Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity.

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2-/- mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2-/- mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and -independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2-/- mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy.

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis.

Interferon-gamma and nitric oxide synthase 2 mediate the aggregation of resident adherent peritoneal exudate cells: implications for the host response to pathogens.

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or "group behavior" of APECs are discussed in the context of host resistance to infectious organisms.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite.

Interferon-gamma (Ifnγ), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifnγ were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifnγ; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifnγ-induced cell death. NO inhibitors greatly reduce Ifnγ-induced ROS; however, ROS inhibitors did not affect the levels of Ifnγ-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifnγ-induced cell death. Further analysis revealed that Ifnγ induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifnγ treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifnγ-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifnγ. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifnγ alone become sensitive to the combination of Ifnγ and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifnγ-mediated cell death. The implications of these findings in the Ifnγ-mediated treatment of malignancies are discussed.

Regulation of chemokines, CCL3 and CCL4, by interferon γ and nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica serovar typhimurium infection.

BACKGROUND: Interferon γ (IFN-γ) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-γ downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-γ and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. METHODS: Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-γ-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-γ(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. RESULTS: IFN-γ repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-γ and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-γ and NOS2 enhanced serum amounts of tumor necrosis factor α and CXCL10 but repressed CCL3 and CCL4. CONCLUSIONS: This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.

Identification of early biomarkers during acetaminophen-induced hepatotoxicity by fourier transform infrared microspectroscopy.

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Gene modulation and immunoregulatory roles of interferon gamma.

Interferon-gamma (IFNgamma) is a central regulator of the immune response and signals via the Janus Activated Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) pathway. Phosphorylated STAT1 homodimers translocate to the nucleus, bind to Gamma Activating Sequence (GAS) and recruit additional factors to modulate gene expression. A bioinformatics analysis revealed that greater number of putative promoters of immune related genes and also those not directly involved in immunity contain GAS compared to response elements (RE) for Interferon Regulatory Factor (IRF)1, Nuclear factor kappa B (NFkappaB) and Activator Protein (AP)1. GAS is present in putative promoters of well known IFNgamma-induced genes, IRF1, GBP1, CXCL10, and other genes identified were TLR3, VCAM1, CASP4, etc. Analysis of three microarray studies revealed that the expression of a subset of only GAS containing immune genes were modulated by IFNgamma. As a significant correlation exists between GAS containing immune genes and IFNgamma-regulated gene expression, this strategy may identify novel IFNgamma-responsive immune genes. This analysis is integrated with the literature on the roles of IFNgamma in mediating a plethora of functions: anti-microbial responses, antigen processing, inflammation, growth suppression, cell death, tumor immunity and autoimmunity. Overall, this review summarizes our present knowledge on IFNgamma mediated signaling and functions.