TLR2, TLR4, CD14, CD11B, and CD11C expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis, and septic shock

ABSTRACT: Bacterial recognition and induced cellular activation are fundamental for the host control of infection, yet the limit between protective and harmful response is still inexact. Forty-one patients were enrolled in this study: 14 with sepsis, 12 with severe sepsis, and 15 with septic shock. Seventeen healthy volunteers (HV) were included as control. The expression of TLR2, TLR4, CD14, CD11b, and CD11c was analyzed on monocytes surface in whole blood. sCD14 was measured in serum, and TNF-alpha, IL-6, and IL-10 cytokine levels were measured in PBMC supernatants after LPS, IL-1beta, and TNF-alpha stimuli by ELISA. An increase in sCD14 and a decreased mCD14 were found in patients as compared with HV (P < 0.001). However, no differences in the expression of TLR2, TLR4, and CD11c were found among the groups. A trend toward differential expression of CD11b was observed, with higher values found in patients with sepsis as compared with HV. A negative regulation of the inflammatory cytokine production was observed in patients with severe sepsis and shock septic in relation to sepsis and HV, regardless of the stimulus. No significant difference in IL-10 production was found among the groups. In this study, we show that the inflammatory response is associated with the continuum of clinical manifestations of sepsis, with a strong inflammatory response in the early phase (sepsis) and a refractory picture in the late phases (severe sepsis and septic shock). Correlation between cell surface receptors and cytokine production after IL-1beta and TNF-alpha stimuli and the observation of a single and same standard response with the different stimulus suggest a pattern of immunology response that is not dependent only on the expression of the evaluated receptors and that is likely to have a regulation in the intracellular signaling pathways.     

Implication of Toll-like receptor and tumor necrosis factor alpha signaling in septic shock

Septic shock is initiated by a systemic inflammatory response to microbial infection that frequently leads to impaired perfusion and multiple organ failure. Because of its high risk of death, septic shock is a major problem particularly for patients in the intensive care unit. In general, bacterial lipopolysaccharide (LPS) is a strong activator of various immune responses and stimulates monocytes/macrophages to release a variety of inflammatory cytokines. However, overproduction of inflammatory factors in response to bacterial infections is known to cause septic shock, similar to that induced by LPS. Studies of LPS-signaling pathways and downstream inflammatory cytokines may have critical implications in the treatment of sepsis. In recent years, there has been significant progress in understanding the signaling pathways activated by LPS and its receptor Toll-like receptor 4 (TLR4), as well as by tumor necrosis factor alpha (TNFalpha), a potent inflammatory cytokine induced by LPS stimulation. This review briefly summarizes our current knowledge of these signaling pathways and critical signal transducers. Characterization of key signal transducers may allow us to identify tractable, novel targets for the therapeutic interventions of sepsis.     

Cell type specific involvement of RIG-I in antiviral responses

Toll-like receptors (TLRs) play an important role in antiviral response by recognizing viral components. Recently, a RNA helicase, RIG-I, is also suggested to recognize viral double-stranded RNA. However, how these molecules contribute to viral recognition in vivo is poorly understood. Here we would like to show that RIG-I is essential for induction of type I interferons (IFNs) after infection with RNA viruses in fibroblasts and conventional dendritic cells (DCs). RIG-I induces type I IFNs by activating IRF3 via IkappaB kinase related kinases. In contrast, plasmacytoid DC, which produce large amounts of IFN-alpha, use the TLR system rather than RIG-I for viral detection. Taken together, RIG-I and the TLR system exert antiviral responses in a cell type specific manner.     

Early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza A (H1N1): speed is life

The need for early antimicrobial therapy is well established for life-threatening bacterial and fungal infections including meningitis and sepsis/septic shock. However, a link between the outcome of serious viral infections and delays in antiviral therapy is not as well recognized. Recently, with the occurrence of the influenza A/H1N1 pandemic of 2009, a large body of data regarding this issue has become available. Studies analysing data from this pandemic have consistently shown that delays in initiation of antiviral therapy following symptom onset are significantly associated with disease severity and death. Optimal survival and minimal disease severity appear to result when antivirals are started as soon as possible after symptom onset.     

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.     

Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice

We have previously identified two antiviral cytokines (interferon [IFN]-gamma and IFN-alpha/beta) that downregulate hepatitis B virus (HBV) replication in the liver of transgenic mice. The cytokine-inducible downstream events that inhibit HBV replication have not been identified. One possible factor is nitric oxide (NO), a pleiotropic free radical with antiviral activity that is produced in the liver by the inducible NO synthase (iNOS). To examine the role of NO in our model, we crossed transgenic mice that replicate HBV with mice that lack a functional iNOS. Importantly, iNOS-deficient mice were almost completely resistant to the noncytopathic inhibitory effect of HBV-specific cytotoxic T lymphocytes on viral replication, an effect that we have shown previously to depend on the intrahepatic induction of IFN-gamma. Conversely, iNOS-deficient mice were not resistant to the antiviral effect of IFN-alpha/beta induced by either polyinosinic-polycytidylic acid complex or by lymphocytic choriomeningitis virus (LCMV) infection. These results indicate that NO mediates the antiviral activity of IFN-gamma, whereas the antiviral activity of IFN-alpha/beta is NO independent. We also compared the relative sensitivity of LCMV to control by NO in these animals. Interestingly, LCMV replicated to higher levels in the liver of iNOS-deficient mice than control mice, indicating that NO controls LCMV replication in the liver, as well as HBV.     

TLR4-induced IFN-gamma production increases TLR2 sensitivity and drives Gram-negative sepsis in mice

Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4–MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-α release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-γ in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection.     

Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense.

To further understand the role of cytokine responses in symptom formation and host defenses in influenza infection, we determined the levels of IL-1beta, IL-2, IL-6, IL-8, IFN-alpha, TGF-beta, and TNF-alpha in nasal lavage fluid, plasma, and serum obtained serially from 19 volunteers experimentally infected with influenza A/Texas/36/91 (H1N1) and correlated these levels with various measures of infection and illness severity. We found that IL-6 and IFN-alpha levels in nasal lavage fluids peaked early (day 2) and correlated directly with viral titers, temperature, mucus production, and symptom scores. IL-6 elevations were also found in the circulation at this time point. In contrast, TNF-alpha responses peaked later (day 3 in plasma, day 4 in nasal fluids), when viral shedding and symptoms were subsiding. Similarly, IL-8 peaked late in the illness course (days 4-6) and correlated only with lower respiratory symptoms, which also occurred late. None of IL-1beta, IL-2, or TGF-beta levels increased significantly. These data implicate IL-6 and IFN-alpha as key factors both in symptom formation and host defense in influenza.     

Two roads diverged: interferon alpha/beta- and interleukin 12-mediated pathways in promoting T cell interferon gamma responses during viral infection

Viral infections induce CD8 T cell expansion and interferon (IFN)-gamma production for defense, but the innate cytokines shaping these responses have not been identified. Although interleukin (IL)-12 has the potential to contribute, IL-12-dependent T cell IFN-gamma has not been detected during viral infections. Moreover, certain viruses fail to induce IL-12, and elicit high levels of IFN-alpha/beta to negatively regulate it. The endogenous factors promoting virus-induced T cell IFN-gamma production were defined in studies evaluating CD8 T cell responses during lymphocytic choriomeningitis virus infections of mice. Two divergent supporting pathways were characterized. Under normal conditions of infections, the CD8 T cell IFN-gamma response was dependent on endogenous IFN-alpha/beta effects, but was IL-12 independent. In contrast, in the absence of IFN-alpha/beta functions, an IL-12 response was revealed and substituted an alternative pathway to IFN-gamma. IFN-alpha/beta-mediated effects resulted in enhanced, but the alternative pathway also promoted, resistance to infection. These observations define uniquely important IFN-alpha/beta-controlled pathways shaping T cell responses during viral infections, and demonstrate plasticity of immune responses in accessing divergent innate mechanisms to achieve similar ultimate goals.     

IFN-alpha gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes.

Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.     

Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1alpha delivery to the liver

Macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) is critical for liver NK cell inflammation and delivery of IFN-gamma to mediate downstream protective responses against murine cytomegalovirus (MCMV) infections. This system was used to evaluate the upstream contribution of the type 1 IFNs, IFN-alpha/beta, in promotion of MIP-1alpha production. Mice deficient in IFN-alpha/beta functions, as a result of mutation in the receptor for these cytokines (IFN-alpha/betaR(-)), were profoundly deficient in MIP-1alpha expression and accumulation of NK cells and macrophages in the liver and had increased sensitivity to MCMV infection. The cytokines themselves were responsible for the immunoregulatory effects, since administration of recombinant IFN-alpha (rIFN-alpha) to immunocompetent mice also induced these changes. IFN-alpha/beta was required for NK cell accumulation during infection, and MIP-1alpha was required for NK cell accumulation in response to administered rIFN-alpha. In vivo trafficking assays demonstrated a requirement for IFN-alpha/betaR signaling for leukocyte localization in, and delivery of MIP-1alpha-producing macrophages to, the liver. These results extend characterization of the cytokine and chemokine cascade required for protection against viral infections in tissues by defining IFN-alpha/beta-dependent mechanisms promoting MIP-1alpha production and the resulting hepatic accumulation of NK cells.     

The cathelicidin-derived tritrpticin enhances the efficacy of ertapenem in experimental rat models of septic shock

Sepsis remains a serious clinical problem despite intense efforts to improve survival. In this study, the efficacy of ertapenem combined with the cathelicidin tritrpticin was investigated in two rat models of septic shock. Main outcome measures were bacterial growth in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; endotoxin, interleukin 6, and tumor necrosis factor alpha concentrations in plasma; and lethality. Adult male Wistar rats were given (1) an intraperitoneal injection of 1 mg Escherichia coli serotype 0111:B4 LPS or (2) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg tritrpticin, 15 mg/kg ertapenem, and 1 mg/kg tritrpticin combined with 15 mg/kg ertapenem. Each group included 20 animals. All compounds significantly reduced bacterial growth and lethality as compared with saline treatment. Treatment with tritrpticin resulted in significant decrease in plasma endotoxin and cytokine levels, whereas ertapenem exerted opposite effect. The combination between tritrpticin and ertapenem proved to be the most effective treatment in reducing all variables measured. In conclusion, tritrpticin enhances ertapenem efficacy in gram-negative septic shock rat models.     

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.     

Type I interferons protect neonates from acute inflammation through interleukin 10-producing B cells

Newborns and infants are highly susceptible to viral and bacterial infections, but the underlying mechanism remains poorly understood. We show that neonatal B cells effectively control the production of proinflammatory cytokines by both neonatal plasmacytoid and conventional dendritic cells, in an interleukin (IL) 10-dependent manner, after Toll-like receptor (TLR) 9 triggering. This antiinflammatory property of neonatal B cells may extend to other TLR agonists (Pam3CSK4, lipopolysaccharide, and R848) and viruses. In the absence of B cells or of CD5(+) B cell subsets, neonatal mice developed stronger inflammatory responses and became lethally susceptible to CpG challenge after galactosamine sensitization, whereas wild-type (WT) mice were resistant. Paradoxically, interferon (IFN)-alpha/beta enhanced the inflammatory response to CpG challenge in adult mice, whereas they helped to control neonatal acute inflammation by stimulating the secretion of IL-10 by neonatal B cells. Finally, WT neonatal B cells rescued IL-10(-/-) neonates from a lethal CpG challenge, whereas IFN-alpha/beta receptor-deficient B cells did not. Our results show that type I IFNs support a negative regulatory role of neonatal B cells on TLR-mediated inflammation, with important implications for neonatal inflammation and infection.     

Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that induces a cytosolic signaling cascade resulting in expression of interferon (IFN)-beta. Although type I IFNs are critical in viral defense, their role in immunity to bacterial pathogens is much less clear. In this study, we addressed the role of type I IFNs by examining the infection of L. monocytogenes in BALB/c mice lacking the type I IFN receptor (IFN-alpha/betaR-/-). During the first 24 h of infection in vivo, IFN-alpha/betaR-/- and wild-type mice were similar in terms of L. monocytogenes survival. In addition, the intracellular fate of L. monocytogenes in macrophages cultured from IFN-alpha/betaR-/- and wild-type mice was indistinguishable. However, by 72 h after inoculation in vivo, IFN-alpha/betaR-/- mice were approximately 1,000-fold more resistant to a high dose L. monocytogenes infection. Resistance was correlated with elevated levels of interleukin 12p70 in the blood and increased numbers of CD11b+ macrophages producing tumor necrosis factor alpha in the spleen of IFN-alpha/betaR-/- mice. The results of this study suggest that L. monocytogenes might be exploiting an innate antiviral response to promote its pathogenesis.     

Natural interferon alpha/beta-producing cells link innate and adaptive immunity

Innate immune responses to pathogens critically impact the development of adaptive immune responses. However, it is not completely understood how innate immunity controls the initiation of adaptive immunities or how it determines which type of adaptive immunity will be induced to eliminate a given pathogen. Here we show that viral stimulation not only triggers natural interferon (IFN)-alpha/beta-producing cells (IPCs) to produce vast amounts of antiviral IFN-alpha/beta but also induces these cells to differentiate into dendritic cells (DCs). IFN-alpha/beta and tumor necrosis factor alpha produced by virus-activated IPCs act as autocrine survival and DC differentiation factors, respectively. The virus-induced DCs stimulate naive CD4(+) T cells to produce IFN-gamma and interleukin (IL)-10, in contrast to IL-3-induced DCs, which stimulate naive CD4(+) T cells to produce T helper type 2 cytokines IL-4, IL-5, and IL-10. Thus, IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-alpha/beta, and subsequently triggering adaptive T cell-mediated immunity by differentiating into DCs. IPCs constitute a critical link between innate and adaptive immunity.     

Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro

To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-alpha, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-alpha, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-alpha, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added 3 d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for interferons in the therapy of HIV infection.     

Bacterial DNA and RNA induce rat cardiac myocyte contraction depression in vitro

Sepsis and septic shock, the systemic immunologic and pathophysiologic response to overwhelming infection, are associated with perturbation of a variety of metabolic cell pathways and with multiple organ failure (MOF) including cardiac depression. This depression has been attributed to the effect of several circulating and locally produced proinflammatory mediators. Recent data suggest that bacterial nucleic acids can produce profound systemic inflammatory responses characterized by circulatory shock in intact animals. In this study, bacterial DNA and RNA derived from pathogenic clinical S. aureus and E. coli isolates are shown to induce early concentration-dependent depression of maximum extent and peak velocity of contraction of electrically paced neonatal rat ventricular myocytes in culture. Significant but more modest depression was generated by a nonpathogenic E. coli isolate. Pretreatment with a DNase or RNase abrogated this effect. Further, synthetic, double-stranded RNA (dsRNA) also induced concentration-dependent depression of myocyte contraction, with the effect also being prevented by pretreatment with RNase. These data suggest that bacterial DNA and RNA may contribute to myocardial depression during bacterial sepsis and septic shock.