L1 monoclonal antibodies and future directions for anti-PD-1/PD-L1 antibodies with combination therapies. Cancer immunotherapy using PD-1/PD-L1 immune checkpoint blockade may require more studies, and this approach may be curative for patients with many types of cancer in the future.

Paeoniflorin ameliorates collagen-induced arthritis via suppressing nuclear factor-κB signalling pathway in osteoclast differentiation.

Paeoniflorin (PF), extracted from the root of Paeonia lactiflora Pall, exhibits anti-inflammatory properties in several autoimmune diseases. Osteoclast, the only somatic cell with bone resorbing capacity, was the direct cause of bone destruction in rheumatoid arthritis (RA) and its mouse model, collagen-induced arthritis (CIA). The objective of this study was to estimate the effect of PF on CIA mice, and explore the mechanism of PF in bone destruction. We demonstrated that PF treatment significantly ameliorated CIA through inflammatory response inhibition and bone destruction suppression. Furthermore, PF treatment markedly decreased osteoclast number through the altered RANKL/RANK/OPG ratio and inflammatory cytokines profile. Consistently, we found that osteoclast differentiation was significantly inhibited by PF through down-regulation of nuclear factor-κB activation in vitro. Moreover, we found that PF suppressed nuclear factor-κB activation by decreasing its translocation to the nucleus in osteoclast precursor cells. Taken together, our new findings provide insights into a novel function of PF in osteoclastogenesis and demonstrate that PF would be a new therapeutic modality as a natural agent for RA treatment and other autoimmune conditions with bone erosion.

Increased frequency of circulating Tfh cells in patients with acute pancreatitis.

Considered as true helper cells for B cells in antibody response, Tfh cells are associated with inflammation and immune abnormality. Acute pancreatitis is an acute abdominal disease characterized by inflammatory response and immune disorder. Thus, our objective was to study the frequency of circulating Tfh cells, together with the Tfh cell-related CD4+ T cells and inflammatory factors in patients with acute pancreatitis. We first examined the frequency of circulating Tfh cell subsets by detecting the expression of CXCR5, PD-1, ICOS and IL-21 by flow cytometry analysis. Then we investigated the abundance of helper T cells and Treg cells. In addition, the plasma level of IgA, IgM, and Tfh cell-related inflammatory factor were detected by cytometric bead array. We showed that the frequency of circulating Tfh cells increased significantly in patients of acute pancreatitis, including CD4+CXCR5+ cells, CD4+CXCR5+PD-1+ cells, CD4+CXCR5+ICOS+ cells, and CD4+CXCR5+IL-21+ cells. Also, increases in plasma IL-1, IL-6, IL-8, IL-17, IL-21 and IgA were observed in patients with acute pancreatitis compared to HCs. This finding indicates that Tfh cells play a vital role in the development and progression of acute pancreatitis that is dependent on IL-6 and IL-21.

Paeoniflorin Ameliorates Experimental Autoimmune Encephalomyelitis via Inhibition of Dendritic Cell Function and Th17 Cell Differentiation.

Paeoniflorin (PF) is a monoterpene glycoside and exhibits multiple effects, including anti-inflammation and immunoregulation. To date, the effect of PF on multiple sclerosis (MS) has not been investigated. In this study, we investigated the effect of PF in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. After administered with PF, the onset and clinical symptoms of EAE mice were significantly ameliorated, and the number of Th17 cells infiltrated in central nervous system (CNS) and spleen was also dramatically decreased. Instead of inhibiting the differentiation of Th17 cells directly, PF influenced Th17 cells via suppressing the expression of costimulatory molecules and the production of interlukin-6 (IL-6) of dendritic cells (DCs) in vivo and in vitro, which may be attributable to the inhibition of IKK/NF-κB and JNK signaling pathway. When naïve CD4+ T cells were co-cultured with PF-treated dendritic cells under Th17-polarizing condition, the percentage of Th17 cells and the phosphorylation of STAT3 were decreased, as well as the mRNA levels of IL-17, RORα, and RORγt. Our study provided insights into the role of PF as a unique therapeutic agent for the treatment of multiple sclerosis and illustrated the underlying mechanism of PF from a new perspective.

Characterization of Sin1 Isoforms Reveals an mTOR-Dependent and Independent Function of Sin1γ.

Sin1 or MAPKAP1 is a key component of mTORC2 signaling complex which is necessary for AKT phosphorylation at the S473 and T450 sites, and also for AKT downstream signaling as well. A number of Sin1 splicing variants have been reported that can produce different Sin1 isoforms due to exon skipping or alternative transcription initiation. In this report, we characterized four Sin1 isoforms, including a novel Sin1 isoform due to alternative 3' termination of the exon 9a, termed Sin1γ. Sin1γ expression can be detected in multiple adult mouse tissues, and it encodes a C-terminal truncated protein comparing to the full length Sin1ß isoform. In contrast to Sin1ß, Sin1γ overexpression in Sin1 deficient mouse embryonic fibroblasts has no significant impact on mTORC2 activity or mTORC2 subunits protein level, although it still can interact with mTORC2 components. More interestingly, Sin1γ was detected in a specific cytosolic location with a distinct feature in structure, and its localization was transiently disrupted during cell cycle. Therefore, Sin1γ is a novel Sin1 isoform and may have distinct properties in cell signaling and intracellular localization from other Sin1 isoforms.

Pygo2 associates with MLL2 histone methyltransferase and GCN5 histone acetyltransferase complexes to augment Wnt target gene expression and breast cancer stem-like cell expansion.

Resent studies have identified Pygopus as a core component of the ß-catenin/T-cell factor (TCF)/lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in ß-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with ß-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wnt-dependent transcriptional program and cancer stem-like cell expansion.

Cytokeratin expression during mouse embryonic and early postnatal mammary gland development.

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6(+) and K8(+)/K14(+) putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation.

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain-containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/beta-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues beta-catenin-induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/beta-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

Abnormal expression, highly efficient detection and novel truncations of midkine in human tumors, cancers and cell lines.

We detected aberrant Midkine (MK) expressions in human insulinoma and pancreatic cancer tissues by immunohistochemistry, revealing its potential role in tumorigenesis/carcinogenesis. With a nested-touchdown PCR program we were able to detect the tMK in all human tumor/cancer tissues and cancer/tumor cell lines. Detection of MK in the peripheral cells and precancerous lesions implies its potential for early cancer/tumor diagnosis. Furthermore, we have discovered two novel truncations of the MK, tMKB and tMKC, respectively, in the disease specimens. Our data not only provide an efficient methodology potentially for clinical application but also shed light on the molecular mechanism underlying the role for MK in tumorigenesis/carcinogenesis.

Transiently truncated and differentially regulated expression of midkine during mouse embryogenesis.

Midkine (MK) is a retinoic acid response cytokine, mostly expressed in embryonic tissues. Aberrant expression of MK was found in numerous cancers. In human, a truncated MK was expressed specifically in tumor/cancer tissues. Here we report the discovery of a novel truncated form of MK transiently expressed during normal mouse embryonic development. In addition, MK is concentrated at the interface between developing epithelium and mesenchyme as well as highly proliferating cells. Its expression, which is closely coordinated with angiogenesis and vasculogenesis, is spatiotemporally regulated with peaks in extensive organogenesis period and undifferentiated cells tailing off in maturing cells, implying its role in nascent blood vessel (endothelial) signaling of tissue differentiation and stem cell renewal/differentiation. Cloning and sequencing analysis revealed that the embryonic truncated MK, in which the conserved domain is in-frame deleted, presumably producing a novel secreted small peptide, is different from the truncated form in human cancer tissues, whose deletion results in a frame-shift mutation. Our data suggest that MK may play a role in epithelium-mesenchyme interactions, blood vessel signaling, and the decision of proliferation vs differentiation. Detection of the transiently expressed truncated MK reveals its novel function in development and sheds light on its role in carcinogenesis.