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Due to welfare concerns and legal restrictions in certain countries, alternatives to wire net floors must be developed in rabbit husbandries. Also, there is a difference in regulations in Europe for laboratory rabbits vs. rabbits bred and kept for meat production. While there are regulations concerning floor design of enclosures for rabbits bred for meat production in many European countries, the European Directive 2010/63 lacks regulations for rabbits used for scientific purposes. This study compares two floors, which meet the Austrian legal requirements for growing rabbits intended for consumption as well as the requirements for laboratory rabbits. The dual use of rabbits bred for meat production and applicable for scientific purposes would avoid the problem of surplus animals of specialized producers for laboratory rabbits. A noryl floor with 12 mm circular holes was compared to a 10 mm slatted plastic floor. Parameters were soiling of cages and animals, parasitic burden, clinical health, and losses using objective scoring. Soiling of cages and animals and coccidial oocytes were significantly higher on the floors with circular holes. Obvious signs of disease showed a non-significant trend to be more frequent in the group with circular holes. This was linked with significantly higher losses. In conclusion, our study clearly shows that the floor with circular hole design cannot be endorsed, although it meets legal requirements. The slatted floor type can be cautiously recommended; however, to assure animal welfare in laboratory rabbits, legal authorities in Europe should take on the responsibility of regulating floor design in this sector.

Enterocytes of the small intestine (SI) play an important role in maintaining systemic lipid levels by regulating dietary lipid absorption and postprandial lipoprotein secretion. An excessive amount of dietary-derived triglycerides (TGs) taken up by the apical side of enterocytes or basolaterally internalized lipoprotein remnants can be transiently stored in cytosolic lipid droplets (cLDs). As mice lacking adipose TG lipase (ATGL) in the SI display massive accumulation of cLDs but also delayed cholesterol absorption, we hypothesized that SI-specific overexpression of ATGL (Atgl iTg) might have beneficial effects on lipid homeostasis in the gut and possibly throughout the body. Here, we demonstrate that Atgl iTg mice had only modestly increased enzymatic activity despite drastically elevated Atgl mRNA levels (up to 120-fold) on chow diet, and was highly induced upon high-fat/high-cholesterol diet (HF/HCD) feeding. Atgl iTg mice showed markedly reduced intestinal TG concentrations after acute and chronic lipid challenge without affecting chylomicron TG secretion. Circulating plasma cholesterol levels were significantly lower in Atgl iTg mice under different feeding conditions, contrasting the accelerated uptake of dietary cholesterol into the circulation after HF/HCD feeding. In the fasted state, gene expression analysis revealed modulation of PPARα and liver X receptor (LXR) target genes by an increased fatty acid release, whereas the decreased plasma cholesterol concentrations in refed mice were more likely due to changes in HDL synthesis and secretion. We conclude that ATGL, in addition to its role in TG catabolism, plays a critical role in whole-body cholesterol homeostasis by modulating PPARα and LXR signaling in intestinal enterocytes.

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.

Objective Failure to properly dispose of glucose in response to insulin is a serious health problem, occurring during obesity and is associated with type 2 diabetes development. Insulin-stimulated glucose uptake is facilitated by the translocation and plasma membrane fusion of vesicles containing glucose transporter 4 (GLUT4), the rate-limiting step of post-prandial glucose disposal. Methods We analyzed the role of Tusc5 in the regulation of insulin-stimulated Glut4-mediated glucose uptake in vitro and in vivo. Furthermore, we measured Tusc5 expression in two patient cohorts. Results Herein, we report that TUSC5 controls insulin-stimulated glucose uptake in adipocytes, in vitro and in vivo. TUSC5 facilitates the proper recycling of GLUT4 and other key trafficking proteins during prolonged insulin stimulation, thereby enabling proper protein localization and complete vesicle formation, processes that ultimately enable insulin-stimulated glucose uptake. Tusc5 knockout mice exhibit impaired glucose disposal and TUSC5 expression is predictive of glucose tolerance in obese individuals, independent of body weight. Furthermore, we show that TUSC5 is a PPARγ target and in its absence the anti-diabetic effects of TZDs are significantly blunted. Conclusions Collectively, these findings establish TUSC5 as an adipose tissue-specific protein that enables proper protein recycling, linking the ubiquitous vesicle traffic machinery with tissue-specific insulin-mediated glucose uptake into adipose tissue and the maintenance of a healthy metabolic phenotype in mice and humans., Molecular Metabolism, 4 (11)

The splenic white pulp is underpinned by poorly characterized stromal cells that demarcate distinct immune cell microenvironments. Here, definition of the embryonic origin and tracing of the differentiation trajectories of fibroblastic reticular cells (FRCs) was enabled by the establishment of FRC-specific fate-mapping in mice. We found that all reticular cell subsets descend from pluripotent progenitors that emerge at embryonic day 19.5 from Sca-1 periarterial progenitors. Commitment of FRC progenitors was concluded during the first week of postnatal life through occupation of niches along developing central arterioles. Single cell transcriptomic analysis facilitated deconvolution of FRC differentiation trajectories and indicated that perivascular reticular cells function both as adult lymphoid organizer cells and mural cell progenitors. Finally, the lymphotoxin-β receptor-independent sustenance of postnatal progenitor stemness unveiled that systemic immune surveillance in the splenic white pulp is governed through subset specification of reticular cells from a pluripotent periarterial progenitor cell.

Summary Intestinal epithelial stem cells (IESCs) control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5+ IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5+ IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury., Graphical Abstract, Highlights • Loss of STAT5 impairs intestinal stem cell proliferation • Activated STAT5 increases intestinal stem cell regeneration • Activation of cytokine-STAT5 signaling mitigates intestinal inflammation, In this article, Han and colleagues link STAT5 signaling to intestinal stem cell replenishment upon injury. They find that depletion of Stat5 leads to decreased stem cell proliferation, sensitizing mice to irradiation. In contrast, activation of STAT5 accelerates crypt regeneration, conferring resistance to gut injury. Therefore, activation of cytokine-induced STAT5 signaling promotes the regenerative capacity of intestinal stem cells to repair gut injury.

SummaryThe stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although the disease can be kept under control using BCR/ABL1 tyrosine kinase inhibitors (TKIs) in most cases, some patients relapse or have resistant disease, so there is a need to identify new therapeutic targets in this malignancy. Recent data suggest that leukemic SCs (LSCs) in CML display the stem-cell (SC)-mobilizing cell surface enzyme dipeptidyl-peptidase IV (DPPIV = CD26) in an aberrant manner. In the present study, we analyzed the effects of the DPPIV blocker vildagliptin as single agent or in combination with the BCR/ABL1 TKI imatinib or nilotinib on growth and survival of CML LSCs in vitro and on LSC engraftment in an in vivo xenotransplantation nonobese diabetic SCID-IL-2Rγ(−/−) (NSG) mouse model. We found that nilotinib induces apoptosis in CML LSCs and inhibits their engraftment in NSG mice. In contrast, no substantial effects were seen with imatinib or vildagliptin. Nevertheless, vildagliptin was found to reduce the “mobilization” of CML LSCs from a stroma cell layer consisting of mouse fibroblasts in an in vitro co-culture model, suggesting reduced disease expansion. However, although vildagliptin and nilotinib produced cooperative effects in individual experiments, overall, no significant effects of coadministered vildagliptin over nilotinib or imatinib treatment alone were seen on the engraftment of CML cells in NSG mice. Gliptins may be interesting drugs in the context of CML and nilotinib therapy, but our preclinical studies did not reveal a major cooperative effect of the drug-combination vildagliptin + nilotinib on engraftment of CML cells in NSG mice.

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, tauopathies, Huntington's disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition.

Peroxiredoxin 6 is an enzyme that detoxifies hydrogen peroxide and various organic peroxides. In previous studies we found strongly increased expression of peroxiredoxin 6 in the hyperproliferative epidermis of wounded and psoriatic skin, suggesting a role of this enzyme in epidermal homeostasis. To address this question, we generated transgenic mice overexpressing peroxiredoxin 6 in the epidermis. Cultured keratinocytes from transgenic mice showed enhanced resistance to the toxicity of various agents that induce oxidative stress. However, overexpression of peroxiredoxin 6 did not affect skin morphogenesis or homeostasis. On skin injury, enhancement of wound closure was observed in aged animals. Most importantly, peroxiredoxin 6 overexpression strongly reduced the number of apoptotic cells after UVA or UVB irradiation. These findings demonstrate that peroxiredoxin 6 protects keratinocytes from cell death induced by reactive oxygen species in vitro and in vivo, suggesting that activation of this enzyme could be a novel strategy for skin protection under stress conditions.

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.

Summary Perforin is a cytotoxic molecule which is found in high concentration at the fetomaternal interface. In this location, perforin is contained in a specialized subset of natural killer cells, called uterine NK cells. By the use of perforin-deficient mice, we studied the function of perforin in the pregnant uterus. Perforin-deficient mice, we studied the function of perforin in the pregnant uterus. Perforin-deficient mice were found to reproduce as efficiently as wild type controls; however, they differed from normal in that the frequency of uterine NK cells was significantly higher and these cells were found to proliferate during more advanced stages of pregnancy. Lipopolysaccharide, which caused a massive increase of fetal resorptions in C57BL/6 control mice when given intraperitoneally on day 6.5 of pregnancy, had the same effect in the perforin-deficient counterpart. This argues against uterine NK cells being effector cells in infection-mediated abortion. Furthermore, no differences in fertility were observed under restricted mating conditions and with severe social stress. However, when embryo transfer was performed, the implantation of blastocysts was achieved in unprepared endometrium of perforin-deficient mice, which was not possible in wild type controls of the same genetic background. This could argue for a role of perforin in the defense of the mother against undue pregnancies.

The beta-amyloid precursor protein (beta APP) gene of the mouse was disrupted by inserting into exon 2 a cassette containing a neomycin resistance gene and a putative transcription termination sequence. Contrary to expectation, brain and other tissues from mice homozygous for the insertion still contained beta APP-specific RNA, albeit at a level 5- to 10-fold lower than wild type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.

STAT5 transcription factors are essential regulators of differentiation, survival and proliferation during hematopoiesis. Hyperactive STAT5 signaling requires enhanced tyrosine phosphorylation (pYSTAT5), which is found in most hematopoietic cancers and is associated with negative prognosis. Importantly, recurrent gain-of-function STAT5 variants have been detected in different diseases with Peripheral T Cell Lymphoma (PTCL) phenotype. We used cS5F, a hyperactive point mutant of STAT5A (S710F), to generate a mouse model expressing the transgene under the vav-promoter from hematopoietic stem cells (HSC). High pYSTAT5 levels in vav-cS5hi mice led to an aggressive expansion of CD8+ T cells being lethal between 25 and 45 weeks of age. The PTCL-like disease was associated with lymphadenopathy, splenomegaly and T cell infiltrations into various organs. The CD8+ T cells were polyclonal, transplantable and expressed CD25, CD44 and CD62L activation markers. Furthermore, the number of active cycling HSCs increased. The expression profile determined by RNA-seq correlated closely with human PTCL. Our results support the concept that enhanced STAT5 signaling drives PTCL and STAT5 represents a target in these life-threatening malignancies. Therefore, we aim to test the effect of a new STAT5 inhibitor on PTCL cell lines.