Primary Cells Research Articles

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells.

Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10-20%) and nuclear transcription (80-90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.

NANOPARTICLE ENCAPSULATION OF THE SPECIALIZED PRO-RESOLVING MEDIATOR MARESIN-2: A NOVEL APPROACH FOR PROMOTING MUCOSAL REPAIR IN THE INTESTINE

Abstract Epithelial cells form protective barriers at mucosal surfaces and play a critical role in maintaining tissue homeostasis. Active inflammation in inflammatory bowel disease (IBD) is associated with epithelial barrier disruption and mucosal erosions/wounds that contribute to the inflammatory response and disease symptoms. Therefore, efficient repair of epithelial erosions/wounds is crucial for restoring intestinal barrier function and resolving mucosal inflammation. In response to injury, epithelial cells proliferate and migrate to cover denuded surfaces and restore critical barrier properties. Resident and recruited immune cells at sites of injury release factors that play an important role in orchestrating mucosal repair. Bioactive lipid mediators released by leukocytes, including leukotrienes and specialized pro-resolving mediators (SPMs), regulate epithelial signaling pathways to influence mucosal repair processes. Liquid chromatography-mass spectrometry (LCMS) analyses of healing colonic mucosal wounds performed to detect SPMs identified high levels of MaR2 in healing colonic wounds. In vitro studies using primary colonic epithelial cells revealed MaR2-driven increases in epithelial wound repair that were potentiated by the pro-inflammatory cytokines TNFα and IFNγ. While MaR2 increased epithelial migration by activating cell matrix adhesion proteins, including Src-paxillin and focal adhesion kinase, we did not observe any influence of MaR2 on the activation of cyclin D1/D2 or on epithelial proliferation. Importantly, in vivo studies demonstrated enhanced colonic mucosal wound repair and recovery from dextran sulfate-induced colitis in mice administered MaR2. Given the thermolabile nature of MaR2 (and associated ultracold storage requirements), thermostable polylactic acid (PLA) nanoparticles (NP) containing MaR2 (PLA MaR2) were generated. PLA MaR2 nanoparticles retained bioactivity following extended storage at room temperature and exhibited potent effects on in vivo colonic biopsy induced wound repair. Taken together these data reveal that thermostable MaR2 containing nanoparticles represent a promising new therapeutic approach for regenerating epithelial barrier function and restoring mucosal homeostasis in individuals with IBD.

INFLAMMATION-TRIGGERED CLAUDIN-23 DOWNREGULATION IMPACTS INTESTINAL EPITHELIAL BARRIER FUNCTION

Abstract Intestinal epithelium maintains gut homeostasis by protecting the underlying tissue compartments from harmful contents. Although it is suggested that epithelial barrier function compromise contributes to the pathogenesis of inflammatory bowel disease (IBD), the underlying mechanisms are not well understood. Claudins (CLDN) are tight junctions (TJ) proteins that determine epithelial barrier properties during homeostasis and whose expression is altered in IBD, thereby contributing to a leaky barrier. We recently identified the expression of an atypical CLDN family member, CLDN23, in the regulation of homeostatic intestinal epithelial barrier function. Analysis of the human colonic crypt-luminal axis revealed increased CLDN23 expression in differentiated intestinal epithelial cells (IEC) facing the lumen compared to the proliferative crypt-base IECs. Complementary cell biologic approaches using IEC-specific Cldn23 knockout mice (Cldn23ERΔIEC) and human colon cell lines revealed that CLDN23 strengthens epithelial barrier function. Mechanistically, CLDN23 promoted redistribution of barrier-forming CLDN3 and CLDN4 from the lateral membrane to the TJ through cis- and trans-interactions, resulting in narrowed CLDN3 and CLDN4 pore diameters. Since inflammation compromises the epithelial barrier and contributes to disease pathogenesis in IBD, we first examined CLDN23 expression in colonic mucosal tissue from IBD individuals. Immunofluorescence and RNA scope labeling revealed that CLDN23 expression was significantly decreased in colonic epithelial cells of IBD individuals. Furthermore, reduced CLDN23 expression was also detected in primary colonic epithelial cells (colonoids) from mice treated with pro-inflammatory cytokines TNFα and IFNγ compared to untreated colonoids. Barrier function assays of control Cldn23f/f and Cldn23ERΔIEC mice-derived colonoids treated with TNFα and IFNγ identified decreased transepithelial electrical resistance in colonoids lacking CLDN23 and treated with these pro-inflammatory cytokines. These findings suggest a protective role for CLDN23 in regulating barrier function under homeostatic and inflammatory conditions.

THE DEVELOPMENT OF HUMAN PRIMARY CULTURE MODELS TO MODEL INFLAMMATORY SIGNALING IN CROHN’S DISEASE AND ULCERATIVE COLITIS

Abstract Intestinal inflammation is a major pathological feature associated with gastrointestinal disorders such as Crohn’s disease and ulcerative colitis (UC). To unravel the complex mechanisms underlying these conditions and to develop effective therapeutic strategies, it is crucial to recapitulate intestinal inflammatory signaling using accurate and biologically relevant experimental systems. To establish primary cell-based models that reprise the inflammatory signaling in Crohn’s and UC, we developed the RepliGut platform. Briefly, we established a biobank of primary intestinal stem cells, derived from healthy full-length intestines from a range of donors of different sexes, ages, and ethnicities. From each donor, intestinal epithelial stem cells were isolated from each region of the small intestine and colon (duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon). The intestinal stem cells were passed to generate large and quality-controlled cell lots of “ready-to-use” vials which are cryopreserved and can be thawed directly onto 12- or 96-well Transwell inserts, generating the RepliGut platform. The RepliGut culture conditions allow for investigation into the physiology of intestinal epithelial cells as either proliferative stem or differentiated cell types and enable studies on focused regional differences in the intestinal tract, while the Transwell platform allows apical and basal media access that facilitates barrier function measures and convenient bi-lateral media sampling. Using this system, Crohn’s and UC-like inflammation can be produced via treatment with the most clinically and therapeutically validated cytokines associated with disease pathophysiology, TNF-α and IFN-γ. Circulating concentrations of both TNF-α and IFN-γ are observed to be elevated in patients with inflammatory bowel diseases and therapeutic antagonists including adalimumab (TNF-α) and JAK inhibitors such as tofacitinib (IFN-γ) represent the current standard of care. On the RepliGut platform, both TNF-α and IFN-γ produced a dose-dependent loss in epithelial barrier function, increased cytokine secretion, and caused cellular death, all of which were mitigated by adalimumab and tofacitinib, respectively. Moreover, at clinically relevant concentrations, combinations of TNF-α and IFN-γ produced synergistic inflammatory response on the intestinal epithelial cultures. In summary, the RepliGut platform provides a convenient and high-throughput platform that reproduces cytokine-induced intestinal inflammation for mechanistic studies and drug screening.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration

Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

P146 Latent activation of the extrinsic coagulation pathway in patients with ulcerative colitis

Abstract Background Ulcerative colitis (UC) is associated with an increased risk of venous thromboembolism. We investigated the correlation of UC severity with latent activation of the extrinsic coagulation pathway and colonic stromal tissue factor (TF) expression. Methods Plasma exosomes, microparticles (EM), TF protein, activities of EM-bound TF (EM-TF) and FVIIa, as well as TF mRNA (F3) in primary colonic stromal cells in culture were measured. Disease extent by Montreal classification, medications, UC clinical activity by partial Mayo score (PMS), endoscopic severity by the MS endoscopic component (eMS; loss of response defines as ≥2), quality of life by the short IBD questionnaire- SIBDQ, complete blood count, CRP, ESR and albumin were retrieved. Statistical significance (p) was assessed with the following tests: Student’s t for 2 groups with normally distributed values, Mann-Whitney U if not, Wilcoxon test for paired data. Correlation of continuous against scale variables was assessed with Pearson’s or Spearman’s r and corresponding p values for linear or logarithmic continuous variables, respectively. Results Plasma was obtained from 38 UC patients (31 males, disease duration 151±25 months, 14 with extensive colitis, 5 current smokers) and 28 healthy blood donors (HBD; 20 males) who served as controls. TF mRNA (F3) was measured in primary colonic stromal cells in culture from 12 UC patients (11 males, disease duration 169±53 months, 8 with extensive colitis, 1 current smoker) and from 7 controls undergoing screening colonoscopy. UC patients had 4- and 3.7- times more exosomes and microparticles than controls, respectively (A). TF protein correlated with PMS (r 0.443), albumin (-0.362), ESR (0.353), PLT (0.575) and endoscopic UC severity (0.468; B). EM-TF activity was 1.6-times higher (C) and correlated to disease extent (E3: 1.9 x E2), albumin (-0.624), endoscopic severity (0.422; D) and SIBDQ (-0.64). Downstream, FVIIa activity was 2.2-times higher. Refractory-to-treatment patients had 5.8-, 1.5-, 2.1- times higher TF protein (E), MP-TF, FVIIa activity, respectively. Within responders, the need for steroids or biologics correlated with 2.2-times higher MP-TF activity. Colonic stromal cells from patients with UC maintained a 2.2-times higher F3 (F), which correlated to PMS (0.56), albumin (Spearman’s r -0.543), endoscopic severity (M3: 8.3 x M2). All the aforementioned were statistically significant with a < 0.05. Conclusion The extent of spontaneous activation of the extrinsic coagulation pathway is associated with clinical and endoscopic UC activity and response to treatment. TF in colonic stromal cells mirrors its systemic activity and indicates the possible contribution of such cells to increased TF plasma bioactivity.

OP22 Topical Sphingosine-1-Phosphate (S1P) Receptor 1 Modulation Regulates Gut Angiogenesis in Inflammatory Bowel Diseases

Abstract Background S1P receptor (S1PR)1/5 modulators have been tested successfully in clinical development programs in ulcerative colitis (UC), leading to their FDA approval. They are believed to act through lymphocyte redistribution in peripheral lymphoid tissue and reduced numbers of circulating lymphocytes, but evidence from other organs suggests potential additional mechanisms of action. Methods A post-hoc analysis of the phase 3 trial of the S1PR1/5 modulator Ozanimod in patients with moderately to severely active UC was performed assessing an association of absolute lymphocyte count (ALC) with efficacy. Expression levels of S1PRs were analyzed in human intestinal tissues and primary cells using qPCR and/or immunohistochemistry (IHC). Effects of S1P, S1PR1/5 modulators and S1P1 knockdown on migration, proliferation, cytokine production, downstream signaling (immunoblot), tube formation of human intestinal microvascular endothelial cells (HIMEC) was evaluated. S1P and S1PR1/5 modulators were examined in a murine matrix plug angiogenesis model and with topical and systemic S1PR1/5 modulator therapy in dextrane sodium sulfate (DSS) colitis in vivo. Results Baseline ALC and relative or absolute ALC reductions were neither correlated with nor predictive of clinical or endoscopic outcomes. In murine DSS colitis topical therapy with a S1PR1/5 modulator ameliorated colitis but did not alter peripheral lymphocyte numbers. S1PR1 gene and protein expression was increased in UC and Crohn’s disease (CD) patients and predominantly expressed on HIMEC. S1P promoted HIMEC migration, proliferation, tube formation, IL-8 secretion, indicative of pro-angiogenic responses in vitro. Modulation of S1PR1/5 and knockdown of S1PR1 markedly inhibited these pro-angiogenic functions in the presence of high concentrations of S1P, but enhanced angiogenesis when low S1P concentrations were present. S1PR5 had no effect on angiogenesis. In the murine plug assay, S1PR1 induced angiogenesis. In acute DSS colitis, treatment of S1PR modulator by enema reduced blood vessels, but left lymphatics unaffected. Conclusion We present a novel mechanism of action of S1PR1 modulators in IBD intestinal tissue, potentially explaining a disconnect between ALC and efficacy observed in a post-hoc analysis of the phase 3 Ozanimod UC trial. The data presented suggest that S1P induced angiogenesis is attenuated by S1PR1 modulation in HIMEC from IBD patients when S1P levels comparable to blood were present. In the presence of S1P at concentrations consistent with lymph fluid angiogenesis was increased. Together with reduced numbers of circulating lymphocytes, this may contribute to efficacy of S1PR1 modulators in IBD patients.

P019 Development of hydrogel systems for targeted rectal delivery of intestinal cells to injured mucosa in inflammatory bowel disease

Abstract Background Crohn’s Disease (CD) and Ulcerative Colitis (UC) are Inflammatory Bowel Diseases (IBD) affecting over 6.8 million people worldwide. CD and UC are chronic and progressive diseases, characterised by destructive inflammation of the intestinal tract. IBD management consists of drugs such as steroids, aminosalicylates, immunosuppressants, and biologics that aim to induce disease maintenance and remission. However, frequent, and systemic drug administration leads to limited local effects, severe side effects, and low patient compliance. Ultimately, about 20% of UC and up to 80% of CD patients require surgical colon removal. Thus, new approaches to locally restore the inflamed mucosa and initiate colonic wound repair are required. Human intestinal organoids (HIOs) are 3D cell aggregates derived from primary tissue or stem cells grown in vitro. They demonstrate a similar composition architecture to the primary intestinal tissue. Studies showed that HIOs can engraft into injured colonic mucosa resulting in wound repair. Thus, delivery of HIOs to damaged intestinal epithelium represents a promising therapeutic option in IBD treatment. The project aims to develop hydrogel systems with embedded HIOs and obtain human-relevant information on their efficacy in the restitution of injured colonic mucosa using an improved and human-relevant organ-on-a-chip technology. Methods A series of thermoresponsive hydrogels were fabricated (based on methylcellulose/hyaluronic acid polymers) and characterised. Following the embedment of HIOs into hydrogels, the morphology and viability of HIOs were determined using microscopy and MTT assays. Inflammation was induced through the administration of LPS to the basolateral side of the gut chip. Hydrogel/HIO systems were applied to the apical chip compartment via flow for 24 hours. Subsequently, hydrogel/HIO systems were removed from the chip, and HIO engraftment into injured mucosa was determined utilising permeability (4 kDa dextran) and cytokine release assays (ELISA IL-8). Results Our findings show that the gels were liquid at 25 °C and solidified at 37 °C and demonstrated low cytotoxicity. Additionally, our results suggest that the hydrogel systems had a restorative and possibly protective effect on the villi structures in the gut chip. Microscopy results clearly demonstrated the presence of fully differentiated villi structures in the chips that were treated with the hydrogel/cell systems (Figure 1B). These villi structures were absent in the not-treated chips (Figure 1A). Conclusion This study suggests that development of organoid-based hydrogels that can serve as a safe, effective, and inexpensive therapy (compared to surgery) and significantly improve the treatment of IBD.

DOP67 MB310: From Clinical FMT to a Phase 1 Study with a Defined, Orally Administered Live Bacterial Therapeutic for Mild Moderate Ulcerative Colitis

Abstract Background The composition of the gut microbiome is a key determinant in inflammatory bowel disease. Consequently, modifying the composition of the gut microbiota has the potential to transform the treatment of ulcerative colitis (UC). The clinical use of faecal microbiota transplantation (FMT) has become widespread, especially for the treatment of C. difficile associated diseases, as a method of changing the composition of the gut microbiome from a diseased to a healthy state. FMT is, by its nature, an undefined and variable therapy due to differences between donors. Microbiotica has taken a "patient-first" discovery approach to identify a defined consortium, which is being developed as a live bacterial therapeutic for treatment of mild-to-moderate UC patients in a Phase 1b clinical trial, the COMPOSER-1 study, in 2024. Methods A clinical trial in UC patients (Costello et al., 2019), in which patients were treated with FMT from healthy donors, showed a 32% clinical remission rate in the active treatment group, as compared to 9% in the placebo arm. Donor, pre-treatment recipient, and post-FMT recipient samples were analysed using shotgun metagenomic sequencing. All samples were analysed using Microbiotica’s precision microbiome analysis platform to identify bacteria associated with patient response. These bacteria were assessed in cellular assays using relevant human cell/cell lines including: Caco2, dendritic cells, M1 macrophages, and CD4+ T-cells. Results We defined a signature of therapeutic response based on bacteria that engrafted from the donor into UC patients and were associated with clinical benefit. A sub-species level analysis identified 8 bacteria, which have been developed into a defined live bacterial therapeutic, MB310. The consortium bacteria enhanced the barrier integrity of an epithelial cell monolayer, and protected the barrier from inflammatory challenge by LPS. In vitro, the bacteria also have an anti-inflammatory effect when incubated with different primary innate immune cells, dendritic cells and M1 macrophages. Specific MB310 bacteria, either directly or via metabolites, are able to regulate T-cell responses. Conclusion In summary, we have identified a consortium of bacteria using patient-first discovery in a successful FMT study that is being developed as a treatment for UC. The consortium of 8 bacteria is able to impact multiple disease-relevant mechanisms including epithelial barrier integrity and immunomodulation. MB310 is being advanced to a first-in-human study in 2024 to explore safety and initial signs of efficacy. Costello, et al. (2019) Jama 321.2: 156-164.

P003 Metabolic Reprogramming in Intestinal Fibrosis: Integrating Metabolomics, Single-Cell Transcriptomics and Isotope Tracing

Abstract Background This study aims to systematically elucidate the metabolic landscape of intestinal fibrosis in Crohn's disease, revealing the key reprogrammed metabolic pathways in the fibrotic microenvironment. Methods We collected normal and fibrosis tissues from 122 Crohn's disease patients undergoing surgery for fibrosis-induced intestinal obstruction, and established DSS/TNBS-induced murine intestinal fibrosis model. To measure metabolites level in normal and fibrotic tissues, untargeted metabolomics was performed. C13-labeled glutamine was used for metabolic tracing to delineate metabolic fluxes in vitro and in vivo. We performed integration analysis by merging metabolomics and metabolic tracing data with established single-cell transcriptomic profiles. Isolation and further validation of human intestinal stromal cells were carried out, and gene expression was assessed using qRT-PCR and immunofluorescence. Results Significant metabolic heterogeneity was observed between normal and fibrotic tissues, with differential metabolites enriched in glucose and amino acid metabolism. Notably, intermediates of glycolysis were significantly increased, while tricarboxylic acid (TCA) cycle intermediates were decreased in fibrotic regions (p<0.05). Amino acids were broadly downregulated in fibrosis, whereas glutamate and glutamine were highly enriched (p<0.05). Mechanistic insights into these changes were provided by isotope tracing, revealing enhanced glycolysis and weakened TCA flux attributed to transition from TCA intermediate α-KG to glutamate. Moreover, catabolism of glutamine to glutamate was enhanced. Glutamate from above pathways converted to proline, which is the ingredient of extracellular matrix (ECM). Single-cell transcriptomic analysis indicated a high enrichment of genes related to glutamine metabolism in stromal cells, with significant upregulation of enzymes such as GLS (Glutaminase) and PYCR1 (Pyrroline-5-carboxylate Reductase 1) involved in glutamine and proline synthesis. Immunofluorescence and qRT-PCR confirmed the upregulation of GLS and PYCR1. Further experiments using primary stromal cells and murine models demonstrated that inhibiting GLS activity reduced ECM synthesis, alleviating intestinal fibrosis. Conclusion In intestinal fibrosis, glycolysis and glutamine catabolism enhanced. Stromal cells utilized the reprogrammed metabolic pathway to synthesize glutamate, thereby promoting pathological ECM anabolism. Targeting the key enzyme GLS represent a potential therapeutic strategy for intestinal fibrosis.

P164 MRT-6160, a VAV1-directed molecular glue degrader, inhibits disease progression and inflammation in a T-cell transfer model of Colitis

Abstract Background VAV1 is an immune-restricted guanine nucleotide exchange factor critical for T-cell receptor (TCR) and B-cell receptor (BCR) signalling. The role of VAV1 in T-cells has been demonstrated in knockout mice, which exhibit decreased effector functions and resistance to autoimmune disease models, and in CRISPR-based screens, where VAV1 has been highlighted as a top hit among positive regulators of T-cell function. Until now, VAV1 has remained undruggable by conventional small molecules. MRT-6160 is a first-in-class molecular glue degrader that specifically targets VAV1 for proteasomal degradation. Given the role of VAV1 in TCR and BCR signalling, MRT-6160-mediated degradation could be an effective therapeutic approach to treating autoimmune disease. Methods We first tested the impact of MRT-6160-mediated human (h)VAV1 degradation on primary human cells using in vitro stimulation-based assays. Oral bioavailability of MRT-6160 and in vivo degradation of murine (m)VAV1 was then examined in mice. We tested the efficacy of MRT-6160 using a T-cell adoptive transfer model of colitis treating recipients of pathogenic CD45RBhigh CD4+ T-cells (8 per group) with anti-TNF (25 mg/kg, Q3D) or MRT-6160 (1 mg/kg, QD) for 41 days following cell transfer. Disease activity index (DAI), comprising stool consistency and weight loss, was assessed every 3 days. At study termination, mesenteric lymph nodes (mLN) were profiled by flow cytometry and colon tissues were assessed for cytokine expression and histopathology. Results Degradation of hVAV1 by MRT-6160 inhibited T-cell activation, proliferation, and cytokine production in a dose-dependent manner. Orally administered MRT-6160 degraded mVAV1 and inhibited colitis disease progression by 85%, reducing average end point DAI scores more than a standard of care control (vehicle, p<0.0001; anti-TNF, p=0.0107). Compared to vehicle treated groups, MRT-6160 treated mice had fewer IL-17A+ (12.6 and 5.3% respectively) and TNF+ (37.6 and 17% respectively) CD4+ T cells in mLN tissue. Colon weight:length ratio was significantly reduced in MRT-6160-treated mice (42.5 mg/cm) compared to vehicle (70.3 mg/cm) and anti-TNF (61.2 mg/cm). Finally, pro-inflammatory cytokine expression in the colon mucosa was reduced in MRT-6160-treated mice compared to vehicle (IL-6: 1305.8 and 682.3 fg/mg; TNF: 175.5 and 66.9 ng/mg; respectively), as well as histopathological evidence of disease. Conclusion MRT-6160 demonstrates strong activity in a preclinical model of colitis reducing DAI, effector cytokine production, and colon tissue damage. These data suggest that MRT-6160-mediated degradation of VAV1 may have therapeutic benefit in IBD patients and warrants further clinical development.

Evidence for hypoxia-induced dysregulated cholesterol homeostasis in preeclampsia: Insights into the mechanisms from human placental cells and tissues.

Preeclampsia (PE) poses a considerable risk to the long-term cardiovascular health of both mothers and their offspring due to a hypoxic environment in the placenta leading to reduced fetal oxygen supply. Cholesterol is vital for fetal development by influencing placental function. Recent findings suggest an association between hypoxia, disturbed cholesterol homeostasis, and PE. This study investigates the influence of hypoxia on placental cholesterol homeostasis. Using primary human trophoblast cells and placentae from women with PE, various aspects of cholesterol homeostasis were examined under hypoxic and hypoxia/reoxygenation (H/R) conditions. Under hypoxia and H/R, intracellular total and non-esterified cholesterol levels were significantly increased. This coincided with an upregulation of HMG-CoA-reductase and HMG-CoA-synthase (key genes regulating cholesterol biosynthesis), and a decrease in acetyl-CoA-acetyltransferase-1 (ACAT1), which mediates cholesterol esterification. Hypoxia and H/R also increased the intracellular levels of reactive oxygen species and elevated the expression of hypoxia-inducible factor (HIF)-2α and sterol-regulatory-element-binding-protein (SREBP) transcription factors. Additionally, exposure of trophoblasts to hypoxia and H/R resulted in enhanced cholesterol efflux to maternal and fetal serum. This was accompanied by an increased expression of proteins involved in cholesterol transport such as the scavenger receptor class B type I (SR-BI) and the ATP-binding cassette transporter G1 (ABCG1). Despite these metabolic alterations, mitogen-activated-protein-kinase (MAPK) signaling, a key regulator of cholesterol homeostasis, was largely unaffected. Our findings indicate dysregulation of cholesterol homeostasis at multiple metabolic points in both the trophoblast hypoxia model and placentae from women with PE. The increased cholesterol efflux and intracellular accumulation of non-esterified cholesterol may have critical implications for both the mother and the fetus during pregnancy, potentially contributing to an elevated cardiovascular risk later in life.

ECCO Grant Dissecting the role of S1PR1 modulator in the mucosal regeneration of Ulcerative Colitis

Abstract Background and Aims Ozanimod, a potent sphingosine 1-phosphate (S1P) 1/5 receptor agonist, first approved for the treatment of adults with moderately to severely active UC in the USA and in the EU, has showed promising results in terms of both tolerability and efficacy reducing inflammatory processes and inducing disease remission. The selectivity of Ozanimod for S1PR1 is 27 times that for S1PR5. Therefore, is plausible that the beneficial effects of Ozanimod are mediated by S1PR1, which is constitutively expressed not only by immune cells, but also by epithelial, endothelial, and stromal cells. However, its function on non-immune cells has been neglected. Based on preliminary data demonstrating a faster recovery of mucosal damage in mice with a conditional deletion of S1PR1 in the epithelia and endothelia compartments, our hypothesis is that the inactivation of S1PR1 not only inhibits immune cell trafficking, but also promotes tissue repair. Based on this background, the main objectives will be to exploit the Ozanimod effects during the process of mucosa healing, and to investigate the molecular pathways activated by Ozanimod on epithelial and endothelial cells. Methods Pre-clinical validation of Ozanimod, in vivo model of UC in association with conditional knockout mice for S1PR1 on epithelial and endothelial cells, stimulation of primary endothelial cells from UC patients and human induced pluripotent stem cell-derived intestinal organoids (iHOs) will be used to address our aims. Transcriptomics, functional assay, immune characterization, WB validation will be performed. Anticipated Impact In short and medium term, we will improve the knowledge on the role of S1PR1 in the processes of mucosal healing and identify the molecular pathways involved in the response to the mucosal damage mediated by the ozanimod in non-immune cells. The results of this study may improve the treatment options for UC patients.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). The WD-NECs from healthy adult donors (N=6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Silencing ANGPTL8 reduces mouse preadipocyte differentiation and insulin signaling

ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.

Endolysosomal two-pore channel 2 plays opposing roles in primary and metastatic malignant melanoma cells.

The ion channel two-pore channel 2 (TPC2), localised on the membranes of acidic organelles such as endo-lysosomes and melanosomes, has been shown to play a role in pathologies including cancer, and it is differently expressed in primary versus metastatic melanoma cells. Whether TPC2 plays a pro- or anti-oncogenic role in different tumour conditions is a relevant open question which we have explored in melanoma at different stages of tumour progression. The behaviour of primary melanoma cell line B16F0 and its metastatic subline B16F10 were compared in response to TPC2 modulation by silencing (by small interfering RNA), knock-out (by CRISPR/Cas9) and overexpression (by mCherry-TPC2 transfected plasmid). TPC2 silencing increased cell migration, epithelial-to-mesenchymal transitionand autophagy in the metastatic samples, but abated them in the silenced primary ones. Interestingly, while TPC2 inactivation failed to affect markers of proliferation in both samples, it strongly enhanced the migratory behaviour of the metastatic cells, again suggesting that in the more aggressive phenotype TPC2 plays a specific antimetastatic role. In line with this, overexpression of TPC2 in B16F10 cells resulted in phenotype rescue, that is, a decrease in migratory ability, thus collectively resuming traits of the B16F0 primary cell line. Our research shows a novel role of TPC2 in melanoma cells that is intriguingly different in initial versus late stages of cancer progression.

Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130.

IL6 is a proinflammatory cytokine that binds to membrane-bound IL6 receptor (IL6R) or soluble IL6R to signal via gp130 in cis or trans, respectively. We tested the hypothesis that sgp130Fc, which is believed to be a selective IL6 trans-signalling inhibitor, is in fact a non-specific inhibitor of gp130 signalling. In human cancer and primary cells, sgp130Fc inhibited IL6, IL11, OSM and CT1 cis-signalling. The IC50 values of sgp130Fc for IL6 and OSM cis-signalling were markedly (20- to 200-fold) lower than the concentrations of sgp130Fc used in mouse studies and clinical trials. sgp130 inhibited IL6 and OSM signalling in the presence of an ADAM10/17 inhibitor and the absence of soluble IL6R or OSMR, with effects that were indistinguishable from those of a gp130 neutralising antibody. These data show that sgp130Fc does not exclusively block IL6 trans-signalling and reveal instead that broad inhibition of gp130 signalling likely underlies its therapeutic effects. This proposes global or modular inhibition of gp130 as a therapeutic approach for treating human disease.

Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.

Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air–liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.