Anti-inflammation Effect Of Combined Adenovirus Mediated Human TLR2 And TLR4 Gene

ObjectiveSepsis is a systemic inflammatory reaction caused by infection,which is marked by acute onset,rapid progress,many complications,high mortality and difficult to control. Lipopolysaccharide(LPS) in outer membrane of gram-negative bacteria cell wall is a major pathogen factor of inflammatory reaction,and LPS toxicity is always a hot research spot. It is reported that Toll-like receptor (TLR) mediated innate immune responses which plays an important role in LPS induced inflammation and is closely related to the intracellular delivery of LPS stimulating signal. It is confirmed that multiple bacterial components of inflammation could activate TLR2 and TLR4 directly and indirectly,then to activate leukocytes. Therefore,blocking or inhibiting the activation of TLR2 and TLR4 may inhibit excessive inflammatory reaction by inhibiting the activation of leukocytes and synthesis or secretion of inflammatory cytokines. Recombinant adenovirus AdTLR2 prepared previously could inhibit the activation of TLR2 on leukocytes membrane competitively. As a result,the expression of inflammatory factors are inhibited which indicates the effect of anti-inflammation. The aim of this study is to construct recombinant adenovirus AdTLR4, which is a recombination of adenovirus vector with TLR4 extracellular domain,as well as observe the anti-inflammatory effects of LPS in vitro and vivo that affected by combined adenovirus AdTLR2 and AdTLR4 to provide a new methed for treatment of clinical inflammation.Methods1. Clone ConstructionThe TLR4 mRNA gene sequences was analyzed according to NCBI site. The TLR4 extracellular domain primers was designed by Oligo software. Polymerase chain reaction (PCR) technique was used to amplify the human TLR4 extracellular domain gene fragment with pUCm-TLR4 plasmid as template. Target gene was sub-cloned to pTA2 vector for the recombination of plasmid pTA2-TLR4 by restriction analysis and sequence identification. 2. Construction of recombinant adenovirusCorrect target fragments were directional cloned to adenovirus shuttle plasmid pAdTrack-CMV for recombinant of plasmid pAdTrack-CMV-TLR4. The positive recon was digested to linear and transferred to competent AdEasier-1 bacteria,and went through homogeneous recombination with adenovirus frame plasmid pAdEasy-1 in BJ5183 for the production of recombinant adenovirus plasmid pAd-TLR4. pAd-TLR4 were analyzed for recombination accuracy,then packaged in 293 cell by liposome fusion and amplified and the protein were purified and tested for virus titer. The mRNA and protein expression of recombinant adenovirus were analyzed by RT-PCR and Western blot.3. Concentration of inflammatory cytokines in LPS treated THP-1 cell transfected by recombinant adenovirusHuman monocytic cells THP-1 were divided into five groups: A group (normal control),B group (control of inflammation:LPS stimulating cell),C group (adenovirus AdTLR2:recombinant adenovirus AdTLR2 infected cell before inflammation),D group (adenovirus AdTLR4:recombinant adenovirus AdTLR2 infected cell before inflammation),E group (recombinant AdTLR2 + AdTLR4:recombinant adenovirus AdTLR2 and AdTLR4 infected cell before inflammation). Cell suspension was collected after 24h. ELISA was assayed to detect the concentration of cytokines TNF-α,IL-1β,IL-6,IL-10 and IL-13 to analyze influence of AdTLR2 and AdTLR4 on cytokines in THP-1 cell serum.4. Concentration of inflammatory cytokines in LPS treated mice transfected by recombinant adenovirus30 BALB/c mice were divided into five groups (n=6) : A group (control : intraperitoneal injection of normal saline),B group (control of the inflammation:intraperitoneal injection of LPS),C group (adenovirus AdTLR2:intraperitoneal injection of recombinant adenovirus AdTLR2 before LPS inflammation),D group (recombinant AdTLR4 : intraperitoneal injection of recombinant adenovirus AdTLR4 before LPS inflammation),E group (recombinant AdTLR2 + AdTLR4:intraperitoneal injection of recombinant adenovirus AdTLR2 and AdTLR 4 before LPS inflammation). serum samples of different experiment groups were collected after 24 h observation,then alteration of cytokines TNF-α,IL-1β,IL-6,IL-10 and IL-13 were tested by ELISA to analyze the influence of AdTLR2 and AdTLR4 on cytokines in serum. Result1. Obtained of the human TLR4 extracellular domain target gene fragment The human TLR4 extracellular domain target gene fragment was obtained by PCR and recombinant plasmid pTA2-TLR4 was successfully structed. Sequence analysis showed that the presence of the cloned fragment was confirmed as 1900bp,the same as TLR4 extracellular domain in GenBank.2. Construction of recombinant adenovirusAfter digestion and sequence,the recombination of adenovirus shuttle plasmid pAdTrack-CMV-TLR4 was successfully obtained . Obvious green fluorescence was observed under fluorescent microscope,with a classical cytopathic effect (CPE) observed after the transfection of pAd-TLR4 in 293 cells,which indicated successful package of adenovirus. Recombinant adenovirus AdTLR4 with a title of 3.2×109 PFU/mL was collected after purified, then the expression of the TLR4 gene and protein were determined by RT-PCR and western blot.3. Concentration of inflammatory cytokines in LPS treated THP-1 cell transfected by recombinant adenovirusAfter LPS inflammatory cells treated with AdTLR2 or AdTLR4 alone,the level of TNF-α,IL-1β,IL-6,IL-10 and IL-13 in cell supernatantlevels were reduced compared with the control of inflammation that had statistic significance. After treatment of AdTLR2 and AdTLR4,above five factor were reduced compared with that treated alone,which had statistic significance(P <0.05) .4. Concentration of inflammatory cytokines in LPS treated mice transfected by recombinant adenovirusNormal control mice had no significant change. After injection of LPS the activity of mice in control group of inflammation was decreased,and loss of appetite with crouching weak and hair mats. When mice were injected intraperitoneally with adenovirus,fewer activities and slightly decreased appetite were found in early days. After treatment with AdTLR2 or AdTLR4 alone,the LPS treated inflammatory mice,compared with the inflammation control group,had lower serum level of TNF-α,IL-1β,IL-6,IL-10 and IL-13. While treated with combination of AdTLR2 and AdTLR4,compared with the groups treated alone,the above five factor were reduced which had statistic significance (P <0.05). Conclusion1. The human TLR4 extracellular domain target gene fragment was successfully obtained,and the target gene was free of base substitution and in accordance with corresponding series in GenBank,which was confirmed by sequencing and homologous comparison.2. Recombinant adenovirus plasmid AdTLR4 carrying TLR4 extracellular domain was successfully constructed. With amplication of 293 cell and purification,recombinant adenovirus with a title of 3.2×109 PFU/mL was produced.3. In vitro , the recombinant adenovirus AdTLR2 and AdTLR4 reduced the concentration of TNF-α,IL-1β,IL-6,IL-10 and IL-13 in supernatant of LPS treated inflammatory cell. And the effect of anti-inflammation was better in combination than AdTLR2 or AdTLR4 alone,suggesting that the recombinant adenovirus could secret sTLR2 and sTLR4 which inhibit the release of inflammatory cytokines to certain anti-inflammatory effects.4. In vivo,the adenovirus AdTLR2 and AdTLR4 reduced the concentration of TNF-α,IL-1β,IL-6,IL-10 and IL-13 in serum of LPS traeted inflammation mice. And combination of the two decreased the concentration of inflammatory cytokines more than that treated alone,suggesting that in vivo the recombinant adenovirus could secret sTLR2 and sTLR4 which effectively inhibit the activation of inflammatory cells,reduce the production of cytokines,and a certain anti-inflammatory effects. It has not been reported at home and abroad.