Summary The cytokine IL17, and signaling via its heterodimeric IL-17RA/IL-17RC receptor, is critical for host defense against extracellular bacterial and fungal pathogens. Polarized lung epithelial cells express IL-17RA and IL-17RC basolaterally. However, their contribution to IL-17-dependent pulmonary defenses in vivo remains to be determined. To address this, we generated mice with conditional deletion of Il17ra or Il17rc in Scgb1a1-expressing club cells, a major component of the murine bronchiolar epithelium. These mice displayed an impaired ability to recruit neutrophils into the airway lumen in response to IL17, a defect in bacterial clearance upon mucosal challenge with the pulmonary pathogen Klebsiella pneumoniae, and substantially reduced epithelial expression of the chemokine Cxcl5. Neutrophil recruitment and bacterial clearance were restored by intranasal administration of recombinant CXCL5. Our data show that IL-17R signaling in the lung epithelium plays a critical role in establishing chemokine gradients that are essential for mucosal immunity against pulmonary bacterial pathogens.

Natural IgM antibodies in diverse species recognize conserved carbohydrates in fungal cell walls and influence early host defense against Pneumocystis in mice.

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17 cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next tested a role for RegIIIγ, an IL-22–inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection, increased accumulation of Th17 cells in the lungs of RegIIIγ−/− and Il22−/− mice was associated with intestinal segmented filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIγ decreased lung inflammatory gene expression and protected Il22−/− mice from weight loss during infection, it had no direct effect on SFB colonization, fungal clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate pulmonary adaptive immune responses.

In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.

Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.

Somatic cell nuclear transfer (SCNT) offers the possibility of preserving endangered species. It is one of the few technologies that avoids the loss of genetic variation and provides the prospect of species continuance, rather than extinction. Nonetheless, there has been a debate over the use of SCNT for preserving endangered species because of abnormal nuclear reprogramming, low efficiency and the involvement of extra mitochondrial DNA (mtDNA) of a different species in live offspring produced by interspecies SCNT. Despite these limitations, live endangered cloned animals have been produced. In the present paper, we describe recent research on the production of cloned embryos derived by fusion of wild felid fibroblast cells with heterospecific domestic cat cytoplasts and their viability after transfer into domestic cat recipients. In addition, we discuss epigenetic events that take place in donor cells and felid cloned embryos and mtDNA inheritance in wild felid clones and their offspring.

Autophagy is a major regulator of pancreatic beta cell homeostasis. Altered autophagic activity has been implicated in the beta cells of patients with type 2 diabetes, and in the beta cells of obese diabetic rodents. Here, we show that autophagy was induced in beta cells by either a high-fat diet or a combined high-fat and high-glucose diet, but not by high-glucose alone. However, a high-glucose intake alone did increase beta cell mass and insulin secretion moderately. Depletion of Atg7, a necessary component of the autophagy pathway, in beta cells by pancreatic intra-ductal AAV8-shAtg7 infusion in C57BL/6 mice, resulted in decreased beta cell mass, impaired glucose tolerance, defective insulin secretion, and increased apoptosis when a combined high-fat and high-glucose diet was given, seemingly due to suppression of autophagy. Taken together, our findings suggest that the autophagy pathway may act as a protective mechanism in pancreatic beta cells during a high-calorie diet.

A thorough understanding of the signaling pathways involved in the regulation of β cell proliferation is an important initial step in restoring β cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced β cell proliferation. Specific deletion of EGFR in the β cells of adult mice impaired β cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult β cell proliferation. Further analyses showed that β cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance β cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal β cell growth.