Effects of Small-Molecule Inhibitors of TNFR2 Signaling Pathways On TNF-Mediated Expansion of CD4+FoxP3+ Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4722-4722
Author(s):  
Xueqiang Wu ◽  
Yan Chen ◽  
Chunsheng Han ◽  
Yiwen Gong ◽  
Dingfang Bu ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Conflicts Of Interest ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
P38 Mapk Inhibitor

Abstract Abstract 4722 TNF is a pleiotropic cytokine with biphasic proinflammatory and immunosuppressive effects. Previous work clearly demonstrated that TNF has the capacity to preferentially activate and promote the proliferative expansion of CD4+FoxP3+ regulatory T cells (Tregs), which represent a basis for the paradoxical anti-inflammatory action of TNF. Our studies also indicate that TNFR2, one of the TNF receptor which is preferentially expressed by Tregs, mediates the Treg-activating effect of TNF. However, the molecular mechanism and signaling pathways mediated Treg-activating effect of TNF remain to be understood. In this study, we first further verified that TNFR2 transduces the activating signal of TNF on Tregs, based on the evidence that 1) human TNF, known to only bind to mouse TNFR1, did not activate mouse Tregs; 2) a blocking anti mouse TNFR2 Ab, but not TNFR1 Ab, dose-dependently inhibited TNF-mediated proliferation of mouse Tregs at concentrations of 2.5–500 ng/ml. We next examined the signaling pathways of TNFR2 leading to the proliferation of Tregs, by using specific small-molecule inhibitors. It is well established that the activation of IKK-NFkB is a major consequence of activation of TNFR2. However, small molecule inhibitors of NFkB signaling pathway, namely BAY11–7082 and Sulfasalazine, did not block TNF-mediated proliferation of Tregs. In contrast, small molecule inhibitors of MAPK signaling pathway, SB203580 (P38 MAPK inhibitor), SP600125 (JNK inhibitor) and PD98059 (Erk1/2 inhibitor), potently suppressed TNF-induced replication of Tregs in a dose-dependent manner. Our results indicate that TNF selectively stimulates the expansion of FoxP3+ Tregs through TNFR2. Activation of MAPK (ERK1/2,P38 and JNK), rather than NFkB, is responsible for this activity of TNF. Disclosures: No relevant conflicts of interest to declare.

AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2866-2866
Author(s):  
Hisayuki Yao ◽  
Eishi Ashihara ◽  
Rina Nagao ◽  
Shinya Kimura ◽  
Hideyo Hirai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Cancer Cell Line ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Abstract 2866 Poster Board II-842 Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells. The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells. We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Multiple Myeloma Generates Regulatory T-Cells in a Contact-Dependent Manner Independent of TGF-Beta and IL-10.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2835-2835
Author(s):  
Sylvia Feyler ◽  
Marie von Lilienfeld-Toal ◽  
Sarah J Jarmin ◽  
Paul Evans ◽  
Mike Short ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Treg Cells ◽  
Tumour Cells ◽  
Cell Generation ◽  
Dependent Manner ◽  
Hdac Inhibition ◽  
Altered Expression

Abstract Abstract 2835 Poster Board II-811 Introduction: We have previously shown that Regulatory T-cells (TReg cells) are increased in the peripheral blood (PB) and bone marrow (BM) of patients with multiple myeloma (MM), related to the stage of disease. Therefore, to investigate whether the tumour cells in MM generate and/or expand TReg cells as a method of immuno-surveillance avoidance, we developed an in vitro model of TReg cell generation. Materials and Methods: PBMCs from PB of healthy donors were co-cultured with human myeloma cell line, U266B, for 7 days. Using a sequential gating strategy, TReg cells identified as CD4+/CD25+/FoxP3+ T-cells were expressed as a percentage of the CD4+ T-cell population. Experiments were repeated after CD25 depletion ± CD4 selection and using transwells to examine cell contact dependence. The tumour generated TReg cells (tTReg cells) were isolated on day 7 by FACS and compared with naturally occurring TReg cells (nTReg cells). Results: Co-culture of unselected PBMC's demonstrated a non-significant increase in TReg cells compared with controls (n=8; controls 3.8±0.8%, co-culture 6.7±1%, p=0.06). However, co-culture of CD4+CD25− T-cells with U266 resulted in a significant increase in tTReg cells (n=15; control 0.6±0.3%; co-culture 31.5±3.6%, p<0.0001). tTReg cell generation was abrogated when contact was prevented (n=7; control 0.17±0.05%; co-culture 30.6±4.9%; addition of transwell 0.1±0.04%, p=0.009). TCR analysis by PCR on tTReg cells revealed a polyclonal nature of those cells. Blocking experiments with anti-IL-10, anti-ICOS-L & anti-TGFb, in addition to the TGFb-specific antagonist, LAP, did not show any reduction in tTReg cell generation. tTReg cells demonstrated full suppressive capabilities in a functional suppression assay. Phenotypically, tTReg cells demonstrated altered expression of key proteins: FoxP3 MFI was significantly lower (925 ± 47 vs. 1960 ± 109 in nTReg cells, p=0.0001), GITR expression was significantly higher (70±4% vs. 10±2.8%, p<0.0001) and PD1 expression was significantly higher (50±9% vs. 5.2±0.8%, p=0.003). CD-62L expression was lower in tTReg cells (88±2% vs. 97±0.5%, p=0.007). When tTReg cells were isolated by FACS on day 7 and stimulated for a further 5 days with IL-2 and CD3/CD28 coated beads, differentiation to FoxP+/IL-17+ and FoxP3−/IL-17+ CD4+ cells was demonstrated with similar efficiency to nTReg cells (2.6±1.8% vs 3.6±2.4% p=0.7 and 4.6±4.0% vs 4.5±3.8% p=0.8, respectively). The addition of U266 pre-treated with thalidomide, lenalidomide and an HDAC inhibitor for 24 hours prior to co-culture demonstrated that thalidomide and HDAC inhibition significantly reduced the generation even at a low concentrations (42.6±24.5% without vs. 20.7±11.9% with thalidomide, p=0.009 and 9.8±3.3% with HDAC inhibition, p=0.003), whereas the addition of lenalidomide did not influence tTReg cell generation. Conclusions: The tumour cells of MM directly generate fully functional TReg cells in a contact dependent manner independent of known inducing cytokines and ligands. This is an active process under epigenetic control. The exact mechanism of induction is under investigation. Disclosures: No relevant conflicts of interest to declare.

Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3353-3353 ◽  
Author(s):  
Jaebok Choi ◽  
Matthew L Cooper ◽  
Kiran R. Vij ◽  
Bing Wang ◽  
Julie Ritchey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Hematopoietic Stem ◽  
Therapeutic Benefits ◽  
In Vivo Administration ◽  
Jak2 Inhibitors

Abstract The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT. We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling. Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells. We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01). Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001). All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. Disclosures DiPersio: Incyte Corporation: Research Funding.

Differential Targeting of Signaling Pathways to Selectively Expand Mouse Regulatory T Cells While Inhibiting Conventional T Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2158-2158
Author(s):  
Atsushi Satake ◽  
Amanda M Schmidt ◽  
Angela Archambault ◽  
Gregory F Wu ◽  
Taku Kambayashi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Signaling Pathways ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Negative Effects ◽  
Tcr Signaling

Abstract Abstract 2158 Regulatory T cells (Tregs) are suppressive T cells with therapeutic potential for ameliorating T cell-mediated diseases. Thus, there has been great interest in revealing the mechanisms by which Tregs proliferate. Recently, we reported that TCR signaling is partially dispensable for Treg proliferation in vivo when exogenous IL-2 is administered. Based on this data, we hypothesized that when given in conjunction with IL-2, pharmacological inhibition of TCR signaling might allow Tregs to expand while simultaneously inhibiting conventional T cell (Tconv) proliferation. Using mutant mice with defective TCR-mediated PLCγ activation, we found that the activation of PLCγ is dispensable for IL-2-mediated Treg proliferation. In contrast, costimulation-derived mTOR signaling was required for IL-2-induced Treg proliferation. We next used Cyclosporine A (CSA; calcineurin inhibitor) and rapamycin (mTOR inhibitor) to differentially target these signaling pathways. Consistent with our hypothesis, while both CSA and rapamycin suppressed antigen-specific Tconv proliferation, only CSA permitted IL-2-induced Treg expansion in vitro and in vivo. Rapamycin, however, did increase the overall Treg:Tconv ratio due to its negative effects on Tconv survival. Given that CSA inhibited antigen-specific Tconv proliferation while maintaining IL-2-induced Treg expansion, we hypothesized that the combination of CSA and IL-2 would be beneficial for attenuating T cell-mediated disease. Indeed, CSA synergized with IL-2 in protection against experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Surprisingly, however, the administration of CSA blocked whereas rapamycin augmented the beneficial effect of IL-2 in graft-versus-host disease (GVHD). These differences potentially results from the overt TCR stimulation that Tregs would receive in the allogeneic (GVHD) vs. syngeneic (EAE) environment. Moreover, inducible Treg (iTreg) generation from allogeneic MHC-stimulated naïve Tconvs contributes greatly to the Treg pool during GVHD. This was consistent with our data showing that rapamycin promotes iTreg generation and allows TCR-enhanced Treg proliferation, whereas CSA inhibited both of these processes. Thus, depending on the disease setting, the signaling pathways contributing to expansion of the Treg pool need to be carefully considered and specifically targeted to increase the Treg:Tconv ratio in treatment of T cell-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

DDR1 and DDR2: a review on signaling pathway and small molecule inhibitors as an anticancer agent

2021 ◽  
Vol 30 (3) ◽  
pp. 535-551
Author(s):  
Gurubasavaraja Swamy Purawarga Matada ◽  
Arka Das ◽  
Prasad Sanjay Dhiwar ◽  
Abhishek Ghara
Keyword(s):  
Signaling Pathway ◽  
Small Molecule ◽  
Anticancer Agent ◽  
Small Molecule Inhibitors

Candida tropicalis induces pro-inflammatory cytokine production, NF-κB and MAPKs pathways regulation, and dectin-1 activation

2018 ◽  
Vol 64 (12) ◽  
pp. 937-944 ◽  
Author(s):  
Zhimin Duan ◽  
Qing Chen ◽  
Rong Zeng ◽  
Leilei Du ◽  
Caixia Liu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Cytokine ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Downstream Signaling ◽  
Tnf Α ◽  
Mitogen Activated Protein

The prevalence of Candida infection induced by non-albicans Candida (NAC) species is increasing. However, as a common NAC species, C. tropicalis has received much less study in terms of host immunity than C. albicans has. In this study, we evaluated the pro-inflammatory cytokine responses evoked by C. tropicalis and determined whether dectin-1 and downstream NF-κB and mitogen-activated protein kinases (MAPKs) signaling pathways played roles in inflammation in human peripheral blood mononuclear cells (PBMCs) and THP-1 macrophage-like cells. Exposure of PBMCs and THP-1 macrophage-like cells to C. tropicalis led to the enhanced gene expression and secretion of TNF-α and IL-6 in a time- and dose-dependent manner. THP-1 macrophage-like cells being challenged by C. tropicalis resulted in the activation of the NF-κB, p38, and ERK1/2 MAPK signaling pathways. We also found that the expression of dectin-1 was increased with C. tropicalis treatment. These data reveal that dectin-1 may play a role in sensing the inflammation response induced by C. tropicalis and that NF-κB and MAPK are involved in the downstream signaling pathways in macrophages.

The defense response of Caenorhabditis elegans to Cutibacterium acnes SK137 via the TIR-1-p38 MAPK signaling pathway

2021 ◽  
Author(s):  
Ayano Tsuru ◽  
Yumi Hamazaki ◽  
Shuta Tomida ◽  
Mohammad Shaokat Ali ◽  
Eriko Kage-Nakadai
Keyword(s):  
Caenorhabditis Elegans ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Defense Response ◽  
Mapk Pathway ◽  
Defense Responses ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Healthy Skin ◽  
Cutibacterium Acnes

Abstract Cutibacterium acnes plays roles in both acne disease and healthy skin ecosystem. We observed that mutations in the tir-1/SARM1 and p38 MAPK cascade genes significantly shortened Caenorhabditis elegans lifespan upon Cutibacterium acnes SK137 infection. Antimicrobial molecules were induced by SK137 in a TIR-1-dependent manner. These results suggest that defense responses against SK137 involve the TIR-1-p38 MAPK pathway in Caenorhabditis elegans.

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