Excessive Neutrophil Infiltration Research Articles

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol treatment inhibits abnormal tumor growth by regulating neutrophil infiltration in a non-small cell lung carcinoma mouse model

Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment.In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97 % reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30 %.In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues.

Mechanism of Jizhi syrup’s prevention and treatment of acute bronchitis based on LPS-iNOS inflammatory mediators’ signalling

Ethnopharmacological relevanceJizhi syrup (JZTJ) is composed of eight medicinal herbs, including Houttuynia cordata, Fagopyrum dibotrys, Ilex chinensis, Ephedra sinica, Aster tataricus, Peucedanum praeruptorum, Citrus aurantium and Glycyrrhiza uralensis. It is mainly used for coughing caused by exogenous wind heat. Symptoms include fever, aversion to cold, chest and diaphragm tightness, cough and sore throat; and acute bronchitis and acute exacerbation of chronic bronchitis with the above symptoms. PurposeThis study aimed to preliminary analyse the chemical components in the liposoluble part of JZTJ, evaluate the anti-inflammatory effect of JZTJ by using six animal and cell models and predict the target and mechanism of acute bronchitis prevention and treatment with JZTJ. MethodsThe chemical components in the liposoluble fraction of JZTJ (extracted by cyclohexane) were quantitatively analysed using gas chromatography-mass spectrometry (GC-MS). Classic non-specific inflammation models and acute bronchitis models were established to systematically evaluate the anti-inflammatory effect of JZTJ. The anti-inflammatory intensity and characteristics of three doses of JZTJ were comprehensively compared on the basis of principal component analysis method at the cellular and overall animal levels. By using lipopolysaccharides (LPSs) as modelling factors, a RAW264.7 macrophage inflammatory response model and a rat acute bronchitis model were created to study the effect of JZTJ on the in-vitro and - vivo LPS-iNOS-inflammatory mediators’ inflammatory signalling pathway to reveal the mechanism of acute bronchitis prevention and treatment by JZTJ at the levels of genes, proteins, and inflammatory mediators. ResultsSeventeen alkane and ester compounds were preliminarily qualitatively identified from the lipid soluble fraction of JZTJ: dibutyl phthalate, tetradecane, ridecane, n-hexadecanoic acid, pentadecane, n-decanoic acid, 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23-hexamethyl-(all-E)-; phenol, 2,2ʹ-methylenebis[6-(1,1-dimethylethyl)-4-methyl-; hexadecane. JZTJ has a significant inhibitory effect on acute non-specific inflammation, specifically inhibiting ‘xylene-induced ear swelling in mice’, ‘acetic acid-induced increased permeability of abdominal capillaries in mice’ and ‘egg white-induced foot swelling in rats’. The above effects are most evident in high doses, followed by medium doses, whereas low doses have poorer or no effects. JZTJ can prevent and treat acute bronchitis induced by LPS in mice and rats, significantly improve the pathological changes in patchy interstitial and alveolar bleeding with excessive neutrophil infiltration and inhibit the release of inflammatory mediators by LPS-induced RAW264.7 macrophages. Its mechanism of action may be by downregulating the phosphorylation level of p-ERK1/2 protein, thereby inhibiting inducible nitric oxide synthase (iNOS) mRNA, tumour necrosis factor (TNF)-α mRNA and IL-1β. The expression levels of genes, such as mRNA and IL-6 mRNA, thereby reducing iNOS, TNF-α and IL-1β. The expression of proteins in the cytoplasm of lung and bronchial tissue cells reduced the release of downstream inflammatory mediators NO and IL-6. ConclusionPreliminary analysis of the chemical components in the lipid soluble fraction of JZTJ can lay the foundation for subsequent research on its effective components. Evaluating the anti-inflammatory effect of JZTJ is helpful for further research on its mechanism of action. The anti-inflammatory effects are exerted by regulating the inflammatory signalling pathway of LPS-iNOS inflammatory mediators, providing a scientific basis for their clinical application.

Necrosis drives susceptibility to Mycobacterium tuberculosis in POLG mtDNA mutator mice.

The genetic and molecular determinants that underlie the heterogeneity of Mycobacterium tuberculosis (Mtb) infection outcomes in humans are poorly understood. Multiple lines of evidence demonstrate that mitochondrial dysfunction can exacerbate mycobacterial disease severity and mutations in some mitochondrial genes confer susceptibility to mycobacterial infection in humans. Here, we report that mutations in mitochondria DNA (mtDNA) polymerase gamma (POLG) potentiate susceptibility to Mtb infection in mice. POLG mutator mtDNA mice fail to mount a protective innate immune response at an early infection timepoint, evidenced by high bacterial burdens, reduced M1 macrophages, and excessive neutrophil infiltration in the lungs. Immunohistochemistry reveals signs of enhanced necrosis in the lungs of Mtb-infected POLG mice and POLG mutator macrophages are hyper-susceptible to extrinsic triggers of necroptosis ex vivo. By assigning a role for mtDNA mutations in driving necrosis during Mtb infection, this work further highlights the requirement for mitochondrial homeostasis in mounting balanced immune responses to Mtb.

Lipid mediators in neutrophil biology: inflammation, resolution and beyond.

Acute inflammation is the body's first defense in response to pathogens or injury. Failure to efficiently resolve the inflammatory insult can severely affect tissue homeostasis, leading to chronic inflammation. Neutrophils play a pivotal role in eradicating infectious pathogens, orchestrating the initiation and resolution of acute inflammation, and maintaining physiological functions. The resolution of inflammation is a highly orchestrated biochemical process, partially modulated by a novel class of endogenous lipid mediators known as specialized pro-resolving mediators (SPMs). SPMs mediate their potent bioactions via activating specific cell-surface G protein-coupled receptors (GPCR). This review focuses on recent advances in understanding the multifaceted functions of SPMs, detailing their roles in expediting neutrophil apoptosis, promoting clearance by macrophages, regulating their excessive infiltration at inflammation sites, orchestrating bone marrow deployment, also enhances neutrophil phagocytosis and tissue repair mechanisms under both physiological and pathological conditions. We also focus on the novel role of SPMs in regulating bone marrow neutrophil functions, differentiation, and highlight open questions about SPMs' functions in neutrophil heterogeneity. SPMs play a pivotal role in mitigating excessive neutrophil infiltration and hyperactivity within pathological milieus, notably in conditions such as sepsis, cardiovascular disease, ischemic events, and cancer. This significant function highlights SPMs as promising therapeutic agents in the management of both acute and chronic inflammatory disorders.

The role of the interleukin-36 axis in generalized pustular psoriasis: a review of the mechanism of action of spesolimab.

Generalized pustular psoriasis (GPP) is a rare, chronic, inflammatory skin disorder characterized by recurrent flares associated with skin erythema, desquamation, and widespread superficial sterile pustules, which may be severe ("lakes of pus"). Systemic symptoms are often present, including malaise, fever, and skin pain. In GPP, innate immune responses are driven by abnormal activation of the interleukin (IL)-36-chemokine-neutrophil axis and excessive neutrophil infiltration. This review highlights the IL-36 pathway in the context of the IL-1 superfamily and describes how unopposed IL-36 signaling can lead to the development of GPP. Targeted inhibition of the IL-36 receptor (IL-36R) is an attractive therapeutic strategy in the treatment of GPP, including flare prevention and sustained disease control. Spesolimab is a first-in-class, humanized, monoclonal antibody that binds specifically to the IL-36R and antagonizes IL-36 signaling. Spesolimab was approved by the US Food and Drug Administration in September 2022 to treat GPP flares in adults and was subsequently approved for GPP flare treatment in other countries across the world. Anti-IL-36R therapy, such as spesolimab, can mitigate flares and address flare prevention in GPP, presumably through rebalancing IL-36 signaling and modulating the pro-inflammatory response of the downstream effectors.

The bidirectional effect of neutrophils on periodontitis model in mice: A systematic review.

To evaluate the regulatory role of neutrophils as the first line of host immune defense in the periodontal microenvironment of mice. A systematic search was performed using PubMed, Web of Science, and ScienceDirect databases for articles published between 2012 and 2023. In this review, articles investigating the effect of neutrophils on alveolar bone resorption in a mouse model of periodontitis were selected and evaluated according to eligibility criteria. Important variables that may influence outcomes were analyzed. Eleven articles were included in this systematic review. The results showed that because of their immune defense functions, the functional homeostasis of local neutrophils is critical for periodontal health. Neutrophil deficiency aggravates alveolar bone loss. However, several studies have shown that excessive neutrophil infiltration is positively correlated with alveolar bone resorption caused by periodontitis in mice. Therefore, the homeostasis of neutrophil function needs to be considered in the treatment of periodontitis. Pooled analysis suggests that neutrophils play a bidirectional role in periodontal tissue remodeling in mouse periodontitis models. Therefore, targeted regulation of local neutrophil function provides a novel strategy for the treatment of periodontitis.

Leptin/obR signaling exacerbates obesity-related neutrophilic airway inflammation through inflammatory M1 macrophages

BackgroundObesity-related asthma is a kind of nonallergic asthma with excessive neutrophil infiltration in the airways. However, the underlying mechanisms have been poorly elucidated. Among the adipokines related to obesity, leptin is related to the inflammatory response. However, little is understood about how leptin acts on the leptin receptor (obR) in neutrophilic airway inflammation in obesity-associated asthma. We explored the inflammatory effects of leptin/obR signaling in an obesity-related neutrophilic airway inflammation mouse model.MethodsWe established a neutrophilic airway inflammation mouse model using lipopolysaccharide (LPS)/ovalbumin (OVA) sensitization and OVA challenge (LPS + OVA/OVA) in lean, obese, or db/db (obR deficiency) female mice. Histopathological, bronchoalveolar lavage fluid (BALF) inflammatory cell, and lung inflammatory cytokine analyses were used to analyze airway inflammation severity. Western blotting, flow cytometry, reverse transcription‐polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the underlying mechanisms. In vitro bone marrow‐derived macrophage (BMDM) and bone marrow-derived neutrophil experiments were performed.ResultsWe found that the serum leptin level was higher in obese than in lean female mice. Compared to LPS/OVA + OVA-treated lean female mice, LPS/OVA + OVA-treated obese female mice had higher peribronchial inflammation levels, neutrophil counts, Th1/Th17-related inflammatory cytokine levels, M1 macrophage polarization levels, and long isoform obR activation, which could be decreased by the obR blockade (Allo-Aca) or obR deficiency, suggesting a critical role of leptin/obR signaling in the pathogenesis of obesity-related neutrophilic airway inflammation in female mice. In in vitro experiments, leptin synergized with LPS/IFN-γ to promote the phosphorylation of the long isoform obR and JNK/STAT3/AKT signaling pathway members to increase M1 macrophage polarization, which was reversed by Allo-Aca. Moreover, leptin/obR-mediated M1 macrophage activity significantly elevated CXCL2 production and neutrophil recruitment by regulating the JNK/STAT3/AKT pathways. In clinical studies, obese patients with asthma had higher serum leptin levels and M1 macrophage polarization levels in induced sputum than non-obese patients with asthma. Serum leptin levels were positively correlated with M1 macrophage polarization levels in patients with asthma.ConclusionsOur results demonstrate leptin/obR signaling plays an important role in the pathogenesis of obesity-related neutrophilic airway inflammation in females by promoting M1 macrophage polarization.Graphical abstract

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury

AbstractAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif–coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI–treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet–neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI–treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with β2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). Esculin inhibits β2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

Hepatic C-X-C chemokine receptor type 6-expressing innate lymphocytes limit detrimental myeloid hyperactivation in acute liver injury.

Acute liver failure (ALF) is characterized by rapid clinical deterioration and high mortality. Acetaminophen (APAP or paracetamol) overdose is a leading cause of ALF, resulting in hepatocellular necrosis with subsequent inflammation, inflicting further liver damage. Infiltrating myeloid cells are early drivers of liver inflammation. However, the role of the abundant population of liver-resident innate lymphocytes, which commonly express the chemokine receptor CXCR6, is incompletely understood in ALF. We investigated the role of CXCR6-expressing innate lymphocytes using the model of acute APAP toxicity in mice deficient in CXCR6 (Cxcr6gfp/gfp). APAP-induced liver injury was strongly aggravated in Cxcr6gfp/gfp mice compared with wild-type counterparts. Immunophenotyping using flow cytometry revealed a reduction in liver CD4+T cells, natural killer (NK) cells, and most prominently, NKT cells, whereas CXCR6 was dispensable for CD8+ T-cell accumulation. CXCR6-deficient mice exhibited excessive neutrophil and inflammatory macrophage infiltration. Intravital microscopy revealed dense cellular clusters of neutrophils in necrotic liver tissue, with higher numbers of clustering neutrophils in Cxcr6gfp/gfp mice. Gene expression analysis linked hyperinflammation in CXCR6 deficiency to increased IL-17 signaling. Although reduced in overall numbers, CXCR6-deficient mice had a shift in NKT cell subsets with increased RORγt-expressing NKT17 cells as a likely source of IL-17. In patients with ALF, we found a prominent accumulation of IL-17-expressing cells. Accordingly, CXCR6-deficient mice lacking IL-17 (Cxcr6gfp/gfpx Il17-/-) had ameliorated liver damage and reduced inflammatory myeloid infiltrates. Our study identifies a crucial role of CXCR6-expressing liver innate lymphocytes as orchestrators in acute liver injury containing IL-17-mediated myeloid cell infiltration. Hence, strengthening the CXCR6-axis or downstream inhibition of IL-17 could yield novel therapeutics in ALF.

Systematic analysis of myocardial immune progression in septic cardiomyopathy: Immune-related mechanisms in septic cardiomyopathy.

The immune infiltration and molecular mechanisms underlying septic cardiomyopathy (SC) have not been completely elucidated. This study aimed to identify key genes related to SC and elucidate the potential molecular mechanisms. The weighted correlation network analysis (WGCNA), linear models for microarray analysis (LIMMA), protein-protein interaction (PPI) network, CIBERSORT, Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), and gene set enrichment analysis (GSEA) were applied to assess the key pathway and hub genes involved in SC. We identified 10 hub genes, namely, LRG1, LCN2, PTX3, E LANE, TCN1, CLEC4D, FPR2, MCEMP1, CEACAM8, and CD177. Furthermore, we used GSEA for all genes and online tools to explore the function of the hub genes. Finally, we took the intersection between differential expression genes (DEGs) and hub genes to identify LCN2 and PTX3 as key genes. We found that immune-related pathways played vital roles in SC. LCN2 and PTX3 were key genes in SC progression, which mainly showed an anti-inflammatory effect. The significant immune cells in cardiomyocytes of SC were neutrophils and M2 macrophages. These cells may have the potential to be prognostic and therapeutic targets in the clinical management of SC. Excessive anti-inflammatory function and neutrophil infiltration are probably the primary causes of SC.

32P Tumor growth inhibition effect of PLAG by regulation of neutrophil infiltration in ICI insensitivity B16F10 melanoma

Malignant melanoma (MM) is a representative tumor with a low survival rate due to its early diagnosis difficulty and poor treatment efficacy. Melanoma usually promotes its growth via tumor-infiltrating neutrophils (TINs), which inhibit the activation of immune cells that prevent cancer cells such as cytotoxic T lymphocytes. Therefore, the best option for MM treatment is to prevent excessive neutrophil infiltration into cancer lessons.

Revealing key regulators of neutrophil function during inflammation by re-analysing single-cell RNA-seq.

Excessive neutrophil infiltration and dysfunction contribute to the progression and severity of hyper-inflammatory syndrome, such as in severe COVID19. In the current study, we re-analysed published scRNA-seq datasets of mouse and human neutrophils to classify and compare the transcriptional regulatory networks underlying neutrophil differentiation and inflammatory responses. Distinct sets of TF modules regulate neutrophil maturation, function, and inflammatory responses under the steady state and inflammatory conditions. In COVID19 patients, neutrophil activation was associated with the selective activation of inflammation-specific TF modules. SARS-CoV-2 RNA-positive neutrophils showed a higher expression of type I interferon response TF IRF7. Furthermore, IRF7 expression was abundant in neutrophils from severe patients in progression stage. Neutrophil-mediated inflammatory responses positively correlate with the expressional level of IRF7. Based on these results, we suggest that differential activation of activation-related TFs, such as IRF7 mediate neutrophil inflammatory responses during inflammation.

High-Dose Mycobacterium tuberculosis H37rv Infection in IL-17A- and IL-17A/F-Deficient Mice.

During experimental tuberculosis (TB), interleukin (IL)-17A appears to be involved in the formation of lung granulomas, possibly through the attraction of neutrophils to the sites of infection. However, the protective impact of cytokine appears to depend on the degree of its induction. Hence, robust production of IL-17A in mice infected with the hypervirulent isolate Mycobacterium tuberculosis (Mtb) HN878 mediates protection, while the cytokine is dispensable for protective immune responses against low-dose infection with the less virulent strain H37rv. Here, we show that after experimental infection with high doses of Mtb H37rv, IL-17A-deficient (−/−) mice exhibited high susceptibility to the infection, which was mediated by the strong accumulation of neutrophils in the infected lung tissue. Accordingly, we observed nearly unrestricted bacterial replication within the neutrophils, indicating that they may serve as a survival niche for Mtb. By use of IL-17A/IL-17F-double-deficient mice, we demonstrated that the susceptibility in the absence of IL-17A is mediated by a compensatory expression of IL-17F, which, however, appeared not to be dependent on neutrophils. Together, our results illustrate the compensatory potential of the Th17-secreted cytokines IL-17A and IL-17F in the context of experimental TB and once again emphasize the detrimental effect of excessive neutrophil infiltration in response to Mtb.

Multimodule characterization of immune subgroups in intrahepatic cholangiocarcinoma reveals distinct therapeutic vulnerabilities

BackgroundImmune microenvironment is well recognized as a critical regulator across cancer types, despite its complex roles in different disease conditions. Intrahepatic cholangiocarcinoma (iCCA) is characterized by a tumor-reactive milieu, emphasizing...

PLAG co-treatment increases the anticancer effect of Adriamycin and cyclophosphamide in a triple-negative breast cancer xenograft mouse model

Chemotherapy induces tumor cell death and inhibits tumor progression, but the accompanying immune responses in the surrounding dying tissue cause significant inflammation. These responses, such as excessive neutrophil infiltration into tumor tissue, are the main causes of resistance to anticancer treatment. The development of drugs that reduce neutrophil infiltration into tumors is necessary to increase the anticancer effect of chemotherapy.Here, we show that the antitumor effect of the chemotherapy AC regimen (Adriamycin and cyclophosphamide) was increased by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) cotreatment in the MDA-MB-231 triple-negative breast cancer xenograft mouse model. Tumor growth was inhibited up to 56% in mice treated with AC and inhibited up to 94% in mice cotreated with AC and PLAG. Side effects of chemotherapy, such as a reduction in body weight, were alleviated in mice cotreated with AC and PLAG. Excessive neutrophil infiltration caused by the AC regimen was successfully cleared in mice cotreated with AC and PLAG.We conclude that PLAG inhibits excessive neutrophil infiltration that aids tumor growth. Reduced neutrophils and increased lymphocytes in PLAG-treated mice can maximize the antitumor effect of the AC regimen and inhibit tumor growth.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001—is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide—a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Endothelial inflammation and neutrophil transmigration are modulated by extracellular matrix composition in an inflammation-on-a-chip model

Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development.

Abstract 13996: Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow is Essential for Myocardial Infarction-Induced Granulopoiesis

Background: Excessive infiltration of neutrophils to the ischemic heart after an acute myocardial infarction (MI) is mediated in-part by the S100A8/A9-NLRP3-IL-1β signaling axis in neutrophils. Despite upregulation of NLRP3 inflammasome signaling components in neutrophils, the serum levels of IL-1β, was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using parabiosis techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in one of the parabiotic mice and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, we studied the molecular mechanisms that govern reverse migration and retention of primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promotes granulopoiesis in the BM and introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited granulopoiesis in the non-infarcted parabionts. MI also promotes greater accumulation/ retention of inflammasome-primed neutrophils in the BM in a neutrophil CXCR4 (C-X-C-motif chemokine receptor 4) and serum S100A8/A9 dependent manner. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveals a new paradigm of how circulatory cells can deliver signaling molecules (e.g., IL-1β) directly to the site of action rather than through systemic release. The absolute requirement of signal-2 in the BM for IL-1β release is consistent with our model where neutrophils undergo programming in the infarcted heart and inertly return to the sites of production (i.e., BM) with cytokine cargo (i.e., IL-1β) to regulate granulopoiesis. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release.