Katharina Brandl

Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits

Infection with antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a dangerous and costly complication of broad-spectrum antibiotic therapy. How antibiotic-mediated elimination of commensal bacteria promotes infection by antibiotic-resistant bacteria is a fertile area for speculation with few defined mechanisms. Here we demonstrate that antibiotic treatment of mice notably downregulates intestinal expression of RegIIIγ (also known as Reg3g), a secreted C-type lectin that kills Gram-positive bacteria, including VRE. Downregulation of RegIIIγ markedly decreases in vivo killing of VRE in the intestine of antibiotic-treated mice. Stimulation of intestinal Toll-like receptor 4 by oral administration of lipopolysaccharide re-induces RegIIIγ, thereby boosting innate immune resistance of antibiotic-treated mice against VRE. Compromised mucosal innate immune defence, as induced by broad-spectrum antibiotic therapy, can be corrected by selectively stimulating mucosal epithelial Toll-like receptors, providing a potential therapeutic approach to reduce colonization and infection by antibiotic-resistant microbes.

MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR→MyD88→AREG/EREG→EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.

Enhanced sensitivity to DSS colitis caused by a hypomorphic Mbtps1 mutation disrupting the ATF6-driven unfolded protein response.

Here, we describe an N-ethyl-N-nitrosourea (ENU)-induced missense error in the membrane-bound transcription factor peptidase site 1 (S1P)-encoding gene (Mbtps1) that causes enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. S1P cleaves and activates cAMP response element binding protein/ATF transcription factors, the sterol regulatory element-binding proteins (SREBPs), and other proteins of both endogenous and viral origin. Because S1P has a nonredundant function in the ATF6-dependent unfolded protein response (UPR), woodrat mice show diminished levels of major endoplasmic reticulum chaperones GRP78 (BiP) and GRP94 in the colon upon DSS administration. Experiments with bone marrow chimeric mice reveal a requirement for S1P in nonhematopoietic cells, without which a diminished UPR and colitis develop.

Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice.

The intestinal mucus layer protects the epithelium from noxious agents, viruses, and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin (Muc) 2, secreted by goblet cells of the intestine. Experimental alcoholic liver disease requires translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. We investigated the roles of the intestinal mucus layer, and in particular Muc2, in development of experimental alcohol‐associated liver disease in mice. We studied experimental alcohol‐induced liver disease, induced by the Tsukamoto‐French method (which involves continuous intragastric feeding of an isocaloric diet or alcohol) in wild‐type and Muc2−/− mice. Muc2−/− mice showed less alcohol‐induced liver injury and steatosis than developed in wild‐type mice. Most notably, Muc2−/− mice had significantly lower plasma levels of lipopolysaccharide than wild‐type mice after alcohol feeding. In contrast to wild‐type mice, Muc2−/− mice were protected from alcohol‐associated microbiome changes that are dependent on intestinal mucins. The antimicrobial proteins regenerating islet‐derived 3 beta and gamma were expressed at significantly higher levels in the jejunum of Muc2−/− mice fed the isocaloric diet or alcohol compared with wild‐type mice. Consequently, Muc2−/− mice showed increased killing of commensal bacteria and prevented intestinal bacterial overgrowth. Conclusion: Muc2−/− mice are protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation, decreasing liver disease. (HEPATOLOGY 2013;)

MyD88 and Type I Interferon Receptor-Mediated Chemokine Induction and Monocyte Recruitment during Listeria monocytogenes Infection

Monocytes play a central role in defense against infection, but the mechanisms promoting monocyte recruitment and activation remain incompletely defined. Defense against Listeria monocytogenes, an intracellular bacterial pathogen, requires in vivo MCP-1 induction and CCR2-dependent recruitment of Ly6Chigh monocytes from bone marrow to sites of infection. Herein, we demonstrate that infection of bone marrow-derived macrophages with virulent L. monocytogenes induces MCP-1 expression in two phases. The first phase is rapid, induces low-level production of MCP-1, and is dependent on TLR/MyD88 signaling. The second phase promotes prolonged, higher level MCP-1 secretion and is dependent on signaling via the type I IFN receptor (IFNAR). Although attenuated L. monocytogenes strains that remain confined to the phagosome trigger TLR/MyD88-mediated signals and induce low-level MCP-1 expression, only cytosol-invasive bacteria promote IFNAR-dependent MCP-1 expression. In vivo, deficiency of either MyD88 or IFNAR signaling does not impair early monocyte emigration from bone marrow and recruitment to infected spleen. Loss of both MyD88 and IFNAR-mediated MCP-1 induction, however, results in deficient Ly6Chigh monocyte recruitment and increased susceptibility to L. monocytogenes infection. Our studies demonstrate that distinct but partially overlapping signal transduction pathways provide redundancy that ensures optimal monocyte recruitment to sites of microbial infection.

Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.

ENU-induced phenovariance in mice: inferences from 587 mutations

BackgroundWe present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.FindingsIn the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.ConclusionsCollectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFkappaB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.

The Tpl2 Mutation Sluggish Impairs Type I IFN Production and Increases Susceptibility to Group B Streptococcal Disease

Sluggish was identified in a population of third generation mice descended from N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes exhibited impaired TNF-α production in response to all TLR ligands tested and displayed impaired type I IFN production in response to TLR7 and TLR9 stimulations. The phenotype was confined to a critical region on mouse chromosome 18 and then ascribed to a T to A transversion in the acceptor splice site of intron 4 at position 13346 of the Map3k8 gene, resulting in defective splicing. The Map3k8Sluggish mutation does not result in susceptibility to viral infections, but Sluggish mice displayed high susceptibility to group B streptococcus infection, with impaired TNF-α and type I IFN production in infected macrophages. Our data demonstrate that the encoded protein kinase Tpl2 plays an essential role in cell signaling in the immune response to certain pathogens.

Increased susceptibility to DNA virus infection in mice with a GCN2 mutation

ABSTRACT The downregulation of translation through eIF2α phosphorylation is a cellular response to diverse stresses, including viral infection, and is mediated by the GCN2 kinase, protein kinase R (PKR), protein kinase-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI). Although PKR plays a major role in defense against viruses, other eIF2α kinases also may respond to viral infection and contribute to the shutdown of protein synthesis. Here we describe the recessive, loss-of-function mutation atchoum (atc) in Eif2ak4, encoding GCN2, which increased susceptibility to infection by the double-stranded DNA viruses mouse cytomegalovirus (MCMV) and human adenovirus. This mutation was identified by screening macrophages isolated from mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. Cells from Eif2ak4atc/atc mice failed to phosphorylate eIF2α in response to MCMV. Importantly, homozygous Eif2ak4atc mice showed a modest increase in susceptibility to MCMV infection, demonstrating that translational arrest dependent on GCN2 contributes to the antiviral response in vivo.