Effect Of TREM-2 Gene Silencing By RNA Interference On Inflammatory Responses In Alveolar Macrophage Of Acute Lung Injury Mice Induced By Lipopolysaccharide

BackgroudAcute lung injury(ALI),mainly caused by infection,is one of the most common critical illnesses of respiratory system.ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by Lipopolysaccharides(LPS)or endotoxin.Alveolar macrophages(AM)are important for airway defense and also the target cells of LPS.AM have some kinds of pattern recognition receptors such as triggering receptors expressed on myeloid cells(TREMs),Toll-like receptor4(TLR4),and scavenger receptors(SR). It is helpful and important to investigate LPS signaling transduction of AM and the blocking effects to understand the mechanism of ALI and find out a new therapy way.TLR4,as a trans-membrane receptor in LPS target cells,is the key molecular in LPS signaling pathway.TLR4 expression and its perfect function directly affect the effects of LPS signaling transduction.Otherwise,excessive inflammatory reaction results in septic shock, multiple organ dysfunction syndrome(MODS)even death.TREM is a newly identified member of the immunoglobulin superfamily of receptors and modulate the innate response either by amplifying or dampening TLR-induced signals,and thus play crucial roles in fine-tuning of the inflammatory response.TREM-1 as an amplifier of TLR4 signaling pathway in the systemic inflammatory response syndrome associated with sepsis.By its capacity to promote inflammation,TREM-1 is an important player in ALI, bacterial infection and sepsis.Unlike TREM-1,which is involved in the amplification of inflammation,TREM-2 is emerging as an important negative regulator of autoimmunity. TREM-2 as an important anti-inflammatory receptor and suggest that modulation of TREM-2 expression or function may be an important way to regulate inflammation.TREM-2 acts a negative regulator by modulating the TLR4 signaling.If the expression of TREM-2mRNA in the alveolar macrophages is reduced,it will results in increased lung injury.This converse result confirms the criticality of TREM-2 in the lung tissue,reveals an anti-inflammatory role in the process,and allows us to direct potential therapies in ALI.RNA interference(RNAi)is a new genetic interference technology,which specially degrades the corresponding sequence mRNA by the mediate of double-stranded RNA(dsRNA) synthesized in vitro or formed in vivo which are diced up 21-23 nucleotide-long small interfering RNA(siRNA)by the enzyme Dicer to induce the post-transcriptional gene silencing. In recent years,RNAi has been found in organisms from epiphytes to plants,and from invertebrates to mammalian animals.Compared with gene knockout,RNAi is simple,time saving and effective.RNAi has now been widely used in studies of gene function.With the breakthrough of RNAi technology in mammalian cells,more and more studies begin to apply the RNAi in the research of functional genomics and gene therapy.ObjectiveIn order to investigate the role of TREM-2 signaling pathway in ALI induced by LPS, eukaryotic expression plasmid vector of short hair RNA(shRNA)targetting mice TREM-2 gene would be designed and constructed to establish a rapid screen system for siRNA targeting TREM-2.Then lentivirus vectors was transfected it into mice AM cells to investigate the inhibitory action to TREM-2 mRNA and protein expression level.To investigate the expression pattern of TREM-2 and its relationship with the secretion of proinflammatory factors during AM cell inflammatory response induced by LPS.In vitro siRNAs were administered to the cultured AM cells before LPS stimulation to study the effect of shRNA targeting TREM-2 gene on inflammatory responses induced by LPS.Expression of TLR4,TREM-1,TREM-2 of AM and cytokine proteins were detected.After transfected from respiratory tract,mice were treated with LPS.Expression of TLR4,TREM-1,TREM-2 of pulmonary tissues and cytokine proteins in BALF were detected.Methods1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 geneFirstly,two RNAi sites targeting mice TREM-2 gene in gene bank were selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed.Secondly,two pairs of oligonucleotides fragments were synthesized and annealed to double strands.Thirdly,the double strands were cloned into plasmid vector pGCsi.Finally,the recombinant plasmids were identified by PCR and sequencing analysis.The three kinds of recombinant plasmids were named pTREM-2-shRNA1,pTREM-2-shRNA2 and pNeg-shRNA.2 Screening siRNA sequence which could suppression TREM-2 expression most effectively in vitroAlveolar macrophages were isolated and cultured,cell morphous and growth condition were observed by inverted microscope.After transfected with transfection complex comprising different proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent for 12h,24h, 48h,72h,96h,transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the optimal proportion of pTREM-2-shRNA plasmid and TransFectin Lipid reagent,and the time point which lead to maximal transfection efficiency for further research.After 48h of being transfected with pTREM-2-shRNA,total RNA was extracted from cells.TREM-2mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR).Total RNA of pCon group(alveolar macrophages transfected with p-GCsi),pNeg group(alveolar macrophages transfected with negative shRNA vector),and non transfected group were also evaluated as control.3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 geneThe mice TREM-2-shRNA segments were obtained from plasmids pTREM-2-shRNA which were constructed at an earlier date by restriction endonucleases digestion and then cloned into the shuttle plasmids pLenti-EGFP-U6 to form recombinant plasmids pLenti-EGFP-TREM -2-shRNA-U6.The recombinant plasmids were co-transfected with genomic plasmids(pLP1,pLP2,pVSV-G)into 293T cells to package recombinant lentivirus which were named Lenti-EGFP-TREM-2-shRNA.Then the recombinant lentivirus were propagated, purified and identified.4 Study on the inhibition of TREM-2 mRNA and protein expression in mice alveolar macrophages mediated by Lenti-EGFP-TREM-2-shRNAMice alveolar macrophages were transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to Polybrene and different time.Transfection efficiency was defected by inverted fluorescence microscope and flow cytometry(FCM)to choose the best MOI,the optimal proportion of lenti to Polybrene,and the time point which lead to maximal transfection efficiency for further research.After 72h of being transfected with Lenti-EGFP-TREM-2-shRNA,total RNA was extracted from alveolar macrophages.TREM-2 mRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by fluorescence-activated cell sorting(FACS).Total RNA and protein of Con group(AM transfected with Lenti-EGFP),Neg group(AM transfected with negative shRNA Lentivirus), and non transfected group were also evaluated as control.5 Expression of TREM-2 in inflammatory responses induced by lipoplysaccharide to alveolar macrophagesAlveolar macrophages were stimulated with the LPS at different concentration(10ng/ml,100ng/ml,1000 ng/ml)for for 0h,3h,6h,12h,24h in vitro.The expression levels of TREM-2 and protein were assayed by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level was evaluated by FACS analysis.Meanwhile,the proinflammatory factors TNF-a,IL-10 in the supernatants of AM were detected by ELISA.6 Effect of RNA interference on TLR4 signaling pathway in alveolar macrophage of mice induced by iipopolysaccharideLenti-EGFP-TREM-2-shRNA were transfected into alveolar macrophage of mice in vitrofor 72h,then the cells were stimulated with the LPS(100ng/ml)for 3h.At 72h post-transfection,the subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and protein expression level were evaluated by FACS analysis,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a,IL-10 in the supernatants of AM.Total RNA and protein of Con+LPS group(AM transfected with Lenti-EGFP),Neg +LPS group(AM transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group and LPS group.7 Experimental studies of Lenti-EGFP-TREM-2-shRNA transfection in vivo before LPS stimulation in miceAfter having been anaesthetized by intraperitoneal injection of 2%sodium pentobarbital (30mg/kg body weight),male C57BL/6 mice were instilled intratracheally via a 50μl of microliter syringes with Lenti-EGFP-TREM-2-shRNA(1×10~8TU/animal,50μl total volume)in vivo.Seven days after transfection,mice were anesthetized and challenged with intratracheal instillation of LPS(E.coli 055:B5;Sigma)(2.5 mg/kg dissolved in 50μl of sterile normal saline) intravenously intratracheally to copy ALI models.The animals were killed after 24h.Cut the pulmonary tissues into microscopic section and examined the section under light microscope after HE stain.Immunohistochemistric and immunofluorescence stain of TREM-2,TLR4和TREM-1.Total RNA and protein were extracted from pulmonary tissues.The subsequent changes in TREM-2mRNA,TLR4mRNA and TREM-1mRNA were detected by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)and protein expression level were evaluated by Western-Blot,respectively.ELISA was applied to examine the secretion of proinflammatory factors TNF-a and IL-10 in the bronchoalveolar lavage fluid (BALF).Total examination of Con+LPS group(mice transfected with Lenti-EGFP),Neg+ LPS group(mice transfected with negative shRNA Lentivirus),and non transfected group were also evaluated as normal group,ALI group and saline control group. Results1 Construction of eukaryotic expression vector for shRNA targeting TREM-2 geneRestriction enzyme digestion and sequencing analysis showed that eukaryotic expression vectors of shRNA targeting TREM-2 gene and negative shRNA had been constructed successfully.Sequencing results revealed that the sequences of the vectors were all right.2 Transfecting with pTREM-2-shRNA into alveolar macrophages in vitro to choose the right sequence to suppress TREM-2 expression2.1 The cultured AM cells were in good condition.2.2 Optimization of the transfection condition of pTREM-2-shRNA to mice alveolar macrophageAfter being transfected with different proportion of plasmids to TransFectin Lipid at different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the proportion of pTREM-2-shRNA to TransFectin Lipid reagent as 0.5μg:1μl at 48h.At this transfection condition,transfection efficiency was(60.75±4.16)%and cells viability was 90.5%.2.3 Study on the inhibitory action of pTREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage48h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA of pTREM-2-shRNA1 and pTREM-2-shRNA2 were significantly suppressed(P＜0.05);the interference efficiency ofpTREM-2-shRNA1 group(45.5%)was higher than that ofpTREM-2-shRNA1 group(33.1%)(P＜0.05);There were no significance difference about TREM-2mRNA expression level of pNeg and pCon group before and after transfection(P＞0.05).So the sequence of siRNAI(5'-GAT GCT GGA GAT CTC TGGG-3',19bp,573-591)was the right sequence to suppress TREM-2 expression.3 Construction of recombinant lentivirus vectors expressing shRNA targeting mice TREM-2 geneRecombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by sequencing analysis and PCR and EGFP expression.4 Inhibition of TREM-2mRNA expression with Lenti-EGFP-TREM-2-shRNA in vitro4.1 Optimization of the transfection condition of Lenti-EGFP-TREM-2-shRNA to AMAfter being transfected with complex of the Multiplicity of Infection(MOI),the optimal ratio of lenti to polybrene and different time,transfection efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the Multiplicity of Infection with twenty,and polybrene with 6μg/ml at 72h.At this transfection condition,transfection efficiency was(87.58±4.02)%and cells viability was 89.2%.So MOI=20 is the best MOI.4.2 Study on the inhibitory action of Lenti-EGFP-TREM-2-shRNA to TREM-2mRNA expression level in alveolar macrophage72h after transfection,total RNA were extracted,and TREM-2mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of TREM-2mRNA with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05), the mRNAexpression was 3.96E+04±6.50E+03,and interference efficiency was 91.43%; There were no significance difference about TREM-2mRNA expression level of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).TREM-2 protein was evaluated by FACS analysis,it showed:Compared with control groups the expression level of TREM-2 protein with Lenti-EGFP-TREM-2-shRNA was significantly suppressed(P＜0.05),protein expression was 13.84±2.87,and suppression efficiency was 90.79%;There were no significance difference about expression level TREM-2 protein of Lenti-Neg and Lenti-EGFP group before and after transfection(P＞0.05).5 TREM-2mRNA expression level of alveolar macrophage in response to LPSAfter stimulation of LPS(100ng/ml)cells were harvested either before or 3h,6h,12h or 24h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found:TREM-2mRNA and protein expression were significantly suppressed at 3h(mRNA 2.96E+05±2.72E+04,protein 106.12±9.47,P＜0.05),even to the lowest point at 6h(mRNA 5.20E+04±1.52E+04,protein 39.13±5.65,P＜0.05)compared with Oh group(mRNA 4.26E+05±6.13E+04,protein 147.36±6.05).There were no significance difference about their expression level of control groups before and after stimulation(P＞0.05).After priming with of LPS by different concentration(0ng/ml,10ng/ml,100ng/ml,1000 ng/ml)cells were harvested at 3h.With total RNA were extracted,TREM-2mRNA was evaluated by FQ-RT-PCR and TREM-2 protein was evaluated by FACS analysis.We found: TREM-2mRNA and protein expression were markedly down-regulated in 100ng/ml group(mRNA 2.26E+05±7.29E+03,protein 106.12±L6.45,P＜0.05),even to the lowest level in 1000 ng/ml group(mRNA 1.10E+05±5.61E+03,protein 75.62±7.32,P＜0.05)compared with normal group(mRNA 4.26E+05±6.33E+04,protein 147.36±7.43).There were no significance difference about their expression level of control group before and after stimulation(P＞0.05).The LPS(100ng/ml)also induced massive secretion of TNF-a and IL-10,which peaked at 3h(compared with Oh,P＜0.05),then reduced rapidly. 6 Lentivirus-mediated TREM-2-shRNA on mouse alveolar macrophages in LPS inflammatory response6.1 shRNA on TREM-2 expression of alveolar macrophageTREM-2mRNA was evaluated by FQ-RT-PCR.In the virus group TREM-2mRNA (2.69E+04±5.50E+03)was significantly lower than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05). TREM-2 protein(11.45±1.09)was consistent with TREM-2 mRNA to FACS analysis.6.2 shRNA on alveolar macrophage expression of TLR4In the virus group TLR4mRNA(4.26E+05±2.93E+04)was significantly higher than that in other groups(P＜0.05).While the control groups were not significantly different compared with the LPS group(P＞0.05).TLR4 protein(140.42±9.53)was consistent with TLR4 mRNA.6.3 shRNA on the alveolar macrophage TREM-1 expressionTREM-1mRNA(1.67E+05±9.22E+03)in virus group was significantly higher than that in the normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.6.4 shRNA on pro-inflammatory factor in alveolar macrophageWe examinated pro-inflammatory factors TNF-a and IL-10 levels in cell supernatants with ELISA.TNF-a(197.15±11.86)and IL-10(872.38±45.67)of virus interference group were significantly higher than those in other groups(P＜0.05,respectively);while the negative control group and blank control group had no significantly different with the LPS groups(P＞0.05).7 Influence of Lentivirus-mediated TREM-2-shRNA on inflammatory response of acute lung injury induced by LPS in mice7.1 Lung pathological changes by fight microscopeALI model mice can be seen clearly swollen lungs.The volume increased and weight of lung increased.Large surface area of lung bleeding was very fragile texture.Bronchoalveolar lavage fluid was like water washed meat.Lentiviral group was more significant heavier than the ALI group.The ALI group could be seen extensive hyperemia,edema,hemorrhage in lung tissue. Alveolar space was narrow and infiltrated by more macrophages.Virus group had been extensive lung lesions.The saline control group of mice lung tissue was basically normal morphology.The blank control group and negative control group changed as same as the ALI group.7.2 Immunohistochemistry and Immunofluorescence assay(to see photoes)7.3 shRNA on TREM-2 expression of pulmonary tissuesWith FQ-RT-PCR on TREM-2 mRNA,we found that:in virus group of TREM-2mRNA (2.66E+04±4.20E+03)was significantly lower than those in other groups(P＜0.05),the mRNAexpression was 2.66E+04±4.20E+03,and interference efficiency was 89.9%.TREM-2 mRNA in the negative control group and blank control group was no significantly different(P＞0.05).To western-blot detection(β-actin as internal control),TREM-2 protein was consistent decline with TREM-2 mRNA.Protein expression was 0.062±0.0152,and interference efficiency was 93.5%.7.4 shRNA on pulmonary tissues expression of TLR4TLR4mRNA in virus group(4.47E+05±1.17E+04)was significantly higher than that in other groups(P＜0.05).The negative control group and blank control group were not significantly different compared with the ALI group(P＞0.05).The protein expression of TLR4 increased with TLR4 mRNA expression in line.7.5 shRNA on TREM-1 expression in lungTREM-1 mRNA(2.84E+05±2.52E+04)in virus group was significantly higher than that in normal group(P＜0.05),but had no significant difference with other groups(P＞0.05).Protein expression was in line with the TREM-1 mRNA.7.6 shRNA on pro-inflammatory factor in BALFELISA assay in BALF showed:TNF-a(139.81±16.28)and IL-10(1281.71±54.75)were significantly higher than those in other group(P＜0.05),while the negative control group and blank control group in promoting content of inflammatory cytokines were not significantly different compared with the ALI group(P＞0.05).Conclusions1 Eukaryotic expression vector of pTREM-2shRNA was constructed successfully.2 TransFectin Lipid can transfect pTREM-2-shRNA into cultured AM efficiently via optimize transfection condition.The siRNA could effectively suppress the expression of TREM-2 mRNA after transfected for 48 hours especially shRNA-1.3 Recombinant lentivirus vectors Lenti-EGFP-TREM-2-shRNA were constructed successfully, which was confirmed by restriction enzyme digestion and PCR and EGFP expression.4 Transfection of Lenti-EGFP-TREM-2-shRNA into AM could inhibit the expression of TREM-2mRNA,subsequently inhibit the protein expression efficiently.5 Expression of TREM-2 can be down-regulated by LPS,which may contribute to the secretion of proinflammatory factors such as TNF-a and IL-10.LPS(100ng/ml)and time point at 3h were choosed to our experiment.6 TREM-2-shRNA transfection can selectively suppress TREM-2 expression,and increase in TLR4-mediated LPS signal transduction efficiency,promote the secretion of pro-inflammatory factors induced by LPS,then amplified the inflammatory response in vitro.It suggested that TREM-2 can inhibit the inflammatory response.7 With TREM-2-shRNA transfection in vivo,lentivirus vectors could be transfected intratracheally safe and effectively.Lenti-EGFP-TREM-2-shRNA could inhibit the expression of TREM-2 mRNA and protein in lung and also increase the expression of the down-stream cytokines like TNF-a and IL-10.Moreover,lung injury was aggravated in mice.This converse result confirms the criticality of TREM-2 in the inflammatory response,and allows us to direct potential therapies in RNAi technology on ALI.