IL-17 Receptor Signaling in the Lung Epithelium Is Required for Mucosal Chemokine Gradients and Pulmonary Host Defense against K. pneumoniae.

The cytokine IL-17, 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 IL-17, 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.

Pulmonary Th17 Antifungal Immunity Is Regulated by the Gut Microbiome.

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.

Novel pneumocystis antigen discovery using fungal surface proteomics.

Pneumonia due to the fungus Pneumocystis jirovecii is a life-threatening infection that occurs in immunocompromised patients. The inability to culture the organism as well as the lack of an annotated genome has hindered antigen discovery that could be useful in developing novel vaccine- or antibody-based therapies as well as diagnostics for this infection. Here we report a novel method of surface proteomics analysis of Pneumocystis murina that reliably detected putative surface proteins that are conserved in Pneumocystis jirovecii. This technique identified novel CD4(+) T-cell epitopes as well as a novel B-cell epitope, Meu10, which encodes a glycosylphosphatidylinositol (GPI)-anchored protein thought to be involved in ascospore assembly. The described technique should facilitate the discovery of novel target proteins for diagnostics and therapeutics for Pneumocystis infection.

Dectin immunoadhesins and pneumocystis pneumonia.

The opportunistic pathogen Pneumocystis jirovecii is a significant cause of disease in HIV-infected patients and others with immunosuppressive conditions. Pneumocystis can also cause complications in treatment following antiretroviral therapy or reversal of immunosuppressive therapy, as the newly reconstituted immune system can develop a pathological inflammatory response to remaining antigens or a previously undetected infection. To target ß-(1,3)-glucan, a structural component of the Pneumocystis cell wall with immune-stimulating properties, we have developed immunoadhesins consisting of the carbohydrate binding domain of Dectin-1 fused to the Fc regions of the 4 subtypes of murine IgG (mIgG). These immunoadhesins bind ß-glucan with high affinity, and precoating the surface of zymosan with Dectin-1:Fc can reduce cytokine production by macrophages in an in vitro stimulation assay. All Dectin-1:Fc variants showed specificity of binding to the asci of Pneumocystis murina, but effector activity of the fusion molecules varied depending on Fc subtype. Dectin-1:mIgG2a Fc was able to reduce the viability of P. murina in culture through a complement-dependent mechanism, whereas previous studies have shown the mIgG1 Fc fusion to increase macrophage-dependent killing. In an in vivo challenge model, systemic expression of Dectin-1:mIgG1 Fc significantly reduced ascus burden in the lung. When administered postinfection in a model of immune reconstitution inflammatory syndrome (IRIS), both Dectin-1:mIgG1 and Dectin-1:mIgG2a Fc reduced hypoxemia despite minimal effects on fungal burden in the lung. Taken together, these data indicate that molecules targeting ß-glucan may provide a mechanism for treatment of fungal infection and for modulation of the inflammatory response to Pneumocystis and other pathogens.

Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina.

Host defense against opportunistic fungi requires coordination between innate and adaptive immunity for resolution of infection. Antibodies generated in mice vaccinated with the fungus Pneumocystis prevent growth of Pneumocystis organisms within the lungs, but the mechanisms whereby antibodies enhance antifungal host defense are poorly defined. Nearly all species of fungi contain the conserved carbohydrates ß-glucan and chitin within their cell walls, which may be targets of innate and adaptive immunity. In this study, we show that natural IgM antibodies targeting these fungal cell wall carbohydrates are conserved across many species, including fish and mammals. Natural antibodies bind fungal organisms and enhance host defense against Pneumocystis in early stages of infection. IgM antibodies influence recognition of fungal antigen by dendritic cells, increasing their migration to draining pulmonary lymph nodes. IgM antibodies are required for adaptive T helper type 2 (Th2) and Th17 cell differentiation and guide B cell isotype class-switch recombination during host defense against Pneumocystis. These experiments suggest a novel role for the IgM isotype in shaping the earliest steps in recognition and clearance of this fungus. We outline a mechanism whereby serum IgM, containing ancient specificities against conserved fungal antigens, bridges innate and adaptive immunity against fungal organisms.

Generation of domestic transgenic cloned kittens using lentivirus vectors.

The efficient use of somatic cell nuclear transfer (SCNT), in conjunction with genetic modification of donor cells provides a general means to add or inactivate genes in mammals. This strategy has substantially improved the efficacy of producing genetically identical animals carrying mutant genes corresponding to specific human disorders. Lentiviral (LV) vectors have been shown to be well suited for introducing transgenes into cells to be used as donor nuclei for SCNT. In the present study, we established an LV vector-based transgene delivery approach for producing live transgenic domestic cats by SCNT. We have demonstrated that cat fetal fibroblasts can be transduced with EGFP-encoding LV vectors bearing various promoters including the human cytomegalovirus immediate early (hCMV-IE) promoter, the human translation elongation factor 1alpha (hEF-1alpha) promoter and the human ubiquitin C (hUbC) promoter. Among the promoters tested, embryos reconstructed with donor cells transduced with a LV-vector bearing the hUbC promoter displayed sustained transgene expression at the blastocyst stage while embryos reconstructed with LV vector-transduced cells containing hCMV-IE-EGFP or hEF-1alpha-EGFP cassettes did not. After transfer of 291 transgenic cloned embryos into the oviducts of eight recipient domestic cats (mean =36.5 +/- 10.1), three (37.5%) were diagnosed to be pregnant, and a total of six embryos (2.1%) implanted. One live male offspring was delivered by Cesarean section on day 64 of gestation, and two kittens were born dead after premature delivery on day 55. In summary, we report the birth of transgenic cloned kittens produced by LV vector-mediated transduction of donor cells and confirm that cloned kittens express the EGFP reporter transgene in all body tissues.

Cloning endangered felids using heterospecific donor oocytes and interspecies embryo transfer.

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.

Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells.

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.

Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells.

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.