Inhibited Neutrophil Infiltration Research Articles

A Two-Pronged Delivery Strategy Disrupting Positive Feedback Loop of Neutrophil Extracellular Traps for Metastasis Suppression.

Neutrophil extracellular traps (NETs) severely affect tumor metastasis through a self-perpetuating feedback loop involving two key steps: (1) mitochondrial aerobic respiration-induced hypoxia promotes NET formation and (2) NETs enhance mitochondrial metabolism to exacerbate hypoxia. Herein, we propose a two-pronged approach with the activity of NET-degrading and mitochondrion-damaging by simultaneously targeting drugs to NETs and tumor mitochondria of this loop. In addition to specifically recognizing and eliminating extant NETs, the NET-targeting nanoparticle also reduces NET-induced mitochondrial biogenesis, thus inhibiting the initial step of the feedback loop and mitigating extant NETs' impact on tumor metastasis. Simultaneously, the mitochondrion-targeting system intercepts mitochondrial metabolism and alleviates tumor hypoxia, inhibiting neutrophil infiltration and subsequent NET formation, which reduces the source of NETs and disrupts another step of the self-amplifying feedback loop. Together, the combination significantly reduces the formation of NET-tumor cell clusters by disrupting the interaction between NETs and tumor mitochondria, thereby impeding the metastatic cascade including tumor invasion, hematogenous spread, and distant colonization. This work represents an innovative attempt to disrupt the feedback loop in tumor metastasis, offering a promising therapeutic approach restraining NET-assisted metastasis.

Thermoresponsive gel containing bilirubin nanoparticles exerts anti-inflammatory effects by inhibiting neutrophil infiltration and augmenting interleukin-10 levels in carrageenan-induced rat paw edema.

Topical corticosteroids treat cutaneous inflammation but have side effects. In earlier studies, bilirubin exhibited anti-inflammatory effect, but its hydrophobicity and poor absorption limit its potential. Synthesis of bilirubin nanoparticles (BNP) and bilirubin nanoparticles gels (BNP gel) to study the anti-inflammatory effect of topical BNP gel against carrageenan-induced rat paw edema in Wistar rats. BNP were synthesized, and BNP gels were prepared by mixing BNP of different concentrations with pluronic F-127 (PF-127). A different group for each formulation was assigned with five rats in each group. After 1 h of carrageenan (1% [w/v]) injection in each group, different gels were applied topically to their respective groups. Paw edema size, percent inflammation, percent edema inhibition, and inhibition time50 were evaluated. Interleukin-10 (IL-10) levels and neutrophil infiltration in rat paw tissue were evaluated by enzyme-linked immunosorbent assay and hematoxylin and eosin, respectively. Synthesized spherical-shaped BNP had negative zeta potential. BNP gels markedly reduced paw edema size and % inflammation as compared to carrageenan and bulk bilirubin gel (Bulk B gel) treated group and significantly increased IL-10 levels and inhibited neutrophil infiltration. BNP gels exhibited a better anti-inflammatory effect than bulk B gel and comparable anti-inflammatory potential with clobetasol.

Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway

This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1β), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1β, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1β, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1β, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.

Mas receptor activation facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice

BackgroundIntracerebral hemorrhage (ICH) is a devastating neurological disease causing severe sensorimotor dysfunction and cognitive decline, yet there is no effective treatment strategy to alleviate outcomes of these patients. The Mas axis-mediated neuroprotection is involved in the pathology of various neurological diseases, however, the role of the Mas receptor in the setting of ICH remains to be elucidated.MethodsC57BL/6 mice were used to establish the ICH model by injection of collagenase into mice striatum. The Mas receptor agonist AVE0991 was administered intranasally (0.9 mg/kg) after ICH. Using a combination of behavioral tests, Western blots, immunofluorescence staining, hematoma volume, brain edema, quantitative-PCR, TUNEL staining, Fluoro-Jade C staining, Nissl staining, and pharmacological methods, we examined the impact of intranasal application of AVE0991 on hematoma absorption and neurological outcomes following ICH and investigated the underlying mechanism.ResultsMas receptor was found to be significantly expressed in activated microglia/macrophages, and the peak expression of Mas receptor in microglia/macrophages was observed at approximately 3–5 days, followed by a subsequent decline. Activation of Mas by AVE0991 post-treatment promoted hematoma absorption, reduced brain edema, and improved both short- and long-term neurological functions in ICH mice. Moreover, AVE0991 treatment effectively attenuated neuronal apoptosis, inhibited neutrophil infiltration, and reduced the release of inflammatory cytokines in perihematomal areas after ICH. Mechanistically, AVE0991 post-treatment significantly promoted the transformation of microglia/macrophages towards an anti-inflammatory, phagocytic, and reparative phenotype, and this functional phenotypic transition of microglia/macrophages by Mas activation was abolished by both Mas inhibitor A779 and Nrf2 inhibitor ML385. Furthermore, hematoma clearance and neuroprotective effects of AVE0991 treatment were reversed after microglia depletion in ICH.ConclusionsMas activation can promote hematoma absorption, ameliorate neurological deficits, alleviate neuron apoptosis, reduced neuroinflammation, and regulate the function and phenotype of microglia/macrophages via Akt/Nrf2 signaling pathway after ICH. Thus, intranasal application of Mas agonist ACE0991 may provide promising strategy for clinical treatment of ICH patients.

Potential Anti-Gouty Arthritis of Citronella Essential Oil and Nutmeg Essential Oil through Reducing Oxidative Stress and Inhibiting PI3K/Akt/mTOR Activation-Induced NLRP3 Activity.

Citronella and Nutmeg are two common spices used for seasoning and medicinal purposes, both of which have significant economic value. This study aimed to investigate whether Citronella essential oil and Nutmeg essential oil (NEO) can ameliorate monosodium urate (MSU)-induced gouty arthritis in rats and the potential mechanisms. The results showed that CEO and NEO reduced swelling and redness at joint sites, inhibited neutrophil infiltration, and limited proinflammatory mediator secretion in mice with MSU-induced gouty arthritis. Based on the results of network pharmacology, molecular docking, and western blotting, CEO and NEO may exert anti-gouty arthritis effects by reducing the expression of reactive oxygen species and oxidative stress and downregulating the phosphorylation of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting the production of the NLRP3 inflammasome and inhibiting the production of inflammatory cytokines. Therefore, these two essential oils show potential for use as adjuvant treatments for gouty arthritis in specific aromatherapy products or food seasonings.

Mechanism of Ganke Granules intervening acute lung injury based on network pharmacology and experimental verification

This study aims to explore the potential mechanism of action in the intervention of acute lung injury(ALI) based on the blood entry components of Ganke Granules in rats and in conjunction with network pharmacology, molecular docking, and animal experimental validation. The blood entry components of Ganke Granules in rats were imported into the SwissTargetPrediction platform to predict drug targets, and ALI-related targets were collected from the disease database. Intersections were taken, and protein-protein interaction(PPI) networks were constructed to screen the core targets, followed by Gene Ontology(GO) functional and Kyoto encyclopedia of genes and gnomes(KEGG) pathway enrichment analyses. A "blood entry components-target-pathway-disease" network was constructed, and the core components for disease intervention based on their topological parameters were screened. Molecular docking was used to predict the binding ability of the core components to key targets. The key targets of Ganke Granules in the intervention of ALI were verified by the lipopolysaccharide(LPS)-induced ALI mouse model. Through PPI topological parameter analysis, the top six key targets of STAT3, SRC, HSP90AA1, MAPK3, HRAS, and MAPK1 related to ALI were obtained. GO functional analysis showed that it was mainly related to ERK1 and ERK2 cascade, inflammatory response, and response to LPS. KEGG analysis showed that the main enrichment pathways were MAPK, neutrophil extracellular trap(NET) formation, and so on. Six core components(schizantherin B, schisandrin, besigomsin, harpagoside, isotectorigenin, and trachelanthamine) were filtered out by the "blood entry components-target-pathway-disease" network based on the analysis of topological parameters. Molecular docking results showed that the six core components and Tectoridin with the highest content in the granules had a high affinity with the key targets of MAPK3, SRC, MAPK1, and STAT3. In vivo experiment results showed that compared with the model group, Ganke Granules could effectively alleviate LPS-induced histopathological injury in the lungs of mice and reduce the percentage of inflammatory infiltration. The total protein content, nitric oxide(NO) level, myeloperoxidase(MPO) content, tumor necrosis factor-α(TNF-α), gamma interferon(IFN-γ), interleukin-1β(IL-1β), interleukin-6(IL-6), vascular endothelial growth factor(VEGF), and chemokine(C-X-C motif) ligand 1(CXCL1) chemokines in bronchoalveolar lavage fluid(BALF) were decreased, and the expression levels of lymphocyte antigen 6G(Ly6G), citrullinated histones 3(Cit-H3), and phosphorylated proteins SRC, ERK1/2, and STAT3 in lung tissue were significantly down-regulated. In conclusion, Ganke Granules could effectively inhibit the inflammatory response of ALI induced by LPS, protect lung tissue, regulate the release of inflammatory factors, and inhibit neutrophil infiltration and NET formation, and the mechanism of action may be related to inhibiting the activation of SRC/ERK1/2/STAT3 signaling pathway.

The capability of Piper betle leaves ethanolic extract to inhibit neutrophil infiltration in sepsis-induced BALB/c Mice

Background: Sepsis due to ESBL-producing Escherichia coli infection requires innovative management to prevent a higher prevalence of meropenem overuse. The antimicrobial effect of Piper betle leaves ethanolic extract has been previously proven and should be tested on sepsis-induced animal models. Objectives: This study assesses the antimicrobial effect of Piper betle leaves ethanolic extract towards the bacterial loads and its anti-inflammatory effect on neutrophil infiltration in the lung, kidney, and liver of sepsis-induced mice. Methods: After sepsis inducement, 3 mg/mL of Piper betle leaves ethanolic extract was administered orally. After the mice died, the bacterial load of the lung, kidney, and liver was measured, while neutrophil infiltration was observed using hematoxylin-eosin staining. Results: There is no significant decreasing the number of bacterial loads in the treatment group but there is a significant decrease in the number of neutrophils infiltration in the treatment group. Conclusion: Single dose administration of Piper betle leaves ethanolic extract is sufficient to inhibit neutrophil infiltration significantly.

Total synthesis and structural modification of the dibenzylbutane lignan LCA as a potent anti-inflammatory agent against LPS-induced acute lung injury

Acute lung injury (ALI) is a serious public health problem associated with high morbidity and mortality. However, few efficacious drugs are clinically available. Inhibition of proinflammatory cytokines is considered to be a promising method for the treatment of inflammatory diseases. Herein, the total synthesis of a dibenzylbutane lignan, 9′-O-di-(E)-feruloyl-meso-5,5′-dimethoxysecoisolariciresinol (LCA), was completed. A series of LCA derivatives were designed and synthesized, and their anti-inflammatory activities were evaluated. Derivative 14r significantly inhibited LPS-induced expression of NO and the proinflammatory cytokines TNF-α, IL-6, and IL-1β in RAW 264.7 cells and inhibited activation of the NF-κB pathway. Compound 14r reduced LPS-induced pulmonary inflammation and ALI in mice. It showed significant protective effects against LPS-induced ALI in mice and significantly reduced levels of proinflammatory cytokines in serum and bronchoalveolar lavage fluid. The ratio of wet weight to dry weight of lung tissue was normalized by compound 14r, which was consistent with suppression of neutrophil infiltration and production of proinflammatory cytokines. Compound 14r reduced the mRNA expression of some proinflammatory cytokines, improved histopathologic changes, and reduced macrophage infiltration in lung tissues. Collectively, these results suggest a new series of LCA derivatives that could be promising anti-inflammatory agents for ALI treatment.

Effect of Cyclosporin H on ischemic injury and neutrophil infiltration in cerebral infarct model of rats via PET imaging.

Brain ischemia-reperfusion injury is a complex process, and neuroinflammation is an important secondary contributing pathological event. Neutrophils play major roles in ischemic neuroinflammation. Once activated, neutrophils express formyl peptide receptors (FPRs), which are special receptors of a class of chemoattractants and may be potential targets to regulate the activity of neutrophils and control cerebral ischemic injury. This study was aimed to explore the ameliorating effect of Cyclosporin H (CsH), a potent FPR antagonist, on brain ischemic injury by inhibiting the activation and migration of neutrophils, and improving cerebral blood flow. We employed a middle cerebral artery occlusion (MCAO) Model on rats and performed behavioral, morphological, and microPET imaging assays to investigate the potential restoring efficacy of CsH on cerebral ischemic damages. Peptide N-cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF), an antagonist to the neutrophil FPR with a high binding affinity, was used for imaging neutrophil distribution. We found that CsH had similar effect with edaravone on improving the neurobehavioral deficient symptoms after cerebral ischemia-reperfusion, and treatment with CsH also alleviated ischemic cerebral infarction. Compared with the MCAO Model group, [18F]FDG uptake ratios of the CsH and edaravone treatment groups were significantly higher. The CsH-treated groups also showed significant increases in [18F]FDG uptake at 144h when compared with that of 24h. This result indicates that like edaravone, treatment with both doses of CsH promoted the recovery of blood supply after cerebral ischemic event. Moreover, MCAO-induced cerebral ischemia significantly increased the radiouptake of [68Ga]Ga-cFLFLF at 72h after ischemia-reperfusion operation. Compared with MCAO Model group, radiouptake values of [68Ga]-cFLFLF in both doses of CsH and edaravone groups were all decreased significantly. These results showed that both doses of CsH resulted in a similar therapeutic effect with edaravone on inhibiting neutrophil infiltration in cerebral infarction. Potent FPR antagonist CsH is promisingly beneficial in attenuating neuroinflammation and improving neurobehavioral function against cerebral infarction. Therefore, FPR may become a novel target for regulating neuroinflammation and improving prognosis for ischemic cerebrovascular disorders.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive. Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Breviscapine Reduces Sepsis-Induced Acute Lung Injury by Targeting CASP8 to Regulate Neutrophil Apoptosis and Inflammation.

Breviscapine has been demonstrated to have beneficial effects in ameliorating acute lung injury (ALI), yet its potential therapeutic value and molecular mechanisms in sepsis-induced ALI remain unexplored. We utilized network pharmacology approach to identify the potential targets and mechanisms of breviscapine in treating sepsis-induced ALI. To construct a murine model of sepsis, we performed cecal ligation and puncture (CLP). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) were employed to respectively determine the pathologic changes and levels of inflammatory factors. Neutrophil count and total protein level in bronchoalveolar lavage fluid (BALF) were detected by corresponding kit. Additionally, we utilized flow cytometry, immunofluorescence, Western blotting, and real-time reverse transcription PCR (qRT-PCR) to detect cell apoptosis, protein expression, and gene expression. Finally, we used ELISA kits to detect the activity of myeloperoxidase (MPO) and caspase-8 (CASP8). Breviscapine was revealed to target 81 potential proteins in the treatment of sepsis-induced ALI, while CASP8 was the most important one as demonstrated by network analysis. In vivo experiments demonstrated that breviscapine effectively reduced the severity of sepsis-induced ALI and inflammation, and significantly suppressed neutrophil infiltration in the lung tissues of CLP mice and promoted neutrophil apoptosis in the peripheral blood. In vitro experiments revealed that lipopolysaccharide (LPS)-induced neutrophil apoptosis was inhibited, and the expression and activity of CASP8 were down-regulated. Breviscapine intervention markedly up-regulated the expression and activity of CASP8, consequently activating neutrophil apoptosis and inhibiting inflammatory response by activating the NF-κB signaling pathway. Breviscapine is remarkably effective in improving sepsis-induced ALI, and its mechanism of action may be to induce neutrophil apoptosis, inhibit inflammatory overreaction and reduce its infiltration in pulmonary tissues by up-regulating the expression and activity of CASP8.

Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway

PurposeThe purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. MethodsIn order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit-lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were determined using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and IF techniques. ResultsSchaftoside at a concentration of 160 μM displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1β, TNF-α, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. ConclusionsSchaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.

Gilaburu (Viburnum opulus L.) fruit extract has potential therapeutic and prophylactic role in a rat model of acetic acid-induced oxidant colonic damage

Ethnopharmacological relevanceUlcerative colitis (UC) which has a global impact on the health care system with its recurrent and incompletely curable characteristics, affects the patients’ quality of life. Gilaburu (GB; Viburnum opulus L.) is a fruit with rich polyphenol ingredient which is used ethnobotanically in Türkiye for medicinal purposes (for example, to pass kidney stones, to treat stomach, heart, and liver diseases, hemorrhages, hypertension, ulcers, common cold, tuberculosis, rheumatic and menstrual pain, and diabetes). On the other hand, the effects of GB in the experimental UC model have not been studied. Aim of the studyThis study aimed to explore the potential antioxidant and anti-inflammatory effects of GB fruit extract in improving acetic acid (AA)-induced UC. Materials and methodsStarting immediately after (AA + GB group) or 1 week before (GB + AA + GB group) the colitis induced by intrarectal AA (5%; v/v) administration, the rats orally received GB (100 mg/kg) once per day for 3 days. The control and AA groups were administered orally saline (1 ml), while the AA + SS group were administered sulfasalazine (SS; 100 mg/kg; orally) as a positive control once per day for 3 days. Distal colonic tissue specimens were obtained for the histological and biochemical [myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), chemiluminescence (CL), caspase-3, 8-hydroxy-2’-deoxyguanosine (8-OHdG), matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β1, smad-3 and cytokine (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ), measurements] evaluations on the 3rd day. ResultsElevated macroscopic and microscopic damage scores, high tissue wet weight values, increased tissue-associated MPO, MDA, CL, caspase-3, 8-OHdG, cytokines (TNF-α, IL-1β, IL-6, IL-8), MMP-9, TGF-β1, smad-3 levels, and decreased GSH values of the AA group were all reversed by GB treatments (AA + GB and GB + AA + GB groups) (p < 0.05–0.001). However, sulfasalazine treatment (AA + SS group) did not change the IL-8, 8-OHdG, MMP-9, and TGF-β1 measurements significantly. ConclusionsGilaburu shows both anti-inflammatory and antioxidant effects against AA-induced colonic damage by suppressing neutrophil infiltration, regulating inflammatory mediators, inhibiting reactive species production, lipid peroxidation, and apoptosis, conserving endogenous antioxidant glutathione, and ameliorating oxidative DNA damage. Since the current ulcerative colitis drugs display limited benefits and adverse side effects, potential therapeutic and/or prophylactic role of gilaburu can be evaluated in ulcerative colitis.

Targeting eIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response.

To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis. The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid-Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting. In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid-Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1β. According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1β expression; and provided a potential treatment for fungal keratitis. This study provides a potential treatment for fungal keratitis in the clinic.

IL4I1-catalyzed tryptophan metabolites mediate the anti-inflammatory function of cytokine-primed human muscle stem cells

Muscle stem cells (MuSCs) have been demonstrated to exert impressive therapeutic efficacy in disease settings through orchestrating inflammatory microenvironments. Nevertheless, the mechanisms underlying the immunoregulatory property of MuSCs remain largely uncharacterized. Here, we showed that interleukin-4-induced-1 (IL4I1), an essential enzyme that catalyzes indole metabolism in humans, was highly expressed in human MuSCs exposed to IFN-γ and TNF-α. Functionally, the MuSCs were found to inhibit the infiltration of neutrophils into sites of inflammation in a IL4I1-dependent manner and thus ameliorate acute lung injury in mice. Mechanistically, the indole metabolites, including indole-3-pyruvic acid (I3P) and indole-3-aldehyde (I3A), produced by IL4I1, acted as ligands to activate aryl hydrocarbon receptor (AHR), leading to augmented expression of TNF-stimulated gene 6 (TSG-6) in inflammatory cytokine-primed MuSCs. Furthermore, I3P administration alone suppressed neutrophil infiltration into damaged lungs. I3P could also reduce the level of reactive oxygen species in neutrophils. Therefore, our study has uncovered a novel mechanism by which MuSCs acquire their immunoregulatory property and may help to develop or optimize MuSC-based therapies for inflammatory diseases.

Flavin adenine dinucleotide (FADH) protects the preterm lung from hyperoxic injury

Background: Bronchopulmonary dysplasia (BPD) is a severe oxidative-stress induced lung disease that primarily affects premature infants requiring long term oxygen supplementation. BPD is characterized by attenuated lung development, fibrosis, inflammation, and airway smooth muscle hyperplasia. Our research objective was to test a novel hypothesis that evaluates whether providing flavin adenine dinucleotide (FADH) can reduce oxidative stress and prevent high oxygen induced lung injury using a mouse model for BPD. FADH is a cofactor for glutathione reductase (GR) enzymatic activity, responsible for recycling the bioavailability of the antioxidant glutathione (GSH) under oxidizing conditions. Methods: We used a hyperoxia-induced lung injury term mouse model (0.85 fraction inspired oxygen; FiO 2 ) to recapitulate the BPD phenotype for experimental study. Briefly, newborn mice were housed in 0.85 FiO 2 (hyperoxic) or 21% O 2 (normoxic) conditions from birth until post-natal day (PN) 14. Following O 2 exposures, FADH (7 μM) or vehicle (saline) treatments were insufflated daily on PN 14-21 (1.5μL/g body weight). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected and evaluated for redox stress, lung injury/alveolar development, and inflammation using commercially available GSH Assay kits (Abcam), standard immunohistochemistry, and LEGENDplex inflammation panels (Biolegend), respectively. Male and female mice were randomly assigned to study groups. Single data comparisons were performed using paired Student’s t-tests. Multiple comparisons were performed using one-way analysis of variance followed by post-hoc testing in Sigma Plot (SPSS Inc). Results: Daily insufflation of FADH improved neonatal lung health following chronic 0.85 FiO 2 exposure, as indicated by measured BALF GSH/GSSG redox potentials (E h ). E h = - 179.10 mV ± 1.85 mV in the FADH treated groups (n=5), wherein vehicle treated lungs were more oxidatively stressed with BALF E h = - 168.77 mV ± 3.64 mV (n=3 independent litters; P=.02). Morphometric analysis of lung development and lung injury scores were significantly improved in 0.85 FiO 2 exposed pups receiving FADH treatment vs. vehicle control pups ( P&lt;.001; n=3 litters). FADH treatments also suppressed neutrophil infiltration (n=10; P&lt;.001) with significant increase in IL-12p70 ( P&lt;.05) and TNF-α ( P&lt;.05; n=3 litters) in hyperoxic lungs. Conclusions: FADH protects the preterm lung from hyperoxic injury, albeit the mechanisms remain unclear. Importantly, we show that FADH increases the bioavailability of the antioxidant GSH, as well as IL12-p70 and TNF-α. The signaling molecules identified have known anti- and pro-inflammatory effects in the lungs and will therefore be explored in future studies as potential therapeutic targets involved in the pathogenesis of high-oxygen induced lung injury. Supported by T35DK103596 (HM) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactobacillus paracasei ATG-E1 improves particulate matter 10 plus diesel exhaust particles (PM10D)-induced airway inflammation by regulating immune responses.

Particulate matter (PM) exposure can adversely affect respiratory function. Probiotics can alleviate the inflammatory responses in respiratory diseases. We examined the protective effects of Lactobacillus paracasei ATG-E1 isolated from the feces of a newborn baby against airway inflammation in a PM10 plus diesel exhaust particle (DEP) (PM10D)-induced airway inflammation model. BALB/c mice were exposed to PM10D by intranasal injection three times at 3-day intervals for 12 days, and L. paracasei ATG-E1 was administered orally for 12 days. Analysis of immune cell population and expression of various inflammatory mediators and gut barrier-related genes were determined in bronchoalveolar lavage fluid (BALF), lung, peyer's patch, and small intestine. A histological analysis of the lungs was performed. In addition, the in vitro safety and their safety in genomic analyses were examined. L. paracasei ATG-E1 was found to be safe in vitro and by genomic analysis. L. paracasei ATG-E1 suppressed neutrophil infiltration and the number of CD4+, CD4+CD69+, CD62L-CD44+high, CD21/35+B220+, and Gr-1+CD11b+ cells, as well as the expression of inflammatory mediators, including chemokine (C-X-C motif) ligand (CXCL)-1, macrophage inflammatory protein (MIP)-2, interleukin (IL)-17a, tumor necrosis factor (TNF)-α, and IL-6 in BALF and lungs in PM10D-induced airway inflammation. It protected against histopathological damage in the lungs of mice with PM10D-induced airway inflammation. L. paracasei ATG-E1 concomitantly increased the expression levels of the gut barrier function-related genes occludin, claudin-1, and IL-10 in the small intestine, with an increased number of CD4+ and CD4+CD25+ immune cells in the peyer's patch. L. paracasei ATG-E1 suppressed immune activation and airway inflammatory responses in the airways and lungs by restoring the lung damage by PM10D. It also regulated intestinal immunity and ameliorated the gut barrier function in the ileum. These results indicate the potential of L. paracasei ATG-E1 as an protective and therapeutic agent against airway inflammation and respiratory diseases.

Protective effects of resolvin D1 in Pseudomonas aeruginosa keratitis

PurposeHere, we explored the protective effects of resolvin D1 (RvD1) in Pseudomonas aeruginosa (PA) keratitis. MethodsC57BL/6 (B6) mice were used as an animal model of PA keratitis. Plate counting and clinical scores were used to assess the severity of the infection and the therapeutic effects of RvD1 in the model. Myeloperoxidase assay was used to detect neutrophil infiltration and activity. Quantitative PCR (qPCR) was used to examine the expression of proflammatory and anti-inflammatory mediators. Immunofluorescence staining and qPCR were performed to identify macrophage polarization. ResultsRvD1 treatment alleviated PA keratitis severity by decreasing corneal bacterial load and inhibiting neutrophil infiltration in the mouse model. Furthermore, RvD1 treatment decreased mRNA levels of TNF-α, IFN-γ, IL-1β, CXCL1, and S100A8/9 while increasing those of IL-1RA, IL-10, and TGF-β1. RvD1 treatment also reduced the aggregation of M1 macrophages and increased that of M2 macrophages. RvD1 provided an auxiliary effect in gatifloxacin-treated mice with PA keratitis. ConclusionBased on these findings, RvD1 may improve the prognosis of PA keratitis by inhibiting neutrophil recruitment and activity, dampening the inflammatory response, and promoting M2 macrophage polarization. Thus, RvD1 may be a potential complementary therapy for PA keratitis.

Abstract 1268: The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus

Abstract Background: Although metformin (Met) is well known to suppress tumor growth, the detailed mechanisms of its anti-tumor effect remain unknown. In recent years, it has been pointed out that tumor-associated neutrophils (TAN) can produce neutrophil extracellular traps (NETs) in the tumor microenvironment (TME) which can tumor invasion and metastasis. Here, in this study, we investigated the effect of Met on tumor-associated NETs and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who received curative surgery in Jichi Medical University Hospital and asked the impact of Met intake on their outcome. In addition, we performed multicolor immunohistochemistry to examine the phenotypes of tumor-infiltrating lymphocytes (TIL), TAN and NETs in surgically removed tissues specimens in 40 (colorectal cancer) CRC patients who had taken Met before surgery and propensity score-matched 40 patients who had not. Results: A total of 1858 patients underwent curative colectomy from April 2010 to March 2021. Among them, 283 patients had T2DM at the time of surgery, and 62 patients had been treated with drugs including Met. The postoperative disease-free survival rate (DFS) was significantly improved in the Met intake group compared with non-intake group (HR=0.21, p&amp;lt;0.01). The number of CD66b(+) TANs significantly decreased in Met-intake group (Median(M)=169 (36-553)/mm2 vs 99 (34-322)/mm2, p&amp;lt;0.01). The number of tumor-associated NETs defined as CD66b(+) cit-H3(+) cells significantly decreased in Met-intake group (M=36 (8-272)/mm2 vs 20 (0-104)/mm2, p&amp;lt;0.01), and tumor-associated NETs/TANs ratio significantly decreased in Met-intake group (29.9% vs 19.8%, p&amp;lt;0.01). The number of CD3(+) TILs significantly increased in Met intake group (M=122 (28-257)/mm2 vs 142 (48-386)/mm2, p&amp;lt;0.05). The number of CD8(+) TILs significantly increased in Met-intake group (M=60 (14-180)/mm2 vs 99 (52-318)/mm2, p&amp;lt;0.01), and CD8/CD3 ratio significantly increased in Met-intake group (51.2% vs 72.1%, p&amp;lt;0.01). In addition, there was a negative correlation between TANs or tumor-associated NETs and CD8 positive TILs in all 80 patients (r=-0.26, p&amp;lt;0.05). Conclusion: Met can inhibit neutrophil infiltration and NETs formation while promote the infiltration of cytotoxic T cells in TME, which may lead to the improvement of the outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Rie Kawashima, Hideyo Miyato, Joji Kitayama, Naohiro Sata. The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1268.