Induction Of Inflammatory Mediators Research Articles

Hepatoprotection of a Standardized Extract of Cultured Lentinula edodes Mycelia against Liver Injury Induced by Ischemia-Reperfusion and Partial Hepatectomy.

A standardized extract of cultured Lentinula edodes mycelia (ECLM, AHCC®) has been shown to have beneficial effects on organ metabolism. ECLM has been indicated to have liver protective properties by suppressing inflammatory responses. The pathogenesis of hepatic ischemia-reperfusion injury is thought to involve the induction of inflammatory mediators. However, whether ECLM affects inflammatory mediators caused by warm hepatic ischemia-reperfusion injury and partial hepatectomy (HIRI+PH) has not been clarified. In this study, we evaluated the protective effects of ECLM against liver damage caused by HIRI+PH. Rats were fed a normal diet (HIRI+PH) or a normal diet with 2% ECLM (HIRI+PH and ECLM) for ten days, then the liver and duodenal ligament were clamped and subjected to 15 min of hepatic ischemia. After 70% hepatectomy, the inflow occlusion was released, and liver and blood samples were collected at 3, 6, and 24 h. The effect of ECLM on mortality induced by 30 min of ischemia and hepatectomy was evaluated. The results showed that ECLM attenuated pathological liver damage, including apoptosis, in the rats treated with HIRI+PH, and decreased serum aminotransferase activity; ECLM decreased mRNA levels of the inflammation-related genes inducible nitric oxide synthase and C-X-C motif chemokine ligand 1, and increased mRNA levels of interleukin 10, an anti-inflammatory cytokine; ECLM increased hepatocyte growth factor mRNA levels and Ki-67 labeled nuclei in the liver at 24 h; ECLM significantly reduced HIRI+PH-induced mortality. In conclusion, ECLM may prevent HIRI+PH-induced liver injury in part by suppressing various inflammatory responses and promoting liver regeneration.

The Wiser Strategy of Using Beta-Agonists in Asthma: Mechanisms and Rationales.

Concerns regarding the safety of beta-2 agonists have led to revisions of the major asthma guidelines to better address these issues. Although these updates allow for a combination of previous and current strategies, they may confuse clinical practitioners. Beta-2 agonists are vital for alleviating asthma symptoms by relaxing smooth muscles; however, they also pose significant risks by inducing pro-inflammatory mediators both in vitro and in vivo. In addition to the risks of overuse and symptom masking, the use of beta-agonists alone at therapeutic doses can worsen airway inflammation and enhance virus-induced inflammation during asthma exacerbation. Inhaled corticosteroids (ICS) can effectively prevent these adverse effects. With new insights into the mechanisms of these adverse events, reserving short-acting beta-agonists for acute symptom relief during exacerbations and only for those who are already on ICS or oral steroids represents a careful approach to using beta-agonists with least adverse effects in patients with asthma. However, a major drawback of this approach is the potential non-compliance with ICS, leading to beta-agonist use without the necessary counteraction by ICS. An optimal strategy, both during and outside exacerbations, would integrate beta-agonists into an anti-inflammatory regimen that includes ICS, ideally combined with the same inhaler to ensure their concurrent use where finances allow. This would maintain the beneficial effects of beta-agonists, such as bronchodilation, while preventing the adverse effects from the induction of inflammatory mediators. This method is aligned with diverse clinical settings, maximizes the safe use of beta-agonists, and supports a comprehensive guideline-compliant management strategy.

Sulforaphane Is Protective against Warm Ischemia/Reperfusion Injury and Partial Hepatectomy in Rats.

Sulforaphane (SFN) has various beneficial effects on organ metabolism. However, whether SFN affects inflammatory mediators induced by warm hepatic ischemia/reperfusion injury (HIRI) is unclear. To investigate the hepatoprotective effects of SFN using an in vivo model of HIRI and partial hepatectomy (HIRI + PH), rats were subjected to 15 min of hepatic ischemia with blood inflow occlusion, followed by 70% hepatectomy and release of the inflow occlusion. SFN (5 mg/kg) or saline was randomly injected intraperitoneally 1 and 24 h before ischemia. Alternatively, ischemia was prolonged for 30 min to evaluate the effect on mortality. The influence of SFN on the associated signaling pathways was analyzed using the interleukin 1β (IL-1β)-treated primary cultured rat hepatocytes. In the HIRI + PH-treated rats, SFN reduced serum liver enzyme activities and the frequency of pathological liver injury, such as apoptosis and neutrophil infiltration. SFN suppressed tumor necrosis factor-alpha (TNF-α) mRNA expression and inhibited nuclear factor-kappa B (NF-κB) activation by HIRI + PH. Mortality was significantly reduced by SFN. In IL-1β-treated hepatocytes, SFN suppressed the expression of inflammatory cytokines and NF-κB activation. Taken together, SFN may have hepatoprotective effects in HIRI + PH in part by inhibiting the induction of inflammatory mediators, such as TNF-α, via the suppression of NF-κB in hepatocytes.

Xenogeneic equine stem cells activate anti-tumor adaptive immunity in a 4T1-based intraductal mouse model for triple-negative breast cancer: proof-of-principle.

Triple-negative breast cancer (TNBC) remains difficult to treat, especially due to ineffective immune responses. Current treatments mainly aim at a cytotoxic effect, whereas (stem) cell therapies are being investigated for their immune stimulatory capacities to initiate the anti-tumor immunity. Here, a thoroughly characterized, homogenous and non-tumorigenic mixture of equine mesenchymal stem cells (eMSCs) harvested from horse peripheral blood as innovative xenogeneic immunomodulators were tested in a 4T1-based intraductal mouse model for TNBC. The eMSCs significantly reduced 4T1 progression upon systemic injection, with induction of inflammatory mediators and T-cell influx in primary tumors, already after a single dose. These xenogeneic anti-cancer effects were not restricted to MSCs as systemic treatment with alternative equine epithelial stem cells (eEpSCs) mimicked the reported disease reduction. Mechanistically, effective eMSC treatment did not rely on the spleen as systemic entrapment site, whereas CD4+ and CD8α+ T-cell infiltration and activation were critical. These results show that eMSCs and potentially also other equine stem cell types can be a valuable TNBC treatment strategy for further (pre)clinical evaluation.

Purification and characterization of a serine protease from organic dust and elucidation of its inductive effects on lung inflammatory mediators.

Organic dust inhalation is associated with the development of respiratory diseases. Serine protease activities in organic dusts were previously reported to contribute to the induction of lung inflammatory mediators however, the identities of the proteases and the mechanisms by which they induce inflammatory mediators are unknown. The goal of this study was to purify and characterize serine protease(s) from organic dust and elucidate mechanisms by which they induce lung inflammatory mediators. A serine protease was purified from poultry organic dust by benzamidine-agarose affinity chromatography. Mass spectrometry and amino-terminal sequence analysis identified the purified protease as chicken trypsin II-P29. Purified protease induced proinflammatory cytokine levels in Beas2B and NHBE epithelial and THP-1 macrophage cells. Treatment with the purified protease increased cellular and mitochondrial reactive oxygen species (ROS) generation. Induction of inflammatory mediators and ROS were suppressed by serine protease inhibitors and antioxidants. Purified protease activated protein kinase C (PKC), mitogen-activated protein kinase (MAPK)1/3 and MAPK14 signaling, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (Stat-3), and chemical inhibitors targeting these pathways suppressed induction of inflammatory mediators. Calcium mobilization studies showed that the purified protease activated protease-activated receptors (PAR) F2R, F2RL1, F2RL2, F2RL3, and F2R and F2RL1 knockdown suppressed the induction of inflammatory mediators. Intranasal instillation of purified protease increased lung chemokine (C-X-C motif) ligand (CXCL)1, interleukin (IL)-6, and tumor necrosis factor (TNF) levels in mice. Our studies have shown that chicken trypsin is a proinflammatory constituent of poultry organic dust, and induces lung inflammatory mediators via increased ROS and PAR activation in a cell signaling pathway involving PKC, MAPK1/3 and MAPK14, and NF-κB and Stat-3.NEW & NOTEWORTHY Inhalation of dust in industrial agricultural operations is linked to the development of lung diseases. Our studies have isolated for the first time a trypsin protease from poultry farm dust and have shown that it stimulates lung inflammation. The protease stimulates the production of oxidants and cell signaling pathways to increase inflammatory mediator production. Targeting trypsin protease in poultry farm environment may be a useful strategy to counter the harmful effects of dust.

WISP-2 modulates the induction of inflammatory mediators and cartilage catabolism in chondrocytes

AbstractWnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1β and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1β-mediated chondrocyte catabolism, that IL-1β and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1β effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1β in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1β in human primary OA chondrocytes in a Wnt/β-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/β-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

MiR-378a regulates keratinocyte responsiveness to interleukin-17A in psoriasis.

BackgroundPsoriasis is an immune‐mediated inflammatory skin disease, in which an interplay between infiltrating immune cells and keratinocytes sustains chronic skin inflammation. Interleukin (IL)‐17A is a key inflammatory cytokine in psoriasis and its main cellular targets are keratinocytes.ObjectivesTo explore the role of miR‐378a in psoriasis.MethodsKeratinocytes obtained from psoriatic skin and healthy epidermis were separated by magnetic sorting, and the expression of miR‐378a was analysed by quantitative polymerase chain reaction. The regulation and function of miR‐378a was studied using primary human keratinocytes. The expression of miR‐378a was modulated by synthetic mimics, and nuclear factor kappa B (NF‐κB) activity and transcriptomic changes were studied. Synthetic miR‐378a was delivered to mouse skin in conjunction with induction of psoriasiform skin inflammation by imiquimod.ResultsWe show that miR‐378a is induced by IL‐17A in keratinocytes through NF‐κB, C/EBP‐β and IκBζ and that it is overexpressed in psoriatic epidermis. In cultured keratinocytes, ectopic expression of miR‐378a resulted in the nuclear translocation of p65 and enhanced NF‐κB‐driven promoter activity even in the absence of inflammatory stimuli. Moreover, miR‐378a potentiated the effect of IL‐17A on NF‐κB nuclear translocation and downstream activation of the NF‐κB pathway. Finally, injection of miR‐378a into mouse skin augmented psoriasis‐like skin inflammation with increased epidermal proliferation and induction of inflammatory mediators. Mechanistically, miR‐378a acts as a suppressor of NFKBIA/IκBζ, an important negative regulator of the NF‐κB pathway in keratinocytes.ConclusionsCollectively, our findings identify miR‐378a as an amplifier of IL‐17A‐induced NF‐κB signalling in keratinocytes and suggest that increased miR‐378a levels contribute to the amplification of IL‐17A‐driven skin inflammation in psoriasis.

Dynamic Regulation of the Nexus Between Stress Granules, Roquin, and Regnase-1 Underlies the Molecular Pathogenesis of Warfare Vesicants.

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals.

The Role of the Stromal Extracellular Matrix in the Development of Pterygium Pathology: An Update.

Pterygium is a benign fibrovascular lesion of the bulbar conjunctiva with frequent involvement of the corneal limbus. Its pathogenesis has been mainly attributed to sun exposure to ultraviolet-B radiation. Obtained evidence has shown that it is a complex and multifactorial process which involves multiple mechanisms such as oxidative stress, dysregulation of cell cycle checkpoints, induction of inflammatory mediators and growth factors, angiogenic stimulation, extracellular matrix (ECM) disorders, and, most likely, viruses and hereditary changes. In this review, we aim to collect all authors’ experiences and our own, with respect to the study of fibroelastic ECM of pterygium. Collagen and elastin are intrinsic indicators of physiological and pathological states. Here, we focus on an in-depth analysis of collagen (types I and III), as well as the main constituents of elastic fibers (tropoelastin (TE), fibrillins (FBNs), and fibulins (FBLNs)) and the enzymes (lysyl oxidases (LOXs)) that carry out their assembly or crosslinking. All the studies established that changes in the fibroelastic ECM occur in pterygium, based on the following facts: An increase in the synthesis and deposition of an immature form of collagen type III, which showed the process of tissue remodeling. An increase in protein levels in most of the constituents necessary for the development of elastic fibers, except FBLN4, whose biological roles are critical in the binding of the enzyme LOX, as well as FBN1 for the development of stable elastin. There was gene overexpression of TE, FBN1, FBLN5, and LOXL1, while the expression of LOX and FBLN2 and -4 remained stable. In conclusion, collagen and elastin, as well as several constituents involved in elastic fiber assembly are overexpressed in human pterygium, thus, supporting the hypothesis that there is dysregulation in the synthesis and crosslinking of the fibroelastic component, constituting an important pathogenetic mechanism for the development of the disease.

CoV-RBD121-NP Vaccine Candidate Protects against Symptomatic Disease following SARS-CoV-2 Challenge in K18-hACE2 Mice and Induces Protective Responses That Prevent COVID-19-Associated Immunopathology.

We developed a SARS-CoV-2 vaccine candidate (CoV-RBD121-NP) comprised of a tobacco mosaic virus-like nanoparticle conjugated to the receptor-binding domain of the spike glycoprotein of SARS-CoV-2 fused to a human IgG1 Fc domain. CoV-RBD121-NP elicits strong antibody responses in C57BL/6 mice and is stable for up to 12 months at 2–8 or 22–28 °C. Here, we showed that this vaccine induces a strong neutralizing antibody response in K18-hACE2 mice. Furthermore, we demonstrated that immunization protects mice from virus-associated mortality and symptomatic disease. Our data indicated that a sufficient pre-existing pool of neutralizing antibodies is required to restrict SARS-CoV-2 replication upon exposure and prevent induction of inflammatory mediators associated with severe disease. Finally, we identified a potential role for CXCL5 as a protective cytokine in SARS-CoV-2 infection. Our results suggested that disruption of the CXCL5 and CXCL1/2 axis may be important early components of the inflammatory dysregulation that is characteristic of severe cases of COVID-19.

211 IFN-γ enhances TWEAK-induced inflammatory responses in psoriasis keratinocytes through suppression of miR-149

Psoriasis is a chronic inflammatory skin disease with disturbed interplay between immune cells and keratinocytes. Here we identify a microRNA-mediated mechanism by which IFN-γ primes keratinocytes to inflammatory stimuli. Treatment with IFN-γ results in a quick and long-lasting suppression of miR-149 in keratinocytes. Depletion of miR-149 in keratinocytes results in wide- spread transcriptomic changes and induction of inflammatory mediators with an enrichment of the TWEAK-pathway. We demonstrate that IFN-γ primes keratinocytes to TWEAK-induced IL-6 expression and this is mediated by suppression of miR-149.

Anti-inflammatory activity of lefamulin versus azithromycin and dexamethasone in vivo and in vitro in a lipopolysaccharide-induced lung neutrophilia mouse model

Several antibiotics demonstrate both antibacterial and anti-inflammatory/immunomodulatory activities and are used to treat inflammatory pulmonary disorders. Lefamulin is a pleuromutilin antibiotic approved to treat community-acquired bacterial pneumonia (CABP). This study evaluated lefamulin anti-inflammatory effects in vivo and in vitro in a lipopolysaccharide-induced lung neutrophilia model in which mouse airways were challenged with intranasal lipopolysaccharide. Lefamulin and comparators azithromycin and dexamethasone were administered 30min before lipopolysaccharide challenge; neutrophil infiltration into BALF and inflammatory mediator induction in lung homogenates were measured 4h postchallenge. Single subcutaneous lefamulin doses (10‒140mg/kg) resulted in dose-dependent reductions of BALF neutrophil cell counts, comparable to or more potent than subcutaneous azithromycin (10‒100mg/kg) and oral/intraperitoneal dexamethasone (0.5/1mg/kg). Lipopolysaccharide-induced pro-inflammatory cytokine (TNF-α, IL-6, IL-1β, and GM-CSF), chemokine (CXCL-1, CXCL-2, and CCL-2), and MMP-9 levels were significantly and dose-dependently reduced in mouse lung tissue with lefamulin; effects were comparable to or more potent than with dexamethasone or azithromycin. Pharmacokinetic analyses confirmed exposure-equivalence of 30mg/kg subcutaneous lefamulin in mice to a single clinical lefamulin dose to treat CABP in humans (150mg intravenous/600mg oral). In vitro, neither lefamulin nor azithromycin had any relevant influence on lipopolysaccharide-induced cytokine/chemokine levels in J774.2 mouse macrophage or human peripheral blood mononuclear cell supernatants, nor were any effects observed on IL-8‒induced human neutrophil chemotaxis. These in vitro results suggest that impediment of neutrophil infiltration by lefamulin in vivo may not occur through direct interaction with macrophages or neutrophilic chemotaxis. This is the first study to demonstrate inhibition of neutrophilic lung infiltration and reduction of pro-inflammatory cytokine/chemokine concentrations by clinically relevant lefamulin doses. This anti-inflammatory activity may be beneficial in patients with acute respiratory distress syndrome, cystic fibrosis, or severe inflammation-mediated lung injury, similar to glucocorticoid (eg, dexamethasone) activity. Future lefamulin anti-inflammatory/immunomodulatory activity studies are warranted to further elucidate mechanism of action and evaluate clinical implications.

Is cyclooxygenase-1 involved in neuroinflammation?

Purpose: Reactive microglia are an important hallmark of neuroinflammation. Reactive microglia release various inflammatory mediators, such as cytokines, chemokines, and prostaglandins, which are produced by enzymes like cyclooxygenases (COX). The inducible COX‐2 subtype has been associated with inflammation, whereas the constitutively expressed COX‐1 subtype is generally considered as a housekeeping enzyme. However, recent evidence suggests that COX‐1 can also be upregulated and may play a prominent role in the brain during neuroinflammation. In this review, we summarize the evidence that supports this involvement of COX‐1. Methods: Five databases were used to retrieve relevant studies that addressed COX‐1 in the context of neuroinflammation. The search resulted in 32 articles, describing in vitro, in vivo, post mortem, and in vivo imaging studies that specifically investigated the COX‐1 isoform under such conditions. Results: Reviewed literature generally indicated that the overexpression of COX‐1 was induced by an inflammatory stimulus, which resulted in an increased production of prostaglandin E2. The pharmacological inhibition of COX‐1 was shown to suppress the induction of inflammatory mediators like prostaglandin E2. Positron emission tomography (PET) imaging studies in animal models confirmed the overexpression of COX‐1 during neuroinflammation. The same imaging method, however, could not detect any upregulation of COX‐1 in patients with Alzheimer's disease. Conclusion: Taken together, studies in cultured cells and living rodents suggest that COX‐1 is involved in neuroinflammation. Most postmortem studies on human brains indicate that the concentration of COX‐1‐expressing microglial cells is increased near sites of inflammation. However, evidence for the involvement of COX‐1 in neuroinflammation in the living human brain is still largely lacking.

Effects of Lipotoxicity in Brain Microvascular Endothelial Cells During Sirt3 Deficiency-Potential Role in Comorbid Alzheimer's Disease.

Silence information regulator 3 (SIRT3) is an NAD+ dependent deacetylase enzyme that enhances the function of key mitochondrial proteins. We have earlier demonstrated that deletion of Sirt3 gene leads to downregulation of metabolic enzymes, mitochondrial dysfunction and neuroinflammation in the brain, the major causes of Alzheimer’s disease (AD). We also reported recently that Sirt3 gene deletion in Alzheimer’s transgenic mice leads to exacerbation of neuroinflammation, amyloid plaque deposition and microglial activation. AD often coexists with other brain lesions caused by comorbidities which can exert their deleterious effects through the neurovascular unit. This unit consists of brain microvascular endothelial cells (BMECs), end feet of astrocytes, and pericytes. BMECs are uniquely different from other vascular endothelial cells because they are glued together by tight-junction proteins. BMECs are in constant contact with circulating factors as they line the luminal side. Therefore, we hypothesized that vascular endothelial injury caused by comorbidities plays a significant role in neuroinflammation. Herein, we investigated the effects of lipotoxicity in BMECs and how Sirt3 deficiency facilitate the deleterious effects of lipotoxicity on them using in vivo and in vitro models. We observed decreases in the levels of SIRT3 and tight junction proteins in the brain samples of western diet-fed APP/PS1 mice. Similar observations were obtained with Alzheimer’s post-mortem samples. Exposure of BEND3 cells, mouse brain-derived Endothelial cells3, to a combination of high glucose and palmitic acid resulted in significant (P < 0.01-P < 0.001) decreases in the levels of SIRT3, claudin-5 and ZO-1. Induction of inflammatory mediators, including Cox-2, CXCL1, RANTES, and GADD45β was also observed in these treated cells. Interestingly, the induction was more with Sirt3-silenced BEND3 cells, suggesting that Sirt3 deficiency exacerbates inflammatory response. Palmitic acid was more potent in inducing the inflammatory mediators. Significant cytotoxicity and changes in microglial morphology were observed when cocultures of Sirt3-silenced BEND3 and Sirt3-silenced BV2 cells were exposed to palmitic acid. Transendothelial electrical resistance measurement with these cocultures suggested decreased barrier integrity. The findings of this study suggest that hyperlipidemia in comorbidities can compromise blood brain barrier integrity by inducing inflammatory mediators and decreasing tight junction proteins in the vascular endothelial cells of the AD brain, leading to activation of microglia.

Influence of Simulated Deep Brain Stimulation on the Expression of Inflammatory Mediators by Human Central Nervous System Cells In Vitro

Deep brain stimulation (DBS) seems to modulate inflammatory processes. Whether this modulation leads to an induction or suppression of inflammatory mediators is still controversially discussed. Most studies of the influence of electrical stimulation on inflammation were conducted in rodent models with direct current stimulation and/or long impulses, both of which differ from the pattern in DBS. This makes comparisons with the clinical condition difficult. We established an in-vitro model that simulated clinical stimulation patterns to investigate the influence of electrical stimulation on proliferation and survival of human astroglial cells, microglia, and differentiated neurons. We also examined its influence on the expression of the inflammatory mediators C-X-C motif chemokine (CXCL)12, CXCL16, CC-chemokin-ligand-2 (CCL)2, CCL20, and interleukin (IL)-1β and IL-6 by these cells using quantitative polymerase chain reaction. In addition, protein expression was assessed by immunofluorescence double staining. In our model, electrical stimulation did not affect proliferation or survival of the examined cell lines. There was a significant upregulation of CXCL12 in the astrocyte cell line SVGA, and of IL-1β in differentiated SH-SY5Y neuronal cells at both messenger RNA and protein levels. Our model allowed a valid examination of chemokines and cytokines associated with inflammation in human brain cells. With it, we detected the induction of inflammatory mediators by electrical stimulation in astrocytes and neurons.

SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages.

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.

HTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host–pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1β-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

Cross-talk between IFN-γ and TWEAK through miR-149 amplifies skin inflammation in psoriasis

Psoriasis is a chronic inflammatory skin disease with disturbed interplay between immune cells and keratinocytes. Astrong IFN-γ signature is characteristic for psoriasis skin, but the role of IFN-γ has been elusive. MicroRNAs are short RNAs regulating gene expression. Our aim was to investigate the role of miR-149 in psoriasis and in the inflammatory responses of keratinocytes. miR-149 expression was measured by quantitative RT-PCR in keratinocytes isolated from healthy skin and lesional and nonlesional psoriasis skin. Synthetic miR-149 was injected intradermally into the back skin of mice, and imiquimod was applied to induce psoriasis-like skin inflammation, which was then evaluated at the morphologic, histologic, and molecular levels. miR-149 was transiently overexpressed or inhibited in keratinocytes in combination with IFN-γ- and/or TNF-related weak inducer of apoptosis (TWEAK)-treatment. Here we report a microRNA-mediated mechanism by which IFN-γ primes keratinocytes to inflammatory stimuli. Treatment with IFN-γ results in a rapid and long-lasting suppression of miR-149 in keratinocytes. Depletion of miR-149 in keratinocytes leads to widespread transcriptomic changes and induction of inflammatory mediators with enrichment of the TWEAK pathway. We show that IFN-γ-mediated suppression of miR-149 leads to amplified inflammatory responses to TWEAK. TWEAK receptor (TWEAKR/Fn14) is identified as a novel direct target of miR-149. The invivo relevance of this pathway is supported by decreased miR-149 expression in psoriasis keratinocytes, as well as by the protective effect of synthetic miR-149 in the imiquimod-induced mouse model of psoriasis. Our data define a new mechanism, in which IFN-γ primes keratinocytes for TWEAK-induced inflammatory responses through suppression of miR-149, promoting skin inflammation.