Klaus Pfeffer

Jak2 Deficiency Defines an EssentialDevelopmental Checkpoint in DefinitiveHematopoiesis

Janus kinases (Jaks) play an important role in signal transduction via cytokine and growth factor receptors. A targeted inactivation of Jak2 was performed. Jak2-/- embryos are anemic and die around day 12.5 postcoitum. Primitive erythrocytes are found, but definitive erythropoiesis is absent. Compared to erythropoietin receptor-deficient mice, the phenotype of Jak2 deficiency is more severe. Fetal liver BFU-E and CFU-E colonies are completely absent. However, multilineage hematopoietic stem cells (CD34low, c-kit(pos)) can be found, and B lymphopoiesis appears intact. In contrast to IFNalpha stimulation, Jak2-/- cells do not respond to IFNgamma. Jak2-/- embryonic stem cells are competent for LIF signaling. The data provided demonstrate that Jak2 has pivotal functions for signal transduction of a set of cytokine receptors required in definitive erythropoiesis.

The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues

Lymphotoxin beta receptor (LTbetaR)-/- mice were created by gene targeting. LTbetaR-/- mice lacked Peyers patches, colon-associated lymphoid tissues, and all lymph nodes. Mucosa patrolling alphaEbeta7high integrin+ T cells were virtually absent. Spleens lost marginal zones; T/B cell segregation and follicular dendritic cell networks were absent. Peanut agglutinin+ cells were aberrantly detectable around central arterioles. In contrast to TNF receptor p55-/- mice, antibody affinity maturation was impaired. Since LTbetaR-/- mice exhibit distinct defects when compared to LTalpha-/- and LTbeta-/- mice, it is suggested that the LTbetaR integrates signals from other TNF family members. Thus, the LTbetaR proves pivotal for the ontogeny of the secondary lymphoid tissues. Furthermore, affinity maturation is dependent on LTalpha1beta2 rather than on LTalpha3.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect

Nicotinic acid (niacin), a vitamin of the B complex, has been used for almost 50 years as a lipid-lowering drug. The pharmacological effect of nicotinic acid requires doses that are much higher than those provided by a normal diet. Its primary action is to decrease lipolysis in adipose tissue by inhibiting hormone-sensitive triglyceride lipase. This anti-lipolytic effect of nicotinic acid involves the inhibition of cyclic adenosine monophosphate (cAMP) accumulation in adipose tissue through a Gi-protein-mediated inhibition of adenylyl cyclase. A G-protein-coupled receptor for nicotinic acid has been proposed in adipocytes. Here, we show that the orphan G-protein-coupled receptor, protein upregulated in macrophages by interferon-γ (mouse PUMA-G, human HM74), is highly expressed in adipose tissue and is a nicotinic acid receptor. Binding of nicotinic acid to PUMA-G or HM74 results in a Gi-mediated decrease in cAMP levels. In mice lacking PUMA-G, the nicotinic acid–induced decrease in free fatty acid (FFA) and triglyceride plasma levels was abrogated, indicating that PUMA-G mediates the anti-lipolytic and lipid-lowering effects of nicotinic acid in vivo. The identification of the nicotinic acid receptor may be useful in the development of new drugs to treat dyslipidemia.NOTE: In the version of this article initially published online, the statements concerning equal author contribution and corresponding authors were incorrect. This mistake has been corrected for the HTML and print versions of the article.

Pretreatment of Mice with Streptomycin Provides a Salmonella enterica Serovar Typhimurium Colitis Model That Allows Analysis of Both Pathogen and Host

ABSTRACT Salmonella enterica subspecies 1 serovar Typhimurium is a principal cause of human enterocolitis. For unknown reasons, in mice serovar Typhimurium does not provoke intestinal inflammation but rather targets the gut-associated lymphatic tissues and causes a systemic typhoid-like infection. The lack of a suitable murine model has limited the analysis of the pathogenetic mechanisms of intestinal salmonellosis. We describe here how streptomycin-pretreated mice provide a mouse model for serovar Typhimurium colitis. Serovar Typhimurium colitis in streptomycin-pretreated mice resembles many aspects of the human infection, including epithelial ulceration, edema, induction of intercellular adhesion molecule 1, and massive infiltration of PMN/CD18+ cells. This pathology is strongly dependent on protein translocation via the serovar Typhimurium SPI1 type III secretion system. Using a lymphotoxin β-receptor knockout mouse strain that lacks all lymph nodes and organized gut-associated lymphatic tissues, we demonstrate that Peyers patches and mesenteric lymph nodes are dispensable for the initiation of murine serovar Typhimurium colitis. Our results demonstrate that streptomycin-pretreated mice offer a unique infection model that allows for the first time to use mutants of both the pathogen and the host to study the molecular mechanisms of enteric salmonellosis.

Partial impairment of cytokine responses in Tyk2-deficient mice.

To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.

Inhibition of natural killer cells results in acceptance of cardiac allografts in CD28-/- mice.

Successful transplantation of allogeneic organs is an important objective in modern medicine. However, sophisticated immune defense mechanisms, primarily evolved to combat infections, often work against medical transplantation. To investigate the roles of natural and adaptive immune responses in transplant rejection, we functionally inactivated key effector systems of the innate (NK cells) and the adaptive immune system (CD28-mediated costimulation of T cells) in mice. Neither of these interventions alone led to acceptance of allogeneic vascularized cardiac grafts. In contrast, inhibition of NK-receptor–bearing cells combined with CD28-costimulation blockade established long-term graft acceptance. These results indicate a concerted interplay between innate and adaptive immune surveillance for graft rejection. Thus we suggest that inactivation of NK-receptor–bearing cells could be a new strategy for successful survival of solid-organ transplants.

Interferon-γ and Tumor Necrosis Factor-α Determine Resistance to Paracoccidioides brasiliensis Infection in Mice

To investigate the role of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-γ (GKO) or TNF-α receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-α levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-α but higher levels of IFN-γ. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO. resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-γ to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-α, acting through the p55 receptor, and IFN-γ mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness.

The Lymphotoxin β Receptor Is Critically Involved in Controlling Infections with the Intracellular Pathogens Mycobacterium tuberculosis and Listeria monocytogenes

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-γ and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LTβR pathway. When mice deficient in LTα or LTβ were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LTαβ heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LTα1β2 heterotrimers (LTβR-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LTβR-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LTβR-KO mice had similar transcript levels of TNF and IFN-γ and recruited similar numbers of CD3+ T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LTβR is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LTβR-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LTα1β2 heterotrimers via the LTβR is an essential prerequisite for containment of intracellular pathogens.

Listeriosis in p47phox−/− and TRp55−/− Mice: Protection Despite Absence of ROI and Susceptibility Despite Presence of RNI

The significance of host defense mechanisms in primary listeriosis in vivo is incompletely understood. Here, we show that tumor necrosis factor receptor p55-/- (TRp55-/-) mice are susceptible to Listeria monocytogenes infection in the presence of leukocyte recruitment, inflammatory cytokine production (including IFNgamma), nitric oxide synthesis, and oxidative burst formation. Mice deficient for oxidative burst (p47[phox-/-] mice) are relatively resistant to listeriosis. Despite activation of these antibacterial effector systems, TRp55-/- phagocytes in vivo are incapable of confining and eradicating L. monocytogenes inside phagolysosomes. Bone marrow chimeras reveal that for eradication of L. monocytogenes, TRp55 is crucially required only on cells from hematopoietic origin. Unexpectedly, prior to death, exocrine pancreatic cells undergo apoptosis in TRp55-/- mice. Collectively, these data demonstrate that in vivo, TRp55 initiates a protective, listericidal mechanism in phagocytes that differs from nitric oxide production and oxidative burst formation and that uncontrolled listeriosis results in necrotizing pancreatitis in TRp55-/- mice.