Adult Apaf-1-deficient mice exhibit male infertility

Release of cytochrome c from the mitochondria, and subsequent binding to apoptotic protease-activating factor-1 (Apaf-1), is a key trigger of apoptotic events. A complex composed of Apaf-1, dATP, and cytochrome c activates a series of cytoplasmic proteases called caspases, leading to apoptotic cell death. We have disrupted the Apaf-1 gene in the mouse. Like previous reports on this knockout model, we find that most Apaf-1 mutants die perinatally and frequently exhibit exencephaly and cranioschesis. We additionally find that the neural lesions that develop in the knockout are due to an excess of neural progenitor cells that manifests as early as embryonic day 9.5 in development. In contrast to previous reports on the Apaf-1 knockout mice, we find that 5% of the mutants successfully survive to adulthood. In these survivors, the brain develops normally, but in males, there is degeneration of spermatogonia resulting in the virtual absence of sperm. Thus, cytochrome c-mediated apoptosis is not absolutely required for normal neural development, but is essential for spermatogenesis. These findings strongly suggest that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death.     

Increased susceptibility of Stat4-deficient and enhanced resistance in Stat6-deficient mice to infection with Trypanosoma cruzi

Although Th1-type responses tend to be associated with resistance to Trypanosoma cruzi infection, mixed Th1 and Th2 cytokine responses are generally observed in both resistant and susceptible mice. To help clarify the role of type 1 and type 2 cytokine responses in immunity to T. cruzi, mice with induced deficiencies in the Stat4 or Stat6 genes were infected with T. cruzi. As expected, Stat4-/- mice deficient in type 1 cytokine responses were highly susceptible to infection, exhibiting increased parasitemia levels relative to wild-type mice and 100% mortality. In contrast, parasitemia levels and survival in Stat6-deficient mice were not different from wild type. The type 1 and type 2 cytokine bias of Stat6- and Stat4-deficient mice, respectively, was confirmed by in situ immunocytochemical analysis of cytokine-producing cells in the tissues of infected mice and by subclass analysis of anti-T. cruzi serum Abs. Notably, both Stat4- and Stat6-deficient mice produced substantial amounts of anti-T. cruzi Abs. Tissues from chronically infected Stat6-deficient mice had little to no evidence of inflammation in the heart and skeletal muscle in contrast to wild-type mice, which exhibited substantial inflammation. In situ PCR analysis of these tissues provided evidence of the persistence of T. cruzi in wild-type mice, but no evidence of parasite persistence in Stat6-deficient mice. These data suggest that type 1 T cells are required for the development of immune control to T. cruzi, but that type 2 T cells contribute to parasite persistence and increased severity of disease.     

Interleukin-1-deficient mice exhibit high sensitivity to gut-derived sepsis caused by Pseudomonas aeruginosa

BACKGROUND: The role of interleukin (IL)-1 in infectious diseases is controversial; some investigators indicated an enhancing effect of IL-1 on host resistance whereas others demonstrated the protective role of IL-1 receptor antagonist in infection. We evaluated the role of endogenous IL-1 in gut-derived sepsis caused by Pseudomonas aeruginosa, by comparing IL-1-deficient mice and wild-type (WT) mice. METHODS: Gut-derived sepsis was induced by intraperitoneal injection of cyclophosphamide after colonization of P. aeruginosa strain D4 in the intestine. RESULTS: The survival rate of IL-1-deficient mice was significantly lower than that of WT mice (P<0.01). Bacterial counts in the liver, mesenteric lymph node and blood were significantly higher in IL-1-deficient mice than in WT mice. Tumor necrosis factor alpha and IL-6 in the liver were significantly higher in IL-1-deficient mice than in WT mice. In vitro, phagocytosis and cytokine production by macrophages were impaired in IL-1-deficient mice compared with WT mice. CONCLUSION: Our results indicate a critical role for IL-1 during gut-derived P. aeruginosa sepsis. The results also suggest that both impairment of cytokine production and phagocytosis by macrophages are caused by IL-1 deficiency and lead to impaired host response.     

Cbl-3-deficient mice exhibit normal epithelial development

Cbl family proteins are evolutionarily conserved ubiquitin ligases that negatively regulate signaling from tyrosine kinase-coupled receptors. The mammalian cbl family consists of c-Cbl, Cbl-b, and the recently cloned Cbl-3 (also known as Cbl-c). In this study, we describe the detailed expression pattern of murine Cbl-3 and report the generation and characterization of Cbl-3-deficient mice. Cbl-3 exhibits an expression pattern distinct from those of c-Cbl and Cbl-b, with high levels of Cbl-3 expression in epithelial cells of the gastrointestinal tract and epidermis, as well as the respiratory, urinary, and reproductive systems. Cbl-3 expression was not detected in nonepithelial cells, but within epithelial tissues, the levels of Cbl-3 expression varied from undetectable in the alveoli of the lungs to very strong in the cecum and colon. Despite this restricted expression pattern, Cbl-3-deficient mice were viable, healthy, and fertile and displayed no histological abnormalities up to 18 months of age. Proliferation of epithelial cells in the epidermises and gastrointestinal tracts was unaffected by the loss of Cbl-3. Moreover, Cbl-3 was not required for attenuation of epidermal growth factor-stimulated Erk activation in primary keratinocytes. Thus, Cbl-3 is dispensable for normal epithelial development and function.     

Ndrg1-deficient mice exhibit a progressive demyelinating disorder of peripheral nerves

NDRG1 is an intracellular protein that is induced under a number of stress and pathological conditions, and it is thought to be associated with cell growth and differentiation. Recently, human NDRG1 was identified as a gene responsible for hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth disease type 4D), which is characterized by early-onset peripheral neuropathy, leading to severe disability in adulthood. In this study, we generated mice lacking Ndrg1 to analyze its function and elucidate the pathogenesis of Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5 weeks of age. However, myelination of Schwann cells in the sciatic nerve was normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness, especially in the hind limbs, but complicated motor skills were retained. In wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells rather than the myelin sheath. These results indicate that NDRG1 deficiency leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for maintenance of the myelin sheaths in peripheral nerves. These mice will be used for future analyses of the mechanisms of myelin maintenance.     

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.     

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

S100A1-deficient male mice exhibit increased exploratory activity and reduced anxiety-related responses

S100 proteins comprise a family of Ca(2+) binding proteins of at least 21 members. They are distinctly expressed in a variety of cell types and tissues and are thought to play unique roles, although they share a high degree of sequence homology and expression overlap. S100A1 is prominently expressed in the heart, where it takes part in Ca(2+)-cycling. Its role in the central nervous system (CNS) is largely unknown. We have generated S100A1-deficient mice by gene trap mutagenesis to study the involvement of S100A1 in the cytoarchitecture of the brain, in learning and memory, and in avoidance-approach behavior. S100A1 knock out (KO) mice develop well and their brains present with normal morphology. In wild type (Wt) mice, S100A1 protein was found in the hippocampus, cerebral cortex and amygdala, and partially co-localized with the astrocyte marker glial fibrillary acidic protein (GFAP) in the stratum radiatum of the hippocampus. Astrocytes and neurons of S100A1KO mice did not differ from those of Wt mice regarding shape, distribution and density. In the water maze, S100A1KO mice performed equally well as Wt, implying that S100A1 is not involved in spatial learning and memory. In avoidance-approach tests, predominantly male S100A1KO mice showed reduced anxiety-like responses and enhanced explorative activities. We conclude that S100A1 plays a role in modulating innate fear and exploration of novel stimuli.     

Altered immune responses and susceptibility to Leishmania major and Staphylococcus aureus infection in IL-18-deficient mice.

IL-18, formerly designated IFN-inducing factor, is a novel cytokine produced by activated macrophages. It synergizes with IL-12 in the induction of the development of Th1 cells and NK cells. To define the biological role of IL-18 in vivo, we have constructed a strain of mice lacking IL-18. Homozygous IL-18 knockout (-/-) mice are viable, fertile, and without evident histopathologic abnormalities. However, in contrast to the heterozygous (+/-) or wild-type (+/+) mice, which are highly resistant to the infection of the protozoan parasite Leishmania major, the IL-18-/- mice are uniformly susceptible. The infected IL-18-/- mice produced significantly lower levels of IFN-gamma and larger amounts of IL-4 compared with similarly infected +/- and +/+ mice. In contrast, when infected with the extracellular Gram-positive bacteria Staphylococcus aureus, the IL-18-/- mice developed markedly less septicemia than similarly infected wild-type (+/+) mice. However, the mutant mice developed significantly more severe septic arthritis than the control wild-type mice. This was accompanied by a reduction in the levels of Ag-induced splenic T cell proliferation, decreased IFN-gamma and TNF-alpha synthesis, but increased IL-4 production by the mutant mice compared with the wild-type mice. These results therefore provide direct evidence that IL-18 is not only essential for the host defense against intracellular infection, but it also plays a critical role in regulating the synthesis of inflammatory cytokines, and therefore could be an important target for therapeutic intervention.     

Apoptosis-linked gene 2-deficient mice exhibit normal T-cell development and function

The apoptosis-linked gene product, ALG-2, is a member of the family of intracellular Ca(2+)-binding proteins and a part of the apoptotic machinery controlled by T-cell receptor (TCR), Fas, and glucocorticoid signals. To explore the physiologic function of ALG-2 in T-cell development and function, we generated mice harboring a null mutation in the alg-2 gene. The alg-2 null mutant mice were viable and fertile and showed neither gross developmental abnormality nor immune dysfunction. Analyses of apoptotic responses of ALG-2-deficient T cells demonstrated that ALG-2 deficiency failed to block apoptosis induced by TCR, Fas, or dexamethasone signals. These findings indicate that ALG-2 is physiologically dispensable for apoptotic responses induced by the above signaling pathways and suggest that other functionally redundant proteins might exist in mammalian cells.     

Gab3-deficient mice exhibit normal development and hematopoiesis and are immunocompetent

Gab proteins are intracellular scaffolding and docking molecules involved in signaling pathways mediated by various growth factor, cytokine, or antigen receptors. Gab3 has been shown to act downstream of the macrophage colony-stimulating factor receptor, c-Fms, and to be important for macrophage differentiation. To analyze the physiological role of Gab3, we used homologous recombination to generate mice deficient in Gab3. Gab3(-/-) mice develop normally, are visually indistinguishable from their wild-type littermates, and are healthy and fertile. To obtain a detailed expression pattern of Gab3, we generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a predominant expression of Gab3 in lymphocytes and bone marrow-derived macrophages. However, detailed analysis demonstrated that hematopoiesis in mice lacking Gab3 is not impaired and that macrophages develop in normal numbers and exhibit normal function. The lack of Gab3 expression during macrophage differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA. Furthermore, Gab3-deficient mice have no major immune deficiency in T- and B-lymphocyte responses to protein antigens or during viral infection. In addition, allergic responses in Gab3-deficient mice appeared to be normal. Together, these data demonstrate that loss of Gab3 does not result in detectable defects in normal mouse development, hematopoiesis, or immune system function.     

ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis

Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share approximately 84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1(-/-) mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1(-/-) mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1(-/-) mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1(-/-) mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1(-/-) mice, with higher IFN-gamma production and lower IL-5 production in MOG35-55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1(-/-) mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE.     

PCSK9-deficient mice exhibit impaired glucose tolerance and pancreatic islet abnormalities

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a liver-secreted plasma enzyme, restricts hepatic uptake of low-density lipoprotein (LDL) cholesterol by promoting the degradation of LDL receptors (LDLR). PCSK9 and LDLR are also expressed in insulin-producing pancreatic islet β cells, possibly affecting the function of these cells. Here we show that, compared to control mice, PCSK9-null male mice over 4 months of age carried more LDLR and less insulin in their pancreas; they were hypoinsulinemic, hyperglycemic and glucose-intolerant; their islets exhibited signs of malformation, apoptosis and inflammation. Collectively, these observations suggest that PCSK9 may be necessary for the normal function of pancreatic islets.     

Genetic control of susceptibility of mice to Rous sarcoma virus tumorigenesis

Studies with congenic resistant strains of mice indicate that susceptibility to Rous sarcoma virus tumorigenesis is influenced by a gene or genes associated with the major histocompatibility complex (H-2). These genes manifest dominant relative susceptibility. Preliminary studies indicate that the CBA/J strain harbors a gene or genes for relative susceptibility, which are recessive. These results are compared with other studies onH-2-associated genes affecting murine viral oncogenesis.     

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.     

MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice

Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.     

Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.