Inflammation In Mice Research Articles

B1 inhomogeneity correction of RARE MRI at low SNR: Quantitative in vivo 19 F MRI of mouse neuroinflammation with a cryogenically-cooled transceive surface radiofrequency probe.

Low SNR in fluorine-19 (19 F) MRI benefits from cryogenically-cooled transceive surface RF probes (CRPs), but strong B1 inhomogeneities hinder quantification. Rapid acquisition with refocused echoes (RARE) is an SNR-efficient method for MRI of neuroinflammation with perfluorinated compounds but lacks an analytical signal intensity equation to retrospectively correct B1 inhomogeneity. Here, a workflow was proposed and validated to correct and quantify 19 F-MR signals from the inflamed mouse brain using a 19 F-CRP. In vivo 19 F-MR images were acquired in a neuroinflammation mouse model with a quadrature 19 F-CRP using an imaging setup including 3D-printed components to acquire co-localized anatomical and 19 F images. Model-based corrections were validated on a uniform 19 F phantom and in the neuroinflammatory model. Corrected 19 F-MR images were benchmarked against reference images and overlaid on in vivo 1 H-MR images. Computed concentration uncertainty maps using Monte Carlo simulations served as a measure of performance of the B1 corrections. Our study reports on the first quantitative in vivo 19 F-MR images of an inflamed mouse brain using a 19 F-CRP, including in vivo T1 calculations for 19 F-nanoparticles during pathology and B1 corrections for 19 F-signal quantification. Model-based corrections markedly improved 19 F-signal quantification from errors > 50% to < 10% in a uniform phantom (p < 0.001). Concentration uncertainty maps ex vivo and in vivo yielded uncertainties that were generally < 25%. Monte Carlo simulations prescribed SNR ≥ 10.1 to reduce uncertainties < 10%, and SNR ≥ 4.25 to achieve uncertainties < 25%. Our model-based correction method facilitated 19 F signal quantification in the inflamed mouse brain when using the SNR-boosting 19 F-CRP technology, paving the way for future low-SNR 19 F-MRI applications in vivo.

Pyruvate kinase M2 (PKM2) interacts with activating transcription factor 2 (ATF2) to bridge glycolysis and pyroptosis in microglia

Pyruvate kinase M2 (PKM2), a glycolytic rate-limiting enzyme, reportedly plays an important role in tumorigenesis and the inflammatory response by regulating the metabolic reprogramming. However, its contribution to microglial activation during neuroinflammation is still unknown. In this study, we observed an enhanced glycolysis level in the lipopolysaccharide (LPS)-activated microglia. Utilizing the glycolysis inhibitor 2-DG, we proved that LPS requires glycolysis to induce microglial pyroptosis. Moreover, the protein expression, dimer/monomer formation, phosphorylation and nuclear translocation of PKM2 were all increased by LPS. Silencing PKM2 or preventing its nuclear translocation by TEPP-46 significantly alleviated the LPS-induced inflammatory response and pyroptosis in microglia. Employing biological mass spectrometry combined with immunoprecipitation technology, we identified for the first time that PKM2 interacts with activating transcription factor 2 (ATF2) in microglia. Inhibition of glycolysis or preventing PKM2 nuclear aggregation significantly reduced the phosphorylation and activation of ATF2. Furthermore, knocking down ATF2 reduced the LPS-induced pyroptosis of microglia. In vivo, we showed the LPS-induced pyroptosis in the cerebral cortex tissues of mice, and first found that an increased PKM2 expression was co-localized with ATF2 in the inflamed mice brain. Collectively, our data suggested for the first time that PKM2, a key rate-limiting enzyme of the Warburg effect, directly interacts with the pro-inflammatory transcription factor ATF2 to bridge glycolysis and pyroptosis in microglia, which might be a pivotal crosstalk between metabolic reprogramming and neuroinflammation in the CNS.

Multimodal Identification by Transcriptomics and Multiscale Bioassays of Active Components in Xuanfeibaidu Formula to Suppress Macrophage-Mediated Immune Response.

Xuanfeibaidu Formula (XFBD) is a Chinese medicine used in the clinical treatment of coronavirus disease 2019 (COVID-19) patients. Although XFBD has exhibited significant therapeutic efficacy in clinical practice, its underlying pharmacological mechanism remains unclear. Here, we combine a comprehensive research approach that includes network pharmacology, transcriptomics, and bioassays in multiple model systems to investigate the pharmacological mechanism of XFBD and its bioactive substances. High-resolution mass spectrometry was combined with molecular networking to profile the major active substances in XFBD. A total of 104 compounds were identified or tentatively characterized, including flavonoids, terpenes, carboxylic acids, and other types of constituents. Based on the chemical composition of XFBD, a network pharmacology-based analysis identified inflammation-related pathways as primary targets. Thus, we examined the anti-inflammation activity of XFBD in a lipopolysaccharide-induced acute inflammation mice model. XFBD significantly alleviated pulmonary inflammation and decreased the level of serum proinflammatory cytokines. Transcriptomic profiling suggested that genes related to macrophage function were differently expressed after XFBD treatment. Consequently, the effects of XFBD on macrophage activation and mobilization were investigated in a macrophage cell line and a zebrafish wounding model. XFBD exerts strong inhibitory effects on both macrophage activation and migration. Moreover, through multimodal screening, we further identified the major components and compounds from the different herbs of XFBD that mediate its anti-inflammation function. Active components from XFBD, including Polygoni cuspidati Rhizoma, Phragmitis Rhizoma, and Citri grandis Exocarpium rubrum, were then found to strongly downregulate macrophage activation, and polydatin, isoliquiritin, and acteoside were identified as active compounds. Components of Artemisiae annuae Herba and Ephedrae Herba were found to substantially inhibit endogenous macrophage migration, while the presence of ephedrine, atractylenolide I, and kaempferol was attributed to these effects. In summary, our study explores the pharmacological mechanism and effective components of XFBD in inflammation regulation via multimodal approaches, and thereby provides a biological illustration of the clinical efficacy of XFBD.

Resorufin-based fluorescent probe with elevated water solubility for visualizing fluctuant peroxynitrite in progression of inflammation

Inflammation is a significant protective response in biological systems and associated with various diseases. Peroxynitrite (ONOO−) as a highly active oxidant participates in the inflammatory process of organisms. Thus, it is necessary to construct novel fluorescent probes for exploring inflammation-related diseases through detecting endogenous ONOO–. Resorufin-based fluorescent probes for testing ONOO− were rare and suffered from poor water solubility. In this work, we elaborately designed three resorufin-based incorporating isatin derivatives probes RF-ITs and successfully obtained two highly selective probes RF-IT-OC and RF-IT-EG for ONOO–. Comparing the other two probes, RF-IT-EG containing triethylene glycol monomethyl ether on isatin moiety displayed better water solubility (3.2 mg/L), faster response rate (60 s), larger signal-to-noise ratio (103-fold) and lower detection limit (87 nM) for monitoring ONOO–. The cells imaging results manifested that probe RF-IT-EG could be applied to trace endogenous ONOO− with inappreciable cytotoxicity. Moreover, the RF-IT-EG was capable of tracking the fluctuation of endogenous ONOO− in LPS-stimulated inflamed mouse leg models. This work will provide a faithful and promising probe for illustrating the roles of ONOO− in various inflammation-related diseases.

Click-Ready Perfluorocarbon Nanoemulsion for 19F MRI and Multimodal Cellular Detection.

We describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling postemulsification functionalization via a click reaction with azide-containing ligands. The addition of DBCO-lipid to the surfactant in PFC nanoemulsions did not affect nanoemulsion size or nanoemulsion stability. As proof-of-concept, fluorescent dye-azides were conjugated to PFC nanoemulsions, demonstrating the feasibility of functionalization the by click reaction. Uptake of the fluorescent PFC by macrophages was demonstrated both in vitro in cultured macrophages and in situ in an acute inflammation mouse model, where fluorescence imaging and 1H/19F magnetic resonance imaging (MRI) were used for in vivo detection. Overall, these data demonstrate the potential of PFC nanoemulsions incorporating DBCO as a versatile platform for generating functionalized probes.

Macrophage-Biomimetic Nanoparticles Ameliorate Ulcerative Colitis through Reducing Inflammatory Factors Expression

Background and Aims: Inflammatory mediator S100A9 is dramatically elevated in ulcerative colitis and correlates with disease severity. S100A9 is a potential molecule to target for the treatment of colitis, but to date, there is no effective targeting method. The aim of this study was to develop a safe and effective nano-delivery system targeting S100A9 and to evaluate its therapeutic efficacy in ulcerative colitis mouse model. Methods: We designed an oral nano-delivery system using poly (lactic acid-glycolic acid) (PLGA)-loaded S100A9 inhibitor tasquinimod to synthesize PLGA-TAS nanoparticles. TLR4-overexpressing macrophage membranes (MMs) were used to wrap the nanoparticles to make MM-PLGA-TAS, which allowed the nanoparticles to acquire the ability to specifically enrich the colitis region. Results: MM-PLGA-TAS was endocytosed by inflammatory phenotype RAW264.7 cells in vitro and can efficiently enrich in inflamed mouse colitis tissue in vivo. A chemically induced ulcerative colitis mouse model was used to evaluate the therapeutic effect of oral MM-PLGA-TAS. MM-PLGA-TAS significantly alleviated the symptoms of ulcerative colitis, and mechanically, MM-PLGA-TAS achieved immunomodulatory and suppressive effects by reducing S100a9 and other cytokines in the colitis region. Conclusion: We describe a convenient, orally targeted colitis drug delivery system that cures the disease in ulcerative colitis mice. This system substantially increases drug accumulation in inflamed colonic tissue, reduces the risk of systemic exposure, and is a promising therapeutic approach against ulcerative colitis.

SMYD2-mediated TRAF2 methylation promotes the NF-κB signaling pathways in inflammatory diseases.

BackgroundThe methylation of lysine residues has been involved in the multiple biological and diseases processes. Recently, some particular non‐histone proteins have been elucidated to be methylated by SMYD2, a SET and MYND domain protein with lysine methyltransferase activity.MethodsSMYD2 was evaluated in synovial tissue and cells derived from rheumatoid arthritis patients. We confirmed TRAF2 could be methylated by SMYD2 using Mass spectrometry, pull‐down, immunoprecipitation, methyltransferase assay, ubiquitination assay, luciferase reporter assays, and western blot analyses. Using loss‐ and gain‐of function studies, we explored the biological functions of SMYD2 in vitro and in vivo. Using acute and chronic inflammation with different mice models to determine the impact of SMYD2.ResultsHere, we first time confirmed that the cytoplasmic protein TRAF2 as the kernel node for NF‐κB signaling pathway could be methylated by SMYD2. SMYD2‐mediated TRAF2 methylation contributed to the durative sensitization of NF‐κB signaling transduction through restraining its own proteolysis and enhancing the activity. In addition, we found knocking down of SMYD2 has different degrees of mitigation in acute and chronic inflammation mice models. Furthermore, as the lysine‐specific demethylase, LSD1 could resist methylation on TRAF2 induced by SMYD2.ConclusionsOur data uncovered an unprecedented cytoplasmic protein network that employed methylation of TRAF2 for the maintenance of NF‐κB activation during inflammatory diseases.

A RAS inhibitor reduces allergic airway remodeling via regulating IL-33-derived type 2 innate lymphoid cells

Purpose: IL-33 is known to induce corticosteroid-resistant eosinophilic inflammation and airway remodeling by activating type 2 innate lymphoid cells (ILC2s). Although the RAS signal pathway plays an important role in IL-33-induced ILC2s activation and airway remodeling, it is not known if RAS inhibitors are effective against refractory asthma. We examined the effects of the RAS inhibitor XRP44X in refractory asthma. Methods: RAS activity were examined by BAL fluid and T-cells isolated from spleen cells in Dermatophagoides pteronyssinus (Dp)-sensitized/challenged acute allergic airway inflammation model. A chronic allergic airway inflammation mouse model was generated by challenged with Dp. XRP44X and/or fluticasone were administrated nasally to different experimental groups. The effects of nasal simultaneous administration of XRP44X or fluticasone were assessed in mice administrated with IL-33 or Dp. Results: RAS activity in CD4+ T cells stimulated by Dp were suppressed by XRP44X. Although fluticasone and XRP44X only improved allergic airway inflammation in mice, XRP44X in combination with fluticasone produced further improvement in not only eosinophilic inflammation but also bronchial subepithelial thickness. XRP44X suppressed IL-5 and IL-13 production from ILC2s, although this effect was not suppressed by fluticasone. IL-33-induced airway inflammation resistant to fluticasone was ameliorated by XRP44X via regulating the accumulation of lung ILC2s. Conclusion: The RAS signal pathway plays a crucial role in allergen-induced airway remodeling associated with ILC2s. XRP44X may have therapeutic potential for refractory asthma.

IL13 Acts Directly on Gastric Epithelial Cells to Promote Metaplasia Development During Chronic Gastritis.

Background & AimsIt is well established that chronic inflammation promotes gastric cancer–associated metaplasia, but little is known regarding the mechanisms by which immune cells and cytokines regulate metaplastic cellular changes. The goals of this study were to identify interleukin 13 (IL13)-producing immune cells, determine the gastric epithelial cell response(s) to IL13, and establish the role(s) of IL13 in metaplasia development.MethodsExperiments used an established mouse model of autoimmune gastritis (TxA23), TxA23×Il4ra-/- mice, which develop gastritis but do not express the IL4/IL13-receptor subunit IL4Rα, and TxA23×Il13-Yfp mice, which express yellow fluorescent protein in IL13-producing cells. Flow cytometry was used to measure IL13 secretion and identify IL13-producing immune cells. Mouse and human gastric organoids were cultured with IL13 to determine epithelial cell response(s) to IL13. Single-cell RNA sequencing was performed on gastric epithelial cells from healthy and inflamed mouse stomachs. Mice with gastritis were administered IL13-neutralizing antibodies and stomachs were analyzed by histopathology and immunofluorescence.ResultsWe identified 6 unique subsets of IL13-producing immune cells in the inflamed stomach. Organoid cultures showed that IL13 acts directly on gastric epithelium to induce a metaplastic phenotype. IL4Rα-deficient mice did not progress to metaplasia. Single-cell RNA sequencing determined that gastric epithelial cells from IL4Rα-deficient mice up-regulated inflammatory genes but failed to up-regulate metaplasia-associated transcripts. Neutralization of IL13 significantly reduced and reversed metaplasia development in mice with gastritis.ConclusionsIL13 is made by a variety of immune cell subsets during chronic gastritis and promotes gastric cancer–associated metaplastic epithelial cell changes. Neutralization of IL13 reduces metaplasia severity during chronic gastritis.

AKAP150 and its Palmitoylation Contributed to Pain Hypersensitivity Via Facilitating Synaptic Incorporation of GluA1-Containing AMPA Receptor in Spinal Dorsal Horn.

The A-kinase anchoring protein 150 (AKAP150) organizes kinases and phosphatases to regulate AMPA receptors (AMPARs) that are pivotal for synaptic plasticity. AKAP150 itself undergoes S-palmitoylation. However, the roles of AKAP150 and its palmitoylation in spinal nociceptive processing remain unknown. In this study, we found that intraplantar injection of complete Freund's adjuvant (CFA) significantly increased the synaptic expression of AKAP150 and caused a reorganization of AKAP150 signaling complex in spinal dorsal horn. Knockdown of AKAP150 or interruption of its interactions with kinases effectively suppressed the CFA-induced synaptic expression of GluA1 subunit of AMPARs. Our data also showed that an upregulation of AKAP150 palmitoylation was involved in the synaptic redistribution of AKAP150. Inhibition of AKAP150 palmitoylation by expression of palmitoylation-defective mutant AKAP150 (C36, 123S) effectively repressed the CFA-induced phosphorylation and synaptic expression of GluA1 subunit, meanwhile, attenuated the development of mechanical allodynia and thermal hyperalgesia. Furthermore, we found that an increased expression of palmitoyl acyltransferase ZDHHC2 contributed to the upregulation of AKAP150 palmitoylation and GluA1 accumulation in inflamed mouse. These data indicated that AKAP150 and its palmitoylation were involved in AMPA receptor-dependent modification of nociceptive transmission, and the manipulations of AKAP150 signaling complex and palmitoylation might serve as potential therapeutic strategies for persistent pain after inflammation.

Anti-inflammatory effect and mechanism of ethanol extract from Saposhnikoviae Radix in LPS-induced inflammation mouse model

This paper aimed to explore the anti-inflammatory effect of ethanol extract from Saposhnikoviae Radix in a lipopolysaccharide(LPS)-induced inflammation mouse model and its regulation of TLR4/NF-κB signaling pathway. The ethanol extract from Saposhnikoviae Radix was separated and purified on the macroporous adsorption resin and its main chemical components were identified by UPLC-QE/MS. The identification results showed that the top ten components of ethanol extract from Saposhnikoviae Radix were mainly chromones and coumarins. A mouse model of inflammation induced by intraperitoneal injection of LPS was used to investigate the anti-inflammatory effects of ethanol extract from Saposhnikoviae Radix after intragastric administration for seven successive days. Mice in all groups except for the control group were treated with intraperitoneal injection of LPS(0.015 g·kg~(-1)) one hour after the last administration, and twelve hours later, the blood was sampled and separated and the broncoalveolar lavage fluid(BALF) was collected. The levels of nitric oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) in mouse serum and BALF were detected by ELISA. The harvested lung tissue was stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by the detection of protein expression levels of related molecules in TLR4/NF-κB signaling pathway by Western blotting. The results showed that the ethanol extract from Saposhnikoviae Radix significantly ameliorated the pathological conditions in lung tissue of model mice, reversed the increase in NO, TNF-α, IL-6, and IL-1β levels of mouse serum and BALF, down-regulated the protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor(MyD88), and phosphorylated nuclear transcription factor κB-p65/nuclear transcription factor κB-p65(P-NF-κB p65/NF-κB p65), and up-regulated the NF-κB inhibitory protein α(IκBα). The ethanol extract from Saposhnikoviae Radix exhibited a good anti-inflammatory effect in the LPS-induced acute inflammation muse model, which might be related to the inhibition of the activation of TLR4/NF-κB inflammatory signaling pathway. Chromones and coumarins have been proved to be the active components for its anti-inflammatory effects.

Machine learning identifies molecular regulators and therapeutics for targeting SARS‐CoV2‐induced cytokine release

Although 15–20% of COVID‐19 patients experience hyper‐inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N‐terminal domain (NTD) of the SARS‐CoV‐2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning‐based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD‐induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA‐approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD‐mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS‐CoV‐2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide‐mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS‐CoV‐2‐mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID‐19.

125I–Amoxicillin preparation as a guide tracer for inflammation detection

Abstract The objective of this study is to label Amoxicillin with radioactive iodine (125I-AC) via direct electrophilic substitution to act as a promising tracer for inflammation imaging. The highest labeling yield of 80% was achieved after studying all the parameters affecting the labeling reaction using Iodogen (IG) as an oxidizing agent. Molecular Modeling Structure was done using MOE program to predict the suitable 125I position. The product structure was established by a cold iodination reaction using Iodine-127. Biological evaluation of (125I-AC) was carried out using groups of inflamed mice with different exogenous agents such as E.Coli and Turpentine oil. The (125I-AC) shows an in vitro stability of about 97% after 24 h.While doing in vivo studies over 4 h, the tracer stability of 99% was observed.

Aqueous Extract from Leaves of Citrus unshiu Attenuates Lipopolysaccharide-Induced Inflammatory Responses in a Mouse Model of Systemic Inflammation.

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.

Rutin, A Natural Inhibitor of IGPD Protein, Partially Inhibits Biofilm Formation in Staphylococcus xylosus ATCC700404 in vitro and in vivo.

Staphylococcus xylosus (S. xylosus) has become an emerging opportunistic pathogen due to its strong biofilm formation ability. Simultaneously, the biofilm of bacteria plays an important role in antibiotic resistance and chronic infection. Here, we confirmed that rutin can effectively inhibit biofilm formation in S. xylosus, of which the inhibition mechanism involves its ability to interact with imidazole glycerol phosphate dehydratase (IGPD), a key enzyme in the process of biofilm formation. We designed experiments to target IGPD and inhibited its activities against S. xylosus. Our results indicated that the activity of IGPD and the amount of histidine decreased significantly under the condition of 0.8 mg/ml rutin. Moreover, the expression of IGPD mRNA (hisB) and IGPD protein was significantly down-regulated. Meanwhile, the results from molecular dynamic simulation and Bio-layer interferometry (BLI) technique showed that rutin could bind to IGPD strongly. Additionally, in vivo studies demonstrated that rutin treatment reduced inflammation and protect mice from acute mastitis caused by S. xylosus. In summary, our findings provide new insights into the treatment of biofilm mediated persistent infections and chronic bacterial infections. It could be helpful to design next generation antibiotics to against resistant bacteria.

SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Towards Drug Delivery Control Using Iron Oxide Nanoparticles in Three-Dimensional Magnetic Resonance Imaging.

The purpose of this paper was to detect and separate the cluster intensity provided by Iron oxide nanoparticles (IO-NPs), in the MRI images, to investigate the drug delivery effectiveness. IO-NPs were attached to the macrophages and inserted into the eye of the inflamed mouse’s calf. The low resolution of MRI and the tiny dimension of the IO-NPs made the situation challenging. IO-NPs serve as a marker, due to their strong intensity in the MRI, enabling us to follow the track of the macrophages. An image processing procedure was developed to estimate the position and the amount of IO-NPs spreading inside the inflamed mouse leg. A fuzzy Clustering algorithm was adopted to select the region of interest (ROI). A 3D model of the femoral region was used for the detection and then the extraction IO-NPs in the MRI images. The results achieved prove the effectiveness of the proposed method to improve the control process of targeted drug delivered. It helps in optimizing the treatment and opens a promising novel research axis for nanomedicine applications.

Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease.

Activation of the Ca2+ activated Cl− channel TMEM16A is proposed as a treatment in inflammatory airway disease. It is assumed that activation of TMEM16A will induce electrolyte secretion, and thus reduce airway mucus plugging and improve mucociliary clearance. A benefit of activation of TMEM16A was shown in vitro and in studies in sheep, but others reported an increase in mucus production and airway contraction by activation of TMEM16A. We analyzed expression of TMEM16A in healthy and inflamed human and mouse airways and examined the consequences of activation or inhibition of TMEM16A in asthmatic mice. TMEM16A was found to be upregulated in the lungs of patients with asthma or cystic fibrosis, as well as in the airways of asthmatic mice. Activation or potentiation of TMEM16A by the compounds Eact or brevenal, respectively, induced acute mucus release from airway goblet cells and induced bronchoconstriction in mice in vivo. In contrast, niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in vivo and in vitro. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the essential transcription factor of Th2-dependent inflammation and goblet cell differentiation, SAM pointed domain-containing ETS-like factor (SPDEF). Activation of TMEM16A in people with inflammatory airway diseases is likely to induce mucus secretion along with airway constriction. In contrast, inhibitors of TMEM16A may suppress pulmonary Th2 inflammation, goblet cell metaplasia, mucus production, and bronchoconstriction, partially by inhibiting expression of SPDEF.

Intravenous Administration of Toll-Like Receptor Inhibitory Peptide 1 is Effective for the Treatment of Systemic Lupus Erythematosus in a Mus musculus Model.

Systemic lupus erythematosus (SLE) is a common chronic autoimmune inflammatory disease According to recent studies, signaling through Toll-like receptor (TLR) protein, which promotes the production of inflammatory cytokines, leads to the development of SLE TLR-inhibitory peptide 1 (TIP1) has been newly identified for the treatment of autoimmune diseases. The effect of TIP1 was analyzed in an SLE mouse model (MRL/lpr) The mice in the control treatment group (n=5) were administered an intravenous injection of phosphate-buffered saline twice weekly, whereas the mice in the TIP1 treatment group (n=6) were administered an intravenous injection of TIP1 (1 nmol/g) twice weekly MRL/mpj mice (n=5) were selected as normal controls The mice were injected for 4 weeks between 14 and 18 weeks of age, followed by assays of their spleen, kidneys, lymph nodes, serum, and urine. The antinuclear antibody and inflammatory cytokine (interferon-α) in the serum as well as levels of albumin in the urine of the mice in the TIP1 treatment group had decreased when compared to those of mice in the control treatment group Kidney inflammation in mice in the TIP1 treatment group was alleviated The mRNA expression levels of TLR7- or TLR9-related downstream signaling molecules also decreased in all organs of the mice in the TIP1 treatment group. Intravenous treatment with TIP1 reduces symptoms and markers of inflammation in MRL/lpr mice Hence, TIP1 is a promising medication for the treatment of SLE.

Abstract 1714: Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1

Abstract Background: Interleukin 12 (IL-12) is a proinflammatory cytokine that plays a central role in regulating both innate and adaptive antitumor immune responses. Clinically, administration of recombinant human IL-12 (rhIL-12) has been limited by a short half-life (t1/2) requiring frequent dosing, causing tachyphylaxis as well as toxic side effects. DF6002/BMS-986415 is an IL-12 Fc-fusion protein designed to have a prolonged t1/2 while retaining equivalent potency to rhIL-12 and promoting sustained IFN-γ response with reduced toxicity. Here we present the preclinical evaluation of DF6002/BMS-986415 ± anti–PD-1. Methods: DF6002/BMS-986415 and rhIL-12 stimulation of IFN-γ release from human and cynomolgus macaque (Cyno) peripheral blood mononuclear cells (PBMCs) and in vivo exposure in Cynos was assessed. As rhIL-12 is inactive in mice, pharmacokinetic, pharmacodynamic, and antitumor effects were evaluated with mDF6006, a mouse surrogate of DF6002/BMS-986415, and recombinant mouse IL-12 (rmIL-12). Tumor growth was evaluated in inflamed (CT26 and MC38) and immunosuppressed (4T1 and B16F10) mouse tumor models (staged ≥ 200 mm3). Results: DF6002/BMS-986415 induced IFN-γ production in human (EC50, 3.35 pM) and Cyno (EC50, 3.09 pM) PBMCs comparable to rhIL-12. DF6002/BMS-986415 was well tolerated at doses yielding greater exposure vs equimolar rhIL-12 in Cynos. In mice, the mouse surrogate mDF6006 vs rmIL-12 yielded a 5-fold increase in t1/2 and a 40-fold greater serum IFN-γ exposure. In contrast to rmIL-12, mDF6006 achieved protracted IFN-γ levels without peak effects associated with increased toxicity; serum IFN-γ Cmax occurred after day 6 and subsided by day 8. mDF6006 was efficacious across mouse tumor models tested. In the CT26 tumor model, mDF6006 (1 µg IP QW) monotherapy showed greater antitumor activity vs equimolar doses of rmIL-12 and led to complete regression (CR) in mice with large tumors (&amp;gt; 800 mm3); immunological memory response was observed in responders rechallenged with fresh tumor cells. In the B16F10 tumor model, mDF6006 (0.5 µg equivalent rmIL-12 SC QW) + anti–PD-1 (10 mg/kg IP Q2W) yielded a higher CR rate (mDF6006 + anti-PD-1, 4 of 10 CR; mDF6006, 1 of 10 CR) and increased survival vs either agent alone. An increase in the number and activation of CD4+ and CD8+ T cells as well as NK cells was observed 4 days after administration of mDF6006 (0.5 µg) in the B16F10 model. Conclusions: DF6002/BMS-986415, a novel IL-12 Fc-fusion protein, retained the full potency of rhIL-12 and showed improved exposure and tolerability; mDF6006 exhibited greater antitumor activity than rmIL-12. These data support the ongoing first-in-human phase 1/2 study evaluating DF6002/BMS-986415 as a novel therapeutic for the treatment of solid tumors in combination with nivolumab (NCT04423029). Citation Format: Eva Gutierrez, Mitchell Bigelow, Colin LaCroix, Patrick Kirby, Lynn Markowitz, Michael Naill, Steve O'Neil, Philip Ansumana Hull, John Engelhardt, Jean-Marie Cuillerott, Ann Cheung, Asya Grinberg, Nicolai Wagtmann. Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1714.