Ralf Kühn

Interleukin-10-deficient mice develop chronic enterocolitis

Interleukin-10 (IL-10) affects the growth and differentiation of many hemopoietic cells in vitro; in particular, it is a potent suppressor of macrophage and T cell functions. In IL-10-deficient mice, generated by gene targeting, lymphocyte development and antibody responses are normal, but most animals are growth retarded and anemic and suffer from chronic enterocolitis. Alterations in intestine include extensive mucosal hyperplasia, inflammatory reactions, and aberrant expression of major histocompatibility complex class II molecules on epithelia. In contrast, mutants kept under specific pathogen-free conditions develop only a local inflammation limited to the proximal colon. These results indicate that the bowel inflammation in the mutants originates from uncontrolled immune responses stimulated by enteric antigens and that IL-10 is an essential immunoregulator in the intestinal tract.

Inducible gene targeting in mice.

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

In Vivo Ablation of Surface Immunoglobulin on Mature B Cells by Inducible Gene Targeting Results in Rapid Cell Death

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.

Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses.

We have characterized the progressive stages of chronic intestinal inflammation that develops spontaneously in specific pathogen-free (SPF) mice with a targeted disruption in the IL-10 gene (IL-10-/-). Our longitudinal studies showed that inflammatory changes first appear in the cecum, ascending and transverse colon of 3-wk-old mutants. As the disease progressed, lesions appeared in the remainder of the colon and in the rectum. Some aged IL-10-/- mice also developed inflammation in the small intestine. Prolonged disease with transmural lesions and a high incidence of colorectal adenocarcinomas (60%) was observed in 6-mo-old mutants. Mechanistic studies have associated uncontrolled cytokine production by activated macrophages and CD4+ Th1-like T cells with the enterocolitis exhibited by IL-10-/- mice. A major role for a pathogenic Th1 response was further suggested by showing that anti-IFNgamma antibody (Ab) treatment significantly attenuated intestinal inflammation in young IL-10-/- mice. When weanlings were treated with IL-10, they failed to develop any signs of intestinal inflammation. Interestingly, IL-10 treatment of adults was not curative but did ameliorate disease progression. Our studies have also shown that inheritable factors strongly influence the disease susceptibility of IL-10-/- mice. In 3-mo-old mutants, intestinal lesions were most severe in IL-10-/- 129/SvEv and IL-10-/- BALB/c strains, of intermediate severity in the IL-10-/- 129 x C57BL/6J outbreds, and least severe in the IL-10-/- C57BL/6J strain.

Essential role for TrkB receptors in hippocampus-mediated learning.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB regulate both short-term synaptic functions and long-term potentiation (LTP) of brain synapses, raising the possibility that BDNF/TrkB may be involved in cognitive functions. We have generated conditionally gene targeted mice in which the knockout of the trkB gene is restricted to the forebrain and occurs only during postnatal development. Adult mutant mice show increasingly impaired learning behavior or inappropriate coping responses when facing complex and/or stressful learning paradigms but succeed in simple passive avoidance learning. Homozygous mutants show impaired LTP at CA1 hippocampal synapses. Interestingly, heterozygotes show a partial but substantial reduction of LTP but appear behaviorally normal. Thus, CA1 LTP may need to be reduced below a certain threshold before behavioral defects become apparent.

Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance.

Previous studies in vivo have shown that IL-10 infusion can prevent lethal endotoxic shock. Mice deficient in the production of IL-10 (IL10T) were used to investigate the regulatory role of IL-10 in the responses to LPS in three experimental systems. In a model of acute endotoxic shock, it was found that the lethal dose of LPS for IL10T mice was 20-fold lower than that for wild type (wt) mice suggesting that endogenous IL-10 determines the amount of LPS which can be tolerated without death. The high mortality rate of IL10T mice challenged with modest doses of LPS was correlated to the uncontrolled production of TNF as treatment with anti-TNF antibody (Ab) resulted in 70% survival. Additional studies suggested that IL-10 mediates protection by controlling the early effectors of endotoxic shock (e.g., TNF alpha) and that it is incapable of directly antagonizing the production and/or actions of late appearing effector molecules (e.g., nitric oxide). We also found that IL10T mice were extremely vulnerable to a generalized Shwartzman reaction where prior exposure to a small amount of LPS primes the host for a lethal response to a subsequent sublethal dose. The priming LPS dose for IL10T mice was 100-fold lower than that required to prime wt mice implying that IL-10 is important for suppressing sensitization. In agreement with this assumption, IL-10 infusion was found to block the sensitization step. Interestingly, IL-10 was not the main effector of endotoxin tolerance as IL10T mice could be tolerized to LPS. Furthermore, IL-10 infusion could not substitute for the desensitizing dose of LPS. These results show that IL-10 is a critical component of the hosts natural defense against the development of pathologic responses to LPS although it is not responsible for LPS-induced tolerance.

Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress

Corticotropin-releasing hormone (CRH) is centrally involved in coordinating responses to a variety of stress-associated stimuli. Recent clinical data implicate CRH in the pathophysiology of human affective disorders. To differentiate the CNS pathways involving CRH and CRH receptor 1 (Crhr1) that modulate behavior from those that regulate neuroendocrine function, we generated a conditional knockout mouse line (Crhr1loxP/loxPCamk2a-cre) in which Crhr1 function is inactivated postnatally in anterior forebrain and limbic brain structures, but not in the pituitary. This leaves the hypothalamic-pituitary-adrenocortical (HPA) system intact. Crhr1loxP/loxPCamk2a-cre mutants showed reduced anxiety, and the basal activity of their HPA system was normal. In contrast to Crhr1 null mutants, conditional mutants were hypersensitive to stress corticotropin and corticosterone levels remained significantly elevated after stress. Our data clearly show that limbic Crhr1 modulates anxiety-related behavior and that this effect is independent of HPA system function. Furthermore, we provide evidence for a new role of limbic Crhr1 in neuroendocrine adaptation to stress.

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP‐mediated gene targeting to disrupt the ubiquitously expressed N‐WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N‐WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N‐WASP‐defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N‐WASP‐defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N‐WASP is dispensible for filopodia formation.

Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases

Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7–4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells.

Inducible gene deletion in astroglia and radial glia—A valuable tool for functional and lineage analysis

Astrocytes are thought to play a variety of key roles in the adult brain, such as their participation in synaptic transmission, in wound healing upon brain injury, and adult neurogenesis. However, to elucidate these functions in vivo has been difficult because of the lack of astrocyte‐specific gene targeting. Here we show that the inducible form of Cre (CreERT2) expressed in the locus of the astrocyte‐specific glutamate transporter (GLAST) allows precisely timed gene deletion in adult astrocytes as well as radial glial cells at earlier developmental stages. Moreover, postnatal and adult neurogenesis can be targeted at different stages with high efficiency as it originates from astroglial cells. Taken together, this mouse line will allow dissecting the molecular pathways regulating the diverse functions of astrocytes as precursors, support cells, repair cells, and cells involved in neuronal information processing.