The Effects Of Cd300lf-gene Modified Dendritic Cells On Airway Inflammation In A Murine Model Of Allergic Asthma

Allergic asthma, characterized by airway hypersensitivity(AHR), cellular infiltration of the airway withpredominantly eosinophils and Th2 cells and increased serum IgE levels.is a chronic inflammatory disorder of the airway caused by manyinflammatory cells. It has been accepted that Tlymphocytes, which predominantly produce Th2 cytokines such as IL-4, IL-13 and IL-5 in response to inhaled antigen, play a major role in the pathogenesis of allergic asthma.CD300 antigen like family member F (Cd3001f) is a type I transmembrane glycoprotein containing an N-terminal signal peptide, aextracellular region with a single IgV-like domain, a transmembrane region, and a cytoplasmic tail with two classical immune-receptor tyrosine-based inhibitory motifs (ITIM) and an immune-receptor tyrosine-based switching motif(ITSM).Based on the fact that both DC-initiatedT-cell proliferation and antigen-specificT-cell responses were enhanced upon blockade of Cd3001f onDCs, and that more potent antigen-specific CD4+and CD8+T-cell responses were elicitedwhen mice were immunized withantigen-pulsed, Cd3001f-silenced DCs, it was confirmed that Cd3001fmediated negative regulationof DC-initiated antigen-specific T-cell responses.In this study,specific primers for the mouse Cd3001f gene containing entire open reading frame (ORF) were designed according to the nucleotide sequence (NM₁45634.3) published in GeneBank. The Cd3001f gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from murine bone marrow-derived dendritic cells (BMDCs) and cloned intopMD 18-T vector, producing the recombinant plasmid pMD-Cd3001f. After bioinformatic analysis of the sequence data, the extracellular domain of Cd3001f gene without the N-terminal signal peptide sequence (amino acid residues from 22 to 185) was amplified by polymerase chain reaction(PCR) from the recombinant plasmid pMD-Cd3001f using other primers, and cloned into the prokaryotic expression vector pGEX-4T-3. The resulting recombinantplasmidpGEX-Cd3001f (22 aa-185 aa) was transformed into E. co//.Rosetta (DE3), and expressed a45.5 kD fusion protein after induction by isopropyl-β-D-thiogalactoside (IPTG). An anti-GST-Cd3001f (22 aa-185 aa)polyclonal antibody was generated byimmunizing subcutaneouslyNewZealand white rabbits with the Cd3001f protein (22 aa-185 aa)purified with the GSTrap FF column, and the result of indirect enzyme-linked immunosorbent assay(ELISA) indicated thatthe titer of the antibody was 1:16384.Human embryonic kidney 293 cells (HEK293)were transiently transfected with aeukaryotic expression plasmid pCDNA-Cd3001f containing the entire Cd3001f gene, and the specificity of the rabbit anti-GST-Cd3001f polyclonal antibody was assessed by both indirect immunofluorescence assay (â…¡A) and Western blot. Results showed that the Cd3001f protein expressed in HEK293cells could be specifically recognized by the rabbit anti-GST-Cd3001f (22 aa-185 aa)polyclonal antibody.According to the siRNA design guidelines, five short hairpin oligonucleotides and complementary strands were designed to target the consensus sequences of mouse Cd3001f gene. The sense and antisense oligonucleotides were annealed, and cloned into the expression vector pLL3.7 containing the mouse U6 promoter. The resultant plasmids.which expressed short hairpin RNA (shRNA) against Cd3001f gene, were cotransfected into HEK293 for 48 hourstogether with eukaryotic expression plasmid pCDNA-Cd3001f using Lipofectamin 2000. Real-time PCR analysis showed that recombinant plasmid pLL-Cd3001f-shRNAe effectively knocked down Cd3001f mRNA by 75.6%. The Cd3001f gene was amplified by PCR from the recombinant plasmid pMD18-Cd3001f using specific primers, and cloned into adenovirus shuttle plasmid pAdTrack-CMV. In addition, the shRNA against Cd3001f gene containing U6 promoter was amplified by PCR from the recombinant plasmid pLL-Cd3001f-shRNAe using specific primers, and cloned into adenovirus shuttle plasmid pAdTrack. Positive recombinant plasmid was digested with Pme I, and transformation into competent BJ5183 cells containing adenoviral bone plasmid pAdEasy-1 for homologous recombination. After digestion with Pac I, the recombinant adenoviral plasmid was transfected into HEK293 cells using Lipofectamine 2000.12-18 days after transfection, recombinant adenovirus Ad-Cd3001f and Ad-Cd3001f-shRNA were generated and amplified sufficiently in HEK293 cells. After purification by CsCl density gradient ultracentrifugation, the titer of the recombinant adenoviruses could reach 1.4×109PFU/ml for Ad-Cd3001f and 2.6×109PFU/ml for Ad-Cd3001f-shRNA, respectively. BMDCs cells infected with recombinant adenoviruses at a multiplicity of infection (MOI) of 75 for 48 hours. Real-time PCR analysis showed that expression of Cd3001f mRNA levels in BMDCs infected with Ad-Cd3001f significantly increased, whereas that in BMDCs infected with Ad-Cd3001f-shRNA reducedby 51.7%. Western blot analysis showed the Cd3001f protein were specifically expressed in BMDCs infected with Ad-Cd3001f.C57BL/6 mice were sensitized with ovalbumin (OVA) emulsified with aluminum hydroxide by an intraperitoneal injection on day Oand 14, respectively. Ten days after last sensitization, mice were challenged with OVA aerosol on 3 successive days. BMDCs culturedfor 5days were infected with recombinant adenovirus Ad-Cd3001f (DC-Cd3001f) and Ad-Cd3001f-shRNA (DC-siCd3001f) at a MOI of 75, respectively and next day the cells were pulsed with OVA for 24 hours. DC-Cd3001for DC-siCd3001f was injected intraperitoneally into mice on day-7and 3, respectively.Twenty-four hours after the last challenge, AHR was measured, and inflammatory cells and cell differentials in bronchoalveolar lavage fluid (BALF) were determined with Wright-Giemsa. The pathological changes in lung tissues were assayed by hematoxylin and eosin (H&E) and periodic acid-Schiff staining (PAS). The levels of IL-4, IL-5, IL-13 and IFN-y in BALF and culture supernatant of splenocytes were measured by ELISA.The percentages of CD4TFoxp3+Tregs and CD4+IL-17A+Th17 in spleen were assessed by flow cytometry analysis. The results showed that the mice transferred by DC-siCd3001f displayeda significant reduction in airway hyperreactivity (AHR) and airway inflammation. Decreased levels of IL-4, IL-5 and IL-13 in BALF and culture supernatant of splenocytes were observed in the mice transferred by DC-siCd3001f.Transfer ofDC-siCd3001finto mice resulted in an increased CD4+FoxP3+Tregs and a decreasedCD4+IL-17A+Thl7in spleen, respectively.In summary, transfer of DC-siCd3001f could significantly attenuate AHR, inhibitairway inflammation, decrease production of Th2 cytokines.and elicitan increased CD4+FoxP3+Tregs and a decreasedCD4+IL-17A+Th17 in spleen, providing a novel immunointervention strategy for treatment of asthma.