Rosavin inhibits neutrophil extracellular traps formation to ameliorate sepsis-induced lung injury by regulating the MAPK pathway

Background: Sepsis is a systemic organ dysfunction caused by infection, and the most affected organ is the lungs. Rosavin, a traditional Tibetan medicine, exerts an impressive anti--inflammatory effect. However, its effects on sepsis-related lung damage have not been investigated. Purpose: This study aimed to investigate the effects of Rosavin on cecal ligation and puncture (CLP)-induced lung injury. Methods: The sepsis mouse model was established by CLP, and the mice were pretreated with Rosavin to explore whether it contributed to the alleviation of lung injury. Hematoxylin-eosin (H&E) stain and lung injury score were used to assess the severity of lung injury. The bronchoalveolar lavage fluid (BALF) inflammatory mediators (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6], IL-1β, and IL-17A) were detected by ELISA. The number of neutrophils in BALF was detected using flow cytometry. The immunofluorescence assay was used to detect histone and myeloperoxidase (MPO) in lung tissues. Then, the western blot was performed to detect the expression of mitogen-activated protein kinase (MAPK) pathways (extracellular regulated kinase [ERK], p-ERK, p38, p-p38, Jun N-terminal kinase 1/2 [JNK1/2], and p-JNK1/2) in lung tissues. Results: We found that Rosavin significantly attenuated sepsis-induced lung injury. Specifically, Rosavin significantly inhibited inflammation response by decreasing the secretion of inflammatory mediators. The level of neutrophil extracellular traps (NETs) and MPO activity in CLP were decreased after administration with Rosavin. Moreover, the western blot showed that Rosavin could suppress NETs formation by inhibiting the MAPK/ERK/p38/JNK signaling pathway. Conclusion: These findings demonstrated that Rosavin inhibited NETs formation to attenuate sepsis-induced lung injury, and the inhibitory effect may be exerted via deregulation of the MAPK pathways (AU)

Host Cell Redox Alterations Promote Latent HIV-1 Reactivation through Atypical Transcription Factor Cooperativity.

Immune cell state alterations rewire HIV-1 gene expression, thereby influencing viral latency and reactivation, but the mechanisms are still unfolding. Here, using a screen approach on CD4+ T cell models of HIV-1 latency, we revealed Small Molecule Reactivators (SMOREs) with unique chemistries altering the CD4+ T cell state and consequently promoting latent HIV-1 transcription and reactivation through an unprecedented mechanism of action. SMOREs triggered rapid oxidative stress and activated a redox-responsive program composed of cell-signaling kinases (MEK-ERK axis) and atypical transcription factor (AP-1 and HIF-1α) cooperativity. SMOREs induced an unusual AP-1 phosphorylation signature to promote AP-1/HIF-1α binding to the latent HIV-1 proviral genome for its activation. Consistently, latent HIV-1 reactivation was compromised with pharmacologic inhibition of oxidative stress sensing or of cell-signaling kinases, and transcription factor's loss of expression, thus functionally linking the host redox-responsive program to viral transcriptional rewiring. Notably, SMOREs induced the redox program in primary CD4+ T cells and reactivated latent HIV-1 in aviremic patient samples alone and in combination with known latency-reversing agents, thus providing physiological relevance. Our findings suggest that manipulation of redox-sensitive pathways could be exploited to alter the course of HIV-1 latency, thus rendering host cells responsive to help achieve a sterilizing cure.

Glycopeptides from Paecilomyces sinensis ameliorate ethanol-induced gastric ulcers via anti-inflammation and the miR-9-5p-MEK/ERK signaling pathway.

The aim of this study was to investigate the immunomodulatory effect and mechanism of the glycopeptides from Paecilomyces sinensis (CPS-II) on ethanol induced ulcers in mice. In this study, histopathological evaluation (H&E staining) and the gastric ulcer score, ulcer index, total acid secretion and gastric pH value were used to determine the anti-ulcer activity. The expression levels of interleukin (IL)-6, interleukin (IL)-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA. The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and epidermal growth factor (PEG2) in serum were measured according to the instructions for the reagents. Western blotting was used to detect the effect of CPS-II on the MEK/ERK pathway. The results showed that CPS-II could inhibit the ulcer score and ulcer index compared with the disease control group. CPS-II could significantly increase gastric pH and decrease gastric acid secretion in mice. The ELISA analysis showed that the expression levels of IL-6 and TNF-α in the CPS-II treatment group were significantly decreased, while the expression levels of IL-10 were significantly increased in the CPS-II treatment group. In the resveratrol treatment group, the content of MDA in serum was decreased, and the level of PEG2 and the activity of SOD in serum were significantly increased, which indicated that CPS-II has immunoregulation and anti-ulcer properties. The CPS-II treatment group could reduce the expression level of miR-9-5p in gastric tissue. pEGFR had been identified as a potential target of miR-9-5p. Western blot analysis showed that CPS-II could up-regulate the relative protein expression of pEGFR/EGFR, pRaf/Raf, pMEK/MEK, pERK/ERK, and ZO-1. The results showed that CPS-II could reduce oxidative stress and inflammatory response by regulating the miR-9-5p-MEK/ERK signaling pathway, thus protecting the gastric mucosa and improving stress gastric ulcers.

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1ß. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study's unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

SUMO enables substrate selectivity by mitogen-activated protein kinases to regulate immunity in plants.

The versatility of mitogen-activated protein kinases (MAPKs) in translating exogenous and endogenous stimuli into appropriate cellular responses depends on its substrate specificity. In animals, several mechanisms have been proposed about how MAPKs maintain specificity to regulate distinct functional pathways. However, little is known of mechanisms that enable substrate selectivity in plant MAPKs. Small ubiquitin-like modifier (SUMO), a posttranslational modification system, plays an important role in plant development and defense by rapid reprogramming of cellular events. In this study we identified a functional SUMO interaction motif (SIM) in Arabidopsis MPK3 and MPK6 that reveals a mechanism for selective interaction of MPK3/6 with SUMO-conjugated WRKY33, during defense. We show that WRKY33 is rapidly SUMOylated in response to Botrytis cinerea infection and flg22 elicitor treatment. SUMOylation mediates WRKY33 phosphorylation by MPKs and consequent transcription factor activity. Disruption of either WRKY33 SUMO or MPK3/6 SIM sites attenuates their interaction and inactivates WRKY33-mediated defense. However, MPK3/6 SIM mutants show normal interaction with a non-SUMOylated form of another transcription factor, SPEECHLESS, unraveling a role for SUMOylation in differential substrate selectivity by MPKs. We reveal that the SUMO proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2 control WRKY33 SUMOylation and demonstrate a role for these SUMO proteases in defense. Our data reveal a mechanism by which MPK3/6 prioritize molecular pathways by differentially selecting substrates using the SUMO-SIM module during defense responses.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

Inflammatory response and MAPK and NF-κB pathway activation induced by natural street rabies virus infection in the brain tissues of dogs and humans.

BACKGROUND: Street rabies virus (RABV) usually infects hosts at peripheral sites and migrates from motor or sensory nerves to the central nervous system. Several studies have found that inflammation is mild in a mouse model of street RABV infection. However, the pathogenetic mechanisms of street RABV in naturally infected dogs or humans are not well understood. METHODS: Brain tissues collected from 3 dogs and 3 humans were used; these tissue samples were collected under the natural condition of rabies-induced death. The inflammatory response and pathway activation in the brain tissue samples of dogs and humans were evaluated by HE, IHC, ARY006, WB and ELISA. The clinical isolate street RABV strains CGS-17 and CXZ-15 from 30 six-week-old ICR mice were used to construct the mouse infection model presented here. RESULTS: Neuronal degeneration and increased lymphocyte infiltration in the cerebral cortex, especially marked activation of microglia, formation of glial nodules, and neuronophagy, were observed in the dogs and humans infected with the street RABV strains. The various levels of proinflammatory chemokines, particularly CXCL1, CXCL12, CCL2, and CCL5, were increased significantly in the context of infection with street RABV strains in dogs and humans in relation to healthy controls, and the levels of MAPK and NF-κB phosphorylation were also increased in dogs and humans with natural infection. We also found that the degrees of pathological change, inflammatory response, MAPK and NF-κB signaling pathway activation were obviously increased during natural infection in dogs and humans compared with artificial model infection in mice. CONCLUSION: The data obtained here provide direct evidence for the RABV-induced activation of the inflammatory response in a dog infection model, which is a relatively accurate reflection of the pathogenic mechanism of human street RABV infection. These observations provide insight into the precise roles of underlying mechanisms in fatal natural RABV infection.

Retinoic Acid-Platinum (II) Complex [RT-Pt(II)] Protects Against Rheumatoid Arthritis in Mice via MEK/Nuclear Factor kappa B (NF-κB) Pathway Downregulation.

BACKGROUND Rheumatoid arthritis (RA) is an inflammatory disorder that is present in approximately 1% of the world's population. This study was aimed to investigate the effect of retinoic acid-platinum (II) complex [RT-Pt(II)] on rheumatoid arthritis (RA) and to explore the mechanism involved. MATERIAL AND METHODS MH7A cell viability was determined by MTT assay and apoptosis was assessed using FACSCalibur flow cytometry. RT-PCR and Western blot assays were used for assessment of mRNA and proteins levels. RESULTS Treatment of rheumatoid arthritis with RT-Pt(II) significantly reduced the levels of IL­1ß, IL-6, IL-8, MMP-1, and MMP-13 in synovial fluid of mice in a dose-dependent manner. The expression of iNOS and COX-2 mRNA and protein in rheumatoid arthritis rats was also significantly inhibited by treatment with RT-Pt(II). The TNF-alpha-induced proliferation of MH7A cells was alleviated by RT-Pt(II) treatment in a concentration-dependent manner. Moreover, RT-Pt(II) treatment induced apoptosis and caused arrest of cell cycle in MH7A cells. The activation of MEK/NF-kappaB pathway was downregulated by RT-Pt(II) treatment in MH7A cells. CONCLUSIONS In summary, the present study demonstrated that RT-Pt(II) inhibits TNF-alpha-induced inflammatory response, suppresses cell viability, and induces apoptosis in rheumatoid arthritis synovial cells. Moreover, RT-Pt(II) exhibited its effect through targeting the MEK/NF-kappaB pathway. Therefore, RT-Pt(II) can be used for the development of treatments for rheumatoid arthritis.

TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching.

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

A closer look at immune-mediated myocarditis in the era of combined checkpoint blockade and targeted therapies.

Immune checkpoint inhibitors (ICI) and tyrosine kinase inhibitors (TKI) have transformed the management of many malignancies. Although rare, immune-mediated myocarditis presents unique clinical challenges due to heterogenous presentation, potential life-threatening consequences, and the time-critical need to differentiate it from other causes of cardiac dysfunction. Increasingly, TKI are being combined with ICI to promote immune modulation and improve efficacy. However, these combinations are associated with more toxicities. This series describes six patients with advanced melanoma who developed immune-mediated myocarditis while receiving an anti-PD-1 antibody or an anti-PD-L1 antibody plus a mitogen-activated protein kinase inhibitor. It provides a review of their heterogenous clinical presentations, investigational findings and treatment outcomes. Presentations ranged from asymptomatic cardiac enzyme elevation to death due to heart failure. We highlight the role of cardiac MRI (CMRI), a sensitive and non-invasive tool for the early detection and subsequent monitoring of myocardial inflammation. Five of the six patients exhibited CMRI changes characteristic of myocarditis, including mid-wall myocardial oedema and late gadolinium enhancement in a non-coronary distribution. Critically, two of these patients had normal findings on echocardiogram. Of the five patients who received immunosuppression, four recovered from myocarditis and one died of cardiac failure. The sixth patient improved with cardiac failure management alone. Three of the four patients responding to ICI derived long-term benefit. Clinical vigilance, prompt multimodal diagnosis and multidisciplinary management are paramount for the treatment of immune-mediated myocarditis.

The dietary freeze-dried fruit powder of Actinidia arguta ameliorates dextran sulphate sodium-induced ulcerative colitis in mice by inhibiting the activation of MAPKs.

In this study, we aimed at investigating the antiinflammatory activity of the freeze-dried fruit powder of Actinidia arguta (FAA) on dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) in mice and the effect of its extract on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. For pharmacodynamic studies, the oral administration of FAA (300 or 600 mg kg-1) could decrease the disease activity index (DAI), reduce the incidence of colon and spleen edemas (caused by inflammation), and alleviate the pathological changes in UC. For research involving biochemical indicators, FAA could decrease the expression of inflammatory markers (such as myeloperoxidase (MPO)) and attenuate the oxidative stress levels. ELISA results revealed that the expressions of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) were downregulated by FAA. Furthermore, the expression levels of the inflammation-induced activation of p38, JNK, and ERK were decreased by FAA. Hence, it was concluded that FAA could alleviate the UC symptoms in mice and the inflammatory response of macrophages via the MAPK signal pathway. Overall, FAA might have the potential to treat UC when used as a dietary supplement.

The diabetes drug semaglutide reduces infarct size, inflammation, and apoptosis, and normalizes neurogenesis in a rat model of stroke.

Stroke is a condition with few medical treatments available. Semaglutide, a novel Glucagon-like peptide-1 (GLP-1) analogue, has been brought to the market as a treatment for diabetes. We tested the protective effects of semaglutide against middle cerebral artery occlusion injury in rats. Animals were treated with 10 nmol/kg bw ip. starting 2 h after surgery and every second day for either 1, 7, 14 or 21 days. Semaglutide-treated animals showed significantly reduced scores of neurological impairments in several motor and grip strength tasks. The cerebral infarction size was also reduced, and the loss of neurons in the hippocampal areas CA1, CA3 and the dentate gyrus was much reduced. Chronic inflammation as seen in levels of activated microglia and in the activity of the p38 MAPK - MKK - c-Jun- NF-κB p65 inflammation signaling pathway was reduced. In addition, improved growth factor signaling as shown in levels of activated ERK1 and IRS-1, and a reduction in the apoptosis signaling pathway C-raf, ERK2, Bcl-2/BAX and Caspase-3 was observed. Neurogenesis had also been normalized by the drug treatment as seen in increased neurogenesis (DCX-positive cells) in the dentate gyrus and a normalization of biomarkers for neurogenesis. In conclusion, semaglutide is a promising candidate for re-purposing as a stroke treatment.

Bee Pollen Extracts Modulate Serum Metabolism in Lipopolysaccharide-Induced Acute Lung Injury Mice with Anti-Inflammatory Effects.

Bee pollen (BP) collected from different floras possesses various potential bioactivities, but the mechanism-related research on anti-inflammatory effects is limited. Here, three types of BP originating from Camellia sinensis L. (BP-Cs), Nelumbo nucifera Gaertn. (BP-Nn), and Brassica campestris L. (BP-Bc) were assessed using molecular and metabolomics methods to determine their anti-inflammatory effects. The differences in polyphenolic abundance of three types of BP extracts were determined by HPLC-DAD/Q-TOF-MS. In vitro anti-inflammatory effects of three BP extracts were evaluated in a lipopolysaccharide (LPS)-induced RAW 264.7 cells model. BP-Cs extract with the most abundant polyphenols was found to be the most effective in reducing inflammation by downregulating inflammatory-related genes expression and blocking the activation of MAPK and NF-κB signaling pathways. Polyphenol-rich BP-Cs was further evaluated for their in vivo anti-inflammatory effect in a LPS-induced acute lung injury mouse model. An UPLC-Q-TOF/MS-based metabolomics approach was applied to analyze metabolite changes in mouse serum. Weshowed that the pretreated BP-Cs extract alleviated inflammation and regulated glycerophospholipid metabolism significantly. Our findings provide a foundation for developing and justifying BP as a potential anti-inflammatory ingredient in functional foods or nutraceutical formulations.

Nobiletin Prevents Trimethylamine Oxide-Induced Vascular Inflammation via Inhibition of the NF-κB/MAPK Pathways.

Dietary choline and its containing foods are biotransformed to trimethylamine (TMA) via gut microbial metabolism. Subsequently, as an intermediate molecule, TMA is quickly transported and oxidized in the liver by hepatic flavin monooxygenases to form trimethylamine oxide (TMAO). TMAO was treated as a waste byproduct from choline metabolism, but recent convincing evidence demonstrated the association between the small molecule TMAO and inflammation-related diseases, including blood vessel inflammation and vascular diseases. The scope of this study is to investigate the preventive effect of nobiletin on TMAO-induced blood vessel inflammation. Our results from Western blot showed that the inhibition of TMAO-induced cardiovascular inflammation was correlated with nobiletin-mediated inhibitory effects on NF-κB and MAPK/ERK related pathways. More specifically, nobiletin prevented the oxidative damage of vascular sites (proximal aorta), inhibited the activity of MAPK/ERK, reduced the expression of NF-κB p65 and phospho-NF-κB p65, and consequently decreased the inflammatory response. Flow cytometry analyses showed that nobiletin decreased TMAO-induced apoptosis of HUVEC cells and counteracted TMAO-induced HUVEC cell proliferation. Results from HE staining and immunohistochemical results also showed that nobiletin reduced the degree of inflammation of the proximal aorta in Sprague-Dawley rats. In summary, nobiletin significantly reduced TMAO-induced vascular inflammation via inhibition of the NF-κB/MAPK pathways.

Nobiletin Protects against Systemic Inflammation-Stimulated Memory Impairment via MAPK and NF-κB Signaling Pathways.

Neuroinflammation has been intensively demonstrated to be related to various neurodegenerative diseases including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD). A natural polymethoxylated flavone, nobiletin (NOB) has been reported to alleviate oxidative stress, insulin resistance, and obesity. In this study, we evaluated the protection effects of NOB on neuroinflammation and memory deficit. Three-month mice were administrated with NOB by oral gavage every day for 6 weeks (100 mg/kg/day); subsequently mice were injected intraperitoneally with lipopolysaccharide (LPS) for 7 days. Results of behavioral tests revealed that NOB dramatically ameliorated LPS-triggered memory deficit regarding synaptic dysfunctions and neuronal loss. Also, NOB suppressed the microglial activation and proinflammatory cytokine secretion, such as COX-2, IL-1ß, TNF-α, and iNOS. Similarly, upon LPS stimulation, pretreatment NOB diminished the secretion of the proinflammatory cytokines in BV-2 microglia cells by exposure to LPS via modulating MAPKs, PI3K/AKT, and NF-κB signaling pathways. In addition, NOB alleviated LPS-amplified redox imbalance, disturbance of mitochondrial membrane potential (MMP), and dampening of the expression of protein related to mitochondrial respiration. The present study provides compelling evidence that NOB decreased LPS-stimulated neuroinflammation and memory impairment through maintaining cellular oxidative balance and blocking the NF-κB transcriptional pathway, illustrating that the nutritional compound NOB may serve as a potential approach to alleviate neuroinflammation-related diseases.

Urolithins Attenuate LPS-Induced Neuroinflammation in BV2Microglia via MAPK, Akt, and NF-κB Signaling Pathways.

Emerging data suggest that urolithins, gut microbiota metabolites of ellagitannins, contribute toward multiple health benefits attributed to ellagitannin-rich foods, including walnuts, red raspberry, strawberry, and pomegranate. However, there is limited data on whether the potential neuroprotective effects of these ellagitannin-rich foods are mediated by urolithins. Herein, we evaluated the potential mechanisms of antineuroinflammatory effects of urolithins (urolithins A, B, and C; 8-methyl-O-urolithin A; and 8,9-dimethyl-O-urolithin C) in BV2 murine microglia in vitro. Nitrite analysis and qRT-PCR suggested that urolithins A and B reduced NO levels and suppressed mRNA levels of pro-inflammatory genes of TNF-α, IL-6, IL-1ß, iNOS, and COX-2 in LPS-treated microglia. Western blot revealed that urolithins A and B decreased phosphorylation levels of Erk1/2, p38 MAPK, and Akt, prevented IκB-α phosphorylation and degradation, and inhibited NF-κB p65 subunit phosphorylation and nuclear translocation in LPS-stimulated microglia. Our results indicated that urolithins A and B attenuated LPS-induced inflammation in BV2 microglia, which may be mediated by inhibiting NF-κB, MAPKs (p38 and Erk1/2), and Akt signaling pathway activation. The antineuroinflammatory activities of urolithins support their role in the potential neuroprotective effects reported for ellagitannin-rich foods warranting further in vivo studies on these ellagitannin gut microbial derived metabolites.

Suppression of Toll-like receptor 2-mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c.

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-ß. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome-lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.

Oleuropein inhibits the IL-1ß-induced expression of inflammatory mediators by suppressing the activation of NF-κB and MAPKs in human osteoarthritis chondrocytes.

Osteoarthritis (OA) is the most common form of joint disease and is widespread in the elderly population and is characterized by erosion of articular cartilage, subchondral bone sclerosis and synovitis. Oleuropein (OL), a secoiridoid, is considered as the most prevalent phenolic component in olive leaves and seeds, pulp and peel of unripe olives and has been shown to have potent anti-inflammatory effects. However, its effects on OA have not been clearly elucidated. This study aimed to assess the effect of OL on human OA chondrocytes. Human OA chondrocytes were pretreated with OL (10, 50 and 100 µM) for 2 h and subsequently stimulated with IL-1ß for 24 h. The production of NO, PGE2, MMP-1, MMP-13, and ADAMTS-5 was evaluated by the Griess reaction and ELISA assays. The messenger RNA (mRNA) expression of COX-2, iNOS, MMP-1, MMP13, ADAMTS-5, aggrecan, and collagen-II was measured by using real-time PCR. The protein expressions of COX-2, iNOS, p65, IκB-α, JNK, p-JNK, ERK, p-ERK, p38, and p-p38 were tested by using western blot. We found that OL significantly inhibited the IL-1ß-induced production of NO and PGE2; expression of COX-2, iNOS, MMP-1, MMP-13, and ADAMTS-5; and degradation of aggrecan and collagen-II. Furthermore, OL dramatically suppressed IL-1ß-stimulated NF-κB and MAPK activation. Immunofluorescence staining demonstrated that OL could suppress IL-1ß-induced phosphorylation of p65 nuclear translocation. These results indicate that the therapeutic effect of OL on OA is accomplished through the inhibition of both NF-κB and MAPK signaling pathways. Altogether, our findings provide the evidence to develop OL as a potential therapeutic agent for patients with OA.

Chemical characterization of a novel polysaccharide ASKP-1 from Artemisia sphaerocephala Krasch seed and its macrophage activation via MAPK, PI3k/Akt and NF-κB signaling pathways in RAW264.7 cells.

The aim of this study was to investigate the molecular mechanism underlying the immunomodulatory effect of the purified Artemisia sphaerocephala Krasch seed polysaccharide (ASKP-1) in RAW264.7 macrophages. Chemical characteristic analysis revealed that ASKP-1 consisted of 14.1% mannose, 56.9% glucose and 19.6% galactose with the average molecular weight of 9.08 × 105 Da and the mixed glycan backbone structure containing 1→4)-Glcp (39.8%), 1→6)-Galp (18.8%), 1→3,6)-Manp (19.6%), 1→)-Glcp (10.8%), 2→6)-Manp (4.0%) and 2→3,5)-Araf (7.0%). In vitro studies showed that ASKP-1 markedly induced the release of cytotoxic molecules (NO and ROS) and secretion of the cytokines (TNF-α, INF-ß, and IL-6) and significantly enhanced the phagocytosis of RAW264.7 macrophages. Furthermore, TLR4 was found to be a recognized target of ASKP-1 and its related mitogen-activated protein (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt, including phosphorylated ERK, JNK, p38 and Akt, were rapidly activated by ASKP-1 in RAW264.7 macrophages. Moreover, ASKP-1 was found to cause the nuclear translocation of the nuclear factor NF-κB subunit p65 and the degradation of IκB-α in RAW264.7 macrophages. All these findings suggest that MAPK, PI3K/Akt and NF-κB pathways are involved in ASKP-1-induced macrophage activation, and ASKP-1 is a potential immunomodulating function food.

Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1ß secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.