In vivo function of airway epithelial TLR2 in host defense against bacterial infection

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2(-/-) BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2(-/-) mice reconstituted 30-40% TLR2 expression compared with TLR2(+/+) cells. Such airway epithelial TLR2 reconstitution in TLR2(-/-) mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2(-/-) mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2(-/-) mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.     

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.     

Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.     

Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-kappaB precursors. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Ikappa-B kinase complex (dmIKK). dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP). Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway. Using inducible expression of dFADD double-stranded RNA, we demonstrate that dFADD is a novel component of the Imd pathway: dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. Our results reinforce the parallels between the Imd and the TNF-R1 pathways.     

In vivo analysis reveals a critical role for neuropilin-1 in cranial neural crest cell migration in chick

The neural crest provides an excellent model system to study invasive cell migration, however it is still unclear how molecular mechanisms direct cells to precise targets in a programmed manner. We investigate the role of a potential guidance factor, neuropilin-1, and use functional knockdown assays, tissue transplantation and in vivo confocal time-lapse imaging to analyze changes in chick cranial neural crest cell migratory patterns. When neuropilin-1 function is knocked down in ovo, neural crest cells fail to fully invade the branchial arches, especially the 2nd branchial arch. Time-lapse imaging shows that neuropilin-1 siRNA transfected neural crest cells stop and collapse filopodia at the 2nd branchial arch entrances, but do not die. This phenotype is cell autonomous. To test the influence of population pressure and local environmental cues in driving neural crest cells to the branchial arches, we isochronically transplanted small subpopulations of DiI-labeled neural crest cells into host embryos ablated of neighboring, premigratory neural crest cells. Time-lapse confocal analysis reveals that the transplanted cells migrate in narrow, directed streams. Interestingly, with the reduction of neuropilin-1 function, neural crest cells still form segmental migratory streams, suggesting that initial neural crest cell migration and invasion of the branchial arches are separable processes.     

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.     

Characterization of murine grancalcin specifically expressed in leukocytes and its possible role in host defense against bacterial infection

Such phagocytic leukocytes as macrophages and neutrophils are the key cellular components of innate immunity. The actin cytoskeleton is essential for their recruitment and activation in infected tissues. We have previously identified p65/L-plastin with Ca(2+)-, calmodulin-, and beta-actin-binding domains in macrophages. In order to further investigate the p65/L-plastin-involved cellular functions, we cloned the cDNA for murine grancalcin, a possible binding partner of p65/L-plastin. According to the sequence, grancalcin is a member of the penta-EF-hand protein family. We prepared recombinant (r) grancalcin for functional studies and found that it exhibited Ca(2+)-dependent precipitation. High-titer antibodies against the protein enabled us to detect intracellular grancalcin. A flow cytometric analysis revealed grancalcin to be highly expressed in macrophages and neutrophils. The protein was particularly abundant in those cells recovered from bacteria-infected sites. Immunohistochemical studies clarified that grancalcin was translocated to the actin cytoskeleton in macrophages upon exposure to bacterial lipopolysaccharide. These findings suggest that grancalcin plays a key role in leukocyte-specific functions that are responsible for host defense.     

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.     

NK cells play a critical protective role in host defense against acute extracellular Staphylococcus aureus bacterial infection in the lung

Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or gammadelta T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.     

Modeling breast cancer in vivo and ex vivo reveals an essential role of Pin1 in tumorigenesis

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.     

The analysis of the defense mechanism against indigenous bacterial translocation in X-irradiated mice

The defense mechanism against indigenous bacterial translocation was studied using a model of endogenous infection in X-irradiated mice. All mice irradiated with 9 Gy died from day 8 to day 15 after irradiation. The death of mice was observed in parallel with the appearance of bacteria from day 7 in various organs, and the causative agent was identified to be Escherichia coli, an indigenous bacterium translocating from the intestine. Decrease in the number of blood leukocytes, peritoneal cells and lymphocytes in Peyer's patches or mesenteric lymph nodes was observed as early as 1 day after irradiation with 6 or 9 Gy. The mitogenic response of lymphocytes from various lymphoid tissues was severely affected as well. The impairment of these parameters for host defense reached the peak 3 days after irradiation and there was no recovery. However, in vivo bactericidal activity of Kupffer cells in mice irradiated with 9 Gy was maintained in a normal level for a longer period. It was suggested that Kupffer cells play an important role in the defense against indigenous bacteria translocating from the intestine in mice.