Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a ...major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10−/− CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.
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•Exposure to bacterial CpG DNA (CpG) expands regulatory lung interstitial macrophages (IMs)•Transfer of WT but not Il10−/− IMs protects from allergen-induced airway inflammation•CpG-induced IMs arise from local and splenic reservoir monocytes•Migration of regulatory IM precursors to the lung does not require CCR2
Exposure to unmethylated CpG DNA (CpG) from bacteria is associated with a reduced risk of developing asthma. Sabatel et al. find that CpG exposure leads to higher numbers of lung interstitial macrophages that prevent allergic inflammation through the production of the regulatory cytokine interleukin-10.
Gene targeting in mice: a review Bouabe, Hicham; Okkenhaug, Klaus
Methods in molecular biology,
01/2013, Letnik:
1064
Journal Article
Odprti dostop
The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. ...The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.
Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with ...varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081.
Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity <50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed.
Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype.
Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs ...against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.
PI3Ks regulate diverse immune cell functions by transmitting intracellular signals from Ag, costimulatory receptors, and cytokine receptors to control cell division, differentiation, survival, and ...migration. In this study, we report the effect of inhibiting the p110δ subunit of PI3Kδ on CD8(+) T cell responses to infection with the intracellular bacteria Listeria monocytogenes. A strong dependency on PI3Kδ for IFN-γ production by CD8(+) T cells in vitro was not recapitulated after Listeria infection in vivo. Inactivation of PI3Kδ resulted in enhanced bacterial elimination by the innate immune system. However, the magnitudes of the primary and secondary CD8 +: T cell responses were reduced. Moreover, PI3Kδ activity was required for CD8(+) T cells to provide help to other responding CD8(+) cells. These findings identify PI3Kδ as a key regulator of CD8(+) T cell responses that integrates extrinsic cues, including those from other responding cells, to determine the collective behavior of CD8(+) T cell populations responding to infection.
Recent reports have highlighted greater complexity, plasticity, and functional diversity of mononuclear phagocytes (MPCs), including monocytes, macrophages, and dendritic cells (DCs), in our organs ...than previously understood. The functions and origins of MPCs resident within healthy organs, especially in the kidney, are less well understood, whereas studies suggest they play roles in disease states distinct from recruited monocytes. We developed an unbiased approach using flow cytometry to analyze MPCs residing in the normal mouse kidney, and identified five discrete subpopulations according to CD11b/CD11c expression as well as F4/80, CD103, CD14, CD16, and CD64 expression. In addition to distinct marker profiles, these subpopulations have different lineages and expression of genes involved in tissue homeostasis, including angiogenesis. Among them, the CD11b(int)CD11c(int) F4/80(high) subpopulation notably exhibited high capacity to produce a representative anti-inflammatory cytokine, IL-10. Each subpopulation had different degrees of both macrophage (phagocytosis) and DC (Ag presentation) capacities, with a tendency to promote differentiation of regulatory T cells, whereas two of these showed expression of transcription factors reported to be highly expressed by classical DCs, and proclivity to exit the kidney following stimulation with LPS. In summary, resident kidney MPCs comprise discrete subpopulations, which cannot be simply classified into the conventional entities, and they produce anti-inflammatory and tissue-homeostatic factors to differing degrees.
In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps ...bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies.
In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme β-lactamase and the fluorescence resonance energy transfer substrate ...coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-β-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.
Many in vitro and in vivo applications for transgenesis require co-expression of heterologous genes. The use of internal ribosome entry sites (IRESs) in dicistronic expression vectors enables the ...expression of two genes controlled by one promoter in target cells or whole organisms. Here we describe the expansion of IRES exploitation to generate multicistronic vectors capable of expressing multiple reporter genes, especially to improve the fluorescence yield of autofluorescent reporter gene products such as green fluorescent protein (GFP). We found that the increase in fluorescence output of GFP is proportional to the number of IRES-GFP repeats in the multicistronic vector. At least four genes can be expressed from a multicistonic vector by using tandem IRES elements, with no significant alteration of the expression level of the cap-dependent translated gene. Moreover, gene expression under the control of multiple IRES element has no effect on the posttranscriptional regulation through 3'-untranslated regions (3'UTR). Thus, endogenous gene expression and regulation, especially those controlled by weak promoters, can be analyzed with our IRES-dependent polycistronic reporter gene expression system.
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