Lox Mice Research Articles

Synaptic GABAA receptor clustering without the γ2 subunit.

Rapid activation of postsynaptic GABAA receptors (GABAARs) is crucial in many neuronal functions, including the synchronization of neuronal ensembles and controlling the precise timing of action potentials. Although the γ2 subunit is believed to be essential for the postsynaptic clustering of GABAARs, synaptic currents have been detected in neurons obtained from γ2(-/-) mice. To determine the role of the γ2 subunit in synaptic GABAAR enrichment, we performed a spatially and temporally controlled γ2 subunit deletion by injecting Cre-expressing viral vectors into the neocortex of GABAARγ2(77I)lox mice. Whole-cell recordings revealed the presence of miniature IPSCs in Cre(+) layer 2/3 pyramidal cells (PCs) with unchanged amplitudes and rise times, but significantly prolonged decays. Such slowly decaying currents could be evoked in PCs by action potentials in presynaptic fast-spiking interneurons. Freeze-fracture replica immunogold labeling revealed the presence of the α1 and β3 subunits in perisomatic synapses of cells that lack the γ2 subunit. Miniature IPSCs in Cre(+) PCs were insensitive to low concentrations of flurazepam, providing a pharmacological confirmation of the lack of the γ2 subunit. Receptors assembled from only αβ subunits were unlikely because Zn(2+) did not block the synaptic currents. Pharmacological experiments indicated that the αβγ3 receptor, rather than the αβδ, αβε, or αβγ1 receptors, was responsible for the slowly decaying IPSCs. Our data demonstrate the presence of IPSCs and the synaptic enrichment of the α1 and β3 subunits and suggest that the γ3 subunit is the most likely candidate for clustering GABAARs at synapses in the absence of the γ2 subunit.

Role of brain stearoyl‐CoA desaturase‐1 in metabolism, obesity, and glucose homeostasis (605.2)

The high prevalence of obesity, coupled with its potential to develop into serious health issues including type II diabetes, insulin resistance and coronary artery disease, reveals it is a significant health problem. To investigate how excess adipose tissue manifests into these health issues, we have observed the effects of a disruption in stearoyl‐CoA desaturase 1 (SCD1), which is involved in the synthesis of endogenous monounsaturated fatty acids from saturated fatty acids. We have previously demonstrated that whole‐body deficiency of SCD1 in mice protects from high‐fat diet‐induced obesity, hepatic steatosis and insulin resistance. However, the tissue‐specific contribution of SCD1 to these phenotypes remains to be fully elucidated. In the current study, we investigated the role of brain SCD1 in mediating the metabolic consequences of a high‐fat diet (HFD). We fed nestin‐cre+;SCD1 flox/flox brain‐specific SCD1 knockout (BKO) mice a HFD for 18 weeks. We observed that both male and female BKO mice weighed significantly less than Lox controls. In addition, male BKO mice had lower hepatic triglyceride levels than Lox mice, and enhanced glucose tolerance relative to Lox mice. Overall, these results indicate brain SCD1 plays a role in HFD‐induced adiposity and glucose homeostasis.

Deletion of IL-4 Receptor Alpha on Dendritic Cells Renders BALB/c Mice Hypersusceptible to Leishmania major Infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.

Abrogation of IL-4 receptor-α-dependent alternatively activated macrophages is sufficient to confer resistance against pulmonary cryptococcosis despite an ongoing Th2 response

In the murine model of pulmonary infection with Cryptococcus neoformans, IL-4 receptor α (IL-4Rα)-dependent polyfunctional T(h)2 cells induce disease progression associated with alternative activation of lung macrophages. To characterize the effector role of IL-4Rα-dependent alternatively activated macrophages (aaMph), we intra-nasally infected mice with genetically ablated IL-4Rα expression on macrophages (LysM(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) littermates. LysM(Cre)IL-4Rα(-/lox) mice were significantly more resistant to pulmonary cryptococcosis with higher survival rates and lower lung burden than non-deficient heterozygous littermates. Infected LysM(Cre)IL-4Rα(-/lox) mice had reduced but detectable numbers of aaMph expressing arginase-1, chitinase-like enzyme (YM1) and CD206. Similar pulmonary expression of inducible nitric oxide synthase was found in LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) control mice, but macrophages from LysM(Cre)IL-4Rα(-/lox) mice showed a higher potential to produce nitric oxide. In contrast to the differences in the macrophage phenotype, pulmonary T(h)2 responses were similar in infected LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice with each mouse strain harboring polyfunctional T(h)2 cells. Consistently, type 2 pulmonary allergic inflammation associated with eosinophil recruitment and epithelial mucus production was present in lungs of both LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice. Our results demonstrate that, despite residual IL-4Rα-independent alternative macrophage activation and ongoing T(h)2-dependent allergic inflammation, abrogation of IL-4Rα-dependent aaMph is sufficient to confer resistance in pulmonary cryptococcosis. This is even evident on a relatively resistant heterozygous IL-4Rα(+/-) background indicating a key contribution of macrophage IL-4Rα expression to susceptibility in allergic bronchopulmonary mycosis.

The expression and role of Lysyl oxidase (LOX) in dentinogenesis

To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation. Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray. No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes. The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Dendritic Cell-Mediated Vaccination Relies on Interleukin-4 Receptor Signaling to Avoid Tissue Damage after Leishmania major Infection of BALB/c Mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.

Lack of IL-4 receptor expression on T helper cells reduces T helper 2 cell polyfunctionality and confers resistance in allergic bronchopulmonary mycosis.

T helper (Th)1 and Th2 cells play decisive roles in the regulation of resistance vs. susceptibility to pulmonary cryptococcosis. To study the function of interleukin (IL)-4 receptor (IL-4R) on Th cells in pulmonary cryptococcosis, we infected mice specifically lacking IL-4Rα on CD4(+) T cells (Lck(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) controls. Lck(Cre)IL-4Rα(-/lox) mice developed enhanced resistance accompanied by reduced pulmonary allergic inflammation and diminished production of the Th2 cytokines IL-4, IL-5, and IL-13 as compared with IL-4Rα(-/lox) mice. Polyfunctional antigen-specific Th2 cells producing simultaneously two or three Th2 cytokines were reduced in infected Lck(Cre)IL-4Rα(-/lox) mice, pointing to a critical role of polyfunctional Th2 cells for disease progression. Reduced Th2 polyfunctionality was associated with fewer pulmonary alternatively activated macrophages. This work is the first direct evidence for a critical contribution of the IL-4R on Th cells to Th2-dependent susceptibility during allergic bronchopulmonary mycosis. Moreover, the data demonstrate that the quality of the Th2 response has an impact on type 2 inflammation. The analysis of polyfunctional Th2 cells may be useful for monitoring the course of the disease.

B Cells That Produce Immunoglobulin E Mediate Colitis in BALB/c Mice

Induction of colitis in mice by administration of oxazolone is mediated by T-helper (Th) 2 cells and has features of human ulcerative colitis. We investigated whether activation of interleukin (IL)-4Rα on T and B cells determines their effector functions and mediates oxazolone-induced colitis. We studied induction of colitis with oxazolone in wild-type mice and those with CD4(+) T cells that did not express IL-4Rα (Lck(cre)IL-4Rα(-/lox)). We also generated mice with B cells that did not express IL-4Rα (mb1(cre)IL-4Rα(-/lox)) and studied induction of colitis. Lck(cre)IL-4Rα(-/lox) mice did not develop colitis in response to oxazolone, and their levels of IL-4, IL-13, and immunoglobulin (Ig) E were reduced. Adoptive transfer of naïve, wild-type CD4(+) Th cells depleted of natural killer T cells to Lck(cre)IL-4Rα(-/lox) mice restored their susceptibility to colitis. In contrast, Lck(cre)IL-4Rα(-/lox) mice maintained their protection against colitis when IL-13-deficient CD4(+) T cells were transferred. These findings indicate that development of colitis involves not only natural killer T-cell functions, but also requires IL-13 production by CD4(+) T helper cells. Mb1(cre)IL-4Rα(-/lox) mice, which cannot produce IgE, were also protected against oxazolone-induced colitis. Blocking IgE binding significantly reduced mast cell numbers in colons and protected wild-type BALB/c mice from the onset of colitis. IL-4 appears to induce CD4(+) Th2 cells to produce IL-13 and B cells to produce IgE, which together mediate oxazolone-induced colitis in mice.

Intestinal epithelial-specific PTEN inactivation results in tumor formation

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear β-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of β-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Tacrolimus-Induced Hypertension

See related article, pp 1167–1175 Hypertension associated with solid-organ transplantation is of major clinical importance, because the degree of posttransplant hypertension has been shown to correlate with reductions in graft viability and survival.1,–,3 Effective blood pressure control with antihypertensives has been shown to enhance graft survival and reduce the risk of future cardiovascular disease and events in transplant recipients.3 The etiology of posttransplant hypertension is varied; however, hypertension produced by immunosuppressant therapy is an important cause of posttransplant-related hypertension. Tacrolimus (FK506) and cyclosporine A are 2 common immunosuppressants that have been shown to affect blood pressure. Thus, elucidation of mechanisms that contribute to posttransplant hypertension has major clinical implications. In general, the type of immune response evoked by a particular set of antigens, such as that associated with organ transplantation, depends on the expansion of specific T-lymphocyte (T-cell) subsets, as well as the duration of antigen exposure, which, in the case of organ transplantation, is life long. Tacrolimus and cyclosporine A limit the immune response via inhibition of calcineurin activity, through similar but slightly different mechanisms. With tacrolimus, the mechanism of calcineurin inhibition involves tacrolimus binding to its cytosolic binding partner, the ubiquitously expressed immunophilin FKBP12. Tacrolimus/FKBP12, along with calcium and calmodulin, forms a complex with calcineurin, thereby inhibiting calcineurin's phosphatase activity. Calcineurin signaling has been shown to regulate expression of a number of cytokines, including interleukin (IL)-2, IL-3, IL-4, and interferon-γ, as well as expression of the IL-2 receptor, which have been implicated in T-cell activation.4 For example, reducing IL-2 signaling with tacrolimus is important in limiting the immune response, because IL-2 promotes the expansion of cytotoxic T cells and has been shown to play an important role in immunologic memory. In this issue of Hypertension , Chiasson et al5 report …

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL-4 receptor α chain (IL-4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL-4Rα-responsive T cells in Schistosoma mansoni egg-induced inflammation was investigated. Egg-induced inflammation in IL-4Rα-responsive BALB/c mice was accompanied with Th2-biased responses, whereas T-cell-specific IL-4Rα-deficient BALB/c mice (iLck(cre)Il4ra(-) (/lox)) developed Th1-biased responses with heightened inflammation. The proportion of Foxp3(+) Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T-cell-specific IL-4Rα-deficient mice. This was due to IL-4-mediated inhibition of CD4(+)Foxp3(+) Treg conversion, demonstrated in adoptively transferred Rag2(-) (/) (-) mice. Interestingly, reduced footpad swelling in Il4ra(-) (/lox) mice was associated with the induction of IL-4 and IL-10-secreting CD4(+)CD25(-)CD103(+)Foxp3(-) cells, confirmed in S. mansoni infection studies. Transfer of IL-4Rα-responsive CD4(+)CD25(-)CD103(+) cells, but not CD4(+)CD25(high) or CD4(+)CD25(-)CD103(-) cells, controlled inflammation in iLck(cre)Il4ra(-) (/lox) mice. The control of inflammation depended on IL-10, as transferred CD4(+)CD25(-)CD103(+) cells from IL-10-deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL-4 signalling in T cells inhibits Foxp3(+) Treg in vivo and promotes CD4(+)CD25(-)CD103(+)Foxp3(-) cells that control S. mansoni egg-induced inflammation via IL-10.

IL-4Rα-responsive smooth muscle cells contribute to initiation of TH2 immunity and pulmonary pathology in Nippostrongylus brasiliensis infections.

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong T(H)2 polarization of the host's immune response. We present data demonstrating N. brasiliensis-driven airway mucus production to be dependent on smooth muscle cell interleukin 4 receptor-α (IL-4Rα) responsiveness. At days 7 and 10 post infection (PI), significant airway mucus production was found in IL-4Rα(-/lox) control mice, whereas global knockout (IL-4Rα(-/-)) and smooth muscle-specific IL-4Rα-deficient mice (SM-MHC(Cre) IL-4Rα(-/lox)) showed reduced airway mucus responses. Furthermore, interleukin (IL)-13 and IL-5 cytokine production in SM-MHC(Cre) IL-4Rα(-/lox) mice was impaired along with a transient reduction in T-cell numbers in the lung. In vitro treatment of smooth muscle cells with secreted N. brasiliensis excretory-secretory antigen (NES) induced IL-6 production. Decreased protein kinase C (PKC)-dependent smooth muscle cell proliferation associated with cell cycle arrest was found in cells stimulated with NES. Together, these data demonstrate that both IL-4Rα and NES-driven responses by smooth muscle cells make important contributions in initiating T(H)2 responses against N. brasiliensis infections.

Expression of IL-4 receptor α on smooth muscle cells is not necessary for development of experimental allergic asthma

Airflow in the lungs of patients with allergic asthma is impaired by excessive mucus production and airway smooth muscle contractions. Elevated levels of the cytokines IL-4 and IL-13 are associated with this pathology. In vitro studies have suggested that IL-4 receptor alpha (IL-4Ralpha) signaling on smooth muscle cells is critical for airway inflammation and airway hyperresponsiveness. To define the contribution of IL-4 and IL-13 to the onset of asthmatic pathology, the role of their key receptor IL-4Ralpha in smooth muscle cells was examined in vivo. By using transgenic smooth muscle myosin heavy chain(cre)IL-4Ralpha(-/lox) mice deficient in IL-4Ralpha in smooth muscle cells, in vivo effects of impaired IL-4Ralpha signaling in smooth muscle cells on the outcome of asthmatic disease were investigated for the first time. Allergic asthma was introduced in mice by repeated sensitization with ovalbumin/aluminum hydroxide on days 0, 7, and 14, followed by intranasal allergen challenge on days 21 to 23. Mice were investigated for the presence of airway hyperresponsiveness, airway inflammation, allergen-specific antibody production, T(h)2-type cytokine responses, and lung pathology. Airway hyperresponsiveness, airway inflammation, mucus production, T(h)2 cytokine production, and specific antibody responses were unaffected in smooth muscle myosin heavy chain(cre)IL-4Ralpha(-/lox) mice compared with control animals. The impairment of IL-4Ralpha on smooth muscle cells had no effect on major etiologic markers of allergic asthma. These findings suggest that IL-4Ralpha responsiveness in airway smooth muscle cells during the early phase of allergic asthma is not, as suggested, necessary for the outcome of the disease.

IL-4Ralpha-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness.

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.

Adipose‐specific deletion of SCD1 does not protect mice from obesity

A critical regulator of lipogenesis is stearoyl‐CoA desaturase 1 (SCD1), which catalyzes the synthesis of monounsaturated fatty acids (MUFA) from saturated fatty acids. To explore the role of SCD1 in the adipose tissue, we used the Cre‐lox system to delete SCD1 from white and brown adipose tissue (AKO) using the aP2‐Cre recombinase transgene. We also generated mice with simultaneous deletion of SCD1 from both adipose and liver (LAKO) or from liver alone (LKO) using the albumin‐Cre transgene. Mice were subjected to high‐fat diet‐induced obesity (DIO; Research Diets 12492) or genetically‐induced obesity using the leptin‐resistant yellow agouti (Ay/a) allele and compared to control mice not expressing Cre recombinase (LOX). Food intakes and changes in body weights were similar amongst the LOX, AKO, LKO and LAKO mice in both models of obesity. Additionally, fat pad mass (epididymal and subcutaneous) was unaffected by adipose and/or liver deletion of SCD1. Immunoblot analysis confirmed deletion of SCD1 and fatty acid composition analysis by gas chromatography revealed reduced MUFA content in the targeted tissues. In light of our previous observations that mice with a whole‐body or skin‐specific deletion of SCD1 are resistant to obesity, the current study suggest that SCD1 deletion from adipose and/or liver is insufficient to elicit protection from obesity.Research supported by funding from NIH‐NIDDK and AHA.

IL-4Rα Responsiveness of Non-CD4 T Cells Contributes to Resistance in Schistosoma mansoni Infection in Pan-T Cell-Specific IL-4Rα-Deficient Mice

Interleukin (IL)-4 and IL-13 are T helper 2 cytokines whose biological functions are induced through a common IL-4 receptor alpha chain (IL-4Ralpha). CD4(+) T cell-specific IL-4Ralpha-mediated signaling drives susceptibility to Leishmania major infection, but is not essential to host survival following Schistosoma mansoni infection. Here we generated a novel mouse model lacking IL-4Ralpha expression specifically on all T cells (iLck(cre)Il4ra(-/lox)), which was compared with CD4(+) T cell-specific IL-4Ralpha-deficient mice (Lck(cre)Il4ra(-/lox)), to investigate the possible roles of IL-4Ralpha responsive non-CD4(+) T cells during either L. major or S. mansoni infection. Our results demonstrate a successful generation of transgene-bearing hemizygous iLck(cre)Il4ra(-/lox) BALB/c mice that have effective deletion of IL-4Ralpha on all T-cell populations. We show that iLck(cre)Il4ra(-/lox) mice infected with L. major developed a healing disease phenotype as previously observed in Lck(cre)Il4ra(-/lox) mice, demonstrating that absence of IL-4Ralpha-responsive non-CD4(+) in addition to CD4(+) T cells does not further affect transformation of BALB/c to a healer phenotype. In acute schistosomiasis, however, iLck(cre)Il4ra(-/lox) mice showed enhanced mortality compared with Il4ra(-/lox) and Lck(cre)Il4ra(-/lox) mice. iLck(cre)Il4ra(-/lox) mice died with similar kinetics to highly susceptible Il4ra(-/-) mice, despite controlling gut inflammation. In addition, iLck(cre)Il4ra(-/lox) mice presented increased liver granuloma sizes, as compared with Lck(cre)Il4ra(-/lox) mice, with similar eosinophils, fibrosis, and liver damage. In conclusion, IL-4Ralpha-responsive non-CD4(+) T cells prolong survival to acute schistosomiasis and contribute to the better control of hepatic granulomatous inflammation.

I025 Smooth muscle specific deletion of the RhoA exchange factor Arhgef1 protects against Ang II-dependent hypertension

Hypertension is one of the most frequent pathology in the industrialized world. Although it is recognized to be dependent on a combination of genetic and environmental factors, its molecular basis remains elusive. A possible candidate is the monomeric G protein RhoA which activates Rho kinase. However, how it is activated and whether it has a causative role in hypertension is still unknown. By in vitro experiments, we provide evidence that Arhgef1 is the RhoA guanine exchange factor specifically responsible for Angiotensin II (Ang II)-induced RhoA/Rho kinase activation in arterial smooth muscle cells. To analyze in vivo the role of Arhgef1, we generated mice lacking Arhgef1 specifically in smooth muscle cells (SM-Arhgef1-KO). We used Arhgef1lox/lox mice, which were mated to SMMHC-CreERT2 to produce SMMHC-CreERT2 ; Arhgef1lox/lox mice (SM-Arhgef1lox/lox). SM-Arhgef1-KO mice were then obtained by treating SM-Arhgef1lox/lox mice with tamoxifen. Remarkably, SM-Arhgef1-KO mice were resistant to hypertension induced by chronic Ang II infusion (sub-cutaneous osmotic pump, 1 μg/kg/min). Furthermore, Arhgef1 deletion after induction of hypertension by Ang II restored normal arterial pressure level. Basal arterial pressure and responses to vasoconstrictors other than Ang II (phenylephrine, U46619, endotheline) were not modified in SM-Arhgef1-KO mice. Our results show that Arhgef1 activation and its downstream RhoA/Rho kinase signaling are central to the development of Ang IIdependent hypertension. We identify Arhgef1 as an alternative potential target for the treatment of hypertension.

K-ras and Wnt Signaling Synergize to Accelerate Prostate Tumorigenesis in the Mouse

Aberrant Ras and Wnt signaling are emerging as key events in the multistep nature of prostate tumorigenesis and progression. Here, we report the generation of a compound model of prostate cancer to define the synergism of activated K-ras (K-ras(+/V12)) and dominant stabilized beta-catenin (Catnb(+/lox(ex3))) in the murine prostate. Recombination of floxed alleles and subsequent expression of oncogenic transgenes was mediated by Cre recombinase expression governed by the composite Probasin (PB) promoter (termed PBCre). Concomitant with elevated mitogen-activated protein kinase (MAPK) signaling, PBCre(+)K-ras(+/V12) mice developed AH at 100 days (100% incidence) and low-grade prostate intraepithelial neoplasia and adenocarcinoma (60% and 7% incidence) by 500 days. PBCre(+)Catnb(+/lox(ex3)) mice showed reduced longevity (average 428 days) and were predisposed to PIN-like keratinized squamous metaplasia at 100 days (100% incidence) and adenocarcinoma (100% incidence) at end-point. These lesions displayed elevated Wnt signaling and basal levels of MAPK signaling. Synchronous activation of K-ras and beta-catenin significantly reduced survival (average 189 days), reflecting accelerated tumorigenesis and the development of invasive carcinoma that displayed activated Wnt and MAPK signaling. Notably, expression of the basal cell marker p63 negatively correlated with tumor grade, resembling human prostate adenocarcinoma. Taken together, our data show that combinatorial oncogenic mutations of K-ras and beta-catenin drive rapid progression of prostate tumorigenesis to invasive carcinoma, characterized by the synergistic elevation of androgen receptor, cyclooxygenase-2, and c-Myc.

Hepatic Stearoyl‐CoA desaturase 1 Deficiency Promotes Liver Inflammation Under High Carbohydrate Low Fat Diet Feeding

High carbohydrate diet causes liver steatosis and inflammation. To understand the role of stearoyl‐CoA desaturase 1 (SCD1) in liver steatosis, we generated liver‐specific SCD1 deficient mouse models (SCD1‐LKO). Previously we showed that SCD1‐LKO mice were protected from liver‐steatosis with long term high carbohydrate feeding. In this study we further analyzed the effects of a short term (10 days) feeding of high‐sucrose‐very‐low‐fat (HSVLF) diet on SCD1‐LKO mice. Surprisingly, the HSVLF feeding led to a dramatic increase in liver inflammation in SCD1‐LKO but not in Lox control mice. Histological analysis on liver sections revealed no sign of steatosis in LKO and Lox mice fed with HSVLF. The expression of macrophage specific genes Emr1 (F4/80) and CD68 in liver was significantly higher in HSVLF‐fed LKO than Lox mice. However, macrophage infiltration was not detected. We subsequently observed an increased liver ER stress response in HSVLF‐fed LKO mice, which was consistent with a dramatic elevation of the plasma ALT level. Furthermore, supplementation of the SCD1 product oleate rescued liver inflammation, ER stress response and liver injury in SCD1‐LKO mice, whereas supplementation of the SCD1 substrate–stearate to HSVLF did not reverse these effects. These data indicate that hepatic SCD1 is both necessary and sufficient to prevent the high carbohydrate low fat diet induced liver inflammation.