Neutrophil Influx Research Articles

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Three Main SCFAs Mitigate Lung Inflammation and Tissue Remodeling Nlrp3-Dependent in Murine HDM-Induced Neutrophilic Asthma.

Neutrophilic asthma is generally defined by poorly controlled symptoms and high levels of neutrophils in the lungs. Short-chain fatty acids (SCFAs) are proposed as nonpharmacological therapy for allergic asthma, but their impact on the neutrophilic asthma lacks evidence. SCFAs regulate immune cell responses and impact the inflammasome NLRP3, a potential pharmacological target for neutrophilic asthma. Here, we explored the capacity of SCFAs to mitigate murine-induced neutrophilic asthma and the contribution of NLRP3 to this asthma. The objective of this study is to analyze whether SCFAs can attenuate lung inflammation and tissue remodeling in murine neutrophilic asthma and NLRP3 contribution to this endotype. Wild-type (WT) C57BL6 mice orotracheally received 10μg of HDM (house dust mite) in 80 μL of saline on days 0, 6-10. To explore SCFAs, each HDM group received 200mM acetate, propionate, or butyrate. To explore NLRP3, Nlrp3 KO mice received the same protocol of HDM. On the 14th day, after euthanasia, bronchoalveolar lavage fluid (BALF) and lungs were collected to evaluate cellularity, inflammatory cytokines, and tissue remodeling. HDM group had increased BALF neutrophil influx, TNF-α, IFN-γ, IL-17A, collagen deposition, and mucus secretion compared to control. SCFAs distinctively attenuate lung inflammation. Only features of tissue remodeling were Nlrp3-dependent such as collagen deposition, mucus secretion, active TGF-β cytokine, and IMs CD206+. SCFAs greatly decreased inflammatory cytokines and tissue remodeling. Only tissue remodeling was dependent on NLRP3. It reveals the potential of SCFAs to act as an additional therapy to mitigate neutrophilic asthma and the NLRP3 contribution to asthma.

Distinct roles for type I and type III interferons in virulent human metapneumovirus pathogenesis.

Human metapneumovirus (HMPV) is an important cause of acute lower respiratory infection in children and adults worldwide. There are four genetic subgroups of HMPV and both neutralizing antibodies and T cells contribute to protection. However, little is known about mechanisms of pathogenesis and most published work is based on a few extensively passaged, laboratory-adapted strains of HMPV. In this study, we isolated and characterized a panel of low passage HMPV clinical isolates representing all four genetic subgroups. The clinical isolates exhibited lower levels of in vitro replication compared to a lab-adapted strain. We compared disease phenotypes using a well-established mouse model. Several virulent isolates caused severe weight loss, lung pathology, airway dysfunction, and fatal disease in mice, which was confirmed in three inbred mouse strains. Disease severity did not correlate with lung viral titer, as virulent strains exhibited restricted replication in the lower airway. Virulent HMPV isolates were associated with markedly increased proinflammatory cytokine production and neutrophil influx; however, depletion of neutrophils or genetic ablation of inflammasome components did not reverse disease. Virulent clinical isolates induced markedly increased type I and type III interferon (IFN) secretion in vitro and in vivo. STAT1/2-deficient mice lacking both type I and type III IFN signaling showed reduced disease severity and increased lung viral replication. Inhibition of type I IFN signaling using a blocking antibody or genetic ablation of the type I IFN receptor reduced pathology with minimal effect on viral replication. Conversely, blockade of type III IFN signaling with a neutralizing antibody or genetic ablation of the IFN-lambda receptor had no effect on pathogenesis but restored viral replication. Collectively, these results demonstrate distinct roles for type I and type III IFN in HMPV pathogenesis and immunity.

Abstract A066: A novel approach for exocrine pancreas transcriptomics reveals the cellular landscape of the pancreas

Abstract Pancreatic acinar cells are responsible for producing large amounts of digestive enzymes, which are essential for nutrient breakdown but make it difficult to capture the transcriptome of individual cells. To address this challenge, we use a reversible fixative that is compatible with 10X Genomics, thereby preserving the acinar transcriptome and capturing cell composition more faithful to histology. Using this method, we transcriptionally characterize all cell types in the healthy mouse pancreas, and track how these populations change during acute pancreatitis and subsequent recovery. We identify immune cells in the healthy and inflamed pancreas at a level of detail previously unappreciated, characterizing T cell subsets including Th2, γδT, and regulatory T cells. Pancreatitis elicits an influx of neutrophils along with activation and proliferation of macrophages and dendritic cells. Observing significant transcriptional changes in acinar cells after pancreatitis, we define an Acinar to Ductal Metaplasia Index (ADMI), a gene signature that can be used to measure deviation from normal acinar identity in bulk or single cell RNA datasets. We corroborate ADMI using orthogonal GeoMx data, and validate its utility in independent published datasets. We are now expanding our study to perform transcriptomics using our fixation method on pancreatic cancer precursor lesions. Together, we aim to characterize cell types in the mouse pancreas under healthy, inflamed, and precursor conditions to establish a reference atlas for future research on the exocrine pancreas in healthy and diseased states. Citation Format: Katherine J. Aney, Woo-Jeong Jeong, Andres F. Vallejo, Ethan Chen, Austin Wang, Stephanie K. Dougan, Kellie Wise, Kirk Jensen, Luciano Martelotto, Sahar Nissim. A novel approach for exocrine pancreas transcriptomics reveals the cellular landscape of the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A066.

The critical role of neutrophil-endothelial cell interactions in sepsis: new synergistic approaches employing organ-on-chip, omics, immune cell phenotyping and in silico modeling to identify new therapeutics.

Sepsis is a global health concern accounting for more than 1 in 5 deaths worldwide. Sepsis is now defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis can develop from bacterial (gram negative or gram positive), fungal or viral (such as COVID) infections. However, therapeutics developed in animal models and traditional in vitro sepsis models have had little success in clinical trials, as these models have failed to fully replicate the underlying pathophysiology and heterogeneity of the disease. The current understanding is that the host response to sepsis is highly diverse among patients, and this heterogeneity impacts immune function and response to infection. Phenotyping immune function and classifying sepsis patients into specific endotypes is needed to develop a personalized treatment approach. Neutrophil-endothelium interactions play a critical role in sepsis progression, and increased neutrophil influx and endothelial barrier disruption have important roles in the early course of organ damage. Understanding the mechanism of neutrophil-endothelium interactions and how immune function impacts this interaction can help us better manage the disease and lead to the discovery of new diagnostic and prognosis tools for effective treatments. In this review, we will discuss the latest research exploring how in silico modeling of a synergistic combination of new organ-on-chip models incorporating human cells/tissue, omics analysis and clinical data from sepsis patients will allow us to identify relevant signaling pathways and characterize specific immune phenotypes in patients. Emerging technologies such as machine learning can then be leveraged to identify druggable therapeutic targets and relate them to immune phenotypes and underlying infectious agents. This synergistic approach can lead to the development of new therapeutics and the identification of FDA approved drugs that can be repurposed for the treatment of sepsis.

Imunoestimulação da resposta broncoalveolar de bezerros vacinados contra complexo respiratório bovino

ABSTRACT: Although intranasal bovine respiratory disease (BRD) vaccines containing live attenuated virus elicit greater stimulation of local humoral immunity response, they can mimic a viral infection, responsible for reducing innate defense during the establishment of vaccine-induced immunity. Probiotics containing Saccharomyces cerevisiae or Enterococcus faecium reduced the occurrence of BRD in neonatal calves challenged with the bovine respiratory syncytial virus (BRSV). Furthermore, the probiotics potentiated the humoral immune response after vaccination in murine models, raising the question of whether they could have the same effect in calves. This study aimed to verify if the probiotic containing E. faecium and S. cerevisiae attenuates the inflammation caused by the vaccine against BRD in the respiratory tract region in calves. Twenty-four healthy Jersey calves, aged 6 to 7 months old, were divided into the groups: control (C), supplemented (S), vaccinated (V), and supplemented and vaccinated (SV), with six animals in each of them. Supplemented groups (S and SV) received S. cerevisiae and E. faecium once a day on D-15 for 51 days (Probios precise®, Ouro Fino®, 2g/day/animal). Vaccinated groups (V and SV) received a single dose of an intranasal BRD vaccine on day 0 (Inforce®, Zoetis®, 1ml/ nostril). The control group was not supplemented or vaccined. Irritation of the respiratory tract and bronchoalveolar (BA) evaluations: cytology, phagocyte function, and IgA were measured on D-15, D3, D7, and D21. The vaccinated groups showed greater irritation of the nasopharynx and trachea. However, only Group V showed a reduction in BA phagocyte function and an increase in cellularity by a neutrophil influx in the BA region. Regarding IgA BA, SV showed the greatest increase, followed by S and V, concerning C. We conclude that isolated supplementation with E. faecium and S. cerevisiae promoted increased production of BA IgA. In association with the vaccine, the supplementation attenuated the inflammation of the respiratory tract produced by the vaccine itself, avoiding the reduction of phagocyte function BA, besides potentiating the humoral immune response of the vaccine containing live attenuated virus against BRD.

Intrahepatic neutrophil accumulation and extracellular trap formation are associated with posthepatectomy liver failure.

Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model. We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease. We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration. Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.

Helicobacter pylori-Induced Angiopoietin-Like 4 Promotes Gastric Bacterial Colonization and Gastritis.

Helicobacter pylori infection is characterized as progressive processes of bacterial persistence and chronic gastritis with features of infiltration of mononuclear cells more than granulocytes in gastric mucosa. Angiopoietin-like 4 (ANGPTL4) is considered a double-edged sword in inflammation-associated diseases, but its function and clinical relevance in H. pylori-associated pathology are unknown. Here, we demonstrate both pro-colonization and pro-inflammation roles of ANGPTL4 in H. pylori infection. Increased ANGPTL4 in the infected gastric mucosa was produced from gastric epithelial cells (GECs) synergistically induced by H. pylori and IL-17A in a cagA-dependent manner. Human gastric ANGPTL4 correlated with H. pylori colonization and the severity of gastritis, and mouse ANGPTL4 from non-bone marrow-derived cells promoted bacteria colonization and inflammation. Importantly, H. pylori colonization and inflammation were attenuated in Il17a -/-, Angptl4 -/-, and Il17a -/- Angptl4 -/- mice. Mechanistically, ANGPTL4 bound to integrin αV (ITGAV) on GECs to suppress CXCL1 production by inhibiting ERK, leading to decreased gastric influx of neutrophils, thereby promoting H. pylori colonization; ANGPTL4 also bound to ITGAV on monocytes to promote CCL5 production by activating PI3K-AKT-NF-κB, resulting in increased gastric influx of regulatory CD4+ T cells (Tregs) via CCL5-CCR4-dependent migration. In turn, ANGPTL4 induced Treg proliferation by binding to ITGAV to activate PI3K-AKT-NF-κB, promoting H. pylori-associated gastritis. Overall, we propose a model in which ANGPTL4 collectively ensures H. pylori persistence and promotes gastritis. Efforts to inhibit ANGPTL4-associated pathway may prove valuable strategies in treating H. pylori infection.

Effects of early treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) on the bronchoalveolar lavage proteome and oxylipids during bovine respiratory syncytial virus (BRSV) infection.

Non-steroidal anti-inflammatory drugs (NSAID) are not recommended for use against pneumonia in humans, but are commonly utilised against bovine respiratory disease. This study aimed to determine if the use of NSAIDs in the early phase of bovine respiratory syncytial virus (BRSV)-infection limits pulmonary inflammation. Four to nine-week old calves were infected with BRSV by aerosol and were treated with either meloxicam intravenously on day (D)4 (n = 5, MEL), acetylsalicylat-DL-lysin intravenously on D4 and D5 (n = 5, ASA), or were left untreated as controls (n = 5, CTR). Clinical signs were monitored daily until necropsy on D7, BRSV-RNA was detected in nasal swabs and bronchoalveolar lavage (BAL) by RT-qPCR, inflammatory cells and proteins were identified in BAL by cytology and label-free quantitative mass spectrometry-based proteomics, respectively, and oxylipids were quantified in BAL and plasma by liquid chromatography tandem mass spectrometry with triple quadrupole mass detectors. The calves developed mild to moderate signs of respiratory disease and, with the exception of one MEL-treated and one ASA-treated calf, limited lung lesions. None of the treatments had a significant effect on virus replication, clinical signs or lung lesion extent. Relative to controls, both treatments initially induced a downregulation of proteins in BAL. Immunoglobulin (Ig)-related proteins, such as the Ig kappa and lambda locus and the joining chain of IgA and IgM, were downregulated in MEL-treated calves compared to controls. In addition, meloxicam induced an increased neutrophil influx in BAL in response to BRSV, possibly related to a reduction in plasma prostaglandin, and to a downregulation of The Liver X Receptor/ Retinoid X Receptor (LXR/RXR), the Farnesoid X Receptor (FXR)/RXR and the 24-Dehydrocholesterol Reductase (DHC24) signalling pathways in the lung. The risk of NSAIDs to increase neutrophil activity during stimulation with BRSV or other toll-like receptor 4 agonists needs to be investigated further. Since augmented neutrophil responses can be detrimental, the results of the present study do not support the use of NSAIDs to prevent the clinical expression of BRSV-infection.

Mrgprb2-dependent Mast Cell Activation Plays a Crucial Role in Acute Colitis

Background & AimsMast cells (MCs) are typically found at mucosal surfaces, where their IgE-dependent activation plays a central role in allergic diseases. Over the past years, signaling through Mas-related G protein-coupled receptor b2 (Mrgprb2) in mice and MRGPRX2 in humans has gained a lot of interest as an alternative MC activation pathway with high therapeutic potential. The aim of this study was to explore the relevance of such IgE-independent, Mrgprb2-mediated signaling in colonic MCs in the healthy and acutely inflamed mouse colon. MethodsMrgprb2 expression and functionality was studied using a genetic labeling strategy combined with advanced microscopic imaging. Furthermore, Mrgprb2 knockout (Mrgprb2-/-) mice were used to determine the role of this pathway in a preclinical dextran sodium sulphate (DSS) colitis model. ResultsWe found that Mrgprb2 acts as a novel MC degranulation pathway in a large subset of connective tissue MCs (CTMCs) in the mouse distal colon. Acute DSS colitis induced a pronounced increase of Mrgprb2-expressing MCs, which were found in close association with Substance P (SP)-positive nerve fibers. Loss of Mrgprb2-mediated signaling impaired DSS-induced neutrophil influx and significantly impacted on acute colitis progression. ConclusionsOur findings uncover a novel, IgE-independent MC degranulation pathway in the mouse colon that plays a central role in acute colitis pathophysiology, mainly by safeguarding acute colitis progression and severity in mice. This pseudo allergic, Mrgprb2-induced signaling is part of a hitherto unconsidered colonic neuro-immune pathway and might have significant potential for the further development of effective therapeutic treatment strategies for gastrointestinal disorders, such as ulcerative colitis.

Single intratracheal administration toxicity study on safety of vapor inhalation of electrolyzed reduced water in rats.

The effects of acute intratracheal administration of electrolyzed reduced water (ERW; alkaline electrolyzed water) were investigated in rats. In this study, no deaths or near-deaths were recorded in either group, namely those treated with ERW or purified water (maximum doses of 900 mg/kg). The main symptoms observed in the rats were decreased spontaneous movements and abnormal breath sounds, which were considered to be transient symptoms caused by intratracheal administration. In addition, low values of alkaline phosphatase, total protein and lactate dehydrogenase were found in BALF tests, but these values were considered to be of low toxicological significance, since they are usually high in the presence of lung inflammation or cellular damage. This suggests that the alkalinity of ERW partially contributes to broken peptide bonds in proteins. There were no significant increases in bronchoalveolar lavage fluid protein in either group. ERW did not cause an increase in the influx of neutrophils, eosinophils, basophils, or lymphocytes, suggesting that intratracheal administration of ERW did not cause lung inflammation. ERW did not cause abnormalities in the body or pathological changes in the lungs. Aggregates of alveolar macrophages, as a measure of inflammation, were observed in both groups. These may be transient symptoms due to intratracheal administration, not due to ERW toxicity. This study confirmed the safety of intratracheal ERW infusion and demonstrated the low risk of acute toxicity for inhalation exposure to ERW aerosol or vapor. Therefore, ERW may be an effective air purifier against viruses or bacteria.

The effect of budesonide delivered by high-frequency oscillatory ventilation on acute inflammatory response in severe lung injury in adult rabbits.

The inflammation present in acute respiratory distress syndrome (ARDS) and thereby associated injury to the alveolar-capillary membrane and pulmonary surfactant can potentiate respiratory failure. Even considering the high mortality rate of severe ARDS, glucocorticoids appear to be a reasonable treatment option along with an appropriate route of delivery to the distal lung. This study aimed to investigate the effect of budesonide therapy delivered intratracheally by high-frequency oscillatory ventilation (HFOV) on lung function and inflammation in severe ARDS. Adult New Zealand rabbits with respiratory failure (P/F<13.3 kPa) induced by intratracheal instillation of hydrochloric acid (HCl, 3 ml/kg, pH 1.5) followed by high tidal ventilation (VT 20 ml/kg) to mimic ventilator-induced lung injury (VILI) were treated with intratracheal bolus of budesonide (0.25 mg/kg, Pulmicort) delivered by HFOV (frequency 8 Hz, MAP 1 kPa, deltaP 0.9 kPa). Saline instead of HCl without VILI with HFOV delivered air bolus instead of therapy served as healthy control. All animals were subjected to lung-protective ventilation for 4 h, and respiratory parameters were monitored regularly. Postmortem, lung injury, wet-to-dry weight ratio, leukocyte shifts, and levels of cytokines in plasma and lung were evaluated. Budesonide therapy improved the lung function (P/F ratio, oxygenation index, and compliance), decreased the cytokine levels, reduced lung edema and neutrophils influx into the lung, and improved lung architecture in interstitial congestion, hyaline membrane, and atelectasis formation compared to untreated animals. This study indicates that HFOV delivered budesonide effectively ameliorated respiratory function, and attenuated acid-induced lung injury in a rabbit model of severe ARDS.

Pleural macrophages translocate to the lung during infection to promote improved influenza outcomes.

Seasonal influenza results in 3 to 5 million cases of severe disease and 250,000 to 500,000 deaths annually. Macrophages have been implicated in both the resolution and progression of the disease, but the drivers of these outcomes are poorly understood. We probed mouse lung transcriptomic datasets using the Digital Cell Quantifier algorithm to predict immune cell subsets that correlated with mild or severe influenza A virus (IAV) infection outcomes. We identified a unique lung macrophage population that transcriptionally resembled small serosal cavity macrophages and whose presence correlated with mild disease. Until now, the study of serosal macrophage translocation in the context of viral infections has been neglected. Here, we show that pleural macrophages (PMs) migrate from the pleural cavity to the lung after infection with IAV. We found that the depletion of PMs increased morbidity and pulmonary inflammation. There were increased proinflammatory cytokines in the pleural cavity and an influx of neutrophils within the lung. Our results show that PMs are recruited to the lung during IAV infection and contribute to recovery from influenza. This study expands our knowledge of PM plasticity and identifies a source of lung macrophages independent of monocyte recruitment and local proliferation.

Azithromycin regulates bacterial virulence and immune response in a murine model of ceftazidime-treated Pseudomonas aeruginosa acute pneumonia.

Pseudomonas aeruginosa (PA) remains one of the leading causes of nosocomial acute pneumonia. The array of virulence factors expressed by PA and the intense immune response associated with PA pneumonia play a major role in the severity of these infections. New therapeutic approaches are needed to overcome the high resistance of PA to antibiotics and to reduce the direct damage to host tissues. Through its immunomodulatory and anti-virulence effects, azithromycin (AZM) has demonstrated clinical benefits in patients with chronic PA respiratory infections. However, there is relatively little evidence in PA acute pneumonia. We investigated the effects of AZM, as an adjunctive therapy combined with ceftazidime (CAZ), in a murine model of PA acute pneumonia. We observed that the combined therapy (i) reduces the weight loss of mice 24 h post-infection (hpi), (ii) decreases neutrophil influx into the lungs at 6 and 24 hpi, while this effect is absent in a LPS-induced pneumonia or when PA is pretreated with antibiotics and mice do not receive any antibiotics, and that (iii) AZM, alone or with CAZ, modulates the expression of PA quorum sensing regulators and virulence factors (LasI, LasA, PqsE, PhzM, ExoS). Our findings support beneficial effects of AZM with CAZ on PA acute pneumonia by both bacterial virulence and immune response modulations. Further investigations are needed to clarify the exact underlying mechanisms responsible for the reduction of the neutrophils influx and to better discriminate between direct immunomodulatory properties of AZM, and indirect effects on neutrophilia resulting from bacterial virulence modulation.

Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response

BackgroundDiesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE.MethodsUsing immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo–keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed.ResultsDE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = −0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo–keto reductases, or epoxide hydrolase protein expression.ConclusionThese in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

The Multicomponent Medicinal Product Hepar Compositum Reduces Hepatic Inflammation and Fibrosis in a Streptozotocin- and High-Fat Diet-Induced Model of Metabolic Dysfunction-Associated Steatotic Liver Disease/Metabolic Dysfunction-Associated Steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD)-formerly known as non-alcoholic fatty liver disease (NAFLD)-is the most common chronic liver disease worldwide. Since there is currently no approved pharmacotherapy for MASLD, there is an urgent unmet need for efficacious therapeutics for this disease. Hepar compositum (HC-24) is a multicomponent medicinal product that consists of 24 natural ingredients. It has been shown to have anti-inflammatory properties in an obesity-associated MASLD mouse model, but its potential to reduce MASLD-associated fibrosis had not been explored before this study. Here, we investigated the hepatic anti-inflammatory and anti-fibrotic potential of HC-24 in a streptozotocin (STZ)- and high-fat diet (HFD)-induced model of MASLD. Mice received a single injection of low-dose STZ at 2 days of age, followed by HFD feeding from 4 to 9 weeks of age. Mice were treated every second day with HC-24 or daily with the positive control telmisartan from 6 to 9 weeks of age. A non-diseased control group was included as a healthy reference. An explorative small-scale pilot study demonstrated that HC-24 improved liver histology, resulting in a lower NAFLD activity score and reduced liver fibrosis. A subsequent full study confirmed these effects and showed that HC-24 reduced hepatic inflammation, specifically reducing T helper cell and neutrophil influx, and decreased hepatic fibrosis (with qualitatively reduced collagen type I and type III immunopositivity) in the absence of an effect on body and liver weight, blood glucose or liver steatosis. These results show that HC-24 has hepatoprotective, anti-inflammatory, and anti-fibrotic properties in an STZ- and HFD-induced model of MASLD/MASH, suggesting that this multicomponent medicine has therapeutic potential for MASLD patients.

Footprint of pancreas infiltrating and circulating immune cells throughout type 1 diabetes development.

Type 1 diabetes (T1D) is defined by immune cell infiltration of the pancreas, in particular the islets of Langerhans, referred to as insulitis, which is especially prominent during the early disease stages in association with decreased beta cell mass. An in-depth understanding of the dynamics and phenotype of the immune cells infiltrating the pancreas and the accompanying changes in their profiles in peripheral blood during T1D development is critical to generate novel preventive and therapeutic approaches, as well as to find biomarkers for the disease process. Using multi-parameter flow cytometry, we explored the dynamic changes of immune cells infiltrating the pancreas and the pancreatic draining lymph nodes (PLN), compared to those in peripheral blood in female and male non-obese diabetic (NOD) mice during T1D progression. The early stages of T1D development were characterized by an influx of innate dendritic cells and neutrophils in the pancreas. While dendritic cells seemed to move in and out (to the PLN), neutrophils accumulated during the pre-symptomatic phase and reached a maximum at 8 weeks of age, after which their numbers declined. During disease progression, CD4+ and CD8+ T cells appeared to continuously migrate from the PLN to the pancreas, which coincided with an increase in beta cell autoimmunity and insulitis severity, and a decline in insulin content. At 12 weeks of age, CD4+ and especially CD8+ T cells in the pancreas showed a dramatic shift from naïve to effector memory phenotype, in contrast to the PLN, where most of these cells remained naïve. A large proportion of pancreas infiltrating CD4+ T cells were naïve, indicating that antigenic stimulation was not necessary to traffic and invade the pancreas. Interestingly, a pre-effector-like T cell dominated the peripheral blood. These cells were intermediates between naïve and effector memory cells as identified by single cell RNA sequencing and might be a potential novel therapeutic target. These time- and tissue-dependent changes in the dynamics and functional states of CD4+ and CD8+ T cells are essential steps in our understanding of the disease process in NOD mice and need to be considered for the interpretation and design of disease-modifying therapies.

Abstract 12972: Trained Innate Immunity Through Toll-Like Receptor 4/Interferon Signaling Establishes Cardioprotection in a Stress-Induced Cardiomyopathy Model

Introduction: Although extensively studied, the mechanisms responsible for the development of cardioprotection following preconditioning stimuli are not known. Our group showed that isoproterenol (ISO) mediated cardiac injury induces potent cardioprotection against a second ISO challenge for up to 5 weeks. The lack of an immune response after the second ISO dose and the durability of protection suggested that trained innate immunity might represent a novel mechanism for cardioprotection. Hypothesis: We hypothesized that ISO confers cardioprotection through trained immunity via Toll-like receptor 4 (TLR4) signaling that is activated by damage-associated molecular patterns released by ISO-induced cell necrosis. Methods: Wild-type C57BL/6J mice were intraperitoneally injected with TLR agonists or diluent (saline), and then 300 mg/kg ISO a week later. Mice were assessed before and after receiving ISO via serum cardiac troponin analysis, flow cytometry, and 2-D echocardiography. Results: The TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, induced cardioprotection as shown by decreased troponin I release (p&lt;0.01), reduced cardiac neutrophil influx (p&lt;0.05), and decreased left ventricle wall motion abnormalities (p&lt;0.05) following ISO injury compared to saline treated controls . RNA sequencing identified interferon signaling as commonly upregulated (p&lt;0.05) in mouse hearts a week after LPS or ISO pre-treatment. Blocking type I/II interferon receptors partially abolished LPS-induced cardioprotection, while pre-treatment with recombinant interferon β+γ conferred cardioprotection as shown by decreased troponin I release post-ISO (p&lt;0.01). Given that β-glucan reverses the epigenetic tolerizing effects of LPS in immune cells, we treated mice with β-glucan following LPS pre-treatment and observed that the cardioprotection to ISO was abolished. Conclusions: Viewed together this study shows for the first time that TLR4/interferon signaling confers cardioprotection against ISO-induced injury. Our findings further suggest that this cardioprotective response may be secondary to TLR4 mediated epigenetic changes that dampen immune responses following tissue injury, consistent with trained innate immunity.

Pathogenesis of Achromobacter xylosoxidans respiratory infections: colonization, persistence, and transcriptome profiling in synthetic cystic fibrosis sputum medium

ABSTRACT Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long infections of the respiratory mucosa caused by a diverse array of opportunists, which are leading causes of morbidity and mortality. In recent years, there has been increased appreciation for the range and diversity of microbes causing CF-related respiratory infections. The introduction of new therapeutics and improved detection methodology has revealed CF-related opportunists such as Achromobacter xylosoxidans ( Ax ). Ax is a Gram-negative bacterial species which is widely distributed in environmental sources and has been increasingly observed in sputa and other samples from pwCF, typically in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors including flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized cultures of CFBE41o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization of cell layers. Ax colonized and persisted in mouse lungs for up to 72 h post infection, with inflammatory consequences that include increased neutrophil influx in the lung, lung damage, cytokine production, and mortality. We also identified genes that are differentially expressed in synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.