NF-κB Activation In Macrophages Research Articles

Haptoglobin buffers lipopolysaccharides to delay activation of NFκB.

It has remained yet unclear which soluble factors regulate the anti-inflammatory macrophage phenotype observed in both homeostasis and tumourigenesis. We show here that haptoglobin, a major serum protein with elusive immunoregulatory properties, binds and buffers bacterial lipopolysaccharides to attenuate activation of NFκB in macrophages. Haptoglobin binds different lipopolysaccharides with low micromolar affinities. Given its abundance, haptoglobin constitutes a buffer for serum-borne lipopolysaccharides, shielding them to safeguard against aberrant inflammatory reactions by reducing the amount of free lipopolysaccharides available for binding to TLR4. Concordantly, NFκB activation by haptoglobin-associated lipopolysaccharides was markedly delayed relative to stimulation with pure lipopolysaccharide. Our findings warrant evaluation of therapeutic benefits of haptoglobin for inflammatory conditions and re-evaluation of purification strategies. Finally, they allow to elucidate mechanisms of enhanced immunosuppression by oncofetal haptoglobin.

Resolvin D2 attenuates LPS-induced macrophage exhaustion.

Early in sepsis, a hyperinflammatory response is dominant, but later, an immunosuppressive phase dominates, and the host is susceptible to opportunistic infections. Anti-inflammatory agents may accelerate the host into immunosuppression, and few agents can reverse immunosuppression without causing inflammation. Specialized pro-resolving mediators (SPMs) such as resolvin D2 (RvD2) have been reported to resolve inflammation without being immunosuppressive, but little work has been conducted to examine their effects on immunosuppression. To assess the effects of RvD2 on immunosuppression, we established a model of macrophage exhaustion using two lipopolysaccharide (LPS) treatments or hits. THP-1 monocyte-derived macrophages were first treated with RvD2 or vehicle for 1 h. One LPS hit increased NF-κB activity 11-fold and TNF-α release 60-fold compared to unstimulated macrophages. RvD2 decreased LPS-induced NF-κB activity and TNF-α production but increased bacterial clearance. Two LPS hits reduced macrophage bacterial clearance and decreased macrophage NF-κB activity (45%) and TNF-α release (75%) compared to one LPS hit, demonstrating exhaustion. RvD2 increased NF-κB activity, TNF-α release, and bacterial clearance following two LPS hits compared to controls. TLR2 inhibition abolished RvD2-mediated changes. In a mouse sepsis model, splenic macrophage response to exogenous LPS was reduced compared to controls and was restored by invivo administration of RvD2, supporting the invitro results. If RvD2 was added to monocytes before differentiation into macrophages, however, RvD2 reduced LPS responses and increased bacterial clearance following both one and two LPS hits. The results show that RvD2 attenuated macrophage suppression invitro and invivo and that this effect was macrophage-specific.

SALMONELLA INFECTION: INTERPLAY BETWEEN THE T3SSS EFFECTORS AND NF-ΚB SIGNALING PATHWAY

Salmonella is an important foodborne pathogen that can evade host immune defense by evolving unique mechanisms. Salmonella manipulate host cell various signaling pathways by delivering specific effectors into target cells to establish infection. The nuclear factor-κB (NF-κB) is an important nuclear transcription factor that regulates the host immune system in Salmonella infection. The Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) encode type III secretion systems (T3SSs), effectors that are associated with the NF-κB signaling pathway through regulate host inflammation response. SPI-1 effectors SipA, SopE, SopE2, and SopB all can activate NF-κB signaling pathway to facilitate Salmonella invasion and intracellular carriage. Studies have shown that T3SS1 and/or T3SS2 effectors such as GtgA, GogA and PipA contain two histidine residues and have metalloprotease activity to control Salmonella replication. These zinc metalloproteases redundantly target the NF-κB subunits p65, RelB, and c-Rel, whereas GogA and GtgA only inhibit NF-κB-dependent gene transcription. The T3SS2 effectors SseK1, SseK2, and SseK3 are death domain-containing proteins with N-linked glycosyltransferase characteristics that can inhibit NF-κB activity by inhibiting IκBα phosphorylation in TNF- α-treated 293ET cells. Among them, SseK1 and SseK3 also suppress Salmonella-induced NF-κB activity in macrophages. SseK3-mediated inhibition of the NF-B signaling pathway is not required for protein 32 containing a tripartite E3-ubiquitin ligase motif. In addition, the SPI-2 T3SS effector SpvD inhibits NF-κB activity by preventing nuclear translocation of p65 through interaction with Exportin-2, but this does not affect IκBα degradation, which ultimately leads to systemic Salmonella growth. However, other effectors SptP, AvrA, IpaJ, SspH1, GtgA, GogA, and SPI-2 encoded SseL, SpvB, SseK1, and GogB all can effectively inhibit NF-κB signaling pathway, and contribute to Salmonella intracellular replication and virulence. In this mini-review, we summarize the special mechanism how NF-κB signaling pathway is regulated by Salmonella T3SSs effectors in the persistent infection of Salmonella, which will further elucidate the pathogenesis of Salmonella.

Effect of Hesperidin on Barrier Function and Reactive Oxygen Species Production in an Oral Epithelial Cell Model, and on Secretion of Macrophage-Derived Inflammatory Mediators during Porphyromonas gingivalis Infection.

Porphyromonas gingivalis is a periodontopathogenic bacterium that can adhere to and colonize periodontal tissues, leading to an inflammatory process, and, consequently, tissue destruction. New therapies using flavonoids, such as hesperidin, are being studied, and their promising properties have been highlighted. The aim of this study was to evaluate the effect of hesperidin on the epithelial barrier function, reactive oxygen species (ROS) production, and on the inflammatory response caused by P. gingivalis in in vitro models. The integrity of the epithelial tight junctions challenged by P. gingivalis was determined by monitoring the transepithelial electrical resistance (TER). P. gingivalis adherence to a gingival keratinocyte monolayer and a basement membrane model were evaluated by a fluorescence assay. A fluorometric assay was used to determine the ROS production in gingival keratinocytes. The level of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) secretion was evaluated by ELISA; to assess NF-κB activation, the U937-3xjB-LUC monocyte cell line transfected with a luciferase reporter gene was used. Hesperidin protected against gingival epithelial barrier dysfunction caused by P. gingivalis and reduced the adherence of P. gingivalis to the basement membrane model. Hesperidin dose-dependently inhibited P. gingivalis-mediated ROS production by oral epithelial cells as well as the secretion of IL-1β, TNF-α, IL-8, MMP-2, and MMP-9 by macrophages challenged with P. gingivalis. Additionally, it was able to attenuate NF-κB activation in macrophages stimulated with P. gingivalis. These findings suggest that hesperidin has a protective effect on the epithelial barrier function, in addition to reducing ROS production and attenuating the inflammatory response associated with periodontal disease.

A frameshift variant in the SIRPB1 gene confers susceptibility to Crohn's disease in a Chinese population.

Background: Crohn's disease (CD), a chronic gastrointestinal inflammatory disease, is increasing in China. With a focus on Han Chinese families with CD, the aim of this study was to find genetic variations that increase CD susceptibility by genome sequencing, genetic association, expression, and functional research. Materials and methods: We performed family-based genome sequencing (WGS) analysis on 24 patients with CD from 12 families and then filtered shared potential causal variants by incorporating association results from meta-analyses of CD GWAS and immunology genes and in silico variant effect prediction algorithms. Replication analyses were performed in an independent cohort including 381 patients with CD and 381 control subjects. Results: There were 92 genetic variants significantly associated with CD in Chinese individuals. Among them, 61 candidate loci were validated in replication analyses. As a result, patients carrying a rare frameshift variant (c.1143_1144insG; p. Leu381_Leu382fs) in gene SIRPB1 had significantly higher risk to develop CD (p = 0.03, OR 4.59, 95% CI 0.98-21.36, 81.82% vs. 49.53%). The frameshift variation induced tyrosine phosphorylation of Syk, Akt, and Jak2, elevated the expression of SIRPB1 at the mRNA and protein levels, activated DAP12, and controlled the activation of NF-κB in macrophages. Additionally, it promoted the synthesis of the pro-inflammatory cytokines IL-1, TNF-, and IL-6. Conclusion: Our results suggest that the rare gain-of-function frameshift variant in SIRPB1 is associated in Han Chinese patients with CD. The functional mechanism of SIRPB1 and its downstream inflammatory pathways was preliminarily explored in CD.

ZNF667 Suppressed LPS-induced Macrophages Inflammation through mTORdependent Aerobic Glycolysis Regulation.

Macrophages participate in all stages of the inflammatory response, and the excessive release of inflammatory mediators and other cytokines synthesized and secreted by macrophages is fundamentally linked to an uncontrolled inflammatory response. The zinc finger 667 (ZNF667) protein, a novel DNAbinding protein, has been shown to play a vital role in oxidative stress. However, none of the target genes in macrophages or the potential roles of ZNF667 have been elucidated to date. The present study was designed to investigate the effects of ZNF667 on LPS-induced inflammation in macrophages. The RAW264.7 macrophage cell line was selected as a model system. Inflammatory response-related gene expression levels and phosphorylation levels of PI3K, AKT, and mTOR were detected in LPS-treated macrophages via RT-PCR and western blotting, respectively. We found that LPS resulted in the up-regulation of ZNF667 in macrophages and a peak response in ZNF667 protein expression levels when used at a concentration of 100 ng/mL. ZNF667 overexpression significantly inhibited the LPS-induced up-regulation of iNOS, and IL-1β mRNA and protein expression levels, together with the secretion of IL-1β, IL-6, and TNF-α. ZNF667 overexpression also inhibited PI3K, AKT, and mTOR hyperphosphorylation and had no effect on the phosphorylation of NF-κB p65, ERK1/2, MAPK p38, and the transcriptional activity of NF-κB in macrophages. The up-regulation of ZNF667 inhibited the levels of expression of HK2 and PFKFB3, glucose consumption, and lactate production in LPS-stimulated macrophages. The up-regulation of mRNA levels of LPS-induced glycolytic genes HK2 and PFKFB3 and the increased mRNA expression of pro-inflammatory cytokines (IL-1β and iNOS) were abolished by hexokinase inhibitor 2-DG in ZNF667-deficient macrophages. Meanwhile, glucose consumption and lactate production were abrogated in macrophages when cells were treated with the specific mTOR inhibitor RPM. Our results demonstrate that ZNF667 suppressed LPS-stimulated RAW264.7 macrophage inflammation by regulating mTOR-dependent aerobic glycolysis.

Sphingosine 1-phosphate receptor 2 mediated early stages of pancreatic and systemic inflammatory responses via NF-kappa B activation in acute pancreatitis

In acute pancreatitis, activation of inflammatory signaling, including the nuclear factor-kappa B (NF-κB) pathway, within acinar cells is known to be an early intracellular event occurring in parallel with pathologic trypsinogen activation. Sphingosine 1-phosphate receptor 2 (S1PR2) plays a critical role in endothelial inflammation, and our previous studies reported that S1PR2 deficiency significantly reduced the inflammatory response in liver injury under cholestasis conditions. However, the role of S1PR2 in inflammatory signaling activation within acinar cells and inflammatory responses during acute pancreatitis has not been elucidated. Here we report that S1PR2 was upregulated in the whole pancreas during acute pancreatitis. Blockade of S1PR2 by pharmacologic inhibition of S1PR2 by JTE-013 or AAV-mediated knockdown of S1PR2 improved the severity of pancreatic injury, as indicated by a significant reduction in inflammation and acinar cells death in acute pancreatitis mice. Moreover, S1PR2 is the predominant S1PRs expressed in pancreatic acinar cells and mediates NF-κB activation and the early inflammatory response within acinar cells under acute pancreatitis conditions via ROCK signaling pathways, not extracellular signal-regulated kinase pathways or p38 mitogen-activated protein kinase pathways. In addition, S1PR2 mediated macrophage NF-κB activation, migration and polarization toward the M1 phenotype. Therefore, these results demonstrated that the S1PR2-mediated early inflammatory response in acinar cells promotes the progression of acute pancreatitis, successfully linking local events to the systematic inflammatory response and leading to a novel therapeutic target for acute pancreatitis aimed at halting the progression of the inflammatory response.4Upw5czRUXogyYiQEsPybuVideo

Notch1 signaling contributes to TLR4-triggered NF-κB activation in macrophages

Macrophages substantially influence the development, progression, and complications of inflammation-driven diseases. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and interaction of Notch signaling with other signaling pathways in macrophages during inflammation, such as the NF-κB pathway. This study confirmed that stimulation with lipopolysaccharide (LPS), a TLR4 ligand, upregulated Notch1 expression in monocyte/macrophage-like RAW264.7 cells and bone marrow-derived macrophages (BMDMs). LPS also induced increased mRNA expression of Notch target genes Notch1 and Hes1 in macrophages, suggesting that TLR4 signaling enhances activation of the Notch pathway. The upregulation of Notch1, Notch1 intracellular domain (NICD), and Hes1 proteins by LPS treatment was inhibited by DAPT, a Notch1 inhibitor. Additionally, the increased TNF-α, IL-6, and IL-1β expression induced by LPS was inhibited by DAPT and rescued by jagged1, a Notch1 ligand. Furthermore, suppression of Notch signaling by DAPT upregulated Cylindromatosis (CYLD) expression but downregulated TRAF6 expression, IκB kinase (IKK) α/β phosphorylation, and subsequently, phosphorylation and degradation of IκB-α, indicating that DAPT inhibited NF-κB activation triggered by TLR-4. Interestingly, DAPT did not inhibit the increased MyD88 expression induced by LPS. Our study findings demonstrate that macrophage stimulation via the TLR4 signaling cascade triggers activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses by activating NF-κB. This effect may be dependent on the CYLD-TRAF6-IKK pathway. Thus, Notch1 signaling may provide a therapeutic target against infectious and inflammation-driven diseases.

Shinjulactone A Blocks Vascular Inflammation and the Endothelial-Mesenchymal Transition

ObjectiveThe endothelial inflammatory response plays an important role in atherogenesis by inducing nuclear factor (NF)κB-dependent cell adhesion molecule expression and monocyte recruitment. Here, we screened for natural ligands and investigated the ability of shinjulactone A to inhibit interleukin-1β (IL-1β)-induced endothelial inflammatory signaling.MethodsThe natural compound library included 880 single compounds isolated from medicinal plants by the Korean Medicinal Material Bank. Primary endothelial cells were pretreated with single compounds before stimulation with IL-1β to induce endothelial inflammation. Endothelial inflammation was measured by assaying NFκB activation and monocyte adhesion. The endothelial-mesenchymal transition (EndMT) was evaluated using cell type-specific marker protein expression and morphology.ResultsShinjulactone A was identified as an efficient blocker of IL-1β -induced NFκB activation, with a half-maximal inhibitory concentration of approximately 1 µM, and monocyte recruitment in endothelial cells. However, it did not affect lipopolysaccharide-induced NFκB activation in macrophages. Compared to Bay 11-782, a well-known NFκB inhibitor that shows considerable cytotoxicity during long-term treatment, shinjulactone A did not affect endothelial cell viability. Furthermore, it also significantly inhibited the EndMT, which is known to promote atherosclerosis and plaque instability.ConclusionWe suggest that shinjulactone A may be an effective and safe drug candidate for atherosclerosis because it targets and inhibits both endothelial inflammation and the EndMT, without impairing NFκB-dependent innate immunity in macrophages.

SOCS2 Suppresses Inflammation and Apoptosis During NASH Progression Through Limiting NF-κB Activation in Macrophages

Background: Inflammation and apoptosis play a crucial role in the progression of nonalcoholic steatohepatitis (NASH). Suppressor of cytokine signaling 2 (SOCS2) is one of classic negative regulators of cytokine signaling, which has recently been described as anti-inflammatory mediators. However, the role of SOCS2 in macrophages during NASH progression and the relationship among SOCS2, inflammation, apoptosis and NASH is largely unknown. Herein, we aimed to study the function of SOCS2 in NASH progression. Methods: We detected SOCS2 expression in macrophages in human subjects without steatosis, with simple steatosis and with NASH to confirm the relationship between SOCS2 and NASH. Free fatty acids was used to establish stress environment in RAW cell lines stably overexpressing or knockdown SOCS2. In vitro and vivo assays also performed to study the molecular function of SOCS2 in NASH progression. Findings: Our human samples and NASH model in vitro illustrated that SOCS2 is decreased in macrophages during NASH progression and is negatively correlated to NASH level. Meanwhile, In vitro assays show SOCS2 overexpression in macrophages suppresses inflammation and apoptosis via inhibiting NF-κB signaling pathway, while SOCS2 knock-down in macrophages caused an increased activation of NF-κB, which can be blocked by ammonium 1-pyrrolidinedithiocarbamate (PDTC). In addition SOCS2 in macrophages also suppresses inflammation via limiting the activation of inflammasomes. Consist with these, our BMT model also confirm the SOCS2 function in macrophages during NASH. Interpretation: Our data strongly indicate that SOCS2 plays a inhibitor of inflammation and apoptosis via NF-κB signal pathway and inflammasome signal pathway in macrophages during NASH. Further studies are required to explore the potential prevention and therapeutic strategies of SOCS2 for this common liver disease. Funding Statement: This work was supported by the National Natural Science Foundation of China (Nos. 81970499 and 81770561), Jiangsu Medical Leading Talent, Innovation Team (No. CXTDA2017033), and Jiangsu Province “333” project (No. BRA2014332) and National Natural Science Foundation of China (Nos. 81972675). Declaration of Interests: The authors declare no conflict of interest relevant to the present manuscript. Ethics Approval Statement: All procedures that involved human sample collection were approved by the tissue bank of The Affiliated Drum Tower Hospital, Nanjing University (Nanjing, China). Informed consent for gene expression analysis of tissue was obtained from each patient before surgery, and the study was approved by our institutional ethics committee. The ethics committe of the Institutional Animal Care and Use Committee (IACUC). First Affiliated Hospital of Nanjing Medical University approved this study, (Ethics Number: 2018-SRFA-034).

Lactucopicrin Inhibits Cytoplasmic Dynein-Mediated NF-κB Activation in Inflammated Macrophages and Alleviates Atherogenesis in Apolipoprotein E-Deficient Mice.

Nuclear factor-κB (NF-κB) activation in macrophages aggravates atherosclerosis. Dietary plant secondary metabolites including sesquiterpene lactone lactucopicrin target multiple organs. This study is focused on the impact of lactucopicrin on NF-κB activation in inflammed macrophages and atherogenesis in a mouse model of atherosclerosis. In LPS-stimulated mouse bone marrow-derived macrophages, lactucopicrin inhibits NF-κB activation, and concomitantly represses the expression of IL-1β, IL-6, and tumor necrosis factor-alpha. This effect is not due to modulation of the inhibitor of NF-κB kinases (IKK) α/β/γ and NF-κB inhibitor α, and NF-κB/p65 DNA binding activity. Instead, the lactucopicrin effect is reliant on the inhibition of cytoplasmic dynein-mediated p65 transportation, a prerequisite step for p65 nuclear translocation. In high-fat diet-fed apolipoprotein E-deficient mice, lactucopicrin consumption dose-dependently reduces plaque area, inhibits plaque macrophage accumulation, attenuates plaque macrophage NF-κB activation, and reduces both plaque and serum inflammatory burden. However, lactucopicrin consumption does not affect the levels of serum lipids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor beta). Dietary lactucopicrin inhibits atherogenesis in mice likely by its anti-inflammatory property. These findings suggest that dietary supplementation with lactucopicrin is a promising strategy to inhibit atherosclerotic cardiovascular disease.

Telocytes Enhances M1 Differentiation and Phagocytosis While Inhibits Mitochondria-Mediated Apoptosis Via Activation of NF-κB in Macrophages

Telocytes (TCs), which are a recently discovered interstitial cell type present in various organs and tissues, perform multiple biological functions and participate in extensive crosstalk with neighboring cells. Endometriosis (EMs) is a gynecological disease characterized by the presence of viable endometrial debris and impaired macrophage phagocytosis in the peritoneal environment. Here, CD34/vimentin-positive TCs were co-cultured with RAW264.7 cells in vitro. M1/M2 differentiation-related markers were detected; phagocytosis, energy metabolism, proliferation, apoptosis, and pathway mechanisms were studied; and the mitochondrial membrane potential (ΔΨm) was measured. Furthermore, in an EMs mouse model, the differentiation of macrophages in response to treatment with TC-conditioned medium (TCM) in vivo was studied. The results showed that upon in vitro co-culture with TCM, RAW264.7 cells differentiated more toward the M1 phenotype with enhancement of phagocytosis, increase in energy metabolism and proliferation owing to reduced the loss of ΔΨm, and suppression of dexamethasone-induced apoptosis. Further, along with the activation of NF-κB, Bcl-2 and Bcl-xl, the expression of Bax, cleaved-caspase9, and cleaved-caspase3 reduced in RAW264.7 cells. In addition, the M1 subtype was found to be the dominant phenotype among tissue and peritoneal macrophages in the EMs model subjected to in vivo TCM treatment. In conclusion, TCs enhanced M1 differentiation and phagocytosis while inhibiting apoptosis via the activation of NF-κB in macrophages, which potentially inhibited the onset of EMs. Our findings provide a potential research target and the scope for developing a promising therapeutic strategy for EMs.

SOCS2 Suppresses Inflammation and Apoptosis during NASH Progression through Limiting NF-κB Activation in Macrophages

Background: Inflammation and apoptosis play a crucial role in the progression of nonalcoholic steatohepatitis (NASH). Suppressor of cytokine signaling 2 (SOCS2) is one of classic negative regulators of cytokine signaling, which has recently been described as anti-inflammatory mediators. However, the role of SOCS2 in macrophages during NASH progression and the relationship among SOCS2, inflammation, apoptosis and NASH is largely unknown. Herein, we aimed to study the function of SOCS2 in NASH progression.Methods: We detected SOCS2 expression in macrophages in human subjects without steatosis, with simple steatosis and with NASH to confirm the relationship between SOCS2 and NASH. Free fatty acids was used to establish stress environment in RAW 264.7 cell lines stably overexpressing or knockdown SOCS2. In vitro and vivo assays also performed to study the molecular function of SOCS2 in NASH progression.Findings: Our human samples illustrated that SOCS2 was decreased in macrophages during NASH progression and was negatively correlated to NASH level. Meanwhile, In vitro assays showed SOCS2 overexpression in macrophages suppressed inflammation and apoptosis via inhibiting NF-κB signaling pathway, while SOCS2 knock-down in macrophages caused an increased activation of NF-κB, which could be blocked by ammonium 1-pyrrolidinedithiocarbamate (PDTC). In addition, SOCS2 in macrophages also suppressed inflammation via limiting the activation of inflammasomes. Consistent with these, our BMT model also confirmed the SOCS2 function in macrophages during NASH.Interpretation: Our data strongly indicate that SOCS2 plays a role in inhibiting inflammation and apoptosis via NF-κB and inflammasome signaling pathway in macrophages during NASH. Further studies are required to explore the potential preventive and therapeutic strategies of SOCS2 for this common liver disease.

Lactate Suppresses Macrophage Pro-Inflammatory Response to LPS Stimulation by Inhibition of YAP and NF-κB Activation via GPR81-Mediated Signaling.

Recent evidence from cancer research indicates that lactate exerts a suppressive effect on innate immune responses in cancer. This study investigated the mechanisms by which lactate suppresses macrophage pro-inflammatory responses. Macrophages [Raw 264.7 and bone marrow derived macrophages (BMDMs)] were treated with LPS in the presence or absence of lactate. Pro-inflammatory cytokines, NF-κB and YAP activation and nuclear translocation were examined. Our results show that lactate significantly attenuates LPS stimulated macrophage TNF-α and IL-6 production. Lactate also suppresses LPS stimulated macrophage NF-κB and YAP activation and nuclear translocation in macrophages. Interestingly, YAP activation and nuclear translocation are required for LPS stimulated macrophage NF-κB activation and TNFα production. Importantly, lactate suppressed YAP activation and nuclear translocation is mediated by GPR81 dependent AMKP and LATS activation which phosphorylates YAP, resulting in YAP inactivation. Finally, we demonstrated that LPS stimulation induces an interaction between YAP and NF-κB subunit p65, while lactate decreases the interaction of YAP and NF-κB, thus suppressing LPS induced pro-inflammatory cytokine production. Our study demonstrates that lactate exerts a previously unknown role in the suppression of macrophage pro-inflammatory cytokine production via GPR81 mediated YAP inactivation, resulting in disruption of YAP and NF-κB interaction and nuclear translocation in macrophages.

Intercellular delivery of NF-κB inhibitor peptide utilizing small extracellular vesicles for the application of anti-inflammatory therapy

Excessive activation of NF-κB in macrophages contributes to the onset and exacerbation of inflammatory disorders. The NEMO binding domain (NBD) peptide is an NF-κB inhibitor peptide that binds to NEMO, one of components of the IκB kinase (IKK) complex, and inhibits the IKK kinase activity, in the cytosol. Because of this property, the NBD peptide is expected to inhibit NF-κB activation in macrophages. In this study, we developed a delivery carrier for NBD based on small extracellular vesicles (sEVs), which are membrane vesicles released from cells. We constructed fusion proteins comprising Gag (an sEV tropic protein) and one, three, or six repeats of NBD peptide (Gag-1NBD, Gag-3NBD, and Gag-6NBD, respectively) to load the NBD peptide to the inner space of the sEVs, and attempted the intracellular delivery of the NBD peptide to macrophages using Gag-NBD-loaded sEVs (nNBD-sEVs). The nNBD-sEVs significantly inhibited LPS-induced phosphorylation of NF-κB pathway-related proteins in macrophages in a repeat number-dependent manner. Moreover, they exerted inhibitory effects on the NF-κB-dependent expression of proinflammatory mediators such as TNFα, CXCL10, iNOS, and NO. Collectively, our results indicate that NBD-containing sEVs can be used for the treatment of inflammatory diseases owing to their ability to effectively deliver peptides to the macrophage cytosol.

Reduced Nhe1 (Na+-H+ Exchanger-1) Function Protects ApoE-Deficient Mice From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysms.

IgE-mediated activation of Nhe1 (Na+-H+ exchanger-1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Ang II (angiotensin II)-induced AAA in Nhe1-insufficient Apoe-/-Nhe1+/- mice and Apoe-/-Nhe1+/+ littermates tested Nhe1 activity in experimental AAA, because Nhe1-/- mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and MMP (matrix metalloproteinase) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker TCTP (translationally controlled tumor protein) but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe-/-Nhe1+/+ mice, such acidic areas were much smaller in lesions from Apoe-/-Nhe1+/- mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and real-time polymerase chain reaction analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.

LETMD1 Regulates Phagocytosis and Inflammatory Responses to Lipopolysaccharide via Reactive Oxygen Species Generation and NF-κB Activation in Macrophages.

LETM1 domain-containing protein 1 (LETMD1), also known as HCCR-1, is a mitochondrial protein and is known to regulate p53 and STAT3 activities in cancer cells. In this study, we present, for the first time (to our knowledge), data indicating that LETMD1 suppresses multiple immune responses in monocyte/macrophage lineage cells and mouse primary macrophages. Attenuation of LETMD1 expression with specific small interfering RNA and short hairpin RNA constructs enhanced LPS-induced expressions of inflammatory mediators in macrophages. In addition, LETMD1 attenuation caused potentiation of phagocytosis as well as migration in a macrophage-like cell line, U937. These enhancing effects were associated with altered activation of signaling adaptors (such as NF-κB, MAPKs, p53, and JAK-STAT) involved in TLR4 signaling. Especially, LETMD1 selectively regulated TLR4-induced NF-κB activation via MyD88 but not via TIR-domain-containing adapter-inducing IFN-β (TRIF). Attenuation of LETMD1 expression caused mitochondrial hyperpolarization and subsequent decrease in ATP production and increase in mitochondrial/cellular reactive oxygen species (ROS) and intracellular calcium levels. LETMD1 attenuation also enhanced LPS-induced expression of NADPH oxidase (NOX) 2, the main producer of cellular ROS in phagocytes, through augmenting IFN regulatory factor 1. Accordingly, treatment with ROS scavenger, NOX2 suppressing agents, or calcium chelators resulted in suppression of LPS-induced cytokine production as well as NF-κB activation in cells with LETMD1 attenuation. These findings reveal a previously unknown function of LETMD1 and provide evidences showing LETMD1 negatively regulates macrophage functions by modulating mitochondrial function, subsequent ROS generation, and NF-κB activation.

Hesperidin methyl chalcone interacts with NFκB Ser276 and inhibits zymosan-induced joint pain and inflammation, and RAW 264.7 macrophage activation.

Arthritis can be defined as a painful musculoskeletal disorder that affects the joints. Hesperidin methyl chalcone (HMC) is a flavonoid with analgesic, anti-inflammatory, and antioxidant effects. However, its effects on a specific cell type and in the zymosan-induced inflammation are unknown. We aimed at evaluating the effects of HMC in a zymosan-induced arthritis model. A dose-response curve of HMC (10, 30, or 100mg/kg) was performed to determine the most effective analgesic dose after intra-articular zymosan stimuli. Knee joint oedema was determined using a calliper. Leukocyte recruitment was performed by cell counting on knee joint wash as well as histopathological analysis. Oxidative stress was measured by colorimetric assays (GSH, FRAP, ABTS and NBT) and RT-qPCR (gp91phox and HO-1 mRNA expression) performed. In vitro, oxidative stress was assessed by DCFDA assay using RAW 264.7 macrophages. Cytokine production was evaluated in vivo and in vitro by ELISA. In vitro NF-κB activation was analysed by immunofluorescence. We observed HMC reduced mechanical hypersensitivity and knee joint oedema, leukocyte recruitment, and pro-inflammatory cytokine levels. We also observed a reduction in zymosan-induced oxidative stress as per increase in total antioxidant capacity and reduction in gp91phox and increase in HO-1 mRNA expression. Accordingly, total ROS production and macrophage NFκB activation were diminished. HMC interaction with NFκB p65 at Ser276 was revealed using molecular docking analysis. Thus, data presented in this work suggest the usefulness of HMC as an analgesic and anti-inflammatory in a zymosan-induced arthritis model, possibly by targeting NFκB activation in macrophages.

Abstract TMIM-085: BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are urgently needed for patients with PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A recently recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) family of proteins. BET proteins such as BRD4 promote oncogenic transcription of genes promoting cell growth, survival and DNA repair. One example is the well-established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). In syngeneic mouse ovarian cancer models, M2-like pro-tumor macrophages are a prominent component of the ovarian cancer tumor microenvironment (TME). NF-κB inhibition can reduce the M2 macrophage population; however, more sustained treatment with a systemic NF-κB inhibitor leads to more ascites and reduced survival time. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in tumors and macrophages that may be beneficial or harmful depending on context. OBJECTIVE: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer cells and peritoneal macrophages. METHODS: Cultured ID8 mouse ovarian cancer cells, PMJ2-PC mouse peritoneal macrophages and immortalized bone marrow-derived macrophages were treated with vehicle, the PARPi olaparib, the BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth in vitro. C57BL/6 mice injected intra-peritoneally (IP) with ID8 cells were treated with JQ1 (30 days, 50mg/kg by IP injection) with volume of ascites fluid, weight of harvested tumor, and number of tumor implants assessed. Markers of apoptosis (cleaved PARP or cleaved caspase-3), and DNA damage (pH2AX) were measured by immunohistochemistry or western blot. NF-κB activity was measured by luciferase assays of a NF-κB reporter plasmid. Expression of M1 (CCL3) and M2 (CD206, arginase-1) macrophage markers was measured by quantitative real-time RT-PCR (QPCR). RESULTS: In culture, JQ1 treatment sensitized ID8 ovarian cancer cells to olaparib-induced growth inhibition, DNA damage and apoptosis. However, despite modest stimulatory effects on DNA damage and apoptosis of long-term JQ1 treatment in ID8 tumors in vivo, JQ1 unexpectedly increased ascites formation without reducing overall tumor burden. In macrophages, there were also contrasting effects between JQ1 treatment in vitro and in vivo. JQ1 alone or combined with olaparib reduced NF-κB activity in cultured macrophage cell lines, and increased expression of CCL3, and reduced CD206 and arginase-1 expression. In contrast, increased ascites due to JQ1 treatment in vivo was accompanied by a pronounced M2 macrophage shift. CONCLUSIONS: Sustained inhibition of NF-κB activity in macrophages and potentially other cells in the ovarian TME could have overall deleterious effects on tumor progression. Our results strongly suggest that the therapeutic effects of BETi in ovarian cancer, and any therapeutic agent with the potential to alter cell function in the tumor microenvironment, need to be more thoroughly tested in the context of functional immune system before being translated to patients. This is particularly relevant since BETi are currently being tested in human patients in early clinical trials. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona E. Yull. BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-085.

METTL3 Attenuates LPS-Induced Inflammatory Response in Macrophages via NF-κB Signaling Pathway.

Methyltransferase-like 3 (METTL3), an RNA N6-methyladenosine (m6A) methyltransferase, is essential for the m6A mRNA modification. As a key enzyme of m6A methylation modification, METTL3 has been implicated in immune and inflammation regulation. However, little is known of the role and underlying mechanism of METTL3 in rheumatoid arthritis (RA). The aim of the present study is to elucidate the function and potential mechanism of METTL3 in RA pathogenesis. We used quantitative real-time polymerase chain reaction to detect the expression of METTL3 in RA patients and controls as well as the macrophage cell line. CCK-8 was used for cell proliferation assay. Enzyme-linked immunosorbent assay (ELISA) was adopted to estimate the generation of IL-6 and TNF-α in macrophages. Western blot and immunofluorescence were applied to evaluate the activation of NF-κB in macrophages. The expression of METTL3 was significantly elevated in patients with RA. It was positively associated with CRP and ESR, two common markers for RA disease activity. Besides, LPS could enhance the expression and biological activity of METTL3 in macrophages, while overexpression of METTL3 significantly attenuated the inflammatory response induced by LPS in macrophages. Moreover, the effect of METTL3 on LPS-induced inflammation in macrophages was dependent on NF-κB. This study firstly demonstrates the critical role of METTL3 in RA, which provides novel insights into recognizing the pathogenesis of RA and a promising biomarker for RA.