Marina Gumenscheimer

The role of the liver in the response to LPS: experimental and clinical findings

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-α induces hepatocyte apoptosis and lethal effects are due to secreted TNF-α and not to cell-associated TNF-α. An exaggerated production of TNF-α has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-γ and IFN-α/β are involved in the macrophage-induced release of TNF-α. The prominent role of LPS and TNF-α in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-α/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-α and interleukin (IL)-1β levels than responders. Moreover, in responders, an equilibrium between IFN-γ and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.

Requirement for TLR9 in the immunomodulatory activity of Propionibacterium acnes.

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-α and IFN-γ in response to LPS, lipid A, synthetic lipopeptide Pam3CysK4, or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9−/−, mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9−/− mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-γ is a key mediator of these effects, and enhanced IFN-γ mRNA expression was absent in spleen and liver of P. acnes-primed TLR9−/− mice, we conclude that TLR9 is required for the induction of IFN-γ by P. acnes.

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans , which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPSresponder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein. MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2— /—) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses

ABSTRACT Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-α) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-α could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-α shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-α doses in TLR4−/− but not in TLR2−/− mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 μg. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-α, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use.

TLR9-Dependent and Independent Pathways Drive Activation of the Immune System by Propionibacterium Acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1–2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders.

Beneficial or Deleterious Effects of a Preexisting Hypersensitivity to Bacterial Components on the Course and Outcome of Infection

ABSTRACT Priming with heat-killed Propionibacterium acnes enhances the sensitivity of mice to lipopolysaccharide (LPS) and other biologically active bacterial components. We show that P. acnes priming has protective and deleterious effects on a subsequent serovar Typhimurium infection. It may result in a complete protection or prolonged survival, or it may accelerate mortality of the infected mice, depending on the number of serovar Typhimurium bacteria administered and on the degree of LPS hypersensitivity at the time of infection. Both effects of P. acnes-induced hypersensitivity are mediated by gamma interferon (IFN-γ) and are based on a differential activation of the innate immune mechanisms which recognize and react against the LPS present in infecting bacteria. In P. acnes-primed mice null for LPS-binding protein (LBP−/− mice), the impaired LPS recognition, due to the absence of LBP, resulted in a higher resistance to serovar Typhimurium infection. A similar P. acnes priming of mice had a protective, but no deleterious effect on a subsequent L. monocytogenes infection. This effect was IFN-γ dependent but independent of LBP.

TNF-α hyper-responses to Gram-negative and Gram-positive bacteria in Propionibacterium acnes primed or Salmonella typhimurium infected mice

IFN-γ-dependent hypersensitivity to LPS is inducible in mice by infection or pre-treatment with killed bacteria. Hypersensitive mice exhibit enhanced inflammatory responses to LPS, including the overproduction of TNF-α. Using Lpsn BALB/c and Lpsd BALB/c/l mice, primed with Propionibacterium acnes or infected with Salmonella typhimurium, we show that concurrently to hypersensitivity to LPS, a hypersensitivity to other constituents of killed Gram-negative or Gram-positive bacteria and to staphylococcal enterotoxin B (SEB) develops. The TNF-α hyper-responses in sensitized mice induced by different Gram-positive bacteria, are generally weaker than those by Gram-negative bacteria and vary significantly, due to the absence of a common, LPS-equivalent component. Using IFN-γR—/— and the respective wild-type mice, we demonstrate that although sensitization to LPS and killed Listeria monocytogenes is exclusively IFN-γ-dependent, an IFN-γ-independent, moderate sensitization to certain TNF-α-inducing constituents in bacteria may develop in parallel.

Role of interferons in LPS hypersensitivity.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-γ-dependent and moderate IFN-γ-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-γ by bacteria. We showed that Gram-negative bacteria induce IFN-γ in mice also by an IFN-β-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-β. Further, IFN-β can be replaced by IFN-α. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-β. Therefore, the IFN-β pathway of IFN-γ induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-α/β-dependent pathway is expected to play a role whenever IFN-α or IFN-β, and IL-18 are produced concomitantly during infection.

Bacteria-induced hypersensitivity to endotoxin

Endotoxin (LPS) hypersensitivity may be induced in mice by live or killed Gram-negative and Gram-positive bacteria. It is characterized by an overproduction of pro-inflammatory cytokines in response to LPS and by an enhanced susceptibility of the mice to the lethal activity of LPS and TNFα. The induction of LPS hypersensitivity by bacteria is mediated by IFN-γ. Consequently, its development can be inhibited by anti-IFN-γ antibodies and is absent in mice with an impaired IFNγ receptor. In sensitized mice, the enhanced activity of LPS is strictly LBP-dependent. Bacteria-treated, LBP-deficient mice exhibit only a marginally enhanced LPS responsiveness. In such mice, the administration of exogenous LBP at the time of LPS challenge restores the LPS hyper-response. Mice hypersensitive to purified LPS are also hypersensitive to whole Gram-negative bacteria. Such mice, however, are only moderately sensitized to Gram-positive bacteria or to the bacterial superantigen SEB. This sensitization, in contrast to the LPS and Gram-negative bacteria hypersensitivity, is IFN-γ independent. The role of LPS hypersensitivity for the outcome of Gram-negative infection is a dual one. At the early stages of infection, hypersensitivity enables the host to sense minute quantities of bacterial antigens and react against the infection. A failure of this early protective mechanism to eliminate the invading bacteria may have disastrous consequences, since the threshold of development of endotoxin shock is considerably lower in the hypersensitive host.