Reduced Leukocyte Recruitment Research Articles

Targeting purinergic signaling in platelets ameliorates the bacterial burden and reduces the cytokine storm in the peritoneal cavity during intra-abdominal sepsis

Sepsis is a complex clinical syndrome of the host response to an infection, involving a pathogenic cytokine storm that drives inflammatory injury in multiple organ systems. The abdomen is a major site of sepsis, contributing to high mortality. Platelets express the purinergic receptors P2Y1 and P2Y12; once activated, these receptors mediate the secretion of pro-inflammatory mediators that contribute to organ damage in sepsis. Systemic blockade of P2Y1 or P2Y12 decreased organ damage and inflammatory cytokine levels in a sex-related manner during sepsis. Here, we hypothesize that blocking P2Y1 or P2Y12 activity enhances pathogen clearance in the peritoneal cavity during intra-abdominal sepsis in a sex-specific manner. Male and female C57BL6/6J mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. P2Y1 or P2Y12 antagonists, MRS2279 (10 mg/kg) and Ticagrelor (30 mg/kg) respectively, were administered intraperitoneally after surgery. The peritoneal cavity fluid (PCF) was collected 24 hours post-surgery by peritoneal lavage. To assess cell recruitment in the cavity, we measured leukocyte and platelet counts using Hemavet® Multispecies Hematology System. To assess inflammation, we measured PCF levels of the cytokines MIP-1α and Rantes as well as bacterial clearance. The ileum of the small intestinal tissue samples were collected 24 hours post-surgery and platelet infiltration was measured by platelet factor 4 (PF4) content in the tissue using an ELISA kit. In response to the P2Y1 or P2Y12 blocker, we noted a significant reduction in the bacteria content in the PCF of CLP mice. Blocking P2Y12 but not P2Y1 reduced leukocyte recruitment, while platelet counts were reduced only with P2Y1 blockade in septic males. The sepsis-induced increase in MIP-1α levels was not noted upon P2Y1 but not P2Y12 blockage in the PCF of septic males. However, Rantes levels in the PCF were reduced for either treatment in males compared to the untreated CLP. The platelet marker PF4 in the ileum was increased during sepsis but reduced after either treatment in sepsis males as compared to the untreated CLP. In female CLP mice, a significant reduction in bacterial burden in the PCF in response to the P2Y1 but not the P2Y12 blocker was shown. Blocking P2Y1 or P2Y12 reduced leukocyte recruitment and platelet counts in female CLP mice. As observed in males, PF4 was increased in the ileum during sepsis but reduced following exposure to either treatment in females. No change was noted for MIP-1α or Rantes levels in the females among all the septic groups. Our data indicate that blocking purinergic signaling and in particular P2Y12 in the peritoneal cavity in males prevents bacterial spreading and diminishes cytokine levels, indicating that it could be an effective treatment for intra-abdominal sepsis. However, in female mice blocking the receptor P2Y1, but not P2Y12 has shown to be optimal. Targeting purinergic signaling is a promising sex-specific therapeutic strategy for sepsis. This work was supported by the Research grant AI156627-01 (EL) from the National Institute of Health. This is the full abstract presented at the American Physiology Summit 2024 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.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

The Role of Vitamin D in Neuroprotection in Multiple Sclerosis: An Update.

Multiple sclerosis (MS) is a complex neurological condition that involves both inflammatory demyelinating and neurodegenerative components. MS research and treatments have traditionally focused on immunomodulation, with less investigation of neuroprotection, and this holds true for the role of vitamin D in MS. Researchers have already established that vitamin D plays an anti-inflammatory role in modulating the immune system in MS. More recently, researchers have begun investigating the potential neuroprotective role of vitamin D in MS. The active form of vitamin D, 1,25(OH)2D3, has a range of neuroprotective properties, which may be important in remyelination and/or the prevention of demyelination. The most notable finding relevant to MS is that 1,25(OH)2D3 promotes stem cell proliferation and drives the differentiation of neural stem cells into oligodendrocytes, which carry out remyelination. In addition, 1,25(OH)2D3 counteracts neurodegeneration and oxidative stress by suppressing the activation of reactive astrocytes and M1 microglia. 1,25(OH)2D3 also promotes the expression of various neuroprotective factors, including neurotrophins and antioxidant enzymes. 1,25(OH)2D3 decreases blood-brain barrier permeability, reducing leukocyte recruitment into the central nervous system. These neuroprotective effects, stimulated by 1,25(OH)2D3, all enhance neuronal survival. This review summarizes and connects the current evidence supporting the vitamin D-mediated mechanisms of action for neuroprotection in MS.

509 AMG487, A CXCR3 Antagonist, changes the Inflammatory Milieu in Familial Hemophagocytic Lymphohistiocytosis (FHL) Hepatitis

OBJECTIVES/GOALS: Familial Hemophagocytic Lymphohistiocytosis (FHL) is a systemic inflammatory disease, causing acute liver failure (ALF). Elevated Interferon gamma (IFN-γ) results in increased hepatic transcription of the chemokines CXCL9 and CXCL10. Inhibition of their receptor CXCR3 may reduce leukocyte recruitment and ameliorate hepatitis. METHODS/STUDY POPULATION: To determine the functional role of the IFN-γ-induced ligands, CXCL9 and CXCL10, in hepatic leukocyte recruitment via CXCR3 we used a prf-/- mouse infected with Lymphocytic Choriomeningitis Virus (LCMV) in our well-established model mimicking human FHL. We used AMG487, a small molecule CXCR3 antagonist, while maintaining intact IFN-γ signaling. Mice were sacrificed 10 days after infection when mice developed features of FHL: cytopenias, organomegaly, elevated serum ferritin and sCD25, and hepatic inflammation. Hepatic inflammation was characterized using flow cytometry, liver histology and noninvasive markers of hepatitis (ALT, liver size). RESULTS/ANTICIPATED RESULTS: AMG487 did not ameliorate the overall disease phenotype with mice developing similar FHL characteristics compared to control, including weight loss, elevation of ALT and sIL-2r as well as degree of thrombocytopenia and anemia. There was significant reduction of recruitment of CXCR3+CD4+ T cells and B cells in mice treated with AMG487. This indicates the importance of CXCR3 receptor in humoral response in FHL hepatitis. In addition, treatment with AMG487 resulted in reduction of CXCR3 expression in hepatic inflammatory monocyte (iMonos) measured by mean fluorescence intensity (MFI). DISCUSSION/SIGNIFICANCE: This is the first pre-clinical experience using AMG487, a small molecule CXCR3 antagonist, to treat FHL hepatitis. AMG487 changed the hepatic inflammatory milieu, reducing CD4 T-cell and B-cell recruitment, as well as CXCR3 expression on iMonos. However, it did not ameliorate FHL hepatitis and other therapeutic approaches should be pursued.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr−/− mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. Importantly, pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr−/− mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.

A microfluidic device for assessment of E-selectin-mediated neutrophil recruitment to inflamed endothelium and prediction of therapeutic response in sickle cell disease

Neutrophil recruitment to the inflamed endothelium is a multistep process and is of utmost importance in the development of the hallmark vaso-occlusive crisis in sickle cell disease (SCD). However, there lacks a standardized, clinically feasible approach for assessing neutrophil recruitment to the inflamed endothelium for individualized risk stratification and therapeutic response prediction in SCD. Here, we describe a microfluidic device functionalized with E-selectin, a critical endothelial receptor for the neutrophil recruitment process, as a strategy to assess neutrophil binding under physiologic flow in normoxia and clinically relevant hypoxia in SCD. We show that hypoxia significantly enhances neutrophil binding to E-selectin and promotes the formation of neutrophil-platelet aggregates. Moreover, we identified two distinct patient populations: a more severe clinical phenotype with elevated lactate dehydrogenase levels and absolute reticulocyte counts but lowered fetal hemoglobin levels associated with constitutively less neutrophil binding to E-selectin. Mechanistically, we demonstrate that the extent of neutrophil activation correlates with membrane L-selectin shedding, resulting in the loss of ligand interaction sites with E-selectin. We also show that inhibition of E-selectin significantly reduces leukocyte recruitment to activated endothelial cells. Our findings add mechanistic insight into neutrophil-endothelial interactions under hypoxia and provide a clinically feasible means for assessing neutrophil binding to E-selectin using clinical whole blood samples, which can help guide therapeutic decisions for SCD patients.

FBXO6 regulates the antiviral immune responses via mediating alveolar macrophages survival.

Inducing early apoptosis in alveolar macrophages is one of the strategies influenza A virus (IAV) evolved to subvert host immunity. Correspondingly, the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 is reported to interact with virus polymerase basic protein 1-frame 2 (PB1-F2) accessory protein to counteract virus-induced apoptosis. Herein, we report that one of the F-box proteins, FBXO6, promotes proteasomal degradation of NLRX1, and thus facilitates IAV-induced alveolar macrophages apoptosis and modulates both macrophage survival and type I interferon (IFN) signaling. We observed that FBXO6-deficient mice infected with IAV exhibited decreased pulmonary viral replication, alleviated inflammatory-associated pulmonary dysfunction, and less mortality. Analysis of the lungs of IAV-infected mice revealed markedly reduced leukocyte recruitment but enhanced production of type I IFN in Fbxo6-/- mice. Furthermore, increased type I IFN production and decreased viral replication were recapitulated in FBXO6 knockdown macrophages and associated with reduced apoptosis. Through gain- and loss-of-function studies, we found lung resident macrophages but not bone marrow-derived macrophages play a key role in the differences FBXO6 signaling pathway brings in the antiviral immune response. In further investigation, we identified that FBXO6 interacted with and promoted the proteasomal degradation of NLRX1. Together, our results demonstrate that FBXO6 negatively regulates immunity against IAV infection by enhancing the degradation of NLRX1 and thus impairs the survival of alveolar macrophages and antiviral immunity of the host.

Pamoic acid is an inhibitor of HMGB1·CXCL12 elicited chemotaxis and reduces inflammation in murine models of Pseudomonas aeruginosa pneumonia

BackgroundHigh-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers.MethodsPAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid.ResultsOur data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation.ConclusionsThis work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.

β-sitosterol ameliorates inflammation and Pseudomonas aeruginosa lung infection in a mouse model

We previously demonstrated that β-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

RvD1 disrupts nociceptor neuron and macrophage activation and neuroimmune communication, reducing pain and inflammation in gouty arthritis in mice.

Gouty arthritis is characterized by an intense inflammatory response to monosodium urate crystals (MSU), which induces severe pain. Current therapies are often ineffective in reducing gout-related pain. Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator with anti-inflammatory and analgesic proprieties. In this study, we evaluated the effects and mechanisms of action of RvD1 in an experimental mouse model of gouty arthritis, an aim that was not pursued previously in the literature. Male mice were treated with RvD1 (intrathecally or intraperitoneally) before or after intraarticular stimulation with MSU. Mechanical hyperalgesia was assessed using an electronic von Frey aesthesiometer. Leukocyte recruitment was determined by knee joint wash cell counting and immunofluorescence. IL-1β production was measured by ELISA. Phosphorylated NF-kB and apoptosis-associated speck-like protein containing CARD (ASC) were detected by immunofluorescence, and mRNA expression was determined by RT-qPCR. CGRP release was determined by EIA and immunofluorescence. MSU crystal phagocytosis was evaluated by confocal microscopy. RvD1 inhibited MSU-induced mechanical hyperalgesia in a dose- and time-dependent manner by reducing leukocyte recruitment and IL-1β production in the knee joint. Intrathecal RvD1 reduced the activation of peptidergic neurons and macrophages as well as silenced nociceptor to macrophage communication and macrophage function. CGRP stimulated MSU phagocytosis and IL-1β production by macrophages. RvD1 downmodulated this phenomenon directly by acting on macrophages, and indirectly by inhibiting CGRP release and CGRP-dependent activation of macrophages. This study reveals a hitherto unknown neuro-immune axis in gouty arthritis that is targeted by RvD1.

Oral administration of botryosphaeran [(1 → 3)(1 → 6)-β-d-glucan] reduces inflammation through modulation of leukocytes and has limited effect on inflammatory nociception.

Several biological activities of the fungal exopolysaccharide (1 → 3)(1 → 6)-β-d-glucan (botryosphaeran) have been described in the literature, but its effects on inflammation have not been evaluated. This study aimed to investigate the action of botryosphaeran on experimental mice models of carrageenan-induced acute pleurisy and acute paw edema, and complete Freund's adjuvant-induced persistent paw edema. All botryosphaeran doses tested (1.0, 2.5, 5.0, and 10.0 mg/kg birth weight [b.w.], orally administered) reduced leukocyte recruitment, nitric oxide (NO) levels, and protein extravasation in the pleural cavity. Botryosphaeran (5 mg/kg b.w.) did not diminish edema and mechanical hyperalgesia in the paw within 4 h; however, cold allodynia was alleviated within the first 2 h. In the persistent paw inflammation model, the effects of daily oral administration of botryosphaeran (5 mg/kg b.w.) were evaluated over 3 and 7 days. The fungal β-glucan significantly reduced the levels of the cytokines, tumor necrosis factor(TNF)-α, interleukin (IL)-6), and IL-10, in the paw homogenates in both protocols, while paw edema and the levels of advanced oxidation protein products (AOPP) only diminished on Day 7. No effect in mechanical hyperalgesia was observed. Oral treatment for 3 or 7 days also decreased the plasma levels of NO, AOPP, TNF-α, and IL-10. On Day 7, the number of leukocytes in the blood was also reduced by this treatment. Importantly, botryosphaeran did not induce inflammation in mice when administered alone over 7 days. This study demonstrated the anti-inflammatory and antinociceptive potential of botryosphaeran in these experimental models, making this fungal β-glucan a new possibility for complementary treating acute and chronic inflammation.

Isorhamnetin Alleviates Airway Inflammation by Regulating the Nrf2/Keap1 Pathway in a Mouse Model of COPD

Chronic obstructive pulmonary disease (COPD) is a severely disabling chronic lung disease characterized by persistent airway inflammation, which leads to limited expiratory airflow that deteriorates over time. Isorhamnetin (Iso) is one of the most important active components in the fruit of Hippophae rhamnoides L. and leaves of Ginkgo biloba L, which is widely used in many pulmonary disease studies because of its anti-inflammatory effects. Here, we investigated the pharmacological action of Iso in CS-induced airway inflammation and dissected the anti-inflammation mechanisms of Iso in COPD mice. A mouse model of COPD was established by exposure to cigarette smoke (CS) and intratracheal inhalation of lipopolysaccharide (LPS). Our results illustrated that Iso treatment significantly reduced leukocyte recruitment and excessive secretion of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and regulated upon activation, normal T-cell expressed and secreted (RANTES) in BALF of CS-induced COPD mice in a dose-dependent manner. This improved airway collagen deposition and emphysema, and further alleviated the decline in lung functions and systemic symptoms of hypoxia and weight loss. Additionally, Iso treatment obviously improves the T lymphocyte dysregualtion in peripheral blood of COPD mice. Mechanistically, Iso may degrade Keap1 through ubiquitination of p62, thereby activating the nuclear factor erythroid 2-related factor (Nrf2) pathway to increase the expression of protective factors, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) 1, and SOD2, in lungs of CS-exposed mice, which plays an anti-inflammatory role in COPD. In conclusion, our study indicates that Iso significantly alleviates the inflammatory response in CS-induced COPD mice mainly by affecting the Nrf2/Keap1 pathway. More importantly, Iso exhibited anti-inflammatory effects comparable with Dex in COPD and we did not observe discernible side effects of Iso. The high safety profile of Iso may make it a potential drug candidate for COPD.

ADAM17/MMP inhibition prevents neutrophilia and lung injury in a mouse model of COVID-19.

Severe coronavirus disease 2019 (COVID‐19) is characterized by lung injury, cytokine storm, and increased neutrophil‐to‐lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID‐19 in infected individuals. Angiotensin‐converting enzyme‐2 (ACE2) is the receptor for SARS‐CoV‐2, the virus causing COVID‐19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID‐19‐related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor‐binding domain of the SARS‐CoV‐2 spike protein (RBD‐S) to mimic lung damage associated with COVID‐19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID‐19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI‐1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM‐1 and VCAM‐1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS‐CoV‐2‐infected individuals to prevent the progression toward severe COVID‐19.

Neutralization of Inflammasome-Processed Cytokines Reduces Inflammatory Mechanisms and Leukocyte Recruitment in the Vasculature of TNF-α-Stimulated Sickle Cell Disease Mice

The chronic inflammatory state associated with sickle cell disease (SCD) incurs pan-cellular activation and the recruitment of leukocytes to the activated endothelium of blood vessels. The resultant rheological alterations and red cell sickling culminate in acute vaso-occlusive processes. Cytokines IL-1β and IL-18, the bio-active products of the activated inflammasome, are elevated in the plasma of SCD patients (ASH Abstract (2016) 128 (22): 854), and a previous study reported that anti-IL1β therapy alleviated reperfusion injury and flow stasis in NY1DD transgenic sickle mice exposed to hypoxia/reoxygenation (ASH Abstract (2011) 118(21): 848). The aim of this study was to determine whether antibodies that neutralize IL-1β and IL-18 could individually, or synergistically, diminish inflammatory processes and leukocyte recruitment in mice with SCD. Townes mice (5-months old; N=4-7 per group) received an i.p. administration of either saline, 200 µg/mouse anti-murine IL-1β (01BSUR), and/or 250 or 500 µg/mouse anti-murine IL-18 (SK113AE-4), or an IgG1 control antibody (iProt105125; 200 or 500 µg/mouse). At 21h after treatments, vaso-occlusive-like processes were induced in mice by the injection of tumor necrosis factor-α (TNF; 0.5μg, i.p.). At 3h after TNF, the cremaster muscles of anesthetized mice were surgically exposed, and leukocyte TNF recruitment and extravasation in venules of the microcirculation were observed using intravital microscopy. Another set of Townes mice (N=4-12 per group) was submitted to the same procedures, with blood sampling for ELISA at 3h post TNF. Optimal concentrations of antibodies were determined by observing leukocyte recruitment by intravital microscopy (doses; 100, 200 µg/mouse anti-IL-1β [N=2; 3, respectively] and 250, 500 µg/mouse anti-IL-18, [N=2 each]) in TNF-stimulated C57BL/6J mice (data not shown). Figure 1 (A-C) demonstrates the extensive recruitment and extravasation of leukocytes that occurs in the microvasculature of Townes mice at 3h post-TNF (saline group). Pre-treatment of mice with either anti-IL-1β or anti-IL-18 significantly abrogated (P<0.01) the TNF-induced adhesion (Fig. 1B) and extravasation (Fig. 1C) of leukocytes in venules, while only anti-IL-18 significantly reduced leukocyte rolling along the venule endothelium (Fig. 1A; P<0.05). In contrast, the administration of 500 µg/mouse (Fig. 1) or 200 µg/mouse (data not shown) of a non-specific IgG1 did not significantly affect TNF-induced leukocyte recruitment/extravasation (P>0.05). The combined use of the anti-IL-1β (200 µg/mouse) together with an intermediate dose of anti-IL-18 (250 µg/mouse) did not further reduce leukocyte recruitment and extravasation in this model, when compared with the effects of anti-IL-1β alone (Fig. 1A-C; P>0.05). Investigating the effects of these biological agents on inflammatory molecules production in TNF-stimulated Townes mice, we found that the administration of anti-IL-1β (200 µg) reduced IL-6 production, a pleiotropic inflammatory molecule that is upregulated by IL-1β (Fig. 1D), as did the combination of anti-IL-1β plus anti-IL-18 (200 µg and 250 µg/mouse, respectively). Pre-treatment of TNF-stimulated SCD mice with anti-IL-1β plus anti-IL-18 decreased Interferon (IFN)-γ production, a molecule that is upregulated synergistically by IL-18 and IL-12 and that mediates early host immune defenses (Fig. 1E). In contrast, anti-inflammatory IL-10 production was not significantly modulated by the pre-treatment of TNF-stimulated mice with these biological agents (Fig. 1F). As such, IL-1β and IL-18 neutralization significantly reduced inflammatory processes and leukocyte recruitment in the microvasculature of mice with SCD, indicating that biological agents that inhibit the effects of inflammasome-processed cytokines may hold potential for reducing vaso-occlusive processes in patients with this disease. [Display omitted] DisclosuresKovarik: Novartis Institutes for Biomedical Research: Current Employment. Costa: Novartis: Consultancy. Conran: Novartis Pharma AG: Research Funding.

Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ1) deletion was lethal, but mice lacking Gnb2 (Gβ2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gβ2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

Insulin Treatment Forces Arteriogenesis in Diabetes Mellitus by Upregulation of the Early Growth Response-1 (Egr-1) Pathway in Mice.

The process of arteriogenesis is severely compromised in patients with diabetes mellitus (DM). Earlier studies have reported the importance of Egr-1 in promoting collateral outward remodeling. However, the role of Egr-1 in the presence of DM in outward vessel remodeling was not studied. We hypothesized that Egr-1 expression may be compromised in DM which may lead to impaired collateral vessel growth. Here, we investigated the relevance of the transcription factor Egr-1 for the process of collateral artery growth in diabetic mice. Induction of arteriogenesis by femoral artery ligation resulted in an increased expression of Egr-1 on mRNA and protein level but was severely compromised in streptozotocin-induced diabetic mice. Diabetes mellitus mice showed a significantly reduced expression of Egr-1 endothelial downstream genes Intercellular Adhesion Molecule-1 (ICAM-1) and urokinase Plasminogen Activator (uPA), relevant for extravasation of leukocytes which promote arteriogenesis. Fluorescent-activated cell sorting analyses confirmed reduced leukocyte recruitment. Diabetes mellitus mice showed a reduced expression of the proliferation marker Ki-67 in growing collaterals whose luminal diameters were also reduced. The Splicing Factor-1 (SF-1), which is critical for smooth muscle cell proliferation and phenotype switch, was found to be elevated in collaterals of DM mice. Treatment of DM mice with insulin normalized the expression of Egr-1 and its downstream targets and restored leukocyte recruitment. SF-1 expression and the diameter of growing collaterals were normalized by insulin treatment as well. In summary, our results showed that Egr-1 signaling was impaired in DM mice; however, it can be rescued by insulin treatment.

Lack of the hyaluronan receptor CD44 affects the course of bacterial otitis media and reduces leukocyte recruitment to the middle ear

BackgroundCD44 is a multifunctional molecule that plays major roles in both leukocyte recruitment and tissue proliferation. Since mucosal hyperplasia and leukocyte infiltration of the middle ear cavity are major features of otitis media, we evaluated the role of CD44 in the pathophysiology and course of this disease in a mouse model of middle ear infection. Expression of genes related to CD44 function were evaluated using gene arrays in wild-type mice. The middle ears of mice deficient in CD44 were inoculated with non-typeable Haemophilus influenzae. Histopathology and bacterial clearance were compared to that seen in wild-type controls.ResultsWe observed strong up-regulation of CD44 and of genes related to its role in leukocyte extravasation into the middle ear, during the course of acute otitis media. Mice deficient in CD44 exhibited reduced early mucosal hyperplasia and leukocyte recruitment, followed by delayed resolution of infection and persistent inflammation.ConclusionsCD44 plays an important role in OM pathogenesis by altering the mucosal growth and neutrophil enlistment. Targeted therapies based on CD44 could be useful adjuncts to the treatment of middle ear infections.

Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii

Little is known about strategies used by pathogens to facilitate CNS invasion. Toxoplasma gondii reaches the CNS by circulating in blood within leukocytes or as extracellular tachyzoites. T. gondii induces EGFR signaling in vitro during invasion of mammalian cells. We examined the effects of endothelial cell EGFR on CNS invasion. Transgenic mice whose endothelial cells expressed a dominant negative (DN) EGFR (inhibits EGFR signaling) exhibited diminished parasite load and histopathology in the brain and retina after T. gondii infection. I.V. administration of infected leukocytes or extracellular tachyzoites led to reduced parasite loads in mice with DN EGFR. This was not explained by enhanced immunity or reduced leukocyte recruitment. Endothelial cell infection is key for CNS invasion. Parasite foci in brain endothelial cells were reduced by DN EGFR. DN EGFR in these cells led to recruitment of the autophagy protein LC3 around T. gondii and spontaneous parasite killing dependent on the autophagy protein ULK1 and lysosomal enzymes. The autophagy inhibitor 3-MA prevented DN EGFR mice from exhibiting reduced CNS invasion. Altogether, EGFR is a novel regulator of T. gondii invasion of neural tissue, enhancing invasion likely by promoting survival of the parasite within endothelial cells.

TNF-Induced Vaso-Occlusive and Inflammatory Processes in Mice with Sickle Cell Anemia Are Abrogated By the Platelet Activation Inhibitor, Prasugrel

Background: Vaso-occlusive processes in sickle cell anemia (SCA) are triggered by inflammatory molecules, along with pancellular activation and the recruitment and adhesion of leukocytes to the endothelium. Interactions between platelets, neutrophils, and erythrocytes are observed in the SCA circulation, but the exact role of platelets in the initiation and propagation of vaso-occlusion is not well understood. Aim: We, herein, evaluated the effects of the inhibition of platelet activation on TNF-α (TNF)-triggered vaso-occlusive processes in the microcirculation of mice with SCA, using prasugrel, a platelet ADP P2Y12 receptor inhibitor. Methods: Chimeric male control (CON) and SCA (SCA) mice received prasugrel (1 mg/kg, by gavage) or vehicle (saline) in a single administration, 1h prior to TNF (0.5 μg/g, i.p). In some experiments, 1 mg/kg prasugrel was administered chronically for 5x per week for 4 weeks. Following i.v. injection of anti-CD41 PE, anti-CD45 PeCy-5 and anti-Ly6G Alexa 488 antibodies for labeling platelets, leukocytes (in general) and neutrophils respectively, the cremaster muscle was exposed and the microcirculation was observed by conventional and confocal intravital microscopy. Results: While the acute administration of prasugrel to CON and SCA mice did not alter the quantity of platelets in the microcirculation (data not shown), it significantly reduced the adhesion of platelets to the vascular wall in both CON animals (12±1.6 vs 5.7±1.4 % adhered of total platelets, n=3; p<0.05) and SCA mice after TNF (36.2±7.1 vs 4.8±2.5 % platelets adhered for w/out and with prasugrel, respectively; n=3; p<0.0001) (see Figure). Furthermore, prasugrel significantly reduced the number of platelet/neutrophil aggregates in TNF-treated CON (2.7±0.4 vs 0.53±0.3 aggregates adhered 100 µm−1, p<0.001 n=3) and SCA mice (3.1±0.7 vs 0.76±0.3 aggregates adhered 100 µm−1, p<0.01 n=3), and also platelet/non-neutrophil leukocytes in CON (1.3±0.7 vs 0.2±0.1 aggregates adhered 100 µm−1, p<0.01 n=3) and SCA mice (2.0±0.3 vs 0.2±0.1 aggregates adhered 100 µm−1, p<0.001 n=3). Alterations in platelet adhesion to the vascular wall and platelet-leukocyte aggregate formation were accompanied by reductions in the recruitment of leukocytes to the venule walls, after prasugrel administration. Prasugrel significantly decreased the rolling, adhesion and extravasation of leukocytes in both CON (4.9±2.7 vs 1.14±0.5 min−1; 9.2±0.8 vs 2.7±0.5 100 µm−1; 1.9±0.3 vs 0.4±0.1 100x50 µm-2; for w/out and with prasugrel, respectively; n=3; p<0.001) and SCA mice (36.0±3.0 vs 3.0±0.6 min−1; 16.6±1.5 vs 6.4±1.2 100 µm−1; 6.9±0.8 vs 1.7±0.4 100x50 µm-2; for w/out and with prasugrel, respectively; n=3; p<0.0001). Acute prasugrel treatment also increased blood flow velocity after TNF stimulation in CON mice (37±10 vs 110±35 mm3/s, p<0.01, n=3) and substantially augmented flow in SCA mice (13±2 vs 94±12 mm3/s, p<0.0001, n=3), approaching flow velocities similar to those observed in control animals that had not received TNF (Data not shown). The survival of SCA animals treated with a single dose of prasugrel increased by 1.3 hours compared to the SCA animals that received the vehicle (n=3, p<0.05). Complimentary experiments demonstrated a similar effect of the chronic administration of prasugrel, which significantly reduced leukocyte recruitment to the vascular wall after TNF in both CON and SCA mice (data not shown, P<0.05). Conclusions: Our findings show that the inhibition of platelet activation by prasugrel, while significantly decreasing the adhesion of platelets to venules walls and the formation of heterocellular aggregates, was able to almost abolish the recruitment of leukocytes to the microcirculation of SCA mice after an inflammatory stimulus. This reduction in platelet and leukocyte vascular recruitment was associated with major improvements in blood flow and in the survival of animals with SCA. Thus, our data suggest that platelet activation and the consequent adhesion of platelets to the vascular wall is a fundamental step in the initiation of the vaso-occlusive process in response to inflammatory stimuli. Our findings support further investigations into the use of drugs that inhibit platelet activation (including prasugrel itself) as a therapeutic approach for SCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.