Intrapulmonary administration of leukotriene B(4) augments neutrophil accumulation and responses in the lung to Klebsiella infection in CXCL1 knockout mice

In prior studies, we demonstrated that 1) CXCL1/KC is essential for NF-κB and MAPK activation and expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine in Klebsiella-infected lungs, and 2) CXCL1 derived from hematopoietic and resident cells contributes to host immunity against Klebsiella. However, the role of CXCL1 in mediating neutrophil leukotriene B(4) (LTB(4)), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. In this study, we investigated 1) the role of CXCL1 in LTB(4), NADPH oxidase, and inducible NO synthase (iNOS) expression in lungs and neutrophils, and 2) whether LTB(4) postinfection reverses innate immune defects in CXCL1(-/-) mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB(4) levels, and decreased ROS and iNOS production in the lungs of CXCL1(-/-) mice after Klebsiella pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB(4) after infection. To treat immune defects in CXCL1(-/-) mice, we intrapulmonarily administered LTB(4). Postinfection, LTB(4) treatment reversed immune defects in CXCL1(-/-) mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB(4) also enhanced myeloperoxidase, H(2)O(2,) RNS production, and bacterial killing in K. pneumoniae-infected CXCL1(-/-) neutrophils. These novel results uncover important roles for CXCL1 in generating ROS and RNS in neutrophils and in regulating host immunity against K. pneumoniae infection. Our findings suggest that LTB(4) could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.     

Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice

Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.     

Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.     

Modulation of phagocyte apoptosis by bacterial pathogens

Phagocytic leukocytes such as neutrophils and macrophages are essential for the innate immune response against invading bacteria. Binding and ingestion of bacteria by these host cells triggers potent anti-microbial activity, including production of reactive oxygen species. Although phagocytes are highly adept at destroying bacteria, modulation of leukocyte apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Whereas induction of macrophage apoptosis by pathogens may adversely affect the host immune response to infection, acceleration of neutrophil apoptosis following phagocytic interaction with bacteria appears essential for the resolution of infection. This idea is supported by the finding that some bacterial pathogens alter normal phagocytosis-induced neutrophil apoptosis to survive and cause disease. This review summarizes what is currently known about modulation of phagocyte apoptosis by bacteria and describes a paradigm whereby bacteria-induced neutrophil apoptosis plays a role in the resolution of infection.     

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle^−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.     

Vascular effects of a common gene variant of extracellular superoxide dismutase in heart failure

A common gene variant of human extracellular superoxide dismutase (ecSOD), in approximately 5% of humans, is associated with increased risk of ischemic heart disease. The purpose of this study was to examine vascular effects of ecSOD with effects of the ecSOD variant (ecSOD(R213G)) in rats with heart failure. Seven weeks after coronary artery ligation, we studied rats with heart failure and sham-operated rats. Adenoviral vectors expressing human ecSOD, ecSOD(R213G), or a control virus were injected intravenously. In the aorta from rats with heart failure, responses to acetylcholine (69 +/- 4% relaxation, means +/- SE) and basal levels of nitric oxide (NO) (vasoconstrictor responses to a NO synthase inhibitor) were greatly impaired, and levels of superoxide and peroxynitrite were increased. Gene transfer of ecSOD restored responses to acetylcholine (92 +/- 2% relaxation) and basal levels of NO to normal and reduced levels of superoxide [from 2.3 +/- 0.2 to 0.9 +/- 0.2 relative light units per second per millimeter squared (RLU x s(-1) x mm(-2))] and peroxynitrite (from 2.4 +/- 0.2 to 0.9 +/- 0.1 RLU x s(-1) x mm(-2)) in the aorta from rats with heart failure. Gene transfer of ecSOD(R213G) produced little or no improvement. Responses to nitroprusside were not different among the groups. Expression of endogenous mRNA for SODs (CuZnSOD, MnSOD, and ecSOD) and endothelial NOS in the aorta was not different among the groups. In contrast to ecSOD, gene transfer of ecSOD(R213G) in rats with heart failure has minimal beneficial effect on oxidative stress, endothelial function, or basal bioavailability of NO. We speculate that greatly diminished efficacy of ecSOD(R213G) in protection against oxidative stress and endothelial dysfunction may contribute to increased risk of cardiovascular disease in humans with ecSOD(R213G).     

Cutting edge: fever-associated temperatures enhance neutrophil responses to lipopolysaccharide: a potential mechanism involving cell metabolism

Although much progress has been made in elucidating the mechanisms underlying the physiological regulation of fever, there is little understanding of the biological utility of fever's thermal component. Considering the evolutionary co-conservation of fever and innate immunity, we hypothesize that fever's thermal component might in general augment innate immune function and, in particular, neutrophil activation. Accordingly, we have evaluated the effect of febrile temperatures on neutrophil function at the single-cell level. We find that reactive oxygen intermediates and NO release are greatly enhanced at febrile temperatures for unstimulated as well as LPS-stimulated adherent human neutrophils. Furthermore, our studies suggest that these changes in oxidant release are linked to upstream changes in NADPH dynamics. Inasmuch as reactive oxygen intermediates and NO production are important elements in innate immune responses to bacterial pathogens, we suggest that the febrile rise in core temperature is a broad-based systemic signaling mechanism to enhance innate immunity.     

Distinct different contributions of the alternative and classical complement activation pathway for the innate host response during sepsis

Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice lacking either the alternative (fD(-/-)) or classical (C1q(-/-)) complement activation pathway. Both knockout mice strains showed a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD(-/-) mice demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q(-/-) mice were already overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD(-/-) mice failed to clear bacteria in a way comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during the onset of sepsis, fD(-/-) mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and blood when compared with both control- and C1q(-/-) mice, indicating a potential loss of control over these immune responses. Further in vitro experiments revealed a strongly increased Nf-κB activation capacity in isolated neutrophils from fD(-/-) mice, supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical pathway.     

Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis.

There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, NG-nitro-L-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L-arginine, suggesting an involvement of the L-arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS(-/-)) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS(-/-) mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS(-/-) mice, in which these mechanisms were potentiated and reverted by nitro and L-arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils.     

IL-4 plays a crucial role in regulating oxidative damage in the liver during schistosomiasis

Liver enlargement and hepatocyte proliferation, normal responses in wild-type (WT) mice infected with the parasitic helminth Schistosoma mansoni, were found to be severely impaired in infected IL-4(-/-) mice. Compared with WT mice, increased levels of O(2)(-), NO, and the more highly reactive ONOO(-) were detected in the liver and produced by lesional cells isolated from liver granulomas of infected IL-4(-/-) mice. Concurrently, antioxidant defenses in the liver, specifically catalase levels, diminished dramatically during the course of infection in these animals. This contrasted to the situation in infected WT mice, where catalase levels remained as high as those in normal mice. Actual levels of reactive oxygen and nitrogen intermediates in the livers of infected IL-4(-/-) animals are thus likely to be considerably higher than those in the livers of infected WT mice. To determine whether these changes contributed to the development of the more severe disease that characterizes infection in the IL-4(-/-) animals, we treated infected IL-4(-/-) mice with uric acid, a potent scavenger of ONOO(-). This resulted in significantly increased hepatocyte proliferation, decreased morbidity, and prolonged survival. Taken together, these data indicate that IL-4 is playing a protective role during schistosomiasis by controlling the tight regulation of the generation of reactive oxygen and nitrogen intermediates in the liver.     

Effect of perfluorocarbon emulsions on function of enzyme systems responsible for generating active forms of oxygen in neutrophils

The ability of the emulsion of perfluoroorganic compounds stabilized with proxanol 268 to affect the functions of peritoneal neutrophils was evaluated. The functional activity of neutrophils was estimated from the intensity of generation of reactive oxygen species using the method of chemiluminescent analysis. The emulsion was shown to suppress the neutrophil responses to phorbol-12-myristate-13-acetate in a dose-dependent manner. No inhibition of the activity of neutrophils in the presence of the emulsion was observed in N-formylmethionylleucylphenylalanine stimulated cells. The data obtained indirectly confirm the suggestion that the perfluoride emulsion inhibits neutrophil NADPH oxidase activity. In the presence of the perfluoride emulsion, myeloperoxidase plays a more important role in the generation of luminescent responses in both N-formylmethionylleucylphenylalanine- and phorbol-12-myristate-13-acetate-stimulated neutrophils. The effect of perfluoride emulsion results in the preferential myeloperoxidase-produced generation of reactive oxygen species in the neutrophil respiratory burst.     

Human-like immune responses in CD46 transgenic mice

Neisseria meningitidis is a major cause of sepsis and/or meningitis. These bacteria normally cause disease only in humans, however, mice expressing human CD46 are susceptible to meningococcal disease. To explain the sensitivity of CD46 transgenic mice to meningococci, we evaluated early immune responses. Stimulation of TNF, IL-6, and IL-10 was stronger in CD46 transgenic mice compared with nontransgenic mice, and resembled human responses. In CD46 transgenic mice, bacterial clearance in blood started at later time points, and neutrophil numbers in blood were lower compared with nontransgenic mice. Further, elevated levels of activated microglia cells and cyclooxygenase-2 were observed in brain of infected CD46 transgenic mice. Intraperitoneal administration of meningococci lead to increased levels of macrophages only in the i.p. cavity of CD46 transgenic mice. Most of the responses were impaired or absent using LPS-deficient meningococci, showing the importance of LPS in the early immune response to meningococcal infection. Taken together, these data demonstrate that responses in mice expressing human CD46 mimic human meningococcal disease in many aspects, and demonstrate novel important links between CD46 and the innate immune system.     

Signaling via platelet-activating factor receptors accounts for the impairment of neutrophil migration in polymicrobial sepsis

Sepsis is a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. Recently, we have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This impairment seems to be mediated by circulating cytokines, chemokines, and NO. Platelet-activating factor (PAF) plays an important role in the orchestration of different inflammatory reactions, including the release of cytokines, chemokines, and free radicals. Using a PAFR antagonist, PCA-4248, and PAFR-deficient mice, we investigated whether signaling via PAFR was relevant for the failure of neutrophils to migrate to the site of infection after lethal sepsis caused by cecum ligation and puncture in mice. In PAFR-deficient mice or mice pretreated with PCA-4248 (5 mg/kg) and subjected to lethal sepsis, neutrophil migration failure was prevented, and bacterial clearance was more efficient. There was also reduced systemic inflammation (low serum cytokine levels), lower nitrate levels in plasma, and higher survival rate. Altogether, the results firmly establish a role for PAFR in mediating the early impairment of neutrophil migration toward the infectious focus. Blockade of PAFR may prevent the establishment of severe sepsis.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Neutrophils that infiltrate the central nervous system regulate T cell responses

Regulation of inflammatory responses is critical to progression of organ-specific autoimmune disease. Although many candidate cell types have been identified, immunoregulatory activity has rarely been directly assayed and never from the CNS. We have analyzed the regulatory capability of Gr-1high neutrophils isolated from the CNS of mice with experimental autoimmune encephalomyelitis. Proportions of neutrophils were markedly increased in the CNS of IFN-gamma-deficient mice. Strikingly, CNS-derived neutrophils, whether or not they derived from IFN-gamma-deficient mice, were potent suppressors of T cell responses to myelin or adjuvant Ags. Neutrophil suppressor activity was absolutely dependent on IFN-gamma production by target T cells, and suppression was abrogated by blocking NO synthase. These data identify an immunoregulatory capacity for neutrophils, and indicate that interplay between IFN-gamma, NO, and activated Gr-1high neutrophils within the target organ determines the outcome of inflammatory and potentially autoimmune T cell responses.     

Pertussis Toxin Stimulates IL-17 Production in Response to Bordetella pertussis Infection in Mice

In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3–4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-α and IFN-γ, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.     

Inducible defense mechanism against nitric oxide in Candida albicans

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.     

Type I IFN modulates host defense and late hyperinflammation in septic peritonitis

TLRs are considered important for the control of immune responses during endotoxic shock or polymicrobial sepsis. Signaling by TLRs may proceed through the adapter proteins MyD88 or TIR domain-containing adaptor inducinng IFN-beta. Both pathways can lead to the production of type I IFNs (IFN-alphabeta). In the present study, the role of the type I IFN pathway for host defense and immune pathology in sepsis was investigated using a model of mixed bacterial peritonitis. Systemic levels of IFN-alphabeta protein were markedly elevated during septic peritonitis. More detailed analyses revealed production of IFN-beta, but not IFN-alpha subtypes, and identified CD11b+ CD11c- macrophage-like cells as major producers of IFN-beta. The results further demonstrate that in IFN-alphabeta receptor I chain (IFNARI)-deficient mice, the early recruitment of neutrophils to the infected peritoneal cavity was augmented, most likely due to an increased local production of MCP-1 and leukotriene B4. In the absence of IFNARI, peritoneal neutrophils also exhibited enhanced production of reactive oxygen intermediates and elevated expression of Mac-1. Conversely, administration of recombinant IFN-beta resulted in reduced leukotriene B4 levels and decreased peritoneal neutrophil recruitment and activation. Analysis of the cytokine response to septic peritonitis revealed that IFNARI deficiency strongly attenuated late, but not early, hyperinflammation. In accordance with these findings, bacterial clearance and overall survival of IFNARI(-/-) mice were improved. Therefore, the present study reveals critical functions of the type I IFN pathway during severe mixed bacterial infections leading to sepsis. The results suggest that type I IFN exerts predominantly adverse effects under these conditions.     

Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.