Inflammatory Insult Research Articles

Inflammatory stimuli induce shedding of heparan sulfate from arterial but not venous porcine endothelial cells leading to differential proinflammatory and procoagulant responses

Endothelial dysfunction is an early event of vascular injury defined by a proinflammatory and procoagulant endothelial cell (EC) phenotype. Although endothelial glycocalyx disruption is associated with vascular damage, how various inflammatory stimuli affect the glycocalyx and whether arterial and venous cells respond differently is unknown. Using a 3D round-channel microfluidic system we investigated the endothelial glycocalyx, particularly heparan sulfate (HS), on porcine arterial and venous ECs. Heparan sulfate (HS)/glycocalyx expression was observed already under static conditions on venous ECs while it was flow-dependent on arterial cells. Furthermore, analysis of HS/glycocalyx response after stimulation with inflammatory cues revealed that venous, but not arterial ECs, are resistant to HS shedding. This finding was observed also on isolated porcine vessels. Persistence of HS on venous ECs prevented complement deposition and clot formation after stimulation with tumor necrosis factor α or lipopolysaccharide, whereas after xenogeneic activation no glycocalyx-mediated protection was observed. Contrarily, HS shedding on arterial cells, even without an inflammatory insult, was sufficient to induce a proinflammatory and procoagulant phenotype. Our data indicate that the dimorphic response of arterial and venous ECs is partially due to distinct HS/glycocalyx dynamics suggesting that arterial and venous thrombo-inflammatory disorders require targeted therapies.

A21 ROLE OF SIALIC ACID O-ACETYLATION IN MAINTAINING MUCUS INTEGRITY AND HOMEOSTASIS OF THE COLONIC MUCOSA

Abstract Background The mucus network provides innate immune defense to protect our gastrointestinal tract from pathogens, and promote homeostasis with our resident microbiota. This network is constituted by the mucin MUC2 (Muc2 in mouse), which is ~80% complex O-linked glycans by weight. Sialic acid (Sia) is a key capping monosaccharide on complex O-glycans which has recently been linked to preserving mucus integrity. Sia can undergo enzymatic modifications including the addition of O-acetyl groups. The 9-O-acetyltransferase CasD1 is responsible for the 9-OAc Sia variants. Functionally, the OAc-modification is known to inhibit microbial sialidase activities which may preserve Sia’s protective roles on mucins. However, the extent of these OAc modifications in human and murine Mucin-2, and how they influence mucus function is unclear. Purpose: To determine whether and how Sia O-acetylation on colonic mucus regulates mucus integrity, host-microbe interactions, and colitis susceptibility. Method We used viral-derived probes that target specific OAc-Sia analogues on mucus on sections from human feces and mouse feces and colon tissues to visualize their spatial arrangement and microbial interaction in situ. For glycomics, OAc-Sia analogues were quantitated on purified human MUC2 and mouse Muc2 by HPLC-MS after derivatization with 4,5-dimethyl-1,2-diaminobenzamine (DMBA). O-glycans were released via non-reductive ammonia-catalyzed β-elimination and analyzed by mass spectrometry. For in vivo work, we generated intestinal epithelial cell-specific Casd1 KO mice (Casd1flox/flox;VillinCre or IEC Casd1-/- mice) and analyzed their mucins. Sialidase activities were quantified in the supernatants of colon fecal materials from WT and IEC Casd1-/- mutants mice using a fluorogenic substrate 4-MU-NeuNAc. Colitis susceptibility was monitored using 1.5% w/v Dextran Sodium Sulfate (DSS). Result(s) We found Sias on both human MUC2 and murine Muc2 were heavily O-acetylated, with ~75% and ~45% of Sias having 9-OAc-based modification in humans and mice respectively, and were distributed throughout the niche and barrier layers of mucus in situ. IEC Casd1-/- mice were viable and healthy with knockdown confirmed by 9-OAc staining, western blot of protein lysates and mucins, and sialylomics. The mucus encapsulation appeared overall intact regardless of OAc status. However, IEC Casd1-/- mice showed heightened susceptibility to 1.5% DSS colitis, linked to thinning of the mucus in IEC Casd1-/- vs WT littermates after challenge. Consistent with the role of OAc Sia in sialidase inhibition, loss of OAc Sia was associated with increased sialidase activities as assessed by heightened 4 MU signal in fecal supernatants in WT vs littermate IEC Casd1-/- mice. O-glycomics also showed reduction in the number of sialylated O-glycan structures upon loss of 9-OAc Sia. Conclusion(s) Sia O-acetylation appears important in maintaining key aspects of Sia-dependent mucus function and protecting from inflammatory insult. Please acknowledge all funding agencies by checking the applicable boxes below: CCC Disclosure of Interest None Declared

A5 A MOUSE MODEL TO UNRAVEL THE PATHOPHYSIOLOGICAL LINK BETWEEN CROHN’S DISEASE AND TYPE-2 DIABETES-ASSOCIATED METABOLIC DISORDERS

Abstract Background Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD), has been recently shown to increase the risk of developing type 2 diabetes (T2D). Moreover, treatment with anti-diabetic drugs has a protective role in preventing the severity and course of CD progression. However, the pathophysiological basis of T2D development in CD remains unclear. Findings have highlighted the contribution of adipose tissue (AT) to the development of chronic inflammatory diseases and have identified parallels between T2D and CD that may provide hints to common mechanisms of disease pathogenesis. Typically, microbial dysbiosis, hyperpermeable intestinal barrier, and intra-abdominal AT accumulation are the common features of both diseases, yet how the interplay of these factors contribute to pathogenesis is not known. Therefore, common pathogenic paradigms underlying both T2D and CD have led us to hypothesize that chronic intestinal inflammation serves as an initiator of AT dysfunction in CD, predisposing individuals to T2D. Further, the lack of appropriate animal models of CD with chronic intestinal inflammation that manifests accumulation of intra-abdominal AT, and extra-intestinal metabolic disorder as observed in CD and T2D patients has been a limitation. Purpose To develop a genetic mouse model to investigate if gut inflammation-mediated microbial dysbiosis and metabolic dysregulation of AT are at the nexus that cause T2D in CD. Method We developed a CD-mouse model, where we challenged Nod2-deficient mice (NOD2 being the strongest genetic risk factor contributing to CD) with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, intraperitoneal insulin and oral glucose tolerance tests, metabolic caging, and MRI imaging of mice were performed. Changes in AT metabolism and microbial infiltration into AT were analyzed by quantitative real-time PCR (qRT-PCR) and/or immunohistochemistry (IHC). Result(s) Our new CD-mouse model revealed increased gut inflammation (TNF and type-I IFN) in Nod2-deficient mice compared to wild-type control mice post-cDSS. Surprisingly, Nod2-deficient mice gained body weight, which was at least in part accounted for by an increased intra-abdominal AT accumulation along with decreased AT fatty-acid metabolism (Cpt1a, Fabp4 expression) and AT browning (Ucp1, Cidea expression, and UCP-1 staining), reduced intestinal goblet cell numbers, increased gut bacterial infiltration within the fat, more insulin resistance and energy expenditure. Conclusion(s) This experimental mouse model mimicking CD-associated T2D will provide insights into how the microbiome-AT axis fuel chronic inflammation-mediated extra-intestinal metabolic disorder and immune dysregulation. Understanding these connections will be transformative, as it will help us devise novel therapeutic strategies to prevent T2D development in progressive CD patients. Disclosure of Interest None Declared

Potential effect of luteolin, epiafzelechin, and albigenin on rats under cadmium-induced inflammatory insult: In silico and in vivo approach.

Introduction: Cadmium(Cd) an industrial poison present abundantly in the environment, causes human toxicity by an inflammatory process. Chronic exposure of cadmium can cause a number of molecular lesions that could be relevant to oncogenesis, through indirect or epigenetic mechanisms, potentially including abnormal activation of oncogenes and suppression of apoptosis by depletion of antioxidants. As induction of cyclooxygenase (COX)-2 is linked to inflammatory processes, use of luteolin, epiafzelechin, and albigenin alone or in different combinations may be used as anti-inflammatory therapeutic agents. Methods: We, herein, performed in silico experiments to check the binding affinity of phytochemicals and their therapeutic effect against COX-2 in cadmium administered rats. Wistar albino rats were given phytochemicals in different combinations to check their anti-inflammatory activities against cadmium intoxication. The level of alanine aminotransferases (ALT), 4-hydroxynonenal (4HNE), 8-hydroxy-2-deoxyguanosine (8-OHdG), tumor necrosis factor-alpha (TNF-α), isoprostanes (IsoP-2α), COX-2, and malondialdehyde (MDA) were estimated with their respective ELISA and spectrophotometric methods. Results: The generated results show that phytocompounds possessed good binding energy potential against COX-2, and common interactive behavior was observed in all docking studies. Moreover, the level of ALT, 4HNE, 8-OHdG, TNF-α, IsoP-2α, malondialdehyde, and COX-2 were significantly increased in rats with induced toxicity compared to the control group, whereas in combinational therapy of phytocompounds, the levels were significantly decreased in the group. Discussion: Taken together, luteolin, epiafzelechin, and albigenin can be used as anti-inflammatory therapeutic agents for future novel drug design, and thus it may have therapeutic importance against cadmium toxicity.

Krill Oil attenuates doxorubicin induced cognitive impairment by inhibiting oxidative stress markers in the brains of wistar rats

Chemobrain is a typical adverse effect of chemotherapeutics agents that affects 15–80 percent of cancer patients. Doxorubicin, an antineoplastic drug, has been associated with severe neurotoxicity manifested as a loss in cognitive skills, most likely due to oxidative stress in brain. Krill oil (KO) is a unique, naturally occurring nutrient derived from a type of Antarctic krill, Euphausia superba that is commonly used as a dietary supplement and is well known for anti-inflammatory, hypolipidemic, anti-depressant and improvement of cognitive function, protection of neurodegeneration, neuroprotective and antioxidant qualities. The current study was aimed at evaluating krill oil's potential neuroprotection and memory improving benefits against Doxorubicin-induced cognitive and neurobiological defects. A novel object recognition task and Morris water maze test were conducted to evaluate the protective effect of krill oil against doxorubicin-induced memory deficits in rats. In female Wistar rats, cognitive impairment was produced by administering ten cycles of Doxorubicin (2.5mg/kg, intra peritoneal, once every five days). Krill oil (50mg/kg and 100mg/kg) was given orally starting one week prior to the doxorubicin treatment and continued throughout the study. Krill treatment (50mg/kg and 100mg/kg) significantly protected against Doxorubicin induced memory impairment. Co-administration of both doses of krill oil resulted in significantly lower the levels of tumour necrosis factor (TNFα), and acetyl cholinesterase activity. Furthermore, doxorubicin-induced oxidative and inflammatory insults in the frontal cortex and hippocampal areas of the brain were prevented by krill oil. In conclusion, krill oil may be a promising adjuvant treatment option for reducing adverse effects associated to doxorubicin treatment.

Human gingival epithelial cells stimulate proliferation, migration, and tube formation of lymphatic endothelial cells invitro.

The aim of this study was to investigate the response of gingival epithelial cells to microbial and inflammatory signals. The gingival epithelial barrier provides the first line of defense and supports tissue homeostasis by maintaining the cross-talk between gingival epithelium, oral microbiota, and immune cells. Lymphatic vessels are essential to sustaining this homeostasis. The gingival epithelial cells have been shown to produce prolymphangiogenic factors during physiologic conditions, but their role in response to microbial and inflammatory signals is unknown. Immortalized human gingival epithelial cells (HGEC) and human dermal lymphatic microvascular endothelial cells (LEC) were cultured. HGEC were exposed to Porphyromonas gingivalis derived-LPS, human IL-1 beta/IL-1F2 protein, or recombinant human IL-6/IL-6R. Levels of vascular growth factors (VEGF-A, VEGF-C, and VEGF-D) in cell supernatants were determined by ELISA. LEC were grown to confluence, and a scratch was induced in the monolayer. Uncovered area was measured up to 48 h after exposure to conditioned medium (CM) from HGEC. Tube formation assays were performed with LEC cocultured with labelled HGEC or exposed to CM. VEGF-A, VEGF-C, and low levels of VEGF-D were constitutively expressed by HGEC. The expression of VEGF-C and VEGF-D, but not VEGF-A, was upregulated in response to proinflammatory mediators. VEGF-C was upregulated in response to P.gingivalis LPS, but not to Escherichia coli LPS. A scratch migration assay showed that LEC migration was significantly increased by CM from HGEC. Both the CM and coculture with HGEC induced significant tube formation of LEC. HGEC can regulate production of lymphangiogenic/angiogenic factors during inflammatory insults and can stimulate proliferation, migration, and tube formation of LEC in vitro in a paracrine manner.

Immunogenic Cell Death Associated Molecular Patterns and the Dual Role of IL17RA in Interstitial Cystitis/Bladder Pain Syndrome.

The unclear etiology and pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS) are responsible for the lack of effective treatment and the poor patient prognosis. Various studies show that chronic inflammation and immune responses are important factors contributing to the pathogenesis of IC/BPS. The process of immunogenic cell death (ICD) involves both the immune response and inflammatory process, and the involvement of ICD in IC/BPS pathogenesis has not been explored. Two IC/BPS transcriptome datasets collected from the Gene Expression Omnibus (GEO) database were used to identify distinct ICD-associated molecular patterns (IAMPs). IAMPs and IC/BPS subtypes were found to be related. The inflammatory immune microenvironments (IIME) in different IAMPs were studied. The potential mechanism by which the interleukin 17 receptor A (IL17RA) influences IC/BPS was examined using in vitro assays. The expression of ICD-related genes (IRGs) was upregulated in IC/BPS bladders, compared with normal bladders. Disease prediction models, based on differentially expressed IRGs, could accurately predict IC/BPS. The IC/BPS patients had two distinct IAMPs, each with its own subtype and clinical features and association with remodeling IIME. IL17RA, a well-established IC/BPS bladder biomarker, mediates both the inflammatory insult and the protective responses. In summary, the current study identified different IAMPs in IC/BPS, which may be involved in the pathogenesis of IC/BPS by remodeling the IIME. The chronic inflammatory process in IC/BPS may be prolonged by IL17RA, which could mediate both pro- and anti-inflammatory responses. The IL17RA-associated pathway may play a significant role in the development of IC/BPS and can be used as a therapeutic target.

SARS-CoV-2 spike protein-induced inflammation underlies proarrhythmia in COVID-19.

Cardiac manifestations are common in severe COVID-19 with 2% of non-ICU patients and 59% of non-survivors impacted. Per recent CDC data, COVID-19 patients are at 1.7 higher risk of arrhythmia. AF, which is the most common arrhythmia, is a frequent complication of acute and long COVID-19. High cardiac expression of the SARS-CoV2 receptor, ACE2, has led to the assumption that infectious virus likely underlies cardiac complications. However, this remains to be verified. We therefore compared the histologic and molecular findings in human cardiac tissue from fatal COVID-19 cases and age-matched controls. Hematoxylin and eosin (H&E) stains revealed histologic changes including multifocal endothelial cell swelling and degeneration plus microvascular thrombi in COVID-19 cases. qRT-PCR and in situ hybridization (ISH) identified trace amounts of viral nucleocapsid RNA and no nucleocapsid protein in the COVID-19 hearts. However, 7/11 COVID-19 hearts were positive for viral spike protein. Further validation by immunohistochemistry (IHC) revealed viral spike protein in all 11 COVID-19 hearts. Co-expression analysis identified viral spike protein mostly in ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Previously, we identified a novel arrhythmia mechanism wherein inflammation-induced vascular endothelial dysfunction triggers cardiomyocyte structural remodeling, particularly of intercalated disc (ID) components including connexin-43 (Cx43) gap junctions and NaV1.5 sodium channels, and thereby promotes arrhythmias. We therefore investigated ID structure in our human samples. Both Cx43 and NaV1.5 showed marked lateral migration in myocytes of COVID-19 hearts, consistent with previous experiments with inflammatory insults in murine hearts. Preliminary results in spike protein-injected mice showed increased arrhythmia burden, as well as structural remodeling and protein migration consistent with human results. These findings suggest that the viral spike protein, and not direct infection by SARS-CoV2, results in the pathophysiology of cardiac dysfunction in fatal COVID-19.

Chronic stress beginning in adolescence decreases spatial memory following an acute inflammatory challenge in adulthood

Prolonged stress beginning in adolescence can contribute to the dysregulation of the neuroendocrine system in adulthood. As the neuroendocrine and neuroimmune systems participate in bi-directional regulatory control, adolescent stress can prime the neuroimmune system to future inflammatory insults. Previous work from our group demonstrates that stress exaggerates the hippocampal response to inflammation, which can lead to deficits in learning and memory. In the current study, we sought to interrogate the interaction between an acute peripheral challenge of lipopolysaccharide (LPS) in male and female Wistar rats with a history of stress beginning in adolescence (CAS). Males from the CAS group were more vulnerable to the peripheral effects of LPS compared to non-stressed males including porphyrin staining and ruffled fur. In contrast, LPS generated similar peripheral effects in females regardless of adolescent stress history. Learning and memory were differentially impacted by LPS as a function of stress history and effects manifested differently when stratified by sex. Males with a history of adolescent stress exhibited deficits in initial learning. Females from the CAS group performed similar to controls during acquisition but exhibited a slight impairment during reversal learning. Males and females with a history of stress displayed memory impairment during the probe assessments as compared to their same-sex control group. We conclude that while stress beginning in adolescence enhanced the vulnerability of learning and memory to an inflammatory challenge, the phenotype of this effect manifested differently in males and females. These data demonstrate a sustained impact of adolescent stress on the neuroimmune system which is sufficient to influence cognitive performance in both sexes.

Gastroschisis Outcomes: Presence of Histologic Chorioamnionitis and the Impact on Time to Full Enteral Feeds and Length of Hospital Stay.

Despite improvements in our ability for early diagnosis and providing supportive care for infants with gastroschisis, it continues to be associated with long length of stay and morbidity. Intestinal dysfunction secondary to chronic inflammatory insult to exposed bowel is well known; however, little research has been done on the impact of acute inflammation in the perinatal period on intestinal function. This study's aim was to investigate the impact of acute chorioamnionitis on the time to achieve full enteral feeds and length of hospital stay. Retrospective chart review of 60 mothers and their infants born with gastroschisis at a Level IV NICU from November 2011 to June 2020 was performed. Infants were divided into two groups based on the presence of histologic chorioamnionitis, and outcomes were compared. The primary outcome was delayed full enteral feeds (full enteral feeds after 28 days of life). The secondary outcomes were differences in their time to achieve full enteral feeds and time to hospital discharge, and prolonged length of hospital stay (discharge after 30 days of life). Univariate and multivariate logistic regression analyses were performed to assess the association between the dependent and the predictor variables. Of the 60 infants enrolled, 23 (38%) had evidence of histologic chorioamnionitis. The median gestational age was 37 weeks. Fifty-four (90%) infants achieved full enteral feeds, with a median time of 24 days. Median length of hospital stay was 31 days. The presence of histologic chorioamnionitis was not associated with delayed full enteral feeds (odds ratio [OR] = 0.79; 95% confidence interval [CI] = 0.14-4.23; p = 0.80) or prolonged length of hospital stay (OR = 0.45; 95% CI = 0.1-0.23; p = 0.32) in the adjusted analysis. Acute placental inflammation during the perinatal period does not impact the infant's time to achieve full feeds or prolong their hospital stay. Larger studies are needed to confirm these findings. · Chronic inflammatory injury to exposed bowel in utero is well known in fetuses with gastroschisis.. · Acute inflammatory injury during perinatal period may impact enteral feeding outcomes.. · No impact of acute placental inflammation on time to full enteral feeds..

Sestrin2 provides cerebral protection through activation of Nrf2 signaling in microglia following subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a neurological emergency characterized by dysfunctional inflammatory response. However, no effective therapeutic options have been reported so far. Microglia polarization has been proposed to exert an essential role in modulating inflammatory response after SAH. Sestrin2 is a stress response protein. Growing evidence has reported that sestrin2 could inhibit M1 microglia and promote M2 microglia polarization. The current study investigated the effects of sestrin2 on microglia phenotype switching and the subsequent brain injury and sought to elucidate the underlying mechanism. We conducted an endovascular perforation SAH model in mice. It was found that sestrin2 was significantly increased after SAH and was mainly distributed in neurons and microglia. Exogenous recombinant human sestrin2 (rh-sestrin2) evidently alleviated inflammatory insults and oxidative stress, and improved neurofunction after SAH. Moreover, rh-sestrin2 increased M2-like microglia polarization and suppressed the number of M1-like microglia after SAH. The protection by rh-sestrin2 was correlated with the activation of Nrf2 signaling. Nrf2 inhibition by ML385 abated the cerebroprotective effects of rh-sestrin2 against SAH and further manifested M1 microglia polarization. In conclusion, promoting microglia polarization from the M1 to M2 phenotype and inducing Nrf2 signaling might be the major mechanism by which sestrin2 protects against SAH insults. Sestrin2 might be a new molecular target for treating SAH.

Triptonide Ameliorates Middle Cerebral Artery Occlusion-induced Cerebral Ischemic Damage in Rats via Regulation of In flammatory Response

Background Cerebral ischemic stroke is the second major cause of mortality worldwide that results in persisting disability and mental agony. Ischemic stroke is induced by the diminished blood circulation to the brain, which can be due to obstruction by arteriosclerosis, fibromuscular dysplasia, or thrombosis. Triptonide is a diterpene triepoxide, purified out of extracts of Tripterygium wilfordii Hook F, and is an emerging target against, for example, cancers and inflammatory insults. Materials and Methods Taking this into consideration, this study was designed to analyze the effect of triptonide on ischemic/reperfusion (I/R) cerebral infarction stroke rats. Results Our study showed that triptonide decreased the infarct volume and brain edema produced by I/R. Moreover, triptonide protected the rats from any neurological deficits, which were analyzed using a five-point scoring system, augmented antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione content, and lowered the activity of acetylcholinesterase. Triptonide also decreased the levels of pro-inflammatory cytokines such as interleukin-1 β (IL-1 β), TNF- α, and IL-6, while it augmented anti-inflammatory cytokines IL-10 and vascular endothelial growth factor. In this study, cerebral infarction stroke rats showed an increase in malondialdehyde levels. Triptonide preserved the normal brain architecture from various neurotoxic effects. Conclusion Thus, triptonide can be targeted for drug discovery in the future to protect against cerebral infarction stroke.

Mesenchymal condensation in tooth development and regeneration: a focus on translational aspects of organogenesis.

The teeth are vertebrate-specific, highly specialized organs performing fundamental functions of mastication and speech, the maintenance of which is crucial for orofacial homeostasis and is further linked to systemic health and human psychosocial well-being. However, with limited ability for self-repair, the teeth can often be impaired by traumatic, inflammatory, and progressive insults, leading to high prevalence of tooth loss and defects worldwide. Regenerative medicine holds the promise to achieve physiological restoration of lost or damaged organs, and in particular an evolving framework of developmental engineering has pioneered functional tooth regeneration by harnessing the odontogenic program. As a key event of tooth morphogenesis, mesenchymal condensation dictates dental tissue formation and patterning through cellular self-organization and signaling interaction with the epithelium, which provides a representative to decipher organogenetic mechanisms and can be leveraged for regenerative purposes. In this review, we summarize how mesenchymal condensation spatiotemporally assembles from dental stem cells (DSCs) and sequentially mediates tooth development. We highlight condensation-mimetic engineering efforts and mechanisms based on ex vivo aggregation of DSCs, which have achieved functionally robust and physiologically relevant tooth regeneration after implantation in animals and in humans. The discussion of this aspect will add to the knowledge of development-inspired tissue engineering strategies and will offer benefits to propel clinical organ regeneration.

Transient Receptor Potential Ankyrin-1-expressing vagus nerve fibers mediate IL-1β induced hypothermia and reflex anti-inflammatory responses

BackgroundInflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines.MethodsTo monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant.ResultsHere, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis.ConclusionThus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity.

Identification of ARMH4 and WIPF3 as human podocyte proteins with potential roles in immunomodulation and cytoskeletal dynamics.

The podocyte is a specialized cell type critically involved in maintaining the selective filtration barrier of the kidney. Podocytes are primary or secondary targets for a multitude of kidney diseases. Despite intense investigation, the transcriptome and proteome of human podocytes remain incompletely characterized. Here, we analyzed publicly available RNA-Seq data from human kidneys (n = 85) to computationally identify potential novel podocyte markers. For confirmation, we used an online histology resource followed by in-house staining of human kidneys and biochemical fractionation of glomeruli. Initial characterization of the novel podocyte transcripts was performed using viral overexpression and mRNA silencing. Several previously unrecognized gene products were identified that correlated to established podocyte markers on the RNA level and that were histologically localized to podocytes. ARMH4 (a.k.a. UT2 or C14orf37) and WIPF3 (a.k.a CR16) were among the hits. We show that these transcripts increase in response to overexpression of the podocyte transcription factor LMX1B. Overexpression of ARMH4 from low endogenous levels in primary kidney epithelial cells reduced the release of the inflammatory mediators IL-1B and IL-8 (CXCL8). The opposite effect was seen in mature human podocytes when ARMH4 was silenced. Overexpression of WIPF3 stabilized N-WASP, known to be required for maintenance of podocyte foot processes, and increased cell motility as shown using a scratch assay. Moreover, data from normal and diseased human kidneys showed that ARMH4 was downregulated in glomerular pathologies, while WIPF3 remained constantly expressed. ARMH4 and WIPF3 are new potential markers of human podocytes, where they may modulate inflammatory insults by controlling cytokine release and contribute to cytoskeletal dynamics, respectively.

Differential expression profiles and functional prediction of circRNA in mice with traumatic heterotopic ossification.

Traumatic heterotopic ossification (HO) is an intractable sequela incited by inflammatory insult. To date, the exact molecular mechanisms of traumatic HO formation remain unclear. Recent studies have indicated that circular RNAs (circRNAs) participate in various human skeletal diseases. Although the formation of HO recapitulates many programs during bone development and remodeling, few data are available concerning whether circRNAs could participate in this pathological osteogenesis. To investigate the differentially expressed circRNAs (DE-circRNAs) in HO formation, microarray assay was performed to analyze the circRNA expression profile in four pairs of mice HO tissues and normal tissues. Then, qRT-PCR was applied to verify the microarray data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed the biological functions of the differentially expressed circRNAs target genes. Cytoscape software was used to construct the circRNA-miRNA-mRNA network for circRNAs with different expression levels as well as the target genes. We demonstrated that 491 circRNAs were significantly differentially expressed in mouse HO tissues by a fold-change ≥ 2 and p-value ≤ 0.05. Among them, the expressions of 168 circRNAs were increased, while 323 were decreased. The expression levels of 10 selected circRNAs were verified successfully by qRT-PCR. GO analysis exhibited that these DE-circRNAs participated in a series of cellular processes. KEGG pathway analysis revealed that multiple upregulated and downregulated pathways were closely related to the DE-circRNAs in HO mice. The circRNA-miRNA-mRNA networks demonstrated that DE-circRNAs may be involved in the pathological osteogenesis of HO through the circRNA-targeted miRNA-mRNA axis. Our study first demonstrated the expression profiles and predicted the potential functions of DE-circRNAs in mice traumatic HO, which may shed new light on the elucidation of mechanisms as well as provide novel potential peripheral biological diagnostic markers and therapeutic targets for traumatic HO.

Immunometabolic factors contributing to obesity-linked hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major public health concern that is promoted by obesity and associated liver complications. Onset and progression of HCC in obesity is a multifactorial process involving complex interactions between the metabolic and immune system, in which chronic liver damage resulting from metabolic and inflammatory insults trigger carcinogenesis-promoting gene mutations and tumor metabolism. Moreover, cell growth and proliferation of the cancerous cell, after initiation, requires interactions between various immunological and metabolic pathways that provide stress defense of the cancer cell as well as strategic cell death escape mechanisms. The heterogenic nature of HCC in addition to the various metabolic risk factors underlying HCC development have led researchers to focus on examining metabolic pathways that may contribute to HCC development. In obesity-linked HCC, oncogene-induced modifications and metabolic pathways have been identified to support anabolic demands of the growing HCC cells and combat the concomitant cell stress, coinciding with altered utilization of signaling pathways and metabolic fuels involved in glucose metabolism, macromolecule synthesis, stress defense, and redox homeostasis. In this review, we discuss metabolic insults that can underlie the transition from steatosis to steatohepatitis and from steatohepatitis to HCC as well as aberrantly regulated immunometabolic pathways that enable cancer cells to survive and proliferate in the tumor microenvironment. We also discuss therapeutic modalities targeted at HCC prevention and regression. A full understanding of HCC-associated immunometabolic changes in obesity may contribute to clinical treatments that effectively target cancer metabolism.

Low-quality protein modulates inflammatory markers and the response to lipopolysaccharide insult: The case of lysine.

The relationship between non-communicable diseases (NCDs) and eating behaviour has long been attributed to a surplus of food and energy. However, the increase in the prevalence of NCDs and their underlying low-grade inflammatory milieu among people of low socioeconomic status (SES) has highlighted the existence of a confounding factor. In this work, we aim to study the effect of lysine deficiency on some inflammatory markers in the absence or presence of an inflammatory insult [lipopolysaccharide (LPS)]. For this purpose, thirty-two five-week-old male Sprague Dawley rats were randomly distributed into four groups: 1) control diet, 2) control diet+LPS, 3) lysine-deficient diet, and 4) lysine-deficient diet+LPS. Groups were only allowed their experimental diets for four weeks, during which LPS (50 µg/kg) or saline injections were administered intraperitoneally three times per week. The study showed that lysine deficiency blunted growth and body compartments development, decreased albumin production and elevated liver c-reactive protein (CRP) expression, independently of interleukins 6 and 1β, the main precursors of CRP. Also, the insufficient levels of lysine in the diet increased hyperactivity and triggered an anxiety-like behaviour, exacerbated with LPS. This work presents evidence that various physiological changes are associated with the absence of a sufficient amount of lysine in the diet and can potentially increase the risk factor for diseases. Thus, the increment in NCDs among the low SES populations, who heavily rely on cereals as a main source of protein, can be, at least partially, blamed on low lysine availability in diets.

Protocatechuic acid reverses myocardial infarction mediated by β-adrenergic agonist via regulation of Nrf2/HO-1 pathway, inflammatory, apoptotic, and fibrotic events.

Myocardial infarction (MI) is an instant ischemic death of cardiomyocytes that remains a major global cause of mortalities. MI is accompanied by oxidative, inflammatory, apoptotic, and fibrotic insults. Protocatechuic acid (PCA) is a polyphenolic compound with various potent biological activities. In this study, we explored the possible cardioprotective role of PCA against isoproterenol (ISO)-mediated MI. Rats were either injected with ISO (85 mg/kg, subcutaneously) or pretreated with PCA (100 or 200 mg/kg, orally). PCA supplementation markedly normalized ISO-induced disturbed cardiac function markers (creatine kinase-MB, lactate dehydrogenase, and troponin T). Notably, PCA administration exerted remarkable increases in glutathione and its derived enzymes, superoxide dismutase, and catalase, as well as decreases in malondialdehyde and nitric oxide levels in the injured cardiac tissue. The molecular findings validated the augmented cellular antioxidative capacity by PCA via increasing the gene expressions of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1. The cardioprotective efficacy of PCA extended to suppress cardiac inflammation as demonstrated by the decreased levels of tumor necrosis factor-alpha, interleukin-1 beta, and nuclear factor kappa B. Additionally, PCA prevented cardiomyocyte loss and fibrosis by decreasing Bax, caspase-3, transforming growth factor-β1 and matrix metalloproteinase-9, and enhancing B-cell lymphoma 2 and tissue inhibitors of metalloproteinase-3. The cardiac histological screening further confirmed the PCA's protective action. The obtained data recommend PCA as an alternative therapeutic agent to attenuate the molecular, biochemical, and histological alterations associated with MI development.

PS-BPB02-3: TARGETING FORMYL PEPTIDE RECEPTORS AS A NOVEL APPROACH TO TREAT HYPERTENSION-INDUCED ADVERSE CARDIOVASCULAR REMODELLING AND DYSFUNCTION

Introduction: Chronic unresolved inflammation has been shown to contribute to the development and progression of hypertension-induced cardiovascular damage. Failure to resolve inflammation exacerbates the risk and severity of cardiovascular disease. Currently, available therapies have limited efficacy in treating hypertension and its associated complications. Formyl peptide receptors (FPR) play a critical role in the regulation of resolution of inflammation. We have previously discovered that a FPR small-molecule prototype Cmpd17b exerts cardioprotective and vasoprotective effects against acute and severe inflammatory cardiac insults, but its impact on a chronic inflammatory insult in cardiovascular diseases, such as hypertension, has not been explored. Aim and hypothesis: To investigate the therapeutic potential of a FPR agonist, Cmpd17b on cardiac and vascular remodelling and function, and blood pressure regulation in hypertensive mice. Methods: Male C57BL/6 mice (12 weeks old) were implanted with a radiotelemetry probe to measure MAP (mean arterial pressure). Hypertensive mice (AngII infused at 0.7 mg/kg/day, subcutaneously) and normotensive mice (saline infused, subcutaneously) underwent 24-hour blood pressure recordings via telemetry at baseline and during 4 weeks daily treatment with Cmpd17b (50 mg/kg/day, intraperioneally) or its vehicle. Sympathetic nervous system (SNS) activity was assessed using power spectral analysis. At week 4, cardiac and vascular function was assessed using B-mode and M-mode echocardiography. At the end of the study, heart and arteries were collected, cardiac and vascular remodelling (fibrosis and hypertrophy) and function, and cutting-edge comparative qualitative proteomics were assessed. Results: Cmpd17b reduced MAP, SNS activity, cardiac and vascular adverse remodelling and dysfunction in hypertensive mice (Table). Cutting-edge quantitative proteomics analysis revealed that hypertension-induced significant adverse structural remodelling (110 proteins in the heart and 60 proteins in the aorta) and mitochondrial dysregulation (38 in the heart and 91 in the aorta), consisted with function and pathology observed. Those changes were prevented by FPR agonism (53 structural cardiac proteins and 38 vascular structural proteins) and (24 cardiac mitochondrial proteins and 36 vascular mitochondrial proteins). Conclusions: Our study demonstrated for the first time that FPR prototype small-molecule Cmpd17b reduced hypertension-induced cardiovascular damage, supporting the development of FPR-based therapy to treat cardiovascular complications in systemic hypertension.