Inflammatory Insult Research Articles

Anti-inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint.

In the two common inflammatory skin diseases, Atopic Dermatitis (AD) and Psoriasis (Ps), keratinocytes (KCs) respond to immune insults through activation of proinflammatory transcription factors (TFs) and their translocation to the cell's nucleus. Therein, the TFs induce expression of genes encoding mediators of skin inflammation. The Nuclear Transport Checkpoint Inhibitors (NTCIs) were developed to regulate nuclear translocation of activated TFs, the essential step of inflammatory response. This new class of cell-penetrating peptide therapeutics controls inflammation caused by allergic, autoimmune, metabolic, and microbial insults. In preclinical model of AD, the treatment with NTCI, cSN50.1 peptide, suppressed the expression of Thymic Stromal Lymphopoietin (TSLP), the key gene in the development of allergic inflammation, among the 15 genes silenced by the NTCI. Here, we report the mechanism of anti-inflammatory action of NTCI in human skin-derived KCs. We aimed to determine whether the NTCI treatment can protect human KCs from harmful inflammatory insults. Human primary KCs were pretreated with NTCI and challenged with the mix of cytokines Tumour Necrosis Factor alpha (TNF-α) and Interleukin (IL)-17A, or with Phorbol 12-Myristate 13-Acetate (PMA), and analysed for nuclear content of TFs and the expression of genes encoding mediators of inflammation. The nuclear import of TFs, Nuclear Factor ĸB (NF-ĸB) and Signal Transduction and Activator of Transcription 3 (STAT3), was inhibited in cells treated with NTCI. The expression of TSLP, along with genes encoding the core mediators of inflammation (TNF, IL1B, and IL6) was suppressed by NTCI. Noteworthy, NTCI silenced genes encoding Granulocyte-Macrophage Colony-Stimulating Factor (CSF2), and chemokine IL-8 (CXCL8), responsible for skin infiltration by the eosinophils and other myelomonocytic cells. The control of inflammatory response in human KCs by NTCI is attributed to the inhibition of nuclear import of proinflammatory TFs. The protection of human KCs by NTCI, adds new perspectives to the completed Phase two clinical trial of the NTCI (AMTX-100 CF) for AD (NCT04313400).

86 Evaluation of dietary Saccharomyces cerevisiae fermentation product on markers of joint inflammation in young, exercising horses following an intra-articular lipopolysaccharide challenge

Abstract Including Saccharomyces cerevisiae fermentation product (SCFP) into the diets of young, exercising horses may prolong their performance career by optimizing the intra-articular environment. Our objective was to evaluate the effect of dietary SCFP on joint inflammation in yearlings undergoing regular exercise, challenged with intra-articular lipopolysaccharide (LPS). Thirty Quarter Horse yearlings (374 ± 25 kg BW; 562 ± 16 d of age; 15 fillies and 15 geldings) were used in a 60-d study to test the hypothesis that dietary SCFP (TruEquine C, Diamond V Mills, Inc.) ameliorates joint inflammation following an acute inflammatory insult. Horses were stratified by BW, age, sex, and randomly assigned to dietary treatments (n = 10/treatment): Control (0), 46, or 92 mg/kg BW SCFP. Treatments were top-dressed onto a custom-formulated concentrate void of added microbials offered at 1% BW/d (as-fed) every 12 h. Horses were individually stalled (3.6 m × 7.3 m) and offered ad libitum Coastal bermudagrass hay. Using a free-stall exerciser, horses were exercised following a progressive workload regimen for 30 min/d, 5 d/wk. On d 46, all horses underwent an intra-articular LPS challenge where each horse had one radial carpal joint randomly assigned to receive either 0.8 mL of a 0.5 ng LPS solution or sterile lactated Ringer’s solution (LRS) as a contralateral control. Synovial fluid samples were obtained pre-injection (h 0) and at 6, 12, 24, and 336 h post-injection and analyzed for prostaglandin E2 (PGE2), a pro-inflammatory prostaglandin via commercial ELISA, and for chemokine-concentration (signaling proteins that direct immune cells; CCL2, and CCL11) and cytokines (inflammatory mediators; TNFα and IL-10) using a multiplex platform via commercial laboratory. Non-normal data (PGE2, IL-10, and CCL2) were log transformed, and all markers were analyzed using MIXED procedure of SAS v9.4. Mean separation was achieved with orthogonal contrasts, and contralateral control carpi were used as a covariate across all hours. There was no effect of SCFP on synovial logPGE2 (P = 0.42), logCCL2 (P = 0.90), CCL11 (P = 0.26), or logIL-10 (P = 0.23). However, there was a treatment × hour interaction for CCL11 (P = 0.04) where the Control had increased concentrations compared with SCFP treatment groups at 6 h post-injection. Furthermore, logIL-10 also had a treatment × hour interaction (P = 0.05) where the 46mg/kg BW group had decreased concentrations at h 12 compared with the Control and 92mg/kg BW group. The main effect of treatment for TNFα (P = 0.04) revealed that the 92mg/kg BW group had less concentrations than the 46mg/kg BW group and tended to have less concentrations than the Control group across all hours. Results indicate that SCFP may help mitigate inflammation markers following an acute intra-articular inflammatory insult.

A193 DECIPHERING THE INFLUENCE OF CROHN’S DISEASE-ASSOCIATED GENETIC RISK FACTOR, NOD2 IN INTESTINAL FIBRO-STENOSIS

Abstract Background Intestinal fibrosis (IF) is a common debilitating complication associated with Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD). IF results in severe strictures that affect the entire gastrointestinal tract, often requiring surgery due to limited or no effective treatment. In general, IF is a consequence of chronic inflammation, excessive extracellular matrix (ECM) deposition, aberrant cellular functions and tissue remodeling involving the innate and adaptive immune system. At the cellular level, fibrosis has been proposed to involve the crosstalk between epithelial cells, mesenchymal stromal cells and immune cells. However, the cascade of events that establish IF is poorly understood as it comprises a dynamic interplay between host genetics, immunity, gut microbiome and aberrant environment insults. Notably, polymorphisms in nucleotide-binding oligomerization domain-containing protein 2 (NOD2; a bonafide cytosolic innate immune receptor), strong genetic risk factors for CD, also increase the incidence of IF. In this context, how NOD2 polymorphisms govern intestinal fibrostenosis in CD remains to be investigated. Aims To investigate how CD-associated risk gene, NOD2 contributes to intestinal fibrostenotic disease Methods We employed a single-cell RNA sequencing (scRNA-seq) approach to explore the cellular landscape in our CD-mouse model, where we challenged Nod2-deficient mice with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, changes in inflammatory, metabolic and fibrotic markers in the gut were analyzed by quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) or western blot analyses. Further, RNA-In-situ hybridization (RNA-ISH) and flow cytometric analyses were performed to validate cell populations contributing to, and the molecular signatures linked with intestinal fibrostenosis. Results We found that Nod2-deficient mice show increased expression of pro-inflammatory cytokines (TNF, IL6) and excessive ECM deposition (fibrosis) in the gut compared to wild-type littermate mice post-cDSS. Curiously, scRNA-seq analysis uncovered altered stromal-immune-cell crosstalk along with the identification of a novel subset of the Dpt+ interstitial stromal population with distinct gene signatures regulated by chronic inflammation. Further, we observed increased senescence (Cdkn2a) mediated trans-differentiation of stroma into fibroblast contributing to IF in Nod2-deficient mice. Conclusions The study outlined will deepen our understanding of cellular and molecular mediators as well as inter and intra-cellular networks that drive IF. This will be transformative in devising therapeutic strategies to prevent IF in progressive CD patients. Funding Agencies CCC, CIHRThe American Association of Immunologist (AAI)

Implications of Inflammatory Processes on a Developing Central Nervous System in Childhood-Onset Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is increasingly affecting pediatric and adult populations. Neuropsychiatric manifestations (ie, cognitive dysfunction and mood disorders) appear to occur with greater severity and poorer prognosis in childhood-onset SLE (cSLE) versus adult-onset SLE, negatively impacting school function, self-management, and psychosocial health, as well as lifelong health-related quality of life. In this review, we describe pathogenic mechanisms active in cSLE, such as maladaptive inflammatory processes and ischemia, which are hypothesized to underpin central phenotypes in patients with cSLE, and the role of alterations in protective central nervous system (CNS) barriers (ie, the blood-brain barrier) are also discussed. Recent findings derived from novel neuroimaging approaches are highlighted because the methods employed in these studies hold potential for identifying CNS abnormalities that would otherwise remain undetected with conventional multiple resonance imaging studies (eg, T2-weighted or fluid-attenuated inversion recovery sequences). Furthermore, we propose that a more robust presentation of neuropsychiatric symptoms in cSLE is in part due to the harmful impact of a chronic inflammatory insult on a developing CNS. Although the immature status of the CNS may leave patients with cSLE more vulnerable to harboring neuropsychiatric manifestations, the same property may represent a greater urgency to reverse the maladaptive effects associated with a proneuroinflammatory state, provided that effective diagnostic tools and treatment strategies are available. Finally, considering the crosstalk among the CNS and other organ systems affected in cSLE, we postulate that a finer understanding of this interconnectivity and its role in the clinical presentation in cSLE is warranted.

Psychiatric Comorbidities of Inflammatory Bowel Disease: It Is a Matter of Microglia's Gut Feeling.

Inflammatory bowel disease (IBD), a common term for Crohn's disease and ulcerative colitis, is a chronic, relapse-remitting condition of the gastrointestinal tract that is increasing worldwide. Psychiatric comorbidities, including depression and anxiety, are more prevalent in IBD patients than in healthy individuals. Evidence suggests that varying levels of neuroinflammation might underlie these states in IBD patients. Within this context, microglia are the crucial non-neural cells in the brain responsible for innate immune responses following inflammatory insults. Alterations in microglia's functions, such as secretory profile, phagocytic activity, and synaptic pruning, might play significant roles in mediating psychiatric manifestations of IBD. In this review, we discuss the role played by microglia in IBD-associated comorbidities.

Innate immunity in peripheral tissues is differentially impaired under normal and endotoxic conditions in aging.

Chronic low-grade inflammation is a hallmark of aging, aka "inflammaging", which is linked to a wide range of age-associated diseases. Immune dysfunction increases disease susceptibility, and increases morbidity and mortality of aging. Innate immune cells, including monocytes, macrophages and neutrophils, are the first responders of host defense and the key mediators of various metabolic and inflammatory insults. Currently, the understanding of innate immune programming in aging is largely fragmented. Here we investigated the phenotypic and functional properties of innate immune cells in various peripheral tissues of young and aged mice under normal and endotoxic conditions. Under the steady state, aged mice showed elevated pro-inflammatory monocytes/macrophages in peripheral blood, adipose tissue, liver, and colon. Under lipopolysaccharide (LPS)-induced inflammatory state, the innate immune cells of aged mice showed a different response to LPS stimulus than that of young mice. LPS-induced immune responses displayed differential profiles in different tissues and cell types. In the peripheral blood, when responding to LPS, the aged mice showed higher neutrophils, but lower pro-inflammatory monocytes than that in young mice. In the peritoneal fluid, while young mice exhibited significantly elevated pro-inflammatory neutrophils and macrophages in response to LPS, aged mice exhibited decreased pro-inflammatory neutrophils and variable cytokine responses in macrophages. In the adipose tissue, LPS induced less infiltrated neutrophils but more infiltrated macrophages in old mice than young mice. In the liver, aged mice showed a more robust increase of pro-inflammatory macrophages compared to that in young mice under LPS stimulation. In colon, macrophages showed relatively mild response to LPS in both young and old mice. We have further tested bone-marrow derived macrophages (BMDM) from young and aged mice, we found that BMDM from aged mice have impaired polarization, displaying higher expression of pro-inflammatory markers than those from young mice. These data collectively suggest that innate immunity in peripheral tissues is impaired in aging, and the dysregulation of immunity is tissue- and cell-dependent. Our findings in the rodent model underscore the complexity of aging immunity. Further investigation is needed to determine whether the immune profile observed in aged mice is applicable in age-associated diseases in humans.

Acute bilateral toxic endothelitis in binge alcohol consumption – A case series

Endothelitis has a vast range of etiologies, including infective, chemical-induced, drug-induced, systemic, and toxic causes. Toxic endothelitis secondary to alcohol binge consumption is rare and is reported less frequently in the literature. We report a series of three cases with bilateral defective vision due to corneal edema and endothelitis following binge alcohol consumption. All cases showed dramatic improvement with frequent topical steroid application alone. We hypothesize the cause for corneal edema as transient suppression of corneal endothelial cell metabolism rather than apoptosis, with later regain of functions following alcohol cessation and control of further inflammatory insults with intensive steroids.

TNF Promoter Hypomethylation Is Associated With Mucosal Inflammation in IBD and Anti-TNF Response

Background and aimsInflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. MethodsWe obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. ResultsTNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. ConclusionsOur study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

Changes in Central Corneal Thickness and Central Macular Thickness following Uncomplicated Small-incision Cataract Surgery.

Cataract surgery is an invasive procedure that causes mechanical and inflammatory insult to the eye. The objective was to study the changes in central corneal thickness (CCT) which is an indirect indicator of corneal endothelial dysfunction and the changes in macular thickness following uncomplicated small incision cataract surgery (SICS) Materials and methods: This was a prospective observational study with a before-and-after design conducted in Reiyukai Eiko Masunaga eye hospital, Banepa, Kavrepalanchok, Nepal. SICS was performed on 68 eyes of 62 patients. Change in CCT and central macular thickness (CMT) from baseline was observed post-surgery on the first day, one week, and six weeks. There were 27 (43.5%) females and 35 males (56.5%) in the study. Mean age was 58.26 ±10 years. This difference of visual acuity between pre and post-operative state was statistically significant. The first post-operative day (POD) and first week post-operative CCT values when compared with preoperative CCT values were statistically significant. However, post-operative CCT values at six weeks were similar to preoperative values. Also, the differences at day one, first week and six weeks post-operative CMT values when compared with preoperative CMT values were statistically significant. This study revealed that there is a significant rise in CCT after SICS which gradually tends to normalize at six weeks. Similarly there is a gradual rise in CMT after SICS persisting even at six weeks. These changes were subtle and there was a marked improvement of visual acuity at six weeks after SICS.

Endogenous GDNF Is Unable to Halt Dopaminergic Injury Triggered by Microglial Activation.

Overactivation of microglial cells seems to play a crucial role in the degeneration of dopaminergic neurons occurring in Parkinson's disease. We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) present in astrocytes secretome modulates microglial responses induced by an inflammatory insult. Therefore, astrocyte-derived soluble factors may include relevant molecular players of therapeutic interest in the control of excessive neuroinflammatory responses. However, in vivo, the control of neuroinflammation is more complex as it depends on the interaction between different types of cells other than microglia and astrocytes. Whether neurons may interfere in the astrocyte-microglia crosstalk, affecting the control of microglial reactivity exerted by astrocytes, is unclear. Therefore, the present work aimed to disclose if the control of microglial responses mediated by astrocyte-derived factors, including GDNF, could be affected by the crosstalk with neurons, impacting GDNF's ability to protect dopaminergic neurons exposed to a pro-inflammatory environment. Also, we aimed to disclose if the protection of dopaminergic neurons by GDNF involves the modulation of microglial cells. Our results show that the neuroprotective effect of GDNF was mediated, at least in part, by a direct action on microglial cells through the GDNF family receptor α-1. However, this protective effect seems to be impaired by other mediators released in response to the neuron-astrocyte crosstalk since neuron-astrocyte secretome, in contrast to astrocytes secretome, was unable to protect dopaminergic neurons from the injury triggered by lipopolysaccharide-activated microglia. Supplementation with exogenous GDNF was needed to afford protection of dopaminergic neurons exposed to the inflammatory environment. In conclusion, our results revealed that dopaminergic protective effects promoted by GDNF involve the control of microglial reactivity. However, endogenous GDNF is insufficient to confer dopaminergic neuron protection against an inflammatory insult. This reinforces the importance of further developing new therapeutic strategies aiming at providing GDNF or enhancing its expression in the brain regions affected by Parkinson's disease.

Impact of continuous testosterone exposure on reproductive physiology, activity, and pain-related behavior in young adult female rats

Testosterone may reduce pain in cisgender women and transgender men. Rodents can provide a useful model for investigating physiological effects of hormone therapy. To this end, continuous-release testosterone or blank (placebo) capsules were implanted s.c. into young adult female rats, and three weeks later rats were either ovariectomized or sham-ovariectomized. Testosterone treatment that mimicked previously reported endogenous levels in males eliminated estrous cycling and decreased uterine weight. Testosterone also significantly increased body weight and suppressed the increases in daily wheel running observed in placebo controls over time. Subsequent ovariectomy or sham-ovariectomy decreased wheel running in all groups, but testosterone-treated rats recovered significantly more quickly than did placebo-treated rats. Neither testosterone nor ovariectomy significantly altered hindpaw mechanical threshold. Two weeks after sham/ovariectomy surgery, injection of Complete Freund Adjuvant (CFA) into one hindpaw reduced wheel running and mechanical threshold in all groups; running significantly decreased from the first to second day after CFA in testosterone- but not in placebo-treated rats. Morphine 1.0 but not 3.2 mg/kg increased CFA-suppressed wheel running similarly in all groups, whereas both doses of morphine increased CFA-suppressed mechanical threshold. These data suggest that weeks-long testosterone treatment with or without ovariectomy may provide a useful physiological model of testosterone therapy as used in human gender transition. Although testosterone administered at levels similar to those in gonadally intact males tended to hasten female rats' recovery from surgery, it did not decrease maximal pain-related behaviors after surgery or hindpaw inflammatory insult, nor did it alter opioid antinociception.

Effect of resistance exercise on secondary inflammatory insult in Alzheimer’s disease models

AbstractBackgroundPhysical exercise improves cognition in human and animal studies associated with Alzheimer’s Disease (AD). However, resistance‐type regimes remain poorly investigated. Acute illness or injury can influence the rate AD progression and trigger accelerated cognitive decline.MethodTo evaluate the role of RE in modulating pre‐existing cognitive impairment, female 3xTg and 5xFAD mice underwent a five‐week RE training protocol. An acute systemic inflammatory insult: laparotomy or intraperitoneally‐injected LPS, was performed to investigate the effect of RE under these challenges. Behavior tests was used to probe for cognition and learning. Brain homogenates were harvested for western blot analysis, qPCR, or post‐fixed in neutral‐buffered formalin solution for immuno‐histochemical exploration.ResultRE showed improved cognitive function, reduced P‐S396‐tau, decreased ionized calcium‐binding adapter molecule 1 (Iba‐1), and decreased inflammatory cytokine transcripts compared to sedentary controls. RE significantly reduced anxiety‐like behavior and cognitive deficit stimulated by acute systemic inflammation episode.ConclusionRE attenuates exaggerated cellular responses and improves cognitive functions in AD and accelerated cognitive impairment triggered by secondary inflammation insult.

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

BackgroundIschemic stroke (IS) occurs when a blood vessel supplying the brain becomes obstructed, resulting in cerebral ischemia. This type of stroke accounts for approximately 87% of all strokes. Globally, IS leads to high mortality and poor prognosis and is associated with neuroinflammation and neuronal apoptosis. D-allose is a bio-substrate of glucose that is widely expressed in many plants. Our previous study showed that D-allose exerted neuroprotective effects against acute cerebral ischemic/reperfusion (I/R) injury by reducing neuroinflammation. Here, we aimed to clarify the beneficial effects D-allose in suppressing IS-induced neuroinflammation damage, cytotoxicity, neuronal apoptosis and neurological deficits and the underlying mechanism in vitro and in vivo.MethodsIn vivo, an I/R model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 N mice, and D-allose was given by intraperitoneal injection within 5 min after reperfusion. In vitro, mouse hippocampal neuronal cells (HT-22) with oxygen–glucose deprivation and reperfusion (OGD/R) were established as a cell model of IS. Neurological scores, some cytokines, cytotoxicity and apoptosis in the brain and cell lines were measured. Moreover, Gal-3 short hairpin RNAs, lentiviruses and adeno-associated viruses were used to modulate Gal-3 expression in neurons in vitro and in vivo to reveal the molecular mechanism.ResultsD-allose alleviated cytotoxicity, including cell viability, LDH release and apoptosis, in HT-22 cells after OGD/R, which also alleviated brain injury, as indicated by lesion volume, brain edema, neuronal apoptosis, and neurological functional deficits, in a mouse model of I/R. Moreover, D-allose decreased the release of inflammatory factors, such as IL-1β, IL-6 and TNF-α. Furthermore, the expression of Gal-3 was increased by I/R in wild-type mice and HT-22 cells, and this factor further bound to TLR4, as confirmed by three-dimensional structure prediction and Co-IP. Silencing the Gal-3 gene with shRNAs decreased the activation of TLR4 signaling and alleviated IS-induced neuroinflammation, apoptosis and brain injury. Importantly, the loss of Gal-3 enhanced the D-allose-mediated protection against I/R-induced HT-22 cell injury, inflammatory insults and apoptosis, whereas activation of TLR4 by the selective agonist LPS increased the degree of neuronal injury and abolished the protective effects of D-allose.ConclusionsIn summary, D-allose plays a crucial role in inhibiting inflammation after IS by suppressing Gal-3/TLR4/PI3K/AKT signaling pathway in vitro and in vivo.

TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization.

Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.

In Vivo CRISPR Screening Platform for Studying Myeloid Inflammation and Clonal Dynamics

Previous experimental approaches using CRISPR to study factors governing hematopoiesis in vivo have been hampered by heterogeneity in cellular expansion during hematopoietic reconstitution. To control for such ‘cellular jackpotting’, we developed an in vivo hematopoietic stem and progenitor (HSPC)-based CRISPR screening system with unique molecular identifiers (UMIs) and applied these techniques to study myeloid inflammation and clonal expansion of Tet2-mutated HSPCs. Transplantation of Cas9-expressing Lin -Sca1 +c-Kit + (LSK) cells transduced with high-diversity UMI-labeled guide-RNAs (gRNAs) enabled sufficient gRNA representation at a genome-wide level after lentiviral integration of one gRNA per cell and controlled for 10 log-fold differences in cellular expansion during hematopoietic reconstitution. Simultaneous comparison of barcoded CRISPR screening with non-barcoded screening revealed that barcoding decreases false-positive biological processes related to cell division while improving sensitivity for detecting biological processes related to transcriptional events and cellular differentiation. To study inflammatory mediators, we applied this CRISPR screening tool towards studying the recruitment of CD101 +Ly6G +CD11b + mature neutrophils during an inflammatory insult, which revealed the majority of genes involved in human congenital neutropenia (Figure A). To demonstrate the reproducibility of this screening system, we performed a targeted transcription factor (TF)-based CRISPR screen, which revealed concordance between the genome-wide screen and the targeted screen. Next, we also applied this screening system to studying dynamics of Tet2-mutated clonal hematopoiesis. To study master regulators of clonal expansion in Tet2 KO, we performed a UMI-based CRISPR screen using chromatic and transcription factors, which revealed Tet3 and Bcor, genes previously identified to cooperate with Tet2 KO to promote myeloproliferation (Figure B). Validation of additional novel genetic hits is currently ongoing. We hypothesize that this screening platform will be useful for dissecting many aspects of hematopoiesis including inflammatory mediators and clonal dynamics of HSPCs.

Inflammatory Memory Restrains Intestinal Stem Cell (ISC) Regeneration after Allogeneic Stem Cell Transplantation (SCT)

Introduction: Intestinal stem cells (ISCs) are responsible for the remarkable ability to maintain intestinal epithelium homeostasis and regeneration throughout life. Inflammatory damage of ISCs underpins injury caused by graft-versus-host disease (GVHD), inflammatory bowel diseases (IBD) and immune check-point blocker mediated colitis. However, it remains unknown whether the ISCs that survive or tolerate inflammation are fully functional and can return to their full functionality after the resolution of ongoing inflammatory insults. Herein we investigated the consequences of inflammation from GVHD on Lgr5+ISCs in multiple well-defined clinically relevant models of gastro-intestinal acute graft-versus-host disease (GI GVHD). Methods: We utilized single cell RNA (scRNA) sequencing to assess transcriptomics, Assay of transposase-accessible chromatin sequencing (ATAC-seq) to assess epigenomics, and functional metabolomics of ISCs in clinically relevant in vivo models of GVHD after major histocompatibility complex (MHC)-disparate BALB/c→C57BL/6 (B6) and MHC matched minor mismatched C3H.sw→B6 models of allogeneic SCT. Ex vivo intestinal organoids cultures, mitochondrial, and functional biochemical assays were utilized to determine the biological relevance of the changes observed from the ‘omic’ analyses and further validated them in vivo by developing novel Lgr5 +ISC specific succinate dehydrogenase A (SDHA) knock-out mice. Results: We examined the transcriptomes of Lgr5+ISCs with scRNA-seq of the intestinal crypts harvested from MHC-disparate BALB/c→B6 recipients on day +7 after allogeneic SCT. Bioinformatic analyses demonstrated upregulation of interferon and inflammation response genes but significant downregulation of genes involved in mitochondrial function, its complexes including complex II (SDH), ATP metabolic process, OXPHOS, and cytoplasmic translation. To assess the impact of metabolic functional gene changes, we harvested and assessed ISCs in ex vivo organoid cultures from transplanted recipients in the absence of ongoing inflammation. The ISCs harvested from GVHD animals demonstrated significantly reduced regeneration, differentiation and oxygen consumption rates (OCRs) with no change in extracellular acidification rates (ECAR) by Seahorse. FACs analysis confirmed reduction in SDHA component of mitochondrial complex II in Lgr5 +ISCs, demonstrating mechanistic cause for reduction in OXPHOS in ISCs. We next generated and utilized Lgr5 + ISC-specific SDHA KO mice as GVHD recipients and found that demonstrated significantly greater mortality when compared to the WT littermate recipients (P<0.01). Biochemical analyses demonstrated increased levels of succinate, a metabolic intermediary with known epigenetic and inflammatory functions, in ISCs harvested from GVHD animals. We therefore hypothesized that the reduction in ability to form functional organoids ex vivo, in the absence of ongoing inflammation, by the ISCs harvested from GVHD hosts is because of the inflammation epigenetic reprogramming induced by succinate mediated changes in DNA methylation (5-mC) of the ISCs in GVHD recipients. Consistent with the hypothesis greater DNA methylation, with alteration of epigenome by ATAC-seq was observed in ISCs sorted from GVHD recipients. Integrative analyses demonstrated correlation between epigenomic changes and transcriptomic changes. Finally, we analyzed whether the inflammation epigenomic induced changes in the GVHD ISCs with serial ex vivo organoid cultures and in vivo by transfer into secondary hosts. Both ex vivo and in vivo studies demonstrated poor regeneration from GVHD ISCs demonstrating retention of maladaptive memory in ISCs following their exposure to inflammation during GVHD. Conclusions: GI GVHD induced inflammation causes not only quantitative loss of ISCs but also induced qualitative changes in the surviving ISCs. Inflammation induced OXPHOS deficiency in Lgr5 + ISCs leads to accumulation of succinate that reprograms the epigenome and restrains their subsequent regeneration potential.

The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

Gastroprotective Effects of Oral Glycosaminoglycans with Sodium Alginate in an Indomethacin-Induced Gastric Injury Model in Rats.

The gastrointestinal (GI) mucosal barrier is often exposed to inflammatory and erosive insults, resulting in gastric lesions. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), chondroitin sulfate (CS), and N-acetylglucosamine (NAG) have shown potential beneficial effects as GI protectants. This study aimed to evaluate the gastroprotective effects of oral GAGs in rats with indomethacin-induced GI lesions. Forty-five Sprague-Dawley rats (8-9 weeks-old, 228 ± 7 g) were included in the study, divided into five study groups, and given, administered orally, either sucralfate (positive control group; PC), NAG (G group), sodium alginate plus HA and CS (AHC group), sodium alginate plus HA, CS, and NAG (AHCG group), or no treatment (negative control group; NC). Animals were administered 12.5 mg/kg indomethacin orally 15 min after receiving the assigned treatment. After 4 h, stomach samples were obtained and used to perform a macroscopic evaluation of gastric lesions and to allow histological assessment of the gastric wall (via H/E staining) and mucous (via PAS staining). The AHCG group showed significant gastroprotective improvements compared to the NC group, and a similar efficacy to the PC group. This combination of sodium alginate with GAGs might, therefore, become a safe and effective alternative to prescription drugs for gastric lesions, such as sucralfate, and have potential usefulness in companion animals.

Naïve Huntington’s disease microglia mount a normal response to inflammatory stimuli but display a partially impaired development of innate immune tolerance that can be counteracted by ganglioside GM1

Chronic activation and dysfunction of microglia have been implicated in the pathogenesis and progression of many neurodegenerative disorders, including Huntington’s disease (HD). HD is a genetic condition caused by a mutation that affects the folding and function of huntingtin (HTT). Signs of microglia activation have been observed in HD patients even before the onset of symptoms. It is unclear, however, whether pro-inflammatory microglia activation in HD results from cell-autonomous expression of mutant HTT, is the response of microglia to a diseased brain environment, or both. In this study, we used primary microglia isolated from HD knock-in (Q140) and wild-type (Q7) mice to investigate their response to inflammatory conditions in vitro in the absence of confounding effects arising from brain pathology. We show that naïve Q140 microglia do not undergo spontaneous pro-inflammatory activation and respond to inflammatory triggers, including stimulation of TLR4 and TLR2 and exposure to necrotic cells, with similar kinetics of pro-inflammatory gene expression as wild-type microglia. Upon termination of the inflammatory insult, the transcription of pro-inflammatory cytokines is tapered off in Q140 and wild-type microglia with similar kinetics. However, the ability of Q140 microglia to develop tolerance in response to repeated inflammatory stimulations is partially impaired in vitro and in vivo, potentially contributing to the establishment of chronic neuroinflammation in HD. We further show that ganglioside GM1, a glycosphingolipid with anti-inflammatory effects on wild-type microglia, not only decreases the production of pro-inflammatory cytokines and nitric oxide in activated Q140 microglia, but also dramatically dampen microglia response to re-stimulation with LPS in an experimental model of tolerance. These effects are independent from the expression of interleukin 1 receptor associated kinase 3 (Irak-3), a strong modulator of LPS signaling involved in the development of innate immune tolerance and previously shown to be upregulated by immune cell treatment with gangliosides. Altogether, our data suggest that external triggers are required for HD microglia activation, but a cell-autonomous dysfunction that affects the ability of HD microglia to acquire tolerance might contribute to the establishment of neuroinflammation in HD. Administration of GM1 might be beneficial to attenuate chronic microglia activation and neuroinflammation.

Transcriptome analysis of the cerebral cortex of acrylamide-exposed wild-type and IL-1β-knockout mice.

Acrylamide is an environmental electrophile that has been produced in large amounts for many years. There is concern about the adverse health effects of acrylamide exposure due to its widespread industrial use and also presence in commonly consumed foods and others. IL-1β is a key cytokine that protects the brain from inflammatory insults, but its role in acrylamide-induced neurotoxicity remains unknown. We reported recently that deletion of IL-1β gene exacerbates ACR-induced neurotoxicity in mice. The aim of this study was to identify genes or signaling pathway(s) involved in enhancement of ACR-induced neurotoxicity by IL-1β gene deletion or ACR-induced neurotoxicity to generate a hypothesis mechanism explaining ACR-induced neurotoxicity. C57BL/6J wild-type and IL-1β KO mice were exposed to ACR at 0, 12.5, 25mg/kg by oral gavage for 7days/week for 4weeks, followed by extraction of mRNA from mice cerebral cortex for RNA sequence analysis. IL-1β deletion altered the expression of genes involved in extracellular region, including upregulation of PFN1 gene related to amyotrophic lateral sclerosis and increased the expression of the opposite strand of IL-1β. Acrylamide exposure enhanced mitochondria oxidative phosphorylation, synapse and ribosome pathways, and activated various pathways of different neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, Huntington disease, and prion disease. Protein network analysis suggested the involvement of different proteins in related to learning and cognitive function, such as Egr1, Egr2, Fos, Nr4a1, and Btg2. Our results identified possible pathways involved in IL-1β deletion-potentiated and ACR-induced neurotoxicity in mice.