Michael W. Marino

Protection from obesity-induced insulin resistance in mice lacking TNF-|[alpha]| function

Obesity is highly associated with insulin resistance and is the biggest risk factor for non-insulin-dependent diabetes mellitus. The molecular basis of this common syndrome, however, is poorly understood. It has been suggested that tumour necrosis factor (TNF)-α is a candidate mediator of insulin resistance in obesity, as it is overexpressed in the adipose tissues of rodents and humans and it blocks the action of insulin in cultured cells and whole animals. To investigate the role of TNF-α in obesity and insulin resistance, we have generated obese mice with a targeted null mutation in the gene encoding TNF-α and those encoding the two receptors for TNF-α. The absence of TNF-α resulted in significantly improved insulin sensitivity in both diet-induced obesity and that resulting for the ob/ob model of obesity. The TNFα-deficient obese mice had lower levels of circulating free fatty acids, and were protected from the obesity-related reduction in the insulin receptor signalling in muscle and fat tissues. These results indicate that TNF-α is an important mediator of insulin resistance in obesity through its effects on several important sites of insulin action.

Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase

The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each others deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2.

Structure of the InlB Leucine-Rich Repeats, a Domain that Triggers Host Cell Invasion by the Bacterial Pathogen L. monocytogenes

The L. monocytogenes protein lnlB activates phosphoinositide 3-kinase and induces phagocytosis in several mammalian cell types. The 1.86 A resolution X-ray crystal structure of the leucine-rich repeat domain of lnlB that is both necessary and sufficient to induce phagocytosis is presented here. The structure supports a crucial role for calcium in host cell invasion by L. monocytogenes and supplies a rationale for its function. Calciums are bound to the protein in an unusually exposed manner that suggests that the metals may act as a bridge between lnlB and mammalian cell surface receptors. The structure also identifies surfaces on the curved and elongated molecule that may constitute additional interaction sites in forming a bacterial-mammalian signaling complex.

TNF-α-dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection

Tumor necrosis factor-α (TNF-α) is well recognized for its role in mediating innate immune responses. However, the mechanisms of TNF-α that influence the adaptive immune response to virus infections are not well understood. In this study, we have investigated the role of TNF-α in activating the cellular and humoral responses to systemic viral challenge with recombinant replication-defective adenovirus (rAd). Evaluation of T cell function in TNF-α-deficient (TNFKO) mice revealed impaired virus-specific proliferation of T cells derived from the draining lymph nodes of the liver. Analysis of dendritic cells (DC) isolated from local draining lymph nodes after systemic challenge showed that DC from TNFKO mice were relatively immature compared with those from strain-matched wild-type mice. In vitro, TNF-α was required to mature DC efficiently during virus-mediated stimulation. Adoptive transfer of primed, mature DC into TNFKO mice restored T cell responses and reconstituted anti-adenovirus antibody responses. Thus, TNF-α plays a significant role in the maturation of DC after adenovirus challenge both in vitro and in vivo, highlighting the importance of this innate cytokine in activating adaptive immunity to viral challenge.

TRAF2 Deficiency Results in Hyperactivity of Certain TNFR1 Signals and Impairment of CD40-Mediated Responses

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.

GW domains of the Listeria monocytogenes invasion protein InlB are SH3‐like and mediate binding to host ligands

InlB, a surface‐localized protein of Listeria monocytogenes, induces phagocytosis in non‐phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q‐R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine‐rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline‐binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q‐R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB‐induced host membrane dynamics with bacterial proximity during invasion.

Visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment.

ABSTRACT Tumor necrosis factor (TNF)-deficient mice were challenged withLeishmania donovani to characterize TNF in the response of visceral intracellular infection to antileishmanial chemotherapy. In wild-type controls (i) liver infection peaked at week 2 and resolved, (ii) discrete liver granulomas developed at weeks 2 to 4 and involuted, and (iii) leishmanicidal responses to antimony (Sb), amphotericin B (AmB), and miltefosine were intact. In TNF knockout (KO) mice (i) initial liver infection was unrestrained, plateaued, and then declined somewhat by week 6, (ii) an absent early granulomatous reaction abruptly accelerated with striking tissue inflammation, widespread hepatic necrosis, and 100% mortality by week 10, and (iii) while the initial response to AmB and miltefosine was intact, killing induced by Sb therapy was reduced by >50%. Although initial AmB treatment during weeks 2 to 3 killed 98% of liver parasites, 75% of AmB-treated KO mice subsequently relapsed and died by week 12; however, additional maintenance AmB preserved long-term survival. These results for a model of visceral infection indicate that endogenous TNF is required early on to control intracellular L. donovani, support granuloma development, and mediate optimal initial effects of Sb and prevent relapse after ordinarily curative AmB treatment. A compensatory, TNF-independent antileishmanial mechanism developed in TNF KO mice; however, its effect was uncontrolled fatal inflammation. Chemotherapeutic elimination of the parasite stimulus reversed the hyperinflammatory response and preserved survival.

Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice Have Impaired Resistance to Blood-Stage Malaria

ABSTRACT The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudiAS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-γ) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-γ levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-α) levels were significantly increased in KO mice and were significantly higher than TNF-α levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-γ and TNF-α production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.

GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation

The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N‐terminal LRR region and C‐terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor‐mediated mode, as opposed to the ligand‐mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q‐R, a host protein implicated in InlB‐mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q‐R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone‐like mimicry of HGF.

Sex Steroids Induce Apoptosis of CD8⁺CD4⁺ Double-Positive Thymocytes via TNF-α

T cell production by the thymus, thymic size, cellularity and output all decrease drastically after puberty. Among the candidates that may mediate this decrease are the sex steroids: Hypersecretion or pharmacological administration of these hormones has long been known to induce thymic hypocellularity, and their depletion yields thymic hypercellularity. Here we show that a typical sex steroid, testosterone, specifically targets CD8+CD4+ double‐positive (DP) thymocytes for apoptosis via TNF‐α Anti‐TNF‐α monoclonal antibodies abrogated testosterone‐induced DP apoptosis, and TNF‐a– / – DP thymocytes were largely resistant to testosterone‐mediated apoptosis in vivo. Testosterone accomplishes this effect by up‐regulating TNF‐α production and by simultaneously sensitizing DP thymocytes to TNF‐α. Thus, TNF‐α is the critical mediator of sex steroid‐induced apoptosis in thymocytes, and its manipulation should provide a point of intervention to modulate T cell production in sex hormone disorders.