Thromboxane Mobilizes Insect Blood Cells to Infection Foci

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.

Indomethacin & SARS-CoV-2: Prospects for clinical management of the inflammatory state

Severe acute respiratory syndrome - (SARS) is a pandemic (called as SARS-CoV-2 or COVID-19), severely affected by transmission and fatal disease caused by unknown coronavirus family of RNA virus (SARS-COV). Humans are under great threat among other species were non-identified. Phenotype can range from asymptomatic to fulminate cytokine storm which leads multiorgan failure resulting death. Still the world is eagerly waiting for antiviral drug to stop the corona virus infection. Previous studies found that indomethacin had ability to inhibit the RNA and DNA virus replication. Objectives . Interleukins (IL), Interferons (IFN) and metabolites like cyclopentane cyclooxygenase (COX 1/COX 2) are active against several RNA viruses. Experts have divided the SARS infection in three phases (Phase-1, 2, 3) based on severity of infection. In phase-3 there was cytokine storm due to exuberant inflammation observed which can damage organs and even fatal. We investigated that effect of indomethacin on COX inhibitor on coronavirus replication and cytokine storm in reducing the hyper inflammatory state. In this article we tried to assess the clinical management of inflammation due to SARS-COV-2 by Indomethacin. Results . Indomethacin can cause relief from the pain on taking deep breath in corona virus infected patients. Indomethacin can be considered safe and effective for prevention and treatment of coronavirus infection also antiviral activity. Conclusion : Indomethacin is a potent inhibitor of SARS CoV-2.

The role of prostaglandins in the realization of respiratory effects of TNF-α in case of hypoxia

At the present time very little is known about interactions between systemic inflammation and control of respiration. The aim of this study was to compare the respiratory effects of the main inflammatory cytokine TNF - α before and after pretreatment with diclofenac, a nonspecific cyclooxygenase (COX) inhibitor. In experiments on anesthetized, tracheostomized rats, pneumotachometry method was used to measure peak airflow and respiratory rate. The ventilatory response to hypoxia was investigated by the rebreathing method. It is shown that an increase in the systemic level of TNF – α causes a significant increase in the minute volume of respiration, tidal volume, the average speed of the inspiratory flow. In contrast the slope of the hypoxic ventilatory response decreased after administration of TNF-α. Diclofenac pretreatment eliminated these respiratory effects of TNF - α. The data indicate that the ability of TNF - α to enhance basal ventilation and to reduce the ventilatory hypoxic response is mediated by the cyclooxygenase pathway. Key words: tumor necrosis factor – α, hypoxia, prostaglandins, peripheral chemoreception, respiration.

RNAseq analysis of treatment-dependent signaling changes during inflammation in a mouse cutaneous wound healing model

Background Despite proven therapeutic effects in inflammatory conditions, the specific mechanisms of phytochemical therapies are not well understood. The transcriptome effects of Traumeel (Tr14), a multicomponent natural product, and diclofenac, a non-selective cyclooxygenase (COX) inhibitor, were compared in a mouse cutaneous wound healing model to identify both known and novel pathways for the anti-inflammatory effect of plant-derived natural products. Methods Skin samples from abraded mice were analyzed by single-molecule, amplification-free RNAseq transcript profiling at 7 points between 12 and 192 h after injury. Immediately after injury, the wounds were treated with either diclofenac, Tr14, or placebo control (n = 7 per group/time). RNAseq levels were compared between treatment and control at each time point using a systems biology approach. Results At early time points (12–36 h), both control and Tr14-treated wounds showed marked increase in the inducible COX2 enzyme mRNA, while diclofenac-treated wounds did not. Tr14, in contrast, modulated lipoxygenase transcripts, especially ALOX12/15, and phospholipases involved in arachidonate metabolism. Notably, Tr14 modulated a group of cell-type specific markers, including the T cell receptor, that could be explained by an overarching effect on the type of cells that were recruited into the wound tissue. Conclusions Tr14 and diclofenac had very different effects on the COX/LOX synthetic pathway after cutaneous wounding. Tr14 allowed normal autoinduction of COX2 mRNA, but suppressed mRNA levels for key enzymes in the leukotriene synthetic pathway. Tr14 appeared to have a broad ‘phytocellular’ effect on the wound transcriptome by altering the balance of cell types present in the wound.

Eicosapentaenoic acid inhibits lipopolysaccharide (LPS)-induced nitrite production and cytokine release from J774 macrophages

Background Eicosapentaenoic acid (EPA), an omega-3 (ω-3) fatty acid, reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT) but the mechanism is not fully understood. Activated macrophages, characterized by cytokine release and increased inducible nitric oxide synthase (iNOS) activity, contribute to atherosclerosis. As both a substrate for and potential inhibitor of cyclooxygenase (COX), EPA may reduce iNOS activity. Purpose The purpose of this study was to evaluate the dose-dependent effects of EPA on nitrite and cytokine release from lipopolysaccharide (LPS)-activated macrophages. Methods Murine J774 macrophages were pretreated with vehicle or EPA at 10, 20 and 40 μM for 2 h, then challenged with LPS at 1.0 μg/ml. After 24 hr, iNOS activity was measured by nitrite production using the Griess assay. EPA was compared to the COX inhibitor diclofenac at 1.0 μg/ml. Levels of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in cell supernatant were measured by immunochemistry using colchicine as a positive control. Results Activated macrophages caused a >4-fold increase in nitrite production (p<0.001) that was reduced by EPA in a dose-dependent manner. EPA decreased nitrite levels by 40, 62 and 77% at 10, 20 and 40 μM, respectively (p<0.01). Diclofenac separately reduced nitrite levels by 40% (p<0.01). EPA also reduced expression of IL-1β and TNF-α by 40% and 31%, respectively (p<0.01), in a manner similar to equimolar colchicine (10 μM). The reductions in IL-1β and TNF-α with EPA were dose-dependent. Conclusions EPA reduced macrophage activation as evidenced by decreased nitrite production and cytokine release similar to other anti-inflammatory agents. These findings indicate a novel effect of EPA on mechanisms of inflammation associated with vascular disease. FUNDunding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Mitochondrial H2Sn-Mediated Anti-Inflammatory Theranostics

The insistent demand for space-controllable delivery, which reduces the side effects of non-steroidal anti-inflammatory drugs (NSAIDs), has led to the development of a new theranostics-based approach for anti-inflammatory therapy. The current anti-inflammatory treatments can be improved by designing a drug delivery system responsive to the inflammatory site biomarker, hydrogen polysulfide (H2Sn). Here, we report a novel theranostic agent 1 (TA1), consisting of three parts: H2Sn-mediated triggering part, a two-photon fluorophore bearing mitochondria targeting unit (Rhodol-TPP), and anti-inflammatory COX inhibitor (indomethacin). In vitro experiments showed that TA1 selectively reacts with H2Sn to concomitantly release both Rhodol-TPP and indomethacin. Confocal-microscopy imaging of inflammation-induced live cells suggested that TA1 is localized in the mitochondria where the H2Sn is overexpressed. The TA1 reacted with H2Sn in the endogenous and exogenous H2Sn environments and in lipopolysaccharide treated inflammatory cells. Moreover, TA1 suppressed COX-2 level in the inflammatory-induced cells and prostaglandin E2 (PGE2) level in blood serum from inflammation-induced mouse models. In vivo experiments with inflammation-induced mouse models suggested that TA1 exhibits inflammation-site-elective drug release followed by significant therapeutic effects, showing its function as a theranostic agent, capable of both anti-inflammatory therapy and precise diagnosis. Theranostic behavior of TA1 is highly applicable in vivo model therapeutics for the inflammatory disease.

Targeting Soluble Epoxide Hydrolase and Cyclooxygenases Enhance Joint Pain Control, Stimulate Collagen Synthesis, and Protect Chondrocytes From Cytokine-Induced Apoptosis

Objective: To determine the symptomatic and disease-modifying capabilities of sEH and COX inhibitors during joint inflammation.Methods: Using a blinded, randomized, crossover experimental design, 6 adult healthy horses were injected with lipopolysaccharide (LPS; 3 μg) from E. coli in a radiocarpal joint and concurrently received the non-selective cyclooxygenase (COX) inhibitor phenylbutazone (2 mg/kg), the sEH inhibitor t-TUCB (1 mg/kg) or both (2 mg/kg phenylbutazone and 0.1, 0.3, and 1 mg/kg t-TUCB) intravenously. There were at least 30 days washout between treatments. Joint pain (assessed via inertial sensors and peak vertical forces), synovial fluid concentrations of prostanoids (PGE2, TxB2), cytokines (IL-1β, IL-6, TNF-α) and biomarkers of collagen synthesis (CPII) and degradation (C2C) were measured at pre-determined intervals over a 48-h period. The anti-apoptotic effect of COX and sEH inhibitors was determined via ELISA technique in primary equine chondrocytes incubated with TNF-α (10 ng/ml) for 24 h. Apoptosis was also determined in chondrocytes incubated with sEH-generated metabolites.Results: Combined COX and sEH inhibition produced significantly better control of joint pain, prostanoid responses, and collagen synthesis-degradation balance compared to each compound separately. When administered separately, pain control was superior with COX vs. sEH inhibition. Cytokine responses were not different during COX and/or sEH inhibition. In cultured chondrocytes, sEH inhibition alone or combined with COX inhibition, but not COX inhibition alone had significant anti-apoptotic effects. However, sEH-generated metabolites caused concentration-dependent apoptosis.Conclusions: Combined COX and sEH inhibition optimize pain control, attenuate loss of articular cartilage matrix during joint inflammation and cytokine-induced chondrocyte apoptosis.

Approaches to Potentiated Neuroprotective Treatment in the Rodent Model of Ischemic Optic Neuropathy

Nonarteritic anterior ischemic optic neuropathy (NAION) commonly causes sudden optic nerve (ON)-related vision loss. The rodent NAION model (rAION) closely resembles NAION in presentation and physiological responses. We identified early rAION-associated optic nerve head (ONH) inflammatory gene expression responses and the anti-inflammatory prostaglandin PGJ2’s effects on those responses. We hypothesized that blocking pro-inflammatory prostaglandin (PGE2) production by inhibiting monoacylglycerol lipase or cyclooxygenase activity and co-administering PGJ2 would potentiate RGC survival following ischemic neuropathy. Deep sequencing was performed on vehicle- and PGJ2-treated ONHs 3d post-rAION induction. Results were compared against responses from a retinal ischemia model. Animals were treated with PGJ2 and MAGL inhibitor KML29, or PGJ2 + COX inhibitor meloxicam. RGC survival was quantified by stereology. Tissue PG levels were quantified by ELISA. Gene expression was confirmed by qPCR. PGJ2 treatment nonselectively reduced inflammatory gene expression post-rAION. KML29 did not reduce PGE2 1d post-induction and KML29 alone increased RGC loss after rAION. Combined treatments did not improve ONH edema and RGC survival better than reported with PGJ2 alone. KML29′s failure to suppress PGE2 ocular synthesis, despite its purported effects in other CNS tissues may result from alternative PG synthesis pathways. Neither KML29 nor meloxicam treatment significantly improved RGC survival compared with vehicle. While exogenous PGJ2 has been shown to be neuroprotective, treatments combining PGJ2 with these PG synthesis inhibitors do not enhance PGJ2’s neuroprotection.