Bernd Echtenacher

Protection from septic shock by neutralization of macrophage migration inhibitory factor.

Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock. High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis. Experiments performed in TNFα knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation. Anti-MIF antibody protected TNFα knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis. Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP. Conversely, co-injection of recombinant MIF and E. coli markedly increased the lethality of peritonitis. Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock. These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention.

The proinflammatory mediator macrophage migration inhibitory factor induces glucose catabolism in muscle

Severe infection or tissue invasion can provoke a catabolic response, leading to severe metabolic derangement, cachexia, and even death. Macrophage migration inhibitory factor (MIF) is an important regulator of the host response to infection. Released by various immune cells and by the anterior pituitary gland, MIF plays a critical role in the systemic inflammatory response by counterregulating the inhibitory effect of glucocorticoids on immune-cell activation and proinflammatory cytokine production. We describe herein an unexpected role for MIF in the regulation of glycolysis. The addition of MIF to differentiated L6 rat myotubes increased synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis. Increased expression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) enhanced F2,6BP production and, consequently, cellular lactate production. The catabolic effect of TNF-alpha on myotubes was mediated by MIF, which served as an autocrine stimulus for F2, 6BP production. TNF-alpha administered to mice decreased serum glucose levels and increased muscle F2,6BP levels; pretreatment with a neutralizing anti-MIF mAb completely inhibited these effects. Anti-MIF also prevented hypoglycemia and increased muscle F2,6BP levels in TNF-alpha-knockout mice that were administered LPS, supporting the intrinsic contribution of MIF to these inflammation-induced metabolic changes. Taken together with the recent finding that MIF is a positive, autocrine stimulator of insulin release, these data suggest an important role for MIF in the control of host glucose disposal and carbohydrate metabolism.

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2–related factor 2 (NRF2)–dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3−/− mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3−/− mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3−/−Il6−/− mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3−/− mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

Tumor Necrosis Factor-Dependent Adhesions as a Major Protective Mechanism Early in Septic Peritonitis in Mice

ABSTRACT The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels. In a model of septic peritonitis—cecal ligation and puncture—TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented. To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase. Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees. Under these experimental conditions, antibiotics prevented death. In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization. These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.

Differences in Innate Defense Mechanisms in Endotoxemia and Polymicrobial Septic Peritonitis

ABSTRACT Loss, reduction, or enhancement of the ability to respond to bacterial lipopolysaccharide (LPS) has no influence on survival of mice in a model of postoperative polymicrobial septic peritonitis induced by cecal ligation and puncture (CLP). This was demonstrated by using either mice with a defective Tlr4 gene, which encodes the critical receptor molecule for LPS responses, or mice deficient for LPS binding protein (LBP) or mice sensitized to LPS byPropionibacterium acnes. Though interleukin-12 (IL-12) and gamma interferon (IFN-γ) play an important role in the sensitivity to LPS as well as in the resistance to several infections, loss of these cytokine pathways does not affect survival after CLP. Thus, neutralization of neither endogenous IL-12 nor IFN-γ altered mortality. In addition, IFN-γ receptor-deficient mice demonstrated the same sensitivity to CLP as mice with a functional IFN-γ receptor. However, administration of IFN-γ at the time of operation or pretreatment of both IFN-γ-sensitive and IFN-γ-resistant mice with IL-12 significantly enhanced mortality. This indicates that in the present infection model activation of innate defense mechanisms is not dependent on LPS recognition and does not require endogenous IL-12 or IFN-γ function. Indeed, exogenous application of these two mediators had deleterious effects.

Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5α. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5α. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

The Putative α-1,2-Mannosyltransferase AfMnt1 of the Opportunistic Fungal Pathogen Aspergillus fumigatus Is Required for Cell Wall Stability and Full Virulence

ABSTRACT Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative α-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A Δafmnt1 mutant is viable and grows normally at 37°C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48°C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48°C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the Δafmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that α-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.

AfMkk2 is required for cell wall integrity signaling, adhesion, and full virulence of the human pathogen Aspergillus fumigatus

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.

Localization of the mouse defense lectin ficolin B in lysosomes of activated macrophages.

Ficolins are pattern-recognition molecules of the innate immune system able to trigger the lectin pathway of the complement activation upon binding to microbial surfaces. In humans, two plasma ficolins have been identified and characterized, whereas a third cell-associated ficolin (M-ficolin) was found on monocyte surfaces. The mouse homologue of M-ficolin is called ficolin B. Although the spatial—temporal expression patterns of mouse ficolins have been described recently, the subcellular localization of ficolin B protein is so far unknown. By using ficolin B-specific antibodies and confocal microscopy, we show that ficolin B is expressed within mouse peritoneal exudate macrophages and is co-localized with Lamp-1, a marker for lysosomes and late endosomes. In addition, the data indicate that ficolin B expression is up-regulated upon macrophage activation.

Approaching the Secrets of N-Glycosylation in Aspergillus fumigatus: Characterization of the AfOch1 Protein

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.