Targeting kinase ITK treats autoimmune arthritis via orchestrating T cell differentiation and function.

IL-2 inducible T cell kinase (ITK) is critical in T helper subset differentiation and its inhibition has been suggested for the treatment of T cell-mediated inflammatory diseases. T follicular helper (Tfh), Th17 and regulatory T cells (Treg) also play important roles in the development of rheumatoid arthritis (RA), while the role of ITK in the development of RA and the intricate balance between effector T and regulatory T cells remains unclear. Here, we found that CD4+ T cells from RA patients presented with an elevated ITK activation. ITK inhibitor alleviated existing collagen-induced arthritis (CIA) and reduced antigen specific antibody production. Blocking ITK kinase activity interferes Tfh cell generation. Moreover, ITK inhibitor effectively rebalances Th17 and Treg cells by regulating Foxo1 translocation. Furthermore, we identified dihydroartemisinin (DHA) as a potential ITK inhibitor, which could inhibit PLC-γ1 phosphorylation and the progression of CIA by rebalancing Th17 and Treg cells. Out data imply that ITK activation is upregulated in RA patients, and therefore blocking ITK signal may provide an effective strategy to treat RA patients and highlight the role of ITK on the Tfh induction and RA progression.

Neuropilin-1 Identifies a New Subpopulation of TGF-ß-Induced Foxp3+ Regulatory T Cells With Potent Suppressive Function and Enhanced Stability During Inflammation.

CD4+Foxp3+ regulatory T cells (Tregs) play a crucial role in preventing autoimmunity and inflammation. There are naturally-derived in the thymus (tTreg), generated extrathymically in the periphery (pTreg), and induced in vitro culture (iTreg) with different characteristics of suppressiveness, stability, and plasticity. There is an abundance of published data on neuropilin-1 (Nrp-1) as a tTreg marker, but little data exist on iTreg. The fidelity of Nrp-1 as a tTreg marker and its role in iTreg remains to be explored. This study found that Nrp-1 was expressed by a subset of Foxp3+CD4+T cells in the central and peripheral lymphoid organs in intact mice, as well as in iTreg. Nrp-1+iTreg and Nrp-1-iTreg were adoptively transferred into a T cell-mediated colitis model to determine their ability to suppress inflammation. Differences in gene expression between Nrp-1+ and Nrp-1-iTreg were analyzed by RNA sequencing. We demonstrated that the Nrp-1+ subset of the iTreg exhibited enhanced suppressive function and stability compared to the Nrp-1- counterpart both in vivo and in vitro, partly depending on IL-10. We found that Nrp-1 is not an exclusive marker of tTreg, however, it is a biomarker identifying a new subset of iTreg with enhanced suppressive function, implicating a potential for Nrp-1+iTreg cell therapy for autoimmune and inflammatory diseases.

Induced, but not natural, regulatory T cells retain phenotype and function following exposure to inflamed synovial fibroblasts.

Aberrant number and/or dysfunction of CD4+Foxp3+ Regulatory T cells (Tregs) are associated with the pathogenesis of rheumatoid arthritis (RA). A previous study has demonstrated that thymus-derived, natural Tregs (nTregs) prefer to accumulate in inflamed joints and transdifferentiate to TH17 cells under the stimulation of inflamed synovial fibroblasts (SFs). In this study, we made a head-to-head comparison of both Treg subsets and demonstrated that induced Tregs (iTregs), but not nTregs, retained Foxp3 expression and regulatory function on T effector cells (Teffs) after being primed with inflamed SFs. In addition, iTregs inhibited proliferation, inflammatory cytokine production, migration, and invasion ability of collagen-induced arthritis (CIA)-SFs in vitro and in vivo. Moreover, we noted that iTregs directly targeted inflamed SFs to treat autoimmune arthritis, while nTregs failed to do this. Thus, manipulation of the iTreg subset may have a greater potential for prevention or treatment of patients with RA.

lncRNA-PDPK2P promotes hepatocellular carcinoma progression through the PDK1/AKT/Caspase 3 pathway.

Hepatocellular carcinoma (HCC) is a malignancy with one of the worst prognoses. Long noncoding RNA (lncRNA) are emerging as an important regulator of gene expression and function, leading to the development of cancer. The aim of this study was to determine the relationship between lncRNA and HCC and to further guide clinical therapy. lncRNA in HCC and adjacent tissues were screened, and the correlation between lncRNA-PDPK2P expression in liver tissues and the pathological characteristics and severity of HCC was assessed. The effects of PDPK2P on HCC proliferation, apoptosis, metastasis, and invasion were also systematically investigated via CCK-8 assay, flow cytometry, scratch wound healing, and transwell assay, respectively. The relationship between PDPK2P and PDK1 was verified by RNA pull-down, rescue experiments and western blot. lncRNA-PDPK2P was highly expressed in HCC tissues with a distinct positive correlation between PDPK2P and PDK1, and the upregulation was clinically associated with a larger tumor embolus, low differentiation, and poor survival. Mechanistically, lncRNA-PDPK2P interacted with PDK1 and promoted HCC progression through the PDK1/AKT/caspase 3 signaling pathway. lncRNA-PDPK2P can promote HCC progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of hepatocellular carcinoma.

Human gingival tissue-derived MSC suppress osteoclastogenesis and bone erosion via CD39-adenosine signal pathway in autoimmune arthritis.

BACKGROUND: Bone destruction is one of many severe complications that occurs in patients with rheumatoid arthritis (RA) and current therapies are unable to cure this manifestation. This study here aims to determine whether GMSC can directly inhibit osteoclast formation and eventually attenuate osteoclastogenesis and bone erosion in an inflammatory milieu. METHOD: GMSC were co-cultured with osteoclast precursors with or without CD39 inhibitor, CD73 inhibitor or adenosine receptors inhibitors pretreatment and osteoclast formation were evaluated in vitro. 2×10^6 GMSC per mouse were transferred to CIA mice and pathology scores, the frequency of osteoclasts, bone erosion in joints were assessed in vivo. FINDING: GMSC but not control cells, markedly suppressed human or mice osteoclastogenesis in vitro. GMSC treatment also resulted in a dramatically decreased level of NF-κB p65/p50 in osteoclasts in vitro. Infusion of GMSC to CIA significantly attenuated the severity of arthritis, pathology scores, frequency of osteoclasts, particularly bone erosion, as well as a decreased expression of RANKL in synovial tissues in vivo. Blockade of CD39/CD73 or adenosine receptors has significantly abrogated the suppressive ability of GMSC in vitro and therapeutic effect of GMSC on bone erosion during CIA in vivo. INTERPRETATION: GMSC inhibit osteoclast formation in vitro and in vivo partially via CD39-CD73-adenosine signals. Manipulation of GMSC may have a therapeutic implication on rheumatoid arthritis and other bone erosion related diseases. FUND: This study was supported by grants from the National Key R&D Program of China (2017YFA0105801 to F.H); the Zhujiang Innovative and Entrepreneurial Talent Team Award of Guangdong Province (2016 ZT 06S 252 to F·H) and National Institutes of Health (R01 AR059103, R61 AR073409 and NIH Star Award to S.G.Z).

Helios but not CD226, TIGIT and Foxp3 is a Potential Marker for CD4+ Treg Cells in Patients with Rheumatoid Arthritis.

BACKGROUND/AIMS: Rheumatoid arthritis (RA) is a progressive, chronic, even disabling systemic autoimmune disease. Imbalance between pathogenic immune cells and immunosuppressive cells is associated with the pathogenesis and development of RA and other autoimmune diseases. As Foxp3 is also expressed on activated CD4+ cells in the presence of inflammation, the identification of Treg cells in patients with RA remains a challenge. METHODS: Comprehensive analyses were carried out by Flow cytometry. Expression of Helios, CD226, T cell immunoreceptor with Ig and ITIM domains clinical samples and healthy controls. RESULTS: We have systemically examined three potential markers, Helios, CD226 and TIGIT, that are possibly related to Treg identification, and found that Helios expression on CD4+Foxp3+cells was decreased and negatively correlated with the disease activity of RA patients, while CD226 and TIGIT both showed elevated expression levels in CD4+Foxp3+cells in RA patients and they were not associated with disease activity of RA patients. CONCLUSION: Taken together, our findings indicate that CD4+CD25hiCD127low/-Foxp3+Helios+ may represent the real Treg cell population in patients with RA.

Negligible Effect of Sodium Chloride on the Development and Function of TGF-ß-Induced CD4+ Foxp3+ Regulatory T Cells.

High-salt diets inhibit the suppressive function of thymus-derived natural regulatory T cells (tTreg). Transforming growth factor ß (TGF-ß)-induced ex vivo regulatory T cells (iTreg) comprise another Treg subset that exhibits similarities and differences with tTreg. Here, we demonstrate that iTregs are completely stable and fully functional under high salt conditions. High salt does not influence the development, differentiation, and functional activities of iTreg but affects Foxp3 stability and function of tTreg in vitro and in vivo. In addition, high salt does not significantly change the transcription profiles of the iTreg signature or pro-inflammatory genes. Therefore, we conclude that iTreg, unlike tTreg, are stable and functional in the presence of high salt. Our findings provide additional evidence that iTreg may have different biological features from tTreg and suggest a greater potential for clinical utility in patients with autoimmune diseases, in which the complicated role of environmental factors, including diet, must be considered.

uPAR promotes tumor-like biologic behaviors of fibroblast-like synoviocytes through PI3K/Akt signaling pathway in patients with rheumatoid arthritis.

Urokinase-type plasminogen activator receptor (uPAR), is a multifunctional receptor on cell surface, widely present in endothelial cells, fibroblasts, and a variety of malignant cells. Current studies have suggested that uPAR overexpressed on synovial tissues or in synovial fluid or plasma in patients with rheumatoid arthritis (RA). However, there are limited researches regarding the role of uPAR on fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLSs) and its underlying mechanisms. Here, our studies show that the expression of uPAR protein was significantly higher in fibroblast-like synoviocytes (FLSs) from RA than those from osteoarthritis or traumatic injury patients. uPAR gene silencing significantly inhibited RA-FLSs cell proliferation, restrained cell transformation from the G0/G1 phase to S phase, aggravated cell apoptosis, interfered with RA-FLSs cell migration and invasion, and reduced activation of the PI3K/Akt signaling pathway, which may be associated with ß1-integrin. Cell supernatants from uPAR gene-silenced RA-FLSs markedly inhibited the migration and tubule formation ability of the HUVECs (a human endothelial cell line). Therefore, we demonstrate that uPAR changes the biological characteristics of RA-FLSs, and affects neoangiogenesis of synovial tissues in patients with RA. All of these may be associated with the ß1-integrin/PI3K/Akt signaling pathway. These results imply that targeting uPAR and its downstream signal pathway may provide therapeutic effects in RA.

In Vivo Attenuation of Antibody-Mediated Acute Renal Allograft Rejection by Ex Vivo TGF-ß-Induced CD4+Foxp3+ Regulatory T Cells.

Antibody-mediated rejection (AMR) has emerged as the major cause of renal allograft dysfunction, and more effective strategies need to be explored for improving transplant outcomes. Regulatory T cells (Tregs), consisting of at least natural and induced Treg subsets, suppress effector responses at multiple levels and play a key role in transplantation tolerance. In this study, we investigated the effect of induced Tregs (iTregs) on preventing antibody-mediated renal injury and rejection in a mouse model. We observed that infusion of iTregs markedly attenuated histological graft injury and rejection and significantly improved renal allograft survival. iTregs exhibited a comprehensive ability to regulate immunological disorders in AMR. First, iTreg treatment decreased the levels of circulating antidonor antibody and the antibody deposition within allografts. Second, iTregs significantly reduced cell infiltration including CD4+ T cells (including Th1, Th17, and Tfh), CD8+IFN-γ+ cells, natural killer cells, B cells, and plasma cells, which are involved in the process of AMR. Our results also highlight a predominance of M1 macrophage infiltration in grafts with acute AMR, and M1 macrophage could be reduced by iTreg treatment. Collectively, our data demonstrate, for the first time, that TGF-ß-induced Tregs can attenuate antibody-mediated acute renal allograft injury through targeting multiple effectors. Thus, use of iTregs in prevention of AMR in clinical practice could be expected.

Volasertib suppresses the growth of human hepatocellular carcinoma in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the sixth most frequent malignant tumor with poor prognosis, and its clinical therapeutic outcome is poor. Volasertib, a potent small molecular inhibitor of polo-like kinase 1 (PLK1), is currently tested for treatment of multiple cancers in the clinical trials. However, the antitumor effect of volasertib on HCC is still unknown. In this study, our data show that volasertib is able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the spindle abnormalities in human HCC cells. Furthermore, volasertib also increases the intracellular reactive oxidative species (ROS) levels, and pretreated with ROS scavenger N-acety-L-cysteine partly reverses volasertib-induced apoptosis. Moreover, volasertib markedly inhibits the subcutaneous xenograft growth of HCC in nude mice. Overall, our study provides new therapeutic potential of volasertib on hepatocellular carcinoma.

TGF-ß-Induced Regulatory T Cells Directly Suppress B Cell Responses through a Noncytotoxic Mechanism.

Foxp3(+) regulatory T cells (Treg) playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases consist of thymus-derived naturally occurring CD4(+)Foxp3(+) Treg cells (nTreg) and those that can be induced ex vivo with TGF-ß (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. The role of iTreg in regulating B cells remains unclear so far. The suppression assays of Treg subsets on activation, proliferation, and Abs production of B cells were measured using a Treg and B cell coculture system in vitro. Transwell and Ab blockade experiments were performed to assess the roles of cell contact and soluble cytokines. Treg were adoptively transferred to lupus mice to assess in vivo effects on B cells. Like nTreg, iTreg subset also directly suppressed activation and proliferation of B cells. nTreg subset suppressed B cell responses through cytotoxic manner related to expression of granzyme A, granzyme B, and perforin, whereas the role of iTreg subset on B cells did not involve in cytotoxic action but depending on TGF-ß signaling. Furthermore, iTreg subset can significantly suppress Ab produced by lupus B cells in vitro. Comparison experiments using autoantibodies microarrays demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo. Our data implicate a role and advantage of iTreg subset in treating B cell-mediated autoimmune diseases, boosting the translational potential of these findings.

Synergistic anticancer effects of triptolide and celastrol, two main compounds from thunder god vine.

Triptolide and celastrol are two main active compounds isolated from Thunder God Vine with the potent anticancer activity. However, the anticancer effect of triptolide in combination with celastrol is still unknown. In the present study, we demonstrated that the combination of triptolide with celastrol synergistically induced cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increased intracellular ROS accumulation in cancer cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine dramatically blocked the apoptosis induced by co-treatment with triptolide and celastrol. Treatment with celastrol alone led to the decreased expressions of HSP90 client proteins including survivin, AKT, EGFR, which was enhanced by the addition of triptolide. Additionally, the celastrol-induced expression of HSP70 and HSP27 was abrogated by triptolide. In the nude mice with xenograft tumors, the lower-dose combination of triptolide with celastrol significantly inhibited the growth of tumors without obvious toxicity. Overall, triptolide in combination with celastrol showed outstanding synergistic anticancer effect in vitro and in vivo, suggesting that this beneficial combination may offer a promising treatment option for cancer patients.

Cyclin-dependent kinase inhibitor dinaciclib potently synergizes with cisplatin in preclinical models of ovarian cancer.

Ovarian cancer is one of the most lethal of woman cancers, and its clinical therapeutic outcome currently is unsatisfied. Dinaciclib, a novel small molecule inhibitor of CDK1, CDK2, CDK5 and CDK9, is assessed in clinical trials for the treatment of several types of cancers. In this study, we investigated the anticancer effects and mechanisms of dinaciclib alone or combined with cisplatin in ovarian cancer. Dinaciclib alone actively induced cell growth inhibition, cell cycle arrest and apoptosis with the increased intracellular ROS levels, which were accompanied by obvious alterations of related proteins such as CDKs, Cyclins, Mcl-1, XIAP and survivin. Pretreatment with N-acety-L-cysteine significantly blocked ROS generation but only partially rescued apoptosis triggered by dinaciclib. Moreover, the combination of dinaciclib with cisplatin synergistically promoted cell cycle arrest and apoptosis, and inhibited the subcutaneous xenograft growth of ovarian cancer in nude mice. Altogether, dinaciclib potently synergizes with cisplatin in preclinical models of ovarian cancer, indicating this beneficial combinational therapy may be a promising strategy for treatment of ovarian cancer.

Trametinib modulates cancer multidrug resistance by targeting ABCB1 transporter.

Overexpression of adenine triphosphate (ATP)-binding cassette (ABC) transporters is one of the main reasons of multidrug resistance (MDR) in cancer cells. Trametinib, a novel specific small-molecule mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, is currently used for the treatment of melanoma in clinic. In this study, we investigated the effect of trametinib on MDR mediated by ABC transporters. Trametinib significantly potentiated the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in cancer cells overexpressed ABCB1, but not ABCC1 and ABCG2. Furthermore, trametinib did not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, trametinib potently blocked the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. Importantly, trametinib remarkably enhanced the effect of vincristine against the xenografts of ABCB1-overexpressing cancer cells in nude mice. The predicted binding mode showed the hydrophobic interactions of trametinib within the large drug binding cavity of ABCB1. Consequently, our findings may have important implications for use of trametinib in combination therapy for cancer treatment.

Sildenafil inhibits the growth of human colorectal cancer in vitro and in vivo.

Colorectal cancer is the third most common human cancer with frequent overexpression of the cGMP-specific phosphodiesterase 5 (PDE5). In the present study, we investigated that the anticancer effect of sildenafil on human colorectal cancer in vitro and in vivo, which is a potent and selective inhibitor of PDE5 for the treatment of erectile dysfunction and pulmonary arterial hypertension in the clinic. Sildenafil significantly induced cell growth inhibition, cell cycle arrest and apoptosis of human colorectal cancer with increased intracellular reactive oxidative specie (ROS) levels, which were accompanied by obvious alterations of related proteins such as CDKs, Cyclins and PARP etc. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse sildenafil-induced ROS accumulation and cell apoptosis. Inhibition of the activity of protein kinase G with KT-5823 could enhance sildenafil-induced apoptosis. Furthermore, sildenafil caused the reduction of xenograft models of human colorectal cancer in nude mice. Overall, these findings suggest that sildenafil has the potential to be used for treatment of human colorectal cancer.

Piperlongumine induces apoptosis and synergizes with cisplatin or paclitaxel in human ovarian cancer cells.

Piperlongumine (PL), a natural alkaloid from Piper longum L., possesses the highly selective and effective anticancer property. However, the effect of PL on ovarian cancer cells is still unknown. In this study, we firstly demonstrate that PL selectively inhibited cell growth of human ovarian cancer cells. Furthermore, PL notably induced cell apoptosis, G2/M phase arrest, and accumulation of the intracellular reactive oxidative species (ROS) in a dose- and time-dependent manner. Pretreatment with antioxidant N-acety-L-cysteine could totally reverse the PL-induced ROS accumulation and cell apoptosis. In addition, low dose of PL/cisplatin or paclitaxel combination therapies had a synergistic antigrowth effect on human ovarian cancer cells. Collectively, our study provides new therapeutic potential of PL on human ovarian cancer.

Resveratrol oligomers for the prevention and treatment of cancers.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally derived phytoalexin stilbene isolated from grapes and other plants, playing an important role in human health and is well known for its extensive bioactivities, such as antioxidation, anti-inflammatory, anticancer. In addition to resveratrol, scientists also pay attention to resveratrol oligomers, derivatives of resveratrol, which are characterized by the polymerization of two to eight, or even more resveratrol units, and are the largest group of oligomeric stilbenes. Resveratrol oligomers have multiple beneficial properties, of which some are superior in activity, stability, and selectivity compared with resveratrol. The complicated structures and diverse biological activities are of significant interest for drug research and development and may provide promising prospects as cancer preventive and therapeutical agents. This review presents an overview on preventive or anticancer properties of resveratrol oligomers.