TNF-α In Dependent Manner Research Articles

Dysregulated myeloid differentiation in colitis is induced by inflammatory osteoclasts in a TNFα-dependent manner

Inflammatory bowel disease (IBD) is characterized by very severe intestinal inflammation associated with extra-intestinal manifestations. One of the most critical ones is bone destruction, which remains a major cause of morbidity and a risk factor for osteopenia and osteoporosis in IBD patients. In various mouse models of IBD, we and other have demonstrated concomitant bone loss due to a significant increase in osteoclast activity. Besides bone resorption, osteoclasts are known to control hematopoietic niches in vivo and modulate inflammatory responses in vitro, suggesting they may participate in chronic inflammation in vivo. Here, using different models of colitis, we showed that osteoclast inhibition significantly reduced disease severity and that induction of osteoclast differentiation by RANKL contributed to disease worsening. Our results demonstrate a direct link between osteoclast activity and myeloid cell accumulation in the intestine during colitis. RNAseq analysis of osteoclasts from colitic mice revealed overexpression of genes involved in the remodeling of hematopoietic stem cell niches. We also demonstrated that osteoclasts induced hematopoietic progenitor proliferation accompanied by a myeloid skewing in the early phases of colitis, which was confirmed in a model of RANKL-induced osteoclastogenesis. Mechanistically, inhibition of TNF-α reduced the induction of myeloid skewing by OCL both in vitro and in vivo. Lastly, we observed that osteoclastic activity and the proportion of myeloid cells in the blood are positively correlated in patients with Crohn’s disease. Collectively, our results shed light on a new role of osteoclasts in colitis in vivo, demonstrating they exert their colitogenic activity through an early action on hematopoiesis, leading to an increase in myelopoiesis sustaining gut inflammation.

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Serum amyloid A proteins reduce bone mass during mycobacterial infections.

Osteopenia has been associated to several inflammatory conditions, including mycobacterial infections. How mycobacteria cause bone loss remains elusive, but direct bone infection may not be required. Genetically engineered mice and morphometric, transcriptomic, and functional analyses were used. Additionally, inflammatory mediators and bone turnover markers were measured in the serum of healthy controls, individuals with latent tuberculosis and patients with active tuberculosis. We found that infection with Mycobacterium avium impacts bone turnover by decreasing bone formation and increasing bone resorption, in an IFNγ- and TNFα-dependent manner. IFNγ produced during infection enhanced macrophage TNFα secretion, which in turn increased the production of serum amyloid A (SAA) 3. Saa3 expression was upregulated in the bone of both M. avium- and M. tuberculosis-infected mice and SAA1 and 2 proteins (that share a high homology with murine SAA3 protein) were increased in the serum of patients with active tuberculosis. Furthermore, the increased SAA levels seen in active tuberculosis patients correlated with altered serum bone turnover markers. Additionally, human SAA proteins impaired bone matrix deposition and increased osteoclastogenesis in vitro. Overall, we report a novel crosstalk between the cytokine-SAA network operating in macrophages and bone homeostasis. These findings contribute to a better understanding of the mechanisms of bone loss during infection and open the way to pharmacological intervention. Additionally, our data and disclose SAA proteins as potential biomarkers of bone loss during infection by mycobacteria.

Abstract TP129: Increased Levels Of Matrix Metalloproteinases-induced BBB Dysfunction May Be Responsible For Increased Post-sich Hematoma Growth In Nicotine-exposed Rats.

Spontaneous intracerebral hemorrhage (sICH) is the deadliest type of stroke, accounting for as high as 28% of all strokes. Tobacco use is one of the major risk factors of sICH, and it also worsens outcomes following sICH. In our earlier study, we observed that nicotine exposure increases TNFα levels in brain vessels, impairs BBB integrity, and increases post-sICH hematoma volume in a TNFα-dependent manner compared to saline-exposed controls. Earlier studies have demonstrated interactions between MMPs and TNFα. Increased levels of matrix metalloproteinases (MMPs) have been found in sICH patients and nicotine-exposed individuals. MMPs are involved in BBB breakdown, edema formation, hematoma expansion, and neuronal loss that follow hematoma development after sICH. In the present study, we tested the hypothesis that nicotine exposure-induced increase in TNFα is responsible for increased post-sICH hematoma volume in a MMP-dependent manner. To test our hypothesis, young male and female rats were exposed to chronic nicotine treatment for 2-3 weeks using osmotic pumps containing either saline or nicotine (4.5 mg/kg/day). Levels of MMPs (2, 3, 9, and 12) and GAPDH were evaluated in brain homogenates using Western blot. Nicotine-treated female rats showed a 37% (100±7 vs. 137±10, p<0.05), 144% (100±18 vs. 244±16, p<0.01), 29% (100±7 vs. 129±9, p<0.05), 22% (100±6 vs. 122±6, p<0.05), and 24% (100±6 vs. 124±7, p<0.05) increase in levels of pro and cleaved forms of MMP-3, cleaved form of MMP-9, pro and cleaved forms of MMP-12, respectively, compared to saline-treated female rats. We are in the process of evaluating levels of MMPs in male samples. Considering the role of MMP-9 in BBB integrity, using both TNFα and MMP-9 inhibitors, we are also in the process of evaluating if TNFα increases post-sICH hematoma growth in nicotine-exposed rats via increased levels of MMP-9. The results so far imply that increased TNFα levels in nicotine-exposed rats may be responsible for increased post-sICH hematoma growth in a MMP-dependent manner. Support: James Esther King Biomedical Research Grant 9JK08 and PK is a recipient of the Lois Pope Neuroscience Summer Research Fellowship.

The effect of ethyl acetate fraction of Marsilea crenata presl. leaves in increasing osterix expression in hFOB 1.19 cells

Marsilea crenata Presl. leaves contain phytoestrogens that is suspected to play a role in increasing bone formation. Bone formation activity is defined by the expression of Osterix, a transcription factor that plays a key role in bone development. The aim of this study is to show that the ethyl acetate fraction of Marsilea crenata Presl. leaves can increase bone formation process in hFOB 1.19 cells in a TNF-α dependent manner, by measurement of Osterix. The hFOB 1.19 cells were grown in 24-well microplates and treated with 10 ng/ml TNF-α for 24 hours. The ethyl acetate fraction of Marsilea crenata Presl. leaves were also added in concentrations of 62.5, 125, and 250 ppm. A positive control was employed, at a concentration of 2.5 g/L of genistein. The expression of osterix was examined using an immunocytochemistry technique with CLSM to assess bone forming activity. The results show that ethyl acetate fraction Marsilea crenata Presl. leaves can boost osterix expression in hFOB 1.19 cells, with optimal concentration was 250 ppm at p<0.005. The ethyl acetate fraction of Marsilea crenata Presl. leaves can increase Osterix in osteoblast hFOB 1.19 cell, indicating improved bone forming activity.

Ephrin Ligands are Upregulated in the Saliva of SARS‐CoV‐2 Infected Patients

BackgroundPatients infected with SARS‐CoV‐2 may develop severe lung disease characterized by respiratory distress, systemic thrombosis, or death. Molecular mechanisms responsible for severe cases of COVID‐19 remain unclear. We investigated the role of the Eph family receptor‐interacting ligands (ephrin‐A1 and ephrin‐B2) in COVID‐19 patients. Ephrin binding to its tyrosine kinase Eph receptor plays important roles in injury and inflammation. Serum levels of ephrin‐A1 ligand correlates positively with COVID‐19 severity (Mendoza et al, CellPress 2021). Based on this recent report, and the emerging role of Ephrin‐Eph signaling in lung injury, we hypothesize that ephrin‐mediated cytokine signaling is upregulated in epithelial and immune cells assayed from saliva samples obtained from seriously ill COVID‐19 patients seeking emergency evaluation and treatment.MethodsSaliva samples were collected from patients (± SARS‐CoV‐2) in the University of Utah Emergency Department (ED) with IRB‐approved written consent. Saliva samples were incubated 1:1 in Streck Preservative (prior to EpCAM and CD45 labeling) or with 0.008% glutaraldehyde (following ELISA and multiplex immunoassays) in order to inactivate virus.ResultsFlow cytometric evaluation of saliva for EpCAM+ and CD45+ cells indicated that SARS‐CoV‐2 positive samples contain both epithelial and immune cell types ( see Fig 1 ). Uninfected saliva samples (control) primarily contained EpCAM+ cells. COVID‐19 samples (n=67 patients) expressed higher levels of type 1 interferon [IFNγ1 and IFNα2 (p< 0.001)] vs control (n=64 patients). Interferon induced protein [IP‐10] was also upregulated in COVID‐19 patients (p<0.001), as well as several cytokines in the interleukin family [IL‐1β, IL‐6, IL‐8 (p<0.001) and IL‐12p70 (p<0.05)]. TNF‐α did not differ significantly between the ± SARS‐CoV‐2 samples evaluated. Using a subset of the +SARS‐CoV‐2 samples (n=20) and uninfected controls obtained from the ED (n=20), we show that both ephrin‐A1 and ephrin‐B2 ligands are significantly elevated in +SARS‐CoV‐2 samples vs. ED control samples (p<0.001). Ephrin‐B2 levels correlated negatively with IL‐12p70 (Pearson Correlation =‐0.53, p=.01).ConclusionSaliva is low risk to obtain and is a biologically relevant sample that can be used to reliably measure changes in epithelial and immune cell responses of COVID‐19 patients. Specifically, our study showed that ephrin‐A1 and ephrin‐B2 ligands are upregulated in the saliva of COVID‐19 patients in a TNF‐α independent manner and that ephrin‐B2 correlates negatively with IL‐12p70 (a potent pro‐inflammatory cytokine). Future studies are aimed at establishing ephrin ligands as a biomarker for severity of lung injury and improving understanding the patho‐physiology of SARS‐CoV‐2 infection.

Systems model identifies baseline cytokine concentrations as potential predictors of rheumatoid arthritis inflammatory response to biologics.

Circulating cytokines are central pathological mediators of inflammatory autoimmune diseases like rheumatoid arthritis. Immunological diversity in patients might contribute to inadequate responses to biological drugs. To address this therapeutic challenge, we developed a mathematical model that simultaneously describes temporal patterns of drug disposition for several biologics and their corresponding targeted cytokines, which were linked to triggering inflammatory responses. A modelling framework was applied to rheumatoid arthritis-relevant cytokines regulating C-reactive protein (CRP) as an inflammatory marker. Clinical data were extracted from the literature for anakinra, canakinumab, infliximab, secukinumab and tocilizumab, along with their corresponding cytokines, interleukin-1 receptor antagonist, IL-1β, tumour necrosis factor α (TNFα), IL-17A and IL-6 receptor (IL-6R). Based on prior knowledge of regulatory mechanisms, cytokines were integrated with CRP profiles. The model well captured all serum concentration-time profiles of cytokines and CRP ratios to respective baselines following drug treatment with good precision. On external validation, reasonable model performance on CRP dynamics, including rebound effects, was confirmed with clinical data not used in model development. Model-based simulations demonstrated that serum infliximab concentrations were accurately recapitulated in both a dose- and baseline TNFα-dependent manner. Furthermore, high baseline profiles of both IL-1β and/or targeted cytokines could be predictors of poor responses to biologics targeting TNFα and IL-6R, although the impact of IL-1β must be carefully interpreted. Our model provides a quantitative platform to guide targeting and dosing strategies, including combination and/or sequential therapy, according to distinct baseline cytokine patterns in rheumatoid arthritis patients.

Abstract 6042: TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer

Abstract Cytotoxic and genotoxic therapies are known to induce the prosurvival Inhibitor-kappaB kinase (IKK)-NF-κB/RELA pathway and DNA repair at the G2/M checkpoint, thereby promoting therapeutic resistance, but the coordination of these mechanisms is unclear. Our recent RNAi screen unexpectedly uncovered possible crosstalk between WEE1, a G2/M checkpoint kinase and the TNFα-IKK-NF-κB prosurvival pathway components. siRNAs targeting WEE1 and IKKs both inhibited TNFα-inducible NF-κB activity in a HNSCC reporter line, suggesting a mechanistic linkage. We investigated this potential connection further and studied the effect of WEE1 inhibition using AZD1775 in head and neck squamous cell carcinoma (HNSCC) cells and mouse xenograft models. Increased expression, phosphorylation and cellular localization of IKK-NF-κB and WEE1-CDC2 axis proteins co-occurred in a subset of HNSCC lines, and were inversely co-modulated by TNFα and WEE1 kinase inhibitor AZD1775. WEE1 co-immunoprecipitated with the IKKα/β and RELA protein complex, and AZD1775 or an IKK inhibitor, IKK16, inhibited phosphorylation and kinase activities, further suggesting that these two axes interact with each other. AZD1775 sensitized HNSCC cells to TNFα-induced cytotoxicity, in part by inhibiting IKK-dependent phosphorylation and nuclear localization of NF-κB activation, and expression of target prosurvival protein BCL-2. Combination of AZD1775 with radiation, a known inducer of TNFα- and DNA-mediated cytotoxicity, potently inhibited HPV+/- HNSCC tumor xenografts. Depleting TNFα abolished the anti-tumor activity of combined therapy with AZD1775 and radiation. The findings of our study reveal that TNFα co-modulates a novel interaction between the prosurvival IKK/NF-κB/RELA and WEE1-CDC2 G2/M checkpoint pathway and that components of both the pathways are inhibited by AZD1775. Our mouse xenograft experiments suggest that AZD1775, when combined with radiation, significantly delays the growth of xenograft HNSCC tumors differing in HPV status in a TNFα-dependent manner. The current findings reveal a novel mechanism whereby crosstalk and co-activation of WEE1- and NF-κB-signaling promotes resistance to TNFα-mediated cytotoxicity, which may be targeted using WEE1 inhibitors. Supported by NIDCD projects ZIA-DC-000016, -73 and 74. Citation Format: Ramya Viswanathan, Zhengbo Hu, Jianghong Chen, Xinping Yang, Angel Huynh, Paul Clavijo, Yi An, Yvette Robbins, Christopher Silvin, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6042.

Dissecting the Mechanism of Intracellular Mycobacterium smegmatis Growth Inhibition by Platelet Activating Factor C-16.

Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C-16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP-1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signaling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signaling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signaling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogs, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.

Migration of murine intestinal dendritic cell subsets upon intrinsic and extrinsic TLR3 stimulation.

Initiation of adaptive immunity to particulate antigens in lymph nodes largely depends on their presentation by migratory dendritic cells (DCs). DC subsets differ in their capacity to induce specific types of immunity, allowing subset-specific DC-targeting to influence vaccination and therapy outcomes. Faithful drug design, however, requires exact understanding of subset-specific versus global activation mechanisms. cDC1, the subset of DCs that excel in supporting immunity toward viruses, intracellular bacteria, and tumors, express uniquely high levels of the pattern recognition receptor TLR3. Using various murine genetic models, we show here that both, the cDC1 and cDC2 subsets of cDCs are activated and migrate equally well in response to TLR3 stimulation in a cell extrinsic and TNF-α dependent manner, but that cDC1 show a unique requirement for type I interferon signaling. Our findings reveal common and differing pathways regulating DC subset migration, offering important insights for the design of DC-based vaccination and therapy approaches.

Modulation of Epidermal γδ T Cell Functionality by Obesity due to Acute versus Chronic TNF-α Reception

Abstract Epidermal γδ T cells exhibit functional plasticity with roles varying from wound healing to tumor cell lysis. Activation of epidermal γδ T cells occurs via the γδ TCR along with costimulation, suggesting that costimulatory ligand expression regulates functional outcome. Data from our lab indicates that the cytokine milieu adds an additional pressure on epidermal γδ T cells to mediate or inhibit a particular functionality. Previous work from our lab shows that obesity impairs epidermal γδ T cell wound repair function in a TNF-α dependent manner. Here we identify TNF-α as a modulator of epidermal γδ T cell function in wound repair or cellular lysis depending on reception of acute or chronic signals. TNF-α signals through two receptors, tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), thus the signaling dynamics are complex and induce varied responses including apoptosis, survival and inflammation. We show that epidermal γδ T cells exhibit low TNFR1 and TNFR2 expression prior to TCR activation. Even with low TNFR expression epidermal γδ T cells produce cytokines and chemokines involved in inflammation and macrophage recruitment in response to acute TNF-α signals without TCR activation. During TCR activation or IL-2 stimulation epidermal γδ T cells to upregulate TNFR2 indicating a reliance on TNFR2 signaling during activation. Alternatively, chronic TNF-α stimulation, similar to levels in obese patients, induces a reduction in epidermal γδ T cell cytotoxicity, proliferation and viability. Normal function is restored with IL-2 addition suggesting the inhibition is not permanent, but instead can be modulated. These studies provide potential targets for regulating epidermal γδ T cell functions such as wound repair and tumor lysis.

Maslinic acid suppresses macrophage foam cells formation: Regulation of monocyte recruitment and macrophage lipids homeostasis

The transformation of macrophages to foam cells is a critical component in atherosclerotic lesion formation. Maslinic acid (MA), a novel natural pentacyclic triterpene, has cardioprotective and anti-inflammatory properties. It is hypothesized that MA can suppress monocyte recruitment to endothelial cells and inhibit macrophage foam cells formation. Previous study shows that MA inhibits inflammatory effects induced by sPLA2-IIA, including foam cells formation. This study elucidates the regulatory effect of MA in monocyte recruitment, macrophage lipid accumulation and cholesterol efflux. Our findings demonstrate that MA inhibits THP-1 monocyte adhesion to HUVEC cells in a TNFα-dependent and independent manner, but it induces trans-endothelial migration marginally at low concentration. MA down-regulates both gene and protein expression on VCAM-1 and MCP-1 in HUVECs. We further showed that MA suppresses macrophage foam cells formation, as indicated from the Oil-Red-O staining and flow cytometric analysis of intracellular lipids accumulation. The effects observed may be attributed to the antioxidant properties of MA where it was shown to suppress CuSO4-induced lipid peroxidation. MA inhibits scavenger receptors SR-A and CD36 expression while enhancing cholesterol efflux. MA enhances cholesterol efflux transporters ABCA1 and ABCG1 genes expression marginally without inducing its protein expression. In this study, MA was shown to target important steps that contribute to foam cell formation, including its ability in reducing monocytes adhesion to endothelial cells and LDL peroxidation, down-regulating scavenger receptors expression as well as enhancing cholesterol efflux, which might be of great importance in the context of atherosclerosis prevention and treatment.

MiR-130a alleviated high-glucose induced retinal pigment epithelium (RPE) death by modulating TNF-α/SOD1/ROS cascade mediated pyroptosis

High-glucose induced retinal pigment epithelium (RPE) death by triggering oxidative stress, however, the underlying mechanisms are still not fully delineated. In this study, the RPE cell line ARPE-19 were treated with different concentrations of glucose, the results showed that high-glucose (50 mM) inhibited cell proliferation, promoted cell apoptosis and reactive oxygen species (ROS) production in a time-dependent manner. Notably, we found that high-glucose (50 mM) increased the expression levels of Caspase-1, Gasdermin D, NLRP3, IL-1β and IL-18 in ARPE-19 cells, which indicated that high-glucose triggered pyroptotic cell death. Further results validated that both ROS scavenger N-acetyl cysteine (NAC) and pyroptosis inhibitor necrosulfonamide (NSA) reversed the effects of high-glucose (50 mM) on ARPE-19 cell proliferation, apoptosis and pyroptosis. In addition, high-glucose (50 mM) significantly decreased the levels of miR-130a and superoxide dismutase (SOD) 1, and promoted tumor necrosis factor (TNF)-α expressions in ARPE-19 cells. Interestingly, upregulation of miR-130a increased SOD1 levels in a TNF-α dependent manner. Furthermore, overexpression of miR-130a abrogated the effects of high-glucose (50 mM) on the above cell functions, which were all reversed by either upregulating TNF-α or knocking down SOD1 in ARPE-19 cells. Taken together, upregulation of miR-130a alleviated the cytotoxic effects of high-glucose (50 mM) on ARPE-19 cells by regulating TNF-α/SOD1/ROS axis mediated pyroptotic cell death.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/β, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell–derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical "moonlighting" functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

Inhibition of Lung Cancer by 2-Methoxy-6-Acetyl-7-Methyljuglone Through Induction of Necroptosis by Targeting Receptor-Interacting Protein 1.

Aims: Most chemotherapeutic agents exploit apoptotic signaling to trigger cancer cell death, which frequently results in drug resistance. Necroptosis, a nonapoptotic form of regulated cell death, offers an alternative strategy to eradicate apoptosis-resistant cancer cells. We previously reported a natural necroptosis inducer 2-methoxy-6-acetyl-7-methyljuglone (MAM) in A549 lung cancer cells. The current study is designed to investigate the detailed necroptotic signaling and its cytotoxicity on drug-resistant cancer cells. Furthermore, in vivo anticancer effects were also evaluated in nude mice model. Results: MAM directly targets receptor-interacting protein 1 (RIP1) kinase in A549 and H1299 cells, which is responsible for reactive oxygen species (ROS, mainly hydrogen peroxide) generation. A positive feedback loop between calcium (Ca2+) and c-Jun N-terminal kinase (JNK) occurred following ROS generation, leading to lysosomal membrane permeabilization and mitochondrial dysfunction. MAM showed similar cytotoxic potency toward cisplatin-resistant A549 (A549/Cis) cells by inducing necroptosis as confirmed by the protective effect of 7-Cl-O-Nec-1 (Nec-1s) and by the morphological characteristics obtained via transmission electron microscopy. Interestingly, tumor necrosis factor alpha (TNFα) was not involved in this process. Intraperitoneal injection of MAM significantly suppressed tumor growth in A549 tumor xenograft without significant body weight loss and multiorgan toxicities. Innovation and Conclusion: Our findings demonstrate that MAM induces necroptosis in A549 and H1299 lung cancer cells by targeting RIP1 kinase and ROS in a TNFα-independent manner. MAM kills A549/Cis cells with similar potency through induction of necroptosis. MAM shows anticancer effect in animal model. The present study raises the therapeutic possibility and strategy to combat cancer by the induction of necroptosis.

Abstract 13: Anti-angiogenic action of leukotriene-C4induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2 (COX-2), which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2 and is often down-regulated in cancer. Interesting enough, CRC patients expressing high levels of Cysteinyl leukotriene receptor 2 (CysLTR2) have a good prognosis and therefore, we investigated a potential link between CysLTR2-signaling and the tumor suppressor 15-PGDH in colon cancer cells. TNF-α is considered as the main regulators of COX-2 and mPGES-1 that contribute to the increased synthesis of PGE2, which is inhibited by overexpressed 15-PGDH. Level of the pro-tumorigenic PGE2 are increased in CRC, previously attributed to increased production via TNF-α mediated COX-2 up-regulation but more recently attributed to decreased catabolism due to down-regulation of 15-PGDH. Elevation of 15-PGDH expression by leukotriene C4 (LTC4), a CysLTR2 ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin and down-regulation of anti-apoptotic marker Bcl-2 with concurrent activation of CASPASE-3 expression. We also observed a dramatic down-regulation of TNF-α on mRNA level and NF-κβ on both mRNA as well as protein level with LTC4 induced 15-PGDH in a TNF-α dependent manner. Moreover, TNF-α regulated anti-angiogenic action of 15-PGDH in HT-29 and Caco-2 colon cancer cells by depleting the mRNA level of MMP-2 and MMP-9 and protein level of VEGFR-1. Furthermore, our preliminary observation also suggested disrupted tube formation in HUVEC with LTC4 induced 15-PGDH which is mediated by TNF-α. Hence, restoration of 15-PGDH expression through CysLTR2-signaling promotes the anti-angiogenic action against colon cancer cells, indicating an anti-tumor as well as the anti-metastatic efficacy of CysLTR2-signaling. Citation Format: Shakti R. Satapathy, Anita Sjölander. Anti-angiogenic action of leukotriene-C4induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 13.

P-196 - Anti-angiogenic action of leukotriene-C4 induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon

Introduction: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2 (COX-2), which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2 and is often down-regulated in cancer. Interesting enough, CRC patients expressing high levels of Cysteinyl leukotriene receptor 2 (CysLTR2) have a good prognosis and therefore, we investigated a potential link between CysLTR2-signaling and the tumor suppressor 15-PGDH in colon cancer cells. In the other hand, TNF-α is considered as the main regulator of COX-2 and mPGES-1 that contribute to the increased synthesis of PGE2, which is inhibited by overexpressed 15-PGDH. Level of the pro-tumorigenic PGE2 is increased in CRC, previously attributed to increased production via TNF-α mediated COX-2 up-regulation but more recently attributed to decreased catabolism due to down-regulation of 15-PGDH. Methods: HT-29 and Caco-2 cells were employed for this study. Unstimulated and LTC4 stimulated cells were compared for various markers through qRT-PCR as well as immunoblotting analysis. Vibrant-DiI labeled HT-29 cells were used for the zebrafish metastasis model. Visualization of the metastatic spread was observed using the light sheet microscopy (SPIM) and tissue sections were analyzed by immunohistochemistry for specific markers. Conditioned media from the unstimulated and LTC4 stimulated HT-29 cells were used to analyze the endothelial tube formation in HUVEC. Results: Elevation of 15-PGDH expression by leukotriene C4 (LTC4), a CysLTR2 ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin and down-regulation of anti-apoptotic marker Bcl-2 with concurrent activation of CASPASE-3 expression. We also observed a dramatic down-regulation of TNF-α on mRNA level and NF-κβ on both mRNA as well as protein level with LTC4 induced 15-PGDH in a TNF-α dependent manner. Moreover, TNF-α regulated anti-angiogenic action of 15-PGDH in HT-29 and Caco-2 colon cancer cells by depleting the mRNA level of MMP-2 and MMP-9 and protein level of VEGFR-1. Furthermore, we also observed disrupted tube formation in HUVEC with LTC4 induced 15-PGDH which is also governed by TNF-α. The angiogenesis study in transgenic zebrafish, Tg(fli1a: EGFP) embryo model suggested significant decrease in early and late metastasis with distinct disruption in the intersegmental vessel in the LTC4 induced 15-PGDH treated group. IHC analysis of Ki-67 exhibited decrease with LTC4 and very interestingly CASPASE-3 showed increase in the stimulated group which supported our in vitro observation. Conclusion: Hence, restoration of 15-PGDH expression through CysLTR2-signaling promotes the anti-angiogenic action against colon cancer cells, indicating an anti-tumor as well as the anti-metastatic efficacy of CysLTR2-signaling.

An Autocrine TNFα-Tumor Necrosis Factor Receptor 2 Loop Promotes Epigenetic Effects Inducing Human Treg Stability In Vitro.

A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro, in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro. Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα–TNFR2 underlying the maintenance of Treg stability in vitro. Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα–TNFR2 loop plays an important role in endorsing Treg stability.

Coexpression of NOS2 and COX2 accelerates tumor growth and reduces survival in estrogen receptor-negative breast cancer

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER- patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER- and TNBC disease.