Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist, SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection.

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

Production of Neutrophil Extracellular Traps Contributes to the Pathogenesis of Francisella tularemia.

Francisella tularensis(Ft) is a highly virulent, intracellular Gram-negative bacterial pathogen. Acute Ft infection by aerosol route causes pneumonic tularemia, characterized by nodular hemorrhagic lesions, neutrophil-predominant influx, necrotic debris, fibrin deposition, and severe alveolitis. Ft suppresses activity of neutrophils by impairing their respiratory burst and phagocytic activity. However, the fate of the massive numbers of neutrophils recruited to the infection site is unclear. Here, we show that Ft infection resulted in prominent induction of neutrophil extracellular traps (NETs) within damaged lungs of mice infected with the live attenuated vaccine strain of Ft(Ft-LVS), as well as in the lungs of domestic cats and rabbits naturally infected with Ft. Further, Ft-LVS infection increased lung myeloperoxidase (MPO) activity, which mediates histone protein degradation during NETosis and anchors chromatin scaffolds in NETs. In addition, Ft infection also induced expression of peptidylarginine deiminase 4, an enzyme that causes citrullination of histones during formation of NETs. The released NETs were found largely attached to the alveolar epithelium, and disrupted the thin alveolar epithelial barrier. Furthermore, Ft infection induced a concentration-dependent release of NETs from neutrophils in vitro. Pharmacological blocking of MPO reduced Ft-induced NETs release, whereas addition of H2O2 (a substrate of MPO) significantly augmented NETs release, thus indicating a critical role of MPO in Ft-induced NETs. Although immunofluorescence and electron microscopy revealed that NETs could efficiently trap Ft bacteria, NETs failed to exert bactericidal effects. Taken together, these findings suggest that NETs exacerbate tissue damage in pulmonary Ft infection, and that targeting NETosis may offer novel therapeutic interventions in alleviating Ft-induced tissue damage.

Combination Therapy Targeting Platelet Activation and Virus Replication Protects Mice against Lethal Influenza Pneumonia.

Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P2Y12 platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.

Neutrophils Induce a Novel Chemokine Receptors Repertoire During Influenza Pneumonia.

Exaggerated host innate immune responses have been implicated in severe influenza pneumonia. We have previously demonstrated that excessive neutrophils recruited during influenza infection drive pulmonary pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. Chemokine receptors (CRs) are essential in the recruitment and activation of leukocytes. Although neutrophils have been implicated in influenza pathogenesis, little is known about their phenotypic changes, including expression of CRs occurring in the infected -lung microenvironment. Here, we examined CC and CXC CRs detection in circulating as well as lung-recruited neutrophils during influenza infection in mice using flow cytometry analyses. Our studies revealed that lung-recruited neutrophils displayed induction of CRs, including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR3, and CXCR4, all of which were marginally induced in circulating neutrophils. CXCR2 was the most predominant CR observed in both circulating and lung-infiltrated neutrophils after infection. The stimulation of these induced CRs modulated neutrophil phagocytic activity, ligand-specific neutrophil migration, bacterial killing, and NETs induction ex vivo. These findings indicate that neutrophils induce a novel CR repertoire in the infectious lung microenvironment, which alters their functionality during influenza pneumonia.

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.

Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

BACKGROUND: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues. METHODS: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application RESULTS: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM). CONCLUSION: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

Arjunolic acid: a novel phytomedicine with multifunctional therapeutic applications.

Herbal plants with antioxidant activities are widely used in Ayurvedic medicine for cardiac and other problems. Arjunolic acid is one such novel phytomedicine with multifunctional therapeutic applications. It is a triterpenoid saponin, isolated earlier from Terminalia arjuna and later from Combretum nelsonii, Leandra chaeton etc. Arjunolic acid is a potent antioxidant and free radical scavenger. The scientific basis for the use of arjunolic acid as cardiotonic in Ayurvedic medicine is proven by its vibrant functions such as prevention of myocardial necrosis, platelet aggregation and coagulation and lowering of blood pressure, heart rate and cholesterol levels. Its antioxidant property combined with metal chelating property protects organs from metal and drug induced toxicity. It also plays an effective role in exerting protection against both type I and type II diabetes and also ameliorates diabetic renal dysfunctions. Its therapeutic multifunctionality is shown by its wound healing, antimutagenic and antimicrobial activity. The mechanism of cytoprotection conferred by arjunolic acid can be explained by its property to reduce the oxidative stress by enhancing the antioxidant levels. Apart from its pathophysiological functions, it possesses dynamic insecticidal property and it is used as a structural molecular framework in supramolecular chemistry and nanoscience. Esters of ajunolic acid function as gelators of a wide variety of organic liquids. Experimental studies demonstrate the versatile effects of arjunolic acid, but still, further investigations are necessary to identify the functional groups responsible for its multivarious effects and to study the molecular mechanisms as well as the probable side effects/toxicity owing to its long-term use. Though the beneficial role of this triterpenoid has been assessed from various angles, a comprehensive review of its effects on biochemistry and organ pathophysiology is lacking and this forms the rationale of this review.

Differential anti-inflammatory and anti-fibrotic activity of transplanted mesenchymal vs. hematopoietic stem cells in carbon tetrachloride-induced liver injury in mice.

Bone marrow stem cells nullify acquired and non-acquired diseases of liver through multiple strategies including antiinflammation. However, little is known about the in vivo mechanism of immunomodulation by stem cells in mediating liver cirrhosis. Mesenchymal stem cells (MSC) or hematopoietic stem cells (HSC) isolated from bone marrow of male mice were transplanted into female mice with acute liver inflammation. Serum levels of liver proteins and aminotransferase as well as hepatic antioxidant enzymes were estimated. Immunostaining for the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), alpha smooth muscle actin (alpha-SMA) and type I collagen proteins was carried out and the expression of these mRNAs was also studied. After post-transplantation, the levels of serum albumin and aminotransferases became normal and the levels of antioxidants were significantly high in the MSC treated mice compared to HSC and control mice. Necrotic cells and invasion of neutrophils were not observed in histological sections of liver of MSC treated mice. Immunostaining showed that IL-6 and TNF-alpha were not expressed in the MSC treated mice when compared to the control and HSC treated mice. alpha-SMA representing activated myofibroblasts and type I collagen were not expressed in MSC treated group. These inflammatory and fibrogenic results were further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The acute inflammation ended with the formation of fibrosis in the HSC and control groups by the uncontrolled immunoreactions. Protection mechanism of MSC therapy against injury and fibrosis in the liver occurs by the suppression of inflammation. Our findings suggest that bone marrow MSC are capable of alleviating the immunoreactions leading to the fibrosis in the liver.