Chymase-Cre; Mcl-1fl/fl Mice Exhibit Reduced Numbers of Mucosal Mast Cells.

Mast cells (MCs) are considered as key effector cells in the elicitation of allergic symptoms, and they are essential players in innate and adaptive immune responses. In mice, two main types of MCs have been described: connective tissue MCs (CTMCs) and mucosal MCs (MMCs). However, little is known about the biological functions of MMCs, which is due to the lack of suitable models to investigate MMCs in vivo. Here, we aimed to generate a mouse model selectively deficient in MMCs. It has been previously described that Cre expressed under the control of the baboon α-chymase promotor is predominantly localized in MMCs. Therefore, we mated α-chymase-Cre transgenic mice with mice bearing a floxed allele of the myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 encodes for an intracellular antiapoptotic factor in MCs; hence, a selective reduction in MMCs was expected. Our results show that this new mouse model contains markedly reduced numbers of MMCs in mucosal tissues, whereas numbers of CTMCs are normal. Thus, Chm-Cre; Mcl-1fl/fl mice are a useful tool for the investigation of the pathophysiological functions of MMCs in vivo.

Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter.

Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato+ dendritic cells, monocytes and neutrophils did not correlate with the tdTomato+ fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4- CD8-) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J mice.

Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene.

Stromal cells in bone marrow (BM) constitute a specific microenvironment supporting the development and maintenance of hematopoietic cells. Adiponectin is a cytokine secreted by adipocytes. Besides its anti-diabetic and anti-atherogenic roles, adiponectin reportedly regulates the development and function of hematopoietic cells in BM. However, it remains unclear whether mesenchymal stromal cells in BM express adiponectin. Here, we show that PDGFRß+VCAM-1+ stromal cells express adiponectin. Lineage tracing revealed that a majority of PDGFRß+VCAM-1+ cells were targeted by an adiponectin promoter-driven Cre (Adipoq-Cre) transgene. Additionally, the Adipoq-Cre transgene targets a minority of osteoblasts at a younger age but larger populations are targeted at an older age. Furthermore, the Adipoq-Cre transgene targets almost all CXCL12-abundant reticular (CAR) cells and most of the stromal cells targeted by the Adipoq-Cre transgene are CAR cells. Finally, deletion of interleukin-7 (IL-7) by the Adipoq-Cre transgene resulted in severe impairment of B lymphopoiesis in BM. These results demonstrate that PDGFRß+VCAM-1+ stromal cells in BM express adiponectin and are targeted by the Adipoq-Cre transgene, suggesting a broader specificity of the Adipoq-Cre transgene.

Spontaneous chondroma formation in CD2-Cre-driven Erk-deficient mice.

Lineage-specific Cre Tg mice are widely used to delineate the functions of genes in a tissue-specific manner. Several T-cell-specific promoter cassettes have been developed; however, the activities of those promoters in non-T cells have not been investigated extensively. Here, we report that CD2-Cre-mediated deletion of Erk proteins by generating CD2-Cre × Erk1-/-Erk2flox/flox (Erk∆CD2-Cre) mice results in abnormal cartilage hyperplasia. Histological analysis revealed that this abnormality is caused by aberrant hyperplasia of chondrocytes. The presence of Erk-deficient T cells is not required for this chondroma formation, as it was similarly observed in the absence of T cells in a CD3ε-deficient background. In addition, adoptive transfer of bone marrow cells from Erk∆CD2-Cre mice to wild-type recipients did not cause chondroma formation, suggesting that Erk-deficient non-immune cells are responsible for this abnormality. By tracing Cre-expressed tissues using a ROSA26-STOP-RFP allele, we found that the chondroma emitted RFP fluorescence, indicating that functional Cre is expressed in hyperplastic chondrocytes in Erk∆CD2-Cre mice. Furthermore, RFP+ chondrocytes were also found in an Erk-sufficient background, albeit without aberrant growth. These results suggest that unexpected expression of CD2-driven Cre in chondrocytes generates Erk-deficient chondrocytes, resulting in hyperplastic cartilage formation. Recently, two independent reports showed that CD4-Cre-mediated Ras-Erk signaling ablation led to similar abnormal cartilage formation (Guittard, G., Gallardo, D. L., Li, W. et al. 2017. Unexpected cartilage phenotype in CD4-Cre-conditional SOS-deficient mice. Front. Immunol. 8:343; Wehenkel, M., Corr, M., Guy, C. S. et al. 2017. Extracellular signal-regulated kinase signaling in CD4-expressing cells inhibits osteochondromas. Front. Immunol. 8:482). Together with these reports, our study suggests that an unexpected link exists between T-like cell and chondrocyte lineages during ontogeny.

Protecting genome integrity during CRISPR immune adaptation.

Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1-Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.

A modular open platform for systematic functional studies under physiological conditions.

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.

Methyl-CpG Binding Protein 2 Regulates Microglia and Macrophage Gene Expression in Response to Inflammatory Stimuli.

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

Dysbiosis and Staphylococcus aureus Colonization Drives Inflammation in Atopic Dermatitis.

Staphylococcus aureus skin colonization is universal in atopic dermatitis and common in cancer patients treated with epidermal growth factor receptor inhibitors. However, the causal relationship of dysbiosis and eczema has yet to be clarified. Herein, we demonstrate that Adam17(fl/fl)Sox9-(Cre) mice, generated to model ADAM17-deficiency in human, developed eczematous dermatitis with naturally occurring dysbiosis, similar to that observed in atopic dermatitis. Corynebacterium mastitidis, S. aureus, and Corynebacterium bovis sequentially emerged during the onset of eczematous dermatitis, and antibiotics specific for these bacterial species almost completely reversed dysbiosis and eliminated skin inflammation. Whereas S. aureus prominently drove eczema formation, C. bovis induced robust T helper 2 cell responses. Langerhans cells were required for eliciting immune responses against S. aureus inoculation. These results characterize differential contributions of dysbiotic flora during eczema formation, and highlight the microbiota-host immunity axis as a possible target for future therapeutics in eczematous dermatitis.

Features of hydrolysis of specific and nonspecific globular proteins and oligopeptides by antibodies against viral integrase from blood of HIV-infected patients.

It was shown previously that, as differentiated from canonical proteases, abzymes against myelin basic protein (MBP) from blood of patients with multiple sclerosis and systemic lupus erythematosus effectively cleaved only MBP, while antibodies (ABs) against integrase (IN) from blood of HIV-infected patients specifically hydrolyzed only IN. In this work, all sites of effective hydrolysis by anti-IN antibodies (IgG and IgM) of 25-mer oligopeptide (OP25) corresponding to MBP were identified using reversed-phase and thin-layer chromatographies and MALDI mass spectrometry. It was found that amino acid sequences of OP25 and other oligopeptides hydrolyzed by anti-MBP abzymes were partially homologous to some fragments of the full sequence of IN. Sequences of IN oligopeptides cleavable by anti-IN abzymes were homologous to some fragments of MBP, but anti-MBP abzymes could not effectively hydrolyze OPs corresponding to IN. The common features of the cleavage sites of OP25 and other oligopeptides hydrolyzed by anti-MBP and anti-IN abzymes were revealed. The literature data on hydrolysis of specific and nonspecific proteins and oligopeptides by abzymes against different protein antigens were analyzed. Overall, the literature data suggest that short OPs, including OP25, mainly interact with light chains of polyclonal ABs, which had lower affinity and specificity to the substrate than intact ABs. However, it seems that anti-IN ABs are the only one example of abzymes capable of hydrolyzing various oligopeptides with high efficiency (within some hours but not days). Possible reasons for the efficient hydrolysis of foreign oligopeptides by anti-IN abzymes from HIV-infected patients are discussed.

Longitudinal analysis of an HLA-B*51-restricted epitope in integrase reveals immune escape in early HIV-1 infection.

OBJECTIVE: To fully define cytotoxic T-lymphocyte (CTL) escape variants of an HLA-B*51-restricted integrase epitope in early HIV-1 infection. DESIGN: Ninety-four longitudinally sampled acute/early HIV-1 subtype B-infected participants were assessed to determine HLA-B*51-restricted LPPVVAKEI (LI9) escape variants. METHODS: LI9 was sequenced at baseline and subsequent time points. Interferon-γ (IFNγ) ELISpot assays were performed using serial log dilutions of variant LI9 peptides to determine the cellular response and functional avidity. RESULTS: There is a significant association between HLA-B*51 expression and an evolving LI9 sequence from baseline to year 1 (P < 0.0001). We detected that the V32I and P30X polymorphisms emerged within HLA-B*51 participants over time. Reversion of the P30S polymorphism was observed by year 1 in one HLA-B*51 participant. LPPIIAKEI and LPSIVAKEI had significantly lower functional avidity compared with LPPVVAKEI and so may be less well recognized by LI9-specific CTLs; a positive IFNγ response to IPSVVAKEI was rarely seen. Functional avidity to wild-type LI9 inversely correlated with viral load (R = 0.448, P = 0.0485). CONCLUSION: Our results provide support for the role of HLA-B*51-restricted CTLs and functional avidity in the control of early HIV-1 infection.

The role of cell types in cytomegalovirus infection in vivo.

Human cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable and the principles which govern these differences cannot be understood without detailed knowledge on tissue specific aspects of HCMV infection. For decades the role of individual cell types during cytomegalovirus infection and disease has been discussed. The pathogenesis of mouse cytomegalovirus (MCMV) mirrors the human infection in many aspects. Although only MCMV infection is studied extensively at the level of organs, the relative contribution of specific cell types to viral pathogenesis in vivo has remained enigmatic. Here we discuss new approaches based on the cre/loxP-system to label nascent virus progeny or to lift a replication block. The salient aspect of this technique is the change of viral genome properties selectively in cells that express cre during infection in vivo. This allowed us to study the role of endothelial cells and hepatocytes for virus dissemination and will permit detailed studies on innate and adaptive immune responses to CMV.

Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions.

We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.

Transduction of human antigen-presenting cells with integrase-defective lentiviral vector enables functional expansion of primed antigen-specific CD8(+) T cells.

Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8(+) T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications.

Maintenance of thymic epithelial phenotype requires extrinsic signals in mouse and zebrafish.

Thymopoiesis strictly depends on proper differentiation of the thymic epithelial anlage. Differentiation of thymic epithelial cells (TECs) is controlled by the Foxn1 transcription factor. The in vivo signals initiating and maintaining Foxn1 expression in the future thymus anlage are unknown. In the mouse, bone morphogenetic protein (BMP) signaling is required for the maintenance of Foxn1 expression in TECs, as shown here by lineage tracing using a Foxn1-driven Cre transgene. Loss of Foxn1 expression after BMP inhibition reverts TECs to a basal state of pharyngeal epithelium unable to support T cell development; it does not divert them into a parathyroid fate. In zebrafish larvae, BMP inhibition likewise causes loss of foxn1 expression in the thymic anlage and subsequent impairment of thymopoiesis. These results indicate an evolutionarily conserved role of BMP signaling in the maintenance of Foxn1 expression.

Development of thymic lymphomas in mice disrupted of Brca2 allele in the thymus.

Germ-line mutations in BRCA2 predispose to early-onset cancer. Homozygous mutant mouse, which has Brca2 truncated in exon 11 exhibit paradoxic occurrence of growth retardation and development of thymic lymphomas. However, due to its large embryonic lethality, cohort studies on the thymic lymphomas were not feasible. With the aid of Cre-loxP system, we demonstrate here that thymus-specific disruption of Brca2 allele without crossing it to p53-mutant background leads to the development of thymic lymphomas. Varying from 16 weeks to 66 weeks after birth, 25% of mice disrupted of Brca2 in the thymus died of thymic lymphomas, whereas previous report did not observe lymphomagenesis using similar Cre-loxP system. Future analysis of thymic lymphomas from these mice presented here will provide information on the cooperative mutations that are required for the BRCA2-associated pathogenesis of cancer.

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus.

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.

Normal hematopoiesis after conditional targeting of RXRalpha in murine hematopoietic stem/progenitor cells.

Because of the retinoic acid receptor-alpha (RARalpha) gene's involvement in acute promyelocytic leukemia, the important role of RARs in hematopoiesis is now well established. However, relatively few studies of hematopoiesis have focused on the role of the retinoid X receptors (RXRs), the obligate heterodimeric partners of the RARs. We sought to establish whether conditional targeting of RXRalpha in early hematopoietic progenitors, ideally to the level of the hematopoietic stem cell (HSC), would compromise hematopoiesis. For hematopoietic targeting of RXRalpha, we characterized IFN-inducible MxCre mice for use in studying the role of RXRalpha in hematopoiesis. We established that MxCre executes recombination of loxP-flanked RXRalpha in hematopoietic progenitors immunophenotypically enriched for HSC, leading to widespread and sustained targeting of RXRalpha in hematopoietic cells. However, we found no evidence of hematologic compromise in mice lacking RXRalpha, suggesting that RXRalpha is dispensable for normal murine hematopoiesis. Nonetheless, RXRalpha null bone marrow cells cultured in methylcellulose form colonies more efficiently than bone marrow cells obtained from control mice. This result suggests that although RXRalpha is not required for murine hematopoiesis, there may be hematopoietic signaling pathways that respond selectively to RXRalpha or settings in which combined expression of RXR (alpha, beta, and gamma) is limiting.

High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

Primate foamy virus Pol proteins are imported into the nucleus.

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127(Pol), p85(RT/RN) and p40(IN) served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.

Generation of Cre recombinase-specific monoclonal antibodies, able to characterize the pattern of Cre expression in cre-transgenic mouse strains.

Transgene-encoded Cre recombinase can target alteration of loxP-tagged genes to specific cell types and developmental stages in mice, depending on the pattern of transgene expression. To facilitate determination of the latter, we have generated monoclonal anti-Cre antibodies which are specific for distinct epitopes on the recombinase and detect Cre both on immunoblots and intracellularly by immunofluorescence. We demonstrate the usefulness of these antibodies by an analysis of Cre expression in mice carrying a cre-transgene under B cell-specific control.