scholarly journals Ceramide kinase regulates TNF-α-induced immune responses in human monocytic cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fatema Al-Rashed ◽  
Zunair Ahmad ◽  
Ashley J. Snider ◽  
Reeby Thomas ◽  
Shihab Kochumon ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Monocytic Cells ◽  
Metabolic Inflammation ◽  
Ceramide Kinase ◽  
Hla Dr ◽  
Tnf Α ◽  
Underlying Mechanisms ◽  
Interfering Rna ◽  
Ceramide 1

AbstractCeramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

scholarly journals Ceramide Kinase Regulates TNF-α-induced Immune Responses in Human Monocytic Cells

2021 ◽  
Author(s):  
Fatema Al-Rashed ◽  
Zunair Ahmad ◽  
Ashley J Snider ◽  
Reeby Thomas ◽  
Shihab Kochumon ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Monocytic Cells ◽  
Metabolic Inflammation ◽  
Ceramide Kinase ◽  
Hla Dr ◽  
Tnf Α ◽  
Underlying Mechanisms ◽  
Interfering Rna ◽  
Ceramide 1

Abstract Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes either by the chemical inhibitor NVP- 231 or by small interfering RNA (siRNA) results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK deficient cells. Phosphorylation of JNK, p38 and NF-κB resulting from TNF-α stimulation was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

scholarly journals Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fatema Al-Rashed ◽  
Zunair Ahmad ◽  
Reeby Thomas ◽  
Motasem Melhem ◽  
Ashley J. Snider ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Neutral Sphingomyelinase ◽  
Tnf Α ◽  
Key Enzyme ◽  
Underlying Mechanisms ◽  
Interfering Rna ◽  
Inflammatory Changes ◽  
Ceramide Production

Abstract Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase-2 (nSMase2: SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1β and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1β and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

scholarly journals Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α

2020 ◽  
Author(s):  
Fatema Al-Rashed ◽  
Zunair Ahmad ◽  
Reeby Thomas ◽  
Motasem Melhem ◽  
Ashley J. Snider ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Neutral Sphingomyelinase ◽  
Phosphodiesterase 3 ◽  
Tnf Α ◽  
Key Enzyme ◽  
Underlying Mechanisms ◽  
Interfering Rna ◽  
Inflammatory Changes ◽  
Ceramide Production

ABSTRACTObesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes /macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1b and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1b and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

A Steroidal Saponin RCE-4 Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses via Blocking PI3K/Akt-Mediated Nf-κB Activation in RAW264.7 Cells

2014 ◽  
Vol 568-570 ◽  
pp. 1901-1906 ◽  
Author(s):  
Cai Hong Bai ◽  
Hai Bo He ◽  
Fan Cheng ◽  
Kun Zou ◽  
Jun Zhi Wang ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Molecular Basis ◽  
Inflammatory Responses ◽  
Antiinflammatory Activity ◽  
Inflammatory Diseases ◽  
Steroidal Saponin ◽  
Promising Candidate ◽  
Akt Phosphorylation ◽  
Tnf Α ◽  
Underlying Mechanisms

Steroidal saponin: (1β,3β,5β,25S)-spirostan-1,3-diol1-[α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside] (RCE-4) is the most abundant and bioactive members in Reineckia carnea, has been reported to possess antiinflammatory activity, but the underlying mechanisms remain largely unknown. The present aim was to study expression of inflammatory cytokines, on the basis of this investigation, the possible mechanism of RCE-4 was elucidated. In the present study, we found that the concentrations of TNF-α, IL-1β and IL-6 released from LPS-stimulated RAW264.7cells significantly increased compared to control (P<0.01, respectively). After pretreatment with RCE-4, the TNF-α, IL-1β and IL-6 levels significantly decreased compared with the LPS group (P<0.05, P<0.01, respectively). Further studies indicated that RCE-4 significantly suppressed Akt phosphorylation and NF-қB activation, and with the dose of RCE-4 increasing; their improvement became more and more strong. Our results showed that RCE-4 inhibited LPS-stimiulated TNF-α, IL-1β, IL-6 productions through the blockage of PI3K/Akt-mediated NF-κB activation. Our findings might provide a molecular basis for the ability of RCE-4 serving as a promising candidate for treating various inflammatory diseases.

scholarly journals The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways

2019 ◽  
Vol 20 (18) ◽  
pp. 4658 ◽  
Author(s):  
Sindhu ◽  
Kochumon ◽  
Shenouda ◽  
Wilson ◽  
Al-Mulla ◽  
...  
Keyword(s):  
Low Grade ◽  
Monocytic Cells ◽  
Signaling Mechanism ◽  
Blocking Antibody ◽  
Metabolic Inflammation ◽  
Qrt Pcr ◽  
Signaling Mechanisms ◽  
Tnf Α ◽  
Low Grade Inflammation ◽  
Tlr4 Receptor

: Chronic low-grade inflammation, also known as metabolic inflammation, is a hallmark of obesity and parallels with the presence of elevated circulatory levels of free fatty acids and inflammatory cytokines/chemokines. CCL4/MIP-1β chemokine plays a key role in the adipose tissue monocyte recruitment. Increased circulatory levels of TNF-α, palmitate and CCL4 are co-expressed in obesity. We asked if the TNF-α/palmitate could interact cooperatively to augment the CCL4 production in human monocytic cells and macrophages. THP-1 cells/primary macrophages were co-treated with TNF-α/palmitate and CCL4 mRNA/protein expression was assessed using qRT-PCR/ELISA. TLR4 siRNA, a TLR4 receptor-blocking antibody, XBlue™-defMyD cells and pathway inhibitors were used to decipher the signaling mechanisms. We found that TNF-α/palmitate co-stimulation augmented the CCL4 expression in monocytic cells and macrophages compared to controls (p < 0.05). TLR4 suppression or neutralization abrogated the CCL4 expression in monocytic cells. Notably, CCL4 cooperative induction in monocytic cells was: (1) Markedly less in MyD88-deficient cells, (2) IRF3 independent, (3) clathrin dependent and (4) associated with the signaling mechanism involving ERK1/2, c-Jun, JNK and NF-κB. In conclusion, TNF-α/palmitate co-stimulation promotes the CCL4 expression in human monocytic cells through the mechanism involving a TLR4-MyD88 axis and MAPK/NF-κB pathways. These findings unravel a novel mechanism of the cooperative induction of CCL4 by TNF-α and palmitate which could be relevant to metabolic inflammation.

scholarly journals Toll-Like Receptor 2 Recognition of the Microsporidia Encephalitozoon spp. Induces Nuclear Translocation of NF-κB and Subsequent Inflammatory Responses

2008 ◽  
Vol 76 (10) ◽  
pp. 4737-4744 ◽  
Author(s):  
Jeffrey Fischer ◽  
Colby Suire ◽  
Hollie Hale-Donze
Keyword(s):  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Host Recognition ◽  
Direct Role ◽  
Necrosis Factor Alpha ◽  
Human Macrophages ◽  
Encephalitozoon Intestinalis ◽  
Intracellular Parasites ◽  
Tnf Α ◽  
Interfering Rna

ABSTRACT Microsporidia are obligate intracellular parasites that are ubiquitous in nature and have been recognized as causing an important emerging disease among immunocompromised individuals. Limited knowledge exists about the immune response against these organisms, and virtually nothing is known about the receptors involved in host recognition. Toll-like receptors (TLR) are pattern recognition receptors that bind to specific molecules found on pathogens and signal a variety of inflammatory responses. In this study, we show that both Encephalitozoon cuniculi and Encephalitozoon intestinalis are preferentially recognized by TLR2 and not by TLR4 in primary human macrophages. This is the first demonstration of host receptor recognition of any microsporidian species. TLR2 ligation is known to activate NF-κB, resulting in inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). We found that the infection of primary human macrophages leads to the nuclear translocation of NF-κB in as early as 1 h and the subsequent production of TNF-α and IL-8. To verify the direct role of TLR2 parasite recognition in the production of these cytokines, the receptor was knocked down in primary human macrophages using small interfering RNA. This knockdown resulted in decreases in both the nuclear translocation of NF-κB and the levels of TNF-α and IL-8 after challenge with spores. Taken together, these experiments directly link the initial inflammatory response induced by Encephalitozoon spp. to TLR2 stimulation in human macrophages.

scholarly journals Dimethyl itaconate inhibits TNF-α induced NF-κB signaling pathway in human epithelial cells

2020 ◽  
Author(s):  
Yalei Zhang ◽  
Xiaobing Deng ◽  
Hao Liang ◽  
Annan Guo ◽  
Kenan Li ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Cysteine Residue ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Nuclear Entry ◽  
Tnf Α ◽  
Novel Strategy ◽  
Dose Dependent ◽  
Dimethyl Itaconate

Abstract Background: Dimethyl itaconate (DMI), a membrane-permeable derivative of itaconate, was found to moderate IL-17-IκBζ-induced skin pathology including psoriasis in mouse experiments . TNF-α induced NF-κB pathway, which controls a variety of immune and inflammatory responses, was also proven to play a crucial role as mediator in psoriasis. However, whether DMI interacts with the TNF-α induced NF-κB pathway remains unclear. Results: Here we show that DMI inhibits TNF-α induced NF-κB transcriptional activities in dose-dependent manner in several human cell lines using dual luciferase assay and blocks the NF-κB nuclear entry. Moreover, DMI potently inhibits IKKβ dependent phosphorylation and degradation of IκBα in TNF-α induced activation of NF-κB pathway. We also demonstrate that DMI covalently binds to cysteine residue in IKKβ, a key regulator in NF-κB pathway, to suppress IKKβ activation and inhibit the canonical NF-κB pathway. Conclusion Our study presents a new mechanism for DMI as an anti-inflammatory agent that may have therapeutic potentials in treating NF-κB related human inflammatory diseases. Our results also suggest that itaconate produced by endogenous IRG1 may regulate NF-κB at post translation modification level, and the IRG1-itaconate-NF-κB axis could be targeted as a novel strategy for the treatment of IRG1-NF-κB mediated diseases.

Load Down-Regulates TNF-Alpha Induced Cartilage Degradation in Part Through NF-KB and P38 Pathways

2007 ◽  
Author(s):  
Valerie M. Wolfe ◽  
Seonghun Park ◽  
Marjana Tomic ◽  
Peter A. Torzilli ◽  
C. T. Christopher Chen
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Interleukin 1 ◽  
Tensile Load ◽  
Cartilage Degradation ◽  
Acute Injury ◽  
Cyclic Compression ◽  
Tnf Α ◽  
Necrosis Factor ◽  
Pro Inflammatory Cytokines

Pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), can induce cartilage degradation after acute injury or in inflammatory diseases [1,2,3,7]. The degradative events are coordinated through the elevation and activation of two classes of enzymes, namely matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS-4 and −5) [1,6]. Prior studies suggested that pro-inflammatory responses induced by IL-1β can be inhibited by tensile load [2] and more recently by cyclic compression [8]. It is, however, not clear whether load affects other cytokines, such as TNF-α. TNF-α is known to bind its receptor (TNFR1) to cause a cascade that ends with degradation of an inhibitor, IκBα, and release of the transcription factor NF-κB [3]. The actions of TNF-α are also known to be affected by at least three NF-κB independent pathways including the p38, ERK, and JNK pathways [4]. The objective of this study was to determine whether cyclic compression could affect TNF-α induced cartilage degradation and to determine the roles of p38, ERK, and JNK pathways in TNF-induced cartilage degradation. We hypothesized that cyclic loading would inhibit the degradative effects caused by TNF-α.

scholarly journals Palmitate-Induced MMP-9 Expression in the Human Monocytic Cells is Mediated through the TLR4-MyD88 Dependent Mechanism

2016 ◽  
Vol 39 (3) ◽  
pp. 889-900 ◽  
Author(s):  
Sardar Sindhu ◽  
Areej Al-Roub ◽  
Merin Koshy ◽  
Reeby Thomas ◽  
Rasheed Ahmad
Keyword(s):  
Underlying Mechanism ◽  
Positive Control ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Monocytic Cells ◽  
Metabolic Inflammation ◽  
Human Monocytic Cell Line ◽  
Tnf Α ◽  
Human Monocytic Cell

Background/Aims: Obese individuals are known to have increased Matrix metalloproteinase (MMP)-9 plasma levels and MMP-9 is reported to play an important role in obesity-associated adipose tissue inflammation. Since in obesity, the levels of circulatory saturated free fatty acid (FFA) palmitate (palimitic acid) are increased and modulate the expression of inflammatory mediators, the role of palmitate in the regulation of MMP-9 remains unclear. Methods: Human monocytic cell line THP-1 and primary monocytes were stimulated with palmitate and TNF-α (positive control). MMP-9 expression was assessed with real time RT-PCR and ELISA. Signaling pathways were studied by using THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. Phosphorylation of NF-kB and c-Jun was analyzed by Western blotting. Results: Here, we provide the evidence that palmitate induces MMP-9 expression at both mRNA (THP-1: 6.8 ± 1.2 Fold; P = 0.01; Primary monocytes: 5.9 ± 0.7 Fold; P = 0.0003) and protein (THP1: 1116 ±14 pg/ml; P<0.001; Primary monocytes: 1426 ± 13.8; P = 0.0005) levels in human monocytic cells. Palmitate-induced MMP-9 secretion was markedly suppressed by neutralizing anti-TLR-4 antibody (P < 0.05). Furthermore, genetic silencing of TLR4 by siRNA also significantly abrogated the palmitate-induced up-regulation of MMP-9. Additionally, MyD88-/- THP-1 cells did not express MMP-9 in response to palmitate treatment. Increased NF-κB/AP-1 activity (P<0.05) was also observed in palmitate-treated THP-1 cells. Conclusion: Altogether, these results show that palmitate induces TLR4-dependent activation of MMP-9 gene expression, which requires the recruitment of MyD88 leading to activation of NF-kB/AP-1 transcription factors. Thus, our findings suggest that the palmitate-induced MMP-9 secretion might be an underlying mechanism of its increased levels in obesity and related metabolic inflammation.

scholarly journals MIP-1α Expression Induced by Co-Stimulation of Human Monocytic Cells with Palmitate and TNF-α Involves the TLR4-IRF3 Pathway and Is Amplified by Oxidative Stress

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1799
Author(s):  
Sardar Sindhu ◽  
Nadeem Akhter ◽  
Ajit Wilson ◽  
Reeby Thomas ◽  
Hossein Arefanian ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Inflammatory Cells ◽  
H2o2 Treatment ◽  
Monocytic Cells ◽  
Metabolic Inflammation ◽  
Genetic Ablation ◽  
Protein Levels ◽  
Inflammatory Protein ◽  
Tnf Α ◽  
In Cells

Metabolic inflammation is associated with increased expression of saturated free fatty acids, proinflammatory cytokines, chemokines, and adipose oxidative stress. Macrophage inflammatory protein (MIP)-1α recruits the inflammatory cells such as monocytes, macrophages, and neutrophils in the adipose tissue; however, the mechanisms promoting the MIP-1α expression remain unclear. We hypothesized that MIP-1α co-induced by palmitate and tumor necrosis factor (TNF)-α in monocytic cells/macrophages could be further enhanced in the presence of reactive oxygen species (ROS)-mediated oxidative stress. To investigate this, THP-1 monocytic cells and primary human macrophages were co-stimulated with palmitate and TNF-α and mRNA and protein levels of MIP-1α were measured by using quantitative reverse transcription, polymerase chain reaction (qRT-PCR) and commercial enzyme-linked immunosorbent assays (ELISA), respectively. The cognate receptor of palmitate, toll-like receptor (TLR)-4, was blunted by genetic ablation, neutralization, and chemical inhibition. The involvement of TLR4-downstream pathways, interferon regulatory factor (IRF)-3 or myeloid differentiation (MyD)-88 factor, was determined using IRF3-siRNA or MyD88-deficient cells. Oxidative stress was induced in cells by hydrogen peroxide (H2O2) treatment and ROS induction was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The data show that MIP-1α gene/protein expression was upregulated in cells co-stimulated with palmitate/TNF-α compared to those stimulated with either palmitate or TNF-α (P < 0.05). Further, TLR4-IRF3 pathway was implicated in the cooperative induction of MIP-1α in THP-1 cells, and this cooperativity between palmitate and TNF-α was clathrin-dependent and also required signaling through c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Notably, ROS itself induced MIP-1α and could further promote MIP-1α secretion together with palmitate and TNF-α. In conclusion, palmitate and TNF-α co-induce MIP-1α in human monocytic cells via the TLR4-IRF3 pathway and signaling involving c-Jun/NF-κB. Importantly, oxidative stress leads to ROS-driven MIP-1α amplification, which may have significance for metabolic inflammation.

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