Peak Inflammation Research Articles

Neuroinflammation in Cryptococcal Post-infectious Inflammatory Response Syndrome (PIIRS) is reduced by Ruxolitinib, a Janus Kinase (JAK) inhibitor.

Abstract Recently, a PIIRS was described in non-HIV infected cryptococcal meningoencephalitis (CM) as a significant cause of neuropathology and mortality. Inflammatory syndromes are particularly severe in neurological infections due to the skull's rigid structure. In the present study, transcriptional profiling of RNA from brains of PIIRS model mice at peak inflammation identified a predominance of STAT1/STAT3-associated downstream genes. Since JAK is known to activate both pathways, we hypothesized that the JAK/STAT inhibitor ruxolitinib could reduce neuro-damaging inflammation from CM. Treatment with ruxolitinib reduced intrathecal STAT1/STAT3-associated gene expression as well as CD44hiCD62loCD4+ effector CD4+ T-cells and MHC II+ inflammatory myeloid cells with improved neuropathology and weight. Based on these data, the first human treatment of PIIRS with ruxolitinib was initiated in 5 patients with residual inflammation despite corticosteroids (CS). Ruxolitinib reduced cerebrospinal fluid (CSF) activated HLA-DR+ CD4+ and CD8+ cells, NK cells, and inflammatory monocytes as well as soluble markers sIL-2R, and IL-10 with a trend in IL-6 on a constant dose of CS. MRI brain imaging also demonstrated significant improvements and therapy was well tolerated. Together, these findings demonstrate a role for the JAK/STAT pathway in PIIRS and support ruxolitinib as a potential adjunctive therapy for further randomized clinical trials of PIIRS as a steroid-sparing therapeutic.

Vagus nerve stimulation enhances remyelination and decreases innate neuroinflammation in lysolecithin-induced demyelination

BackgroundCurrent treatments for Multiple Sclerosis (MS) poorly address chronic innate neuroinflammation nor do they offer effective remyelination. The vagus nerve has a strong regulatory role in inflammation and Vagus Nerve Stimulation (VNS) has potential to affect both neuroinflammation and remyelination in MS. ObjectiveThis study investigated the effects of VNS on demyelination and innate neuroinflammation in a validated MS rodent model. MethodsLysolecithin (LPC) was injected in the corpus callosum (CC) of 46 Lewis rats, inducing a demyelinated lesion. 33/46 rats received continuously-cycled VNS (cVNS) or one-minute per day VNS (1minVNS) or sham VNS from 2 days before LPC-injection until perfusion at 3 days post-injection (dpi) (corresponding with a demyelinated lesion with peak inflammation). 13/46 rats received cVNS or sham from 2 days before LPC-injection until perfusion at 11 dpi (corresponding with a partial remyelinated lesion). Immunohistochemistry and proteomics analyses were performed to investigate the extend of demyelination and inflammation. ResultsImmunohistochemistry showed that cVNS significantly reduced microglial and astrocytic activation in the lesion and lesion border, and significantly reduced the Olig2+ cell count at 3 dpi. Furthermore, cVNS significantly improved remyelination with 57.4 % versus sham at 11 dpi. Proteomic gene set enrichment analyses showed increased activation of (glutamatergic) synapse pathways in cVNS versus sham, most pronounced at 3 dpi. ConclusioncVNS improved remyelination of an LPC-induced lesion. Possible mechanisms might include modulation of microglia and astrocyte activity, increased (glutamatergic) synapses and enhanced oligodendrocyte clearance after initial injury.

Activation of ETAR and ETBR in myocardial tissue characterizes heart failure induced by experimental autoimmune myocarditis

BackgroundEndothelial dysfunction is characterized by an imbalance between endothelium-derived vasodilatory and vasoconstrictive effects and may play an important role in the development of heart failure. An increasing number of studies have shown that endothelial-derived NO-mediated vasodilation is attenuated in heart failure patients. However, the role of endothelin-1 (ET-1) in heart failure remains controversial due to its different receptors including ET-1 receptor type A (ETAR) and ET-1 receptor type B (ETBR). The aim of this study was to determine whether ET-1 and its receptors are activated and to explore the role of ETAR and ETBR in heart failure induced by myocarditis.MethodsWe constructed an animal model of experimental autoimmune myocarditis (EAM) with porcine cardiac myosin. Twenty rats were randomized to the control group (3 weeks, n = 5), the extended control group (8 weeks, n = 5), the EAM group (3 weeks, n = 5), the extended EAM group (8 weeks, n = 5). HE staining was used to detect myocardial inflammatory infiltration and the myocarditis score, Masson’s trichrome staining was used to assess myocardial fibrosis, echocardiography was used to evaluate cardiac function, ELISA was used to detect serum NT-proBNP and ET-1 concentrations, and immunohistochemistry and western blotting were used to detect ETAR and ETBR expression in myocardial tissue of EAM-induced heart failure. Subsequently, a model of myocardial inflammatory injury in vitro was constructed to explore the role of ETAR and ETBR in EAM-induced heart failure.ResultsEAM rats tended to reach peak inflammation after 3 weeks of immunization and developed stable chronic heart failure at 8 weeks after immunization. LVEDd and LVEDs were significantly increased in the EAM group compared to the control group at 3 weeks and 8 weeks after immunization while EF and FS were significantly reduced. Serum NT-proBNP concentrations in EAM (both 3 weeks and 8 weeks) were elevated. Therefore, EAM can induce acute and chronic heart failure due to myocardial inflammatory injury. Serum ET-1 concentration and myocardial ETAR and ETBR protein were significantly increased in EAM-induced heart failure in vivo. Consistent with the results of the experiments in vivo, ETAR and ETBR protein expression levels were significantly increased in the myocardial inflammatory injury model in vitro. Moreover, ETAR gene silencing inhibited inflammatory cytokine TNF-α and IL-1β levels, while ETBR gene silencing improved TNF-α and IL-1β levels.ConclusionsET-1, ETAR, and ETBR were activated in both EAM-induced acute heart failure and chronic heart failure. ETAR may positively regulate EAM-induced heart failure by promoting myocardial inflammatory injury, whereas ETBR negatively regulates EAM-induced heart failure by alleviating myocardial inflammatory injury.

PIEZO1 expression at the glio-vascular unit adjusts to neuroinflammation in seizure conditions

Mechanosensors are emerging players responding to hemodynamic and physical inputs. Their significance in the central nervous system remains relatively uncharted. Using human-derived brain specimens or cells and a pre-clinical model of mesio-temporal lobe epilepsy (MTLE), we examined how the mRNA levels of the mechanosensitive channel PIEZO1 adjust to disease-associated pro-inflammatory trajectories.In brain tissue micro-punches obtained from 18 drug-resistant MTLE patients, PIEZO1 expression positively correlated with pro-inflammatory biomarkers TNFα, IL-1β, and NF-kB in the epileptogenic hippocampus compared to the adjacent amygdala and temporal cortex tissues. In an experimental MTLE model, hippocampal Piezo1 and cytokine expression levels were increased post-status epilepticus (SE) and during epileptogenesis. Piezo1 expression positively correlated with Tnfα, Il1β, and Nf-kb in the hippocampal foci. Next, by combining RNAscope with immunohistochemistry, we identified Piezo1 in glio-vascular cells. Post-SE and during epileptogenesis, ameboid IBA1 microglia, hypertrophic GFAP astrocytes, and damaged NG2DsRed pericytes exhibited time-dependent patterns of increased Piezo1 expression. Digital droplet PCR analysis confirmed the Piezo1 trajectory in isolated hippocampal microvessels in the ipsi and contralateral hippocampi. The combined examinations performed in this model showed Piezo1 expression returning towards basal levels after the epileptogenesis-associated peak inflammation. From these associations, we next asked whether pro-inflammatory players directly regulate PIEZO1 expression. We used human-derived brain cells and confirmed that endothelium, astrocytes, and pericytes expressed PIEZO1. Exposure to human recombinant TNFα or IL1β upregulated NF-kB in all cells. Furthermore, TNFα induced PIEZO1 expression in a dose and time-dependent manner, primarily in astrocytes.This exploratory study describes a spatiotemporal dialogue between PIEZO1 brain cell-mechanobiology and neuro-inflammatory cell remodeling. The precise functional mechanisms regulating this interplay in disease conditions warrant further investigation.

Time-Course Lipidomics of Ornithine-Induced Severe Acute Pancreatitis Model Reveals the Free Fatty Acids Centered Lipids Dysregulation Characteristics.

The relationship between the type and intensities of lipids of blood and pancreas and the pathological changes in the pancreas during severe acute pancreatitis (SAP) remains unclear. In our study, we employed a rat model of SAP induced through intraperitoneal ornithine injections. We collected serum and pancreas samples at various time points (0-144 h) for histopathological and biochemical assessments, followed by lipidomic analyses using LC-MS/MS or in situ mass spectrometry imaging (MSI) To discern changes over time or at specific points, we employed time-course and univariate analyses for lipid screening, respectively. Our findings indicated that the peak inflammation in the Orn-SAP model occurred within the 24-30 h timeframe, with evident necrosis emerging from 24 h onwards, followed by regeneration starting at 48 h. Time-course analysis revealed an overall decrease in glycerophospholipids (PEs, PCs, LPEs, LPCs), while CEs exhibited an increase within the pancreas. Univariate analysis unveiled a significant reduction in serum TAGs containing 46-51 carbon atoms at 24 h, and CERs in the pancreas significantly increased at 30 h, compared with 0 h. Moreover, a substantial rise in TAGs containing 56-58 carbon atoms was observed at 144 h, both in serum and pancreas. MSI demonstrated the CERs containing saturated mono-acyl chains of 16 and 18 carbon atoms influenced pancreatic regeneration. Tracing the origin of FFAs hydrolyzed from pancreatic glycerophospholipids and serum TAGs during the early stages of inflammation, as well as FFAs utilized for CEs and CERs synthesis during the repair phase, may yield valuable strategies for diagnosing and managing SAP.

Evolving acidic microenvironments during colitis provide selective analgesic targets for a pH-sensitive opioid.

Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Ultrasonic-controlled “explosive” hydrogels to precisely regulate spatiotemporal osteoimmune disturbance

Spatiotemporal Immune disorder is a key factor leading to the failure of bone tissue healing. It is of vital importance to accurately suppress excessive peak immune response within 24-48 h of the injury and so regulate the spatiotemporal osteoimmune disturbance of bones. In this study, Ultrasound Controlled “Explosive” (UCE) hydrogels were prepared from gelatin-hyaluronic acid methacrylate hydrogels loaded with resveratrol nanobubbles produced by double emulsification through a condensation reaction. Such materials innovatively enable ultrasound-controlled RES release for precise regulation of spatiotemporal osteoimmune disorders. Under an ultrasonic power level of 1.5 W/cm2, the rate of effectively released RES through the blast of UCE hydrogels reached 38.14 %. And compared with the control group, the in vivo inhibition of inflammation and osteogenesis effects of UCE hydrogels were more effective, respectively. As suggested by the results, the excessive local inflammatory response was inhibited by the release of resveratrol, the temporospatial disorder of bone immune was precisely regulated, and as a result, the process of bone repair was accelerated. Altogether, this study confirms that the newly created UCE Hydrogels effectively promote bone repair by intervening peak inflammation during the early phase of fracture healing.

Reset of Inflammatory Priming of Joint Tissue and Reduction of the Severity of Arthritis Flares by Bromodomain Inhibition.

We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model. BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo. Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs. Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Abstract P1105: Inflammatory Bowel Disease Induced Autonomic Dysregulation Increases The Risk For Atrial Fibrillation

Introduction: Ulcerative colitis causes inflammation of the innermost lining of the colon and rectum. The disease has a relapsing and remitting course and is accompanied by extraintestinal manifestations such as an increased risk for atrial fibrillation (AF). Here we will test the hypothesis that during active colitis, dysregulation of parasympathetic signaling increases the propensity for atrial arrhythmic events. Methods: Dextran Sulfate Sodium (DSS: 3%, 7days) treated mice (C57BL/6) exhibit disrupted intestinal barrier function that mimics the disease phenotype of active colitis. Changes in atrial electrophysiology was quantified in-vivo (EKG) and in the Langendorff (LD)-perfused heart (atrial electrograms) during peak inflammation (DSS A ) and remission (DSS R ). Results: During peak inflammation, structural remodeling was reflected in a decreased heart weight to tibia length ratio (Control (CTL A ): 9.9±0.4 mg/mm, n=10; DSS A : 8.8±0.3 mg/mm, n=12; p<0.05) whereas electrophysiological atrial remodeling was supported by a prolonged P-wave duration (CTL A : 25.5±0.6 ms, n=10; DSS A : 31.2±0.6 ms, n=24; p<0.0001) and shortened atrial effective refractory period (CTL A : 27.3±1.7 ms, n=4; DSS A : 19.3±0.6 ms, n=4; p<0.01). In comparison to CTL A , DSS A mice exhibited autonomic dysregulation reflected in an attenuated heart rate (HR) variability and a decreased change in HR in response to the muscarinic receptor blocker atropine (CTL A : 7.6±1.8 %, n=5; DSS A : 2.2±0.7 %, n=7; p<0.01). The decreased vagal tone in DSS A coincided with an exaggerated response to the parasympathomimetic carbachol (CCh, 150ng/g) that resulted in a larger decrease in HR (in vivo: CTL A : -12.3±3.1 %, n=10; DSS A : -46.0±5.6 %, n=10; p<0.0001) and increased number of spontaneous arrhythmic events (CTL A : 1.7±0.6 events/min, n=10; DSS A : 9.8±3.8 events/min, n=10; p<0.05) in vivo and in the LD-configuration. The susceptibility for pacing induced AF trended to be increased in DSS A animals. Structural and electrophysiological changes were reversible upon remission. Conclusion: Active colitis induces transient structural and electrophysiological remodeling. We propose that dysregulation of cholinergic signaling drives the increased propensity for atrial arrhythmia.

Effective Spatio-Temporal Regimes for Wound Treatment by Way of Macrophage Polarization: A Mathematical Model

Wound healing consists of a sequence of biological processes often grouped into different stages. Interventions applied to accelerate normal wound healing must take into consideration timing with respect to wound healing stages in order to maximize treatment effectiveness. Macrophage polarization from M1 to M2 represents a transition from the inflammatory to the proliferative stage of wound healing. Accelerating this transition may be an effective way to accelerate wound healing; however, it must be induced at the appropriate time. We search for an optimal spatio-temporal regime to apply wound healing treatment in a mathematical model of wound healing. In this work we show that to maximize effectiveness, treatment must not be applied too early or too late with respect to peak inflammation. We also show that the effective spatial distribution of treatment depends on the heterogeneity of the wound surface. In conclusion, this research provides a possible optimal regime of therapy that focuses on macrophage activity and a hypothesis of treatment outcome to be tested in future experiments. Finding optimal regimes for treatment application is a first step toward the development of intelligent algorithms for wound treatment that minimize healing time.

A model of infection and immune response to low dose radiation

Purpose Low dose radiation therapy (LDRT) using doses in the range of 30–150 cGy has been proposed as a means of mitigating the pneumonia associated with COVID-19. However, preliminary results from ongoing clinical trials have been mixed. The aim of this work is to develop a mathematical model of the viral infection and associated systemic inflammation in a patient based on the time evolution of the viral load. The model further proposes an immunomodulatory response to LDRT based on available data. Inflammation kinetics are then explored and compared to clinical results. Methods The time evolution of a viral infection, inflammatory signaling factors, and inflammatory response are modeled by a set of coupled differential equations. Adjustable parameters are taken from the literature where available and otherwise iteratively adjusted to fit relevant data. Simple functions modeling both the suppression of pro-inflammatory signal factors and the enhancement of anti-inflammatory factors in response to low doses of radiation are developed. The inflammation response is benchmarked against C-reactive protein (CRP) levels measured for cohorts of patients with severe COVID-19. Results The model fit the time-evolution of viral load data, cytokine data, and inflammation (CRP) data. When LDRT was applied early, the model predicted a reduction in peak inflammation consistent with the difference between the non-surviving and surviving cohorts. This reduction of peak inflammation diminished as the application of LDRT was delayed. Conclusion The model tracks the available data on viral load, cytokine levels, and inflammatory biomarkers well. An LDRT effect is large enough in principle to provide a life-saving immunomodulatory effect, though patients treated with LDRT already near the peak of their inflammation trajectory are unlikely to see drastic reductions in that peak. This result potentially explains some discrepancies in the preliminary clinical trial data.

Abstract 14228: Mechanism of Atrial Fibrillation in an Animal Model of Inflammatory Bowel Disease

Introduction: Ulcerative colitis (UC) is an i nflammatory bowel disease with a relapsing and remitting course. UC has extraintestinal manifestations and UC patients exhibit twice the risk for atrial fibrillation (AF) during acute disease activity. The mechanism of AF due to UC remains to be determined. Hypothesis: During acute UC a dysregulation of vagal signaling increases a patients propensity for AF. Methods: Mice (male, C57BL/6) were treated with Dextran Sulfate Sodium (DSS: 3%) supplemented drinking water for 7days that disrupts intestinal barrier function and mimics the disease phenotype of acute UC. Changes in atrial electrophysiology were quantified in vivo (EKG), on the whole heart (Langendorff configuration) and the cellular level (Fluo-4AM) during peak inflammation (DSS A ) and remission (DSS R ). Results: DSS A mice exhibited an attenuated heart rate (HR) in vivo (CTL: 491 ± 11 bpm, n=10; DSS A : 448 ± 11 bpm, n=16; p<0.05) and in excised perfused hearts (Langendorff configuration: CTL: 321 ± 15 bpm, n = 3; DSS A : 256 ± 17 bpm, n = 4; p < 0.05). The P-wave duration was prolonged (in vivo, CTL: 25.5 ± 0.6 ms, n=10; DSS A : 30.2 ± 0.5 ms, n=16; p < 0.0001) and HR variability time-domain measure RR-corrected SDNN was attenuated (CTL: 11.3 ± 1.1 ms, n=10; DSS A : 8.2 ± 0.7 ms, n=16; p < 0.05). The latter is an indicator for an attenuated vagal tone in DSS A mice and coincided with an increased sensitivity to the vagal agonist Carbachol in-vivo (CCh 150 ng/g, change in HR: CTL: -3.8 ± 4.4 %, n=6; DSS A : -42.2 ± 4.8 %, n=10; p < 0.0001). After burst pacing DSS A hearts exhibited a prolonged RR-corrected sinus node recovery time (CTL: 11.2 ± 1.4 ms, n=3; DSS A : 25.5 ± 2.1 ms, n=4; p < 0.0001), an increased AF propensity, and prolonged duration of AF episodes (CTL: 0.8 ± 0.17 s, n=3; DSS A : 2.4 ± 0.31 s, n=4; p < 0.05) even in the absence of CCh. No changes in atrial myocyte Ca handling properties were determined. After remission HR, P-wave duration, and HRV in DSS R returned to control values. Conclusions: The data suggests that acute UC increases the risk for AF likely as a consequence of an attenuated sinus rhythm and atrial conduction velocity where vagal stimuli could exacerbate the risk for reentry of excitation. If these changes are the direct consequence of the attenuated vagal tone remains to be determined.

Prostaglandin-E2 receptor-4 stimulant rescues cardiac malfunction during myocarditis and protects the heart from adverse ventricular remodeling after myocarditis

Cardioprotective effect of prostaglandin-E2 receptor-4 (EP4) stimulation on the ischemic heart has been demonstrated. Its effect on the heart affected by myocarditis, however, remains uncertain. In this study, we investigated therapeutic effect of EP4 stimulant using a mouse model of autoimmune myocarditis (EAM) that progresses to dilated cardiomyopathy (DCM). EP4 was present in the hearts of EAM mice. Treatment with EP4 agonist (ONO-0260164: 20 mg/kg/day) improved an impaired left ventricular (LV) contractility and reduction of blood pressure on day 21, a peak myocardial inflammation. Alternatively, DCM phenotype, characterized by LV dilation, LV systolic dysfunction, and collagen deposition, was observed on day 56, along with activation of matrix metalloproteinase (MMP)-2 critical for myocardial extracellular matrix disruption, indicating an important molecular mechanism underlying adverse ventricular remodeling after myocarditis. Continued treatment with ONO-0260164 alleviated the DCM phenotype, but this effect was counteracted by its combination with a EP4 antagonist. Moreover, ONO-0260164 inhibited in vivo proteolytic activity of MMP-2 in association with up-regulation of tissue inhibitor of metalloproteinase (TIMP)-3. EP4 stimulant may be a promising and novel therapeutic agent that rescues cardiac malfunction during myocarditis and prevents adverse ventricular remodeling after myocarditis by promoting the TIMP-3/MMP-2 axis.

Resolvin D2 Promotes the Resolution of Lung Inflammation in Allergen-Induced Asthma

Abstract Background: Asthma is a chronic inflammatory disease without complete resolution. The production of specialized pro-resolving mediators (SPMs) is defective in asthma, so they represent a potential therapeutic opportunity. We hypothesize that docosahexaenoic acid derived resolvin D2 (RvD2) can promote the resolution of asthma. Methods: BALB/c mice were challenged intra-tracheally with house dust mite (HDM). Bronchoalveolar lavage (BAL) fluid was collected at predefined protocol endpoints for peak inflammation and during resolution. To determine impact on resolution, some mice were given RvD2 (100 ng, intra-nasally) for 2 days after the last HDM challenge, and the resolution index for eosinophils and other markers were calculated. Results: Airway HDM challenge led to marked increases in BAL eosinophils and BAL fluid pro-inflammatory cytokine and chemokine levels. Histology demonstrated changes in airway inflammation and mucous cell metaplasia in HDM-challenged lungs. NF-κB expression was increased in the lungs of HDM allergic mice. Compared to vehicle, treatment with RvD2 significantly decreased BAL inflammatory macrophage and eosinophil counts, and accelerated the resolution of HDM-evoked inflammation with a marked decrease in the resolution index for BAL eosinophils from 5 days to 1 day. Interestingly, RvD2 attenuation of eosinophils specifically targeted a specific eosinophil population, but not the “resident” eosinophils. The impact of RvD2 on NF-kB activity and its GPR18 receptor-mediated actions is under current investigation to determine mechanisms for RvD2’s pro-resolving actions. Conclusion: RvD2 promotes resolution of allergen-induced inflammation in a murine model of allergic asthma.

Outcomes Associated with the Use of Renin-Angiotensin-Aldosterone System Blockade in Hospitalized Patients with SARS-CoV-2 Infection.

Data regarding the benefits or harm associated with the continuation of Angiotensin Converting Enzyme Inhibitors (ACEIs) and Angiotensin II Receptor Blockers (ARBs), especially the impact on inflammation, in hypertensive, hospitalized patients with COVID-19 in the United States is unclear. This is a single-center cohort study of sequentially hospitalized patients with COVID-19 at Stony Brook University Medical Center from March 7, 2020 to April 1, 2020, inclusive of these dates. Data collection included history of known comorbidities, medications, vital signs and laboratory values (admission and during the hospitalization). Outcomes include inflammatory burden (composite scores for multiple markers of inflammation), acute kidney injury (AKI), admission to the intensive care unit (ICU), need for invasive mechanical ventilation, and mortality. Of the 300 patients in the study cohort, 80 patients (26.7%) had history of ACEI or ARB use prior to admission, with 61.3% (49/80) of these patients continuing the medications during hospitalization. Multivariable analysis revealed that the history of ACEI or ARB use prior to hospitalization was not associated with worse outcomes. In addition, the continuation of these agents during hospitalization was not associated with an increase in adverse outcomes and predicted fewer ICU admissions (OR=0.25, 0.08-0.81) with a decrease in the severity of inflammatory burden (peak CRP (6.9±3.1mg/dl, p=0.03) and peak inflammation score (2.3±1.1unit reduction, p=0.04)). Use of ACEI or ARBs prior to hospitalization was not associated with adverse outcomes in COVID-19 and the therapeutic benefits of continuing ACEI or ARB in hospitalized patients with COVID-19 was not offset by adverse outcomes.

Resolvin D2 Promotes the Resolution of Allergen-Induced Lung Inflammation

Abstract Background Asthma is a chronic inflammatory disease without complete resolution. The production of specialized pro-resolving mediators (SPMs) is defective in asthma, so they represent a potential therapeutic opportunity. We hypothesize that docosahexaenoic acid derived resolvin D2 (RvD2) can promote the resolution of asthma. Methods BALB/c mice were challenged intra-tracheally with house dust mite (HDM). Bronchoalveolar lavage (BAL) fluid was collected at predefined protocol endpoints for peak inflammation and during resolution. To determine impact on resolution, some mice were given RvD2 (100 ng, intra-nasally) for 2 days after the last HDM challenge, and the resolution index for eosinophils and other markers were calculated. Results Airway HDM challenge led to marked increases in BAL eosinophils and BAL fluid pro-inflammatory cytokine and chemokine levels. Histology demonstrated changes in airway inflammation and mucous cell metaplasia in HDM-challenged lungs. NF-κB and Nrf2 expression were increased the lungs of HDM allergic mice. Compared to vehicle, treatment with RvD2 significantly decreased BAL inflammatory macrophage and eosinophil counts, and accelerated the resolution of HDM-evoked inflammation with a marked decrease in the resolution index for BAL eosinophils from 5 days to 1 day. Interestingly, RvD2 attenuation of eosinophils specifically targeted the inflammatory eosinophil population, but not the resident eosinophils. The impact of RvD2 on cytokine expression and NF-kB and Nrf2 activity is under current investigation to determine mechanisms for RvD2’s pro-resolving actions. Conclusion RvD2 promotes resolution of allergen-induced inflammation in a murine model of allergic asthma.

Liver fibrosis and biliary proliferation are enhanced in the rat model of inflammatory bowel disease

BackgroundPatients who suffer from Inflammatory Bowel Disease (IBD) often experience concurrent conditions such as lymphatic dysfunction and bone loss. Additionally, liver and biliary abnormalities are frequently observed, including primary sclerosing cholangitis (PSC), autoimmune hepatitis, and cholelithiasis. The pathogenesis is unknown but is presumed to be secondary to chronic inflammation, which drives intrahepatic inflammation and fibrogenesis. The lymphatic system, via transport of inflammatory stimuli, is a likely route by which the regional inflammatory changes in the bowel effect distal organs such as the liver. Therefore, the aim of this study was to evaluate changes in biliary damage and senescence, ductular reaction (DR), and liver inflammation and fibrosis in an experimental rat model of inflammatory bowel disease.MethodsStudies were performed in male rats receiving a 2,4,6‐trinitrobenzene sulfonic acid (TNBS) enemas at 30mg/kg of body weight, once a week, for 4 weeks. Lymph, livers, isolated cholangiocytes, and hepatic stellate cells were collected. Ductular reaction was evaluated by immunohistochemistry (IHC) for CK19 in paraffin embedded liver sections and cellular senescence was determined by SA‐β‐gal staining. Gene expression was measured using qPCR for p16, CCL2 and p21 in total liver and laser‐captured microscopy (LCM) isolated cholangiocytes. Liver fibrosis was assessed via Sirius Red staining and qPCR for the following genes; α‐SMA, TGFβ‐1, FN‐1, and collagen1a1 in total liver, HSCs, and pure cholangiocytes. To determine the level of inflammation, IHC for CD68 and MHCII was performed, while gene expression was measured using qPCR for inflammatory/senescence markers (IL‐1β, IL‐6, IL‐33 and TNF‐α) in total liver and LCM isolated cholangiocytes. Immunofluorescence was used to visualize the expression of TLR4 in the intact bile ducts of total liver sections. Lymph was used for proteomic analysis post‐instillation at peak inflammation, to identify potential inflammatory signals that may precipitate the changes that were noted.ResultsRats treated with TNBS had increased DR, collagen deposition, and fibrotic gene expression in both cholangiocytes and HSCs; enhanced liver inflammation with increased CD68 positive macrophages in liver sections; increased biliary senescence and biliary expression of senescence markers; and increased biliary expression of TLR4. There was a strong correlation between the increase in inflammatory pathways in the lymph from IBD animals, with marked increases in pathways associated with bacterial translocation and lipid metabolism.ConclusionWe observed increased biliary ductular reaction that was associated with increased liver inflammation/fibrosis similar to the co‐morbidity of PSC reported in IBD patients.Support or Funding InformationNIH RO1 DK110035

Enteric Glia Contribute to Visceral Hypersensitivity Through Interactions with Nociceptors

Enteric glia regulate gut motility, secretions, and neuroinflammation through intercellular signaling mechanisms that involve connexin‐43 (Cx43) hemichannels. Glial Cx43 signaling is active during the acute phase of colitis when the sensitization of nociceptors occurs, but how glia contribute to nociceptor sensitization is not known. Here, we tested the hypothesis that alterations to intercellular signaling between glia and nociceptors mediated by Cx43 contribute to the sensitization of nociceptors during gut inflammation. We used the dinitrobenzene sulfonic acid (DNBS) model of acute colitis and tested the effects on enteric glia using immunohistochemistry, multiplex immunoassays, and ethidium bromide dye uptake to measure glial Cx43 channel activity. Specific interactions between glia and nociceptors were assessed using the glial specific GFAP::hM3Dq DREADD mouse model in combination with visceromotor reflex recordings and pERK expression, and by directly recording nociceptor nerve fiber activity in TRPV1‐GCaMP5g‐tdT mice. DNBS colitis drove an increase in proinflammatory cytokines including IL6, TNFα, and IL17, and IL1β showed a 5‐fold increase (p<0.05) at peak inflammation. Enteric glia contribute to IL1β production and immunolabeling data showed a 40% increase in glial IL1β (p<0.05) that was confirmed by RNAseq data (2‐fold, p<0.05). The pro‐inflammatory mediators IL1β, IL6, INFy, and TNFα increased glial dye uptake under basal conditions and IL1β and IL6 potentiated ADP‐stimulated dye uptake by 24% and 53%, respectively (p<0.0001), in a Cx43‐dependent manner. The selective activation of glial cells with clozapine‐N‐oxide in GFAP::hM3Dq mice increased visceromotor responses to colorectal distensions (p<0.05) and drove pERK expression in TRPV1+ sensory nerve fibers in the myenteric plexus. Neither IL1β nor glial activation alone significantly affected Ca2+ responses to capsaicin in TRPV1+ nerve fibers in the colon. However, glial activation in the presence of IL1β significantly potentiated nociceptor Ca2+ responses to capsaicin and this effect required signaling through Cx43 hemichannels. Our data show that enteric glia can affect the sensitivity visceral nociceptors in the context of inflammation through mechanisms that include the potentiation of glial Cx43 hemichannel function by IL1β and the subsequent sensitization of TRPV1 channels expressed by sensory neurons.Support or Funding InformationNational Institute of Health through NIDDK: R01DK103723, R01DK120862

L‐Plastin, a Novel Regulator of Microglial Activation in Parkinson’s disease

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease worldwide behind Alzheimer’s disease. One prominent feature of PD is the marked loss of dopaminergic and motor dysfunction. Currently, there are no therapies to effectively slow disease progression. Neuroinflammation is a major contributing factor in neurodegeneration diseases including PD. Microglia, a type of glial cell, are primary regulators of neuroinflammation and these cells are capable of assuming activation states ranging from inflammatory to anti‐inflammatory and reparative. Thus methods to combat microglial inflammation or promote microglial anti‐inflammatory and reparative effects show promise as treatments to slow neurodegeneration. L‐plastin (LPL) is an acting bundling protein and other research has shown that LPL plays a role in osteoclast function, T‐cell activation, and it may be involved in the NLRP3 inflammasome activation in macrophages. It has been shown in osteoclasts, that LPL phosphorylation at serine 5 plays a role in sealing zone formation. Using a peptide inhibitor to block phosphorylation of serine 5 on LPL prevented sealing zone formation and inhibited osteoclast‐mediated bone resorption. Given the evidence that LPL may play a role in inflammasome activation in macrophages, we investigated if LPL may play a role in microglial activation. To assess the function of LPL in the microglial inflammatory response, we utilized qPCR, Western blot, and immunofluorescence techniques. We show here that lipopolysaccharide (LPS) treatment induces LPL phosphorylation at serine 5, as well as increased Lcp1 mRNA expression. Furthermore, using a TAT‐fused peptidomimetic inhibitor of LPL phosphorylation significantly reduced LPS‐induced inflammatory gene (Il‐1b, Il‐6, Tnfa) expression. For this study, an acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model was also used. Male (10‐week‐old) C57BL/6N mice were treated with four injections (one every two hours) of either saline or 15mg/kg of MPTP. Mice were then sacrificed two days after the last injection and striatum and hindbrain were isolated. Two days after MPTP treatment is when peak inflammation occurs in the brain. We found that Lcp1 mRNA expression was significantly increased in the striatum of MPTP‐treated mice compared to saline‐treated mice. These results suggest that LPL may play a role in the microglial inflammatory response, and that LPL inhibition may be a novel way to combat neuroinflammation and neurodegeneration. We are currently staining for phosphorylated LPL in the microglia of MPTP treated mice. Furthermore, we have obtained LPL KO mice to assess the in vivo function of LPL in mouse models of PD.Support or Funding InformationKent State University Biomedical Sciences