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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: J Thorac Cardiovasc Surg. 2015 Oct 14;151(2):499–506. doi: 10.1016/j.jtcvs.2015.09.125

VALIDATING THE USE OF SIRNA AS A NOVEL TECHNIQUE FOR CELL SPECIFIC TARGET GENE KNOCKDOWN IN LUNG ISCHEMIA-REPERFUSION INJURY

Heather E Merry 1, Patrick Phelan 1, Billanna Hwang 1,2,*, Michael S Mulligan 1,2
PMCID: PMC4727997  NIHMSID: NIHMS745007  PMID: 26687890

Abstract

Objective

Short interfering RNA is an effective method for target gene knockdown. However, concerns surround their design, administration, efficacy, specificity and immunostimulatory potential. Though uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell specific target gene knockdown in our model of lung ischemia reperfusion injury.

Methods

Dose response experiments were performed and three distinct sequences of TLR4, TLR2, and MyD88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector and non-coding short interference RNA controls were utilized. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media was assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake.

Results

Target protein expression was significantly reduced using short interference RNA. However, TLR4 knockdown was isolated to alveolar macrophages and biotin labeling confirmed TLR4 short interference RNA localization to alveolar macrophages. There was significant knockdown of TLR4 expression in cultured cells treated with TLR4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence utilized.

Conclusions

Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of their role in the development of lung ischemia reperfusion injury.

Keywords: siRNA, Lung reperfusion injury, TLR4, TLR2

Introduction

RNA interference with short interfering RNA (siRNA) is powerful research tool for the selective, transient post-transcriptional knockdown of gene expression both in vivo and in vitro.1-3 siRNA consists of short, doubled-stranded RNA duplexes, 21-25 nucleotide base pairs in length, designed to specifically target and cleave the mRNA of the gene of interest, thereby transiently preventing protein expression of the target gene.4 Several distinct advantages with siRNA use include its efficacy and specificity, versatile array of prospective targets and broad therapeutic potential.2,4,5 While in vivo siRNA use has been studied in models targeting multiple organ systems, including the lung, it has yet to be comprehensively validated in a model of lung ischemia reperfusion injury (LIRI). Many in vitro studies have shown the diverse applicability and success of siRNA in multiple cell lines, whereas in vivo translation has been impaired in part by the lack of cell specific targeting, particularly endothelial and epithelial siRNA transfection.6-8 Having previously identified the central importance of the alveolar macrophage (AM) in LIRI,9 it would be highly desirable if we were effective in selectively transfecting the AM in vivo. Intravenously administered siRNA has been shown to localize to resident tissue inflammatory cells, including kupffer cells in the liver, peritoneal macrophages, and alveolar macrophages (AM) in the lung,7,10-12 Therefore, we were indeed optimistic that we would be successful in transfecting the AM in the lung in our model of LIRI.

LIRI remains a significant clinical problem following lung transplantation in up to 25% of recipients and increases the risks of acute and chronic rejection, bronchiolitis obliterans and has been found to adversely affect early post-transplant mortality.13-16 Understanding the cell-specific inflammatory signaling mechanisms relevant to LIRI may lead to the identification of strategies aimed at alleviating this form of lung injury. Our warm ischemia model of LIRI in the rat is not only relevant, but provides for the evaluation of LIRI outcomes with a predictable time of onset that may allow for practical pretreatment. Our lab and others have identified several site-specific mediators and characterized the central importance of early activation of the AM in the development of LIRI.9,17,18 In addition, toll-like receptor-4 (TLR4), toll-like receptor-2 (TLR2) and myeloid differentiation factor-88 (MyD88) have emerged as potential upstream regulators of the inflammatory response in LIRI.19,20 The predictable time of onset and transient nature of siRNA administration make it an attractive tool for in vivo examination of the mechanistic role these upstream regulators have in the development of LIRI.

Despite widespread adoption of RNA interference, there are several concerns related to its use. Some authors urge caution when interpreting results from experiments that have not addressed what is broadly referred to as off-target effects (OTE). 21-22 Specifically, OTE include silencing of non-target genes, activation of non-specific immune responses, notably the interferon (IFN) response, toxicity and saturation of a cells native mRNA processing thereby affecting its ability to function normally. 21-22 siRNA manufacturers have developed strategies to deal with some aspects of OTE, such as siRNA design and control siRNA, but they are independent of individual experimental conditions and model systems.

The purpose of this study was to develop a comprehensive approach to validate the novel use of siRNA in vivo in our well developed warm ischemia model of LIRI in the rat and in vitro in our cell culture based model of hypoxia and reoxygenation (HR). Our strategy includes consideration and evaluation of siRNA design, dose response, uptake or localization, as well as efficacy and specificity, particularly focusing on minimizing off-target effects such as the IFN response.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise specified.

siRNA Design and Transfection

The siRNAs used in this study were obtained from Invitrogen (Carlsbad, California). Three unique siRNA duplexes were designed to target TLR2, TLR4, and MyD88 mRNA. Sequences containing 5’-UGUGU-3’ or 5’-GUCCUUCAA-3’, which are known to activate the innate immune response (IFN production), were not incorporated.23 In addition, scrambled, non-coding (nonsense) siRNA that contains limited sequence homology to rat, mouse or human genome served as control siRNA. For in vivo transfection, siRNA was administered in two equally divided doses, 24 and 48 hours prior to undergoing the in situ ischemia-reperfusion protocol. 10nM of siRNA(based on dose response experiments, see Figure 3) was diluted in 250μl of sterile saline and combined with 100μl/kg of lipofectamine 2000 (Invitrogen) diluted in 250μl of sterile saline. After 15 minutes of incubation, the lipid associated siRNA was rapidly injected into the penile vein. For in vitro AM transfection, 100pmol of siRNA was diluted in 50μl of growth media per well and incubated for 15 minutes. This was then mixed with 3μl of lipofectamine diluted in 50μl of growth media. The cell culture media was then changed and supplemented with siRNA-lipid mixture. The cells incubated with the primer for at least 6 hours, after which time the media was changed. Type 2 pneumocytes (T2P) and pulmonary artery endothelial cells (PAEC) siRNA transfection was as described above for AM except 1μl of lipofectamine was used. Additional animals and cells were treated with biotin labeled TLR4 siRNA and assessed for localization and uptake after 2 and 6 hours, respectively.

Figure 3. siRNA Dose Response.

Figure 3

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TLR4 siRNA sequence-2 treatment in concentrations of 0, 1, 10, 20, and 50nM, reduced TLR4 expression by 8% (p>0.05), 42% (p>0.05), 80% (p<0.01), 81% (p<0.001), and 76% (p<0.01), respectively, compared to non-coding siRNA controls 48 hours after the initial intravenous TLR4 siRNA administration (n=3). siRNA = short interference Ribonucleic Acid, TLR = Toll-Like Receptor.

Ischemia-Reperfusion (IR) Protocol

Pathogen free adult male Long-Evans rats (Harlan Sprague Dawley, Indianapolis, Indiana), weighing 275-300 grams, were used for all in vivo experiments. Approval for all experimental protocols was granted by the University of Washington Animal Care Committee. Animals received humane care in compliance with the “Principles of Laboratory Animal Care” established by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” developed by the Institute of Laboratory Animal Resources and published by the national Institute of Health (NIH Publication No. 86-23, revised 1996).

A well established, in situ warm rat ischemia-reperfusion model was utilized with 90 minutes ischemia and 4 hours of reperfusion.9,18 At the end of reperfusion, a midline laparotomy and sternotomy were performed for blood sampling and excision of heart-lung block.

Cellular Harvest and Hypoxia-Reoxygenation (HR)

AM were isolated and cultured as we have described previously.24 Cell counts and viability were assessed by standard trypan blue exclusion methods. Cells were plated at a density of 1,000,000 cells/well and allowed to quiesce overnight. T2P were harvested as described previously by Dobbs.25 PAEC will be isolated, cultured, and purified as described previously by our group.17 HR was performed as previously described.24 In brief, once T2P or PAEC have reached confluence, or AM have quiesced for at least 6 hours, the cells were placed in a hypoxic chamber (FiO2 0.5%) for 90 minutes. The media equilibrates with the hypoxic environment in less than 5 minutes. Cells were then removed from the hypoxic chamber and placed in a normoxic (FiO2 21%) incubator for up to 4 hours. Negative controls remained in the normoxic incubator for up to 6 hours.

Experimental Groups

Non-coding siRNA control animals and cells received intravenous non-coding scrambled siRNA prior to undergoing IR or HR, respectively. Negative control animals and cells did not receive siRNA treatment or undergo IR or HR. Separate animal and cell treatment groups received either TLR2, TLR4, or MyD88 siRNA, lipid vector alone or normal saline alone prior to undergoing IR or HR, respectively.

Markers of Lung Injury

Left lung permeability index was determined as described previously.9,18 I125-radiolabeled bovine serum albumin (NEN Life Sciences, Boston, MA) was intravenously injected 5 minutes prior to removal of the hilar clamp and a scintillation counter used to quantitate radioactivity. Permeability index was calculated as follows: Permeability index = left lung (cpm)/1 mL blood (cpm). Myeloperoxidase (MPO) content was used to quantitate neutrophil accumulation in the lungs as described previously.9,18 Briefly, lung samples were homogenized, sonicated, and the supernatants recovered and the change in absorbance at 460 nm wavelength was recorded after mixing 50 μL of each sample with 1.45 ml of assay buffer. Left lung ronchoalveolar lavage (BAL) was performed after harvesting the heart lung block by placing a clamp across the right hilum and using the intratracheal angiocatheter to lavaged the lung with 3 ml of cold sterile saline as described previously.9,18 The recovered lavage fluid is centrifuged at 1,800 rpm for ten minutes at 4°C to pellet the cells. The supernatant was frozen for cytokine analysis and the pellet resuspended in 10 mL of sterile PBS. One milliliter of resuspended cells were stained with Gill's solution and counted using a hemacytometer (Hausser Scientific, Horsham, PA).

Protein analysis for Cytokine Content by ELISA

Media from cell cultures and serum from rats pretreated with siRNA was collected 1, 6, and 24 hours after siRNA administration and processed as described above (serum) or as previously published (media)3 and then analyzed for IFNγ and IFNβ content via sandwich ELISA.24 Similarly, media from cell cultures was collected after undergoing HR and analyzed for cytokine-induced neutrophil chemoattractant (CINC) content via ELISA as above. Samples and standards were run in triplicate.

Western Blot Analysis

Total protein was extracted from whole left lung homogenates and cultured cells after 15 minutes of reperfusion and western blot analysis performed as previously described using 40 μg of protein and TLR2, TLR4 or MyD88-specific antibody (Cell Signaling, Beverly, MA). Densitometry was performed to assess relative differences between groups using Image J (version 1.2; Cybernetics Corp, Silver Springs, Maryland).

Immunohistochemistry Protocol

Whole lung tissue specimens were fixed in 4% paraformaldehyde, processed, sectioned, and stained previously described.12,34,40 Stained sections were examined using Image J software (Version 1.2).

Statistical Analysis

All data is presented as mean values + the standard error of the mean. Comparisons among groups were made using one-way ANOVA. Bonferroni's method was used to adjust for multiple comparisons. A post-hoc two-tailed Student's t-test was performed to assess statistical differences between individual groups, which was defined for all tests as a p< 0.05.

RESULTS

We developed a specific, comprehensive approach using siRNA in vivo and in vitro to characterize signaling pathways and cellular responses in LIRI. Two hours after treatment in the in vivo in situ model of lung ischemia-reperfusion, appreciable uptake of the TLR4 siRNA was only present in alveolar macrophages. There was no appreciable uptake in other lung cell types including rat pulmonary endothelial cells and type 2 pneumocytes (Figure 1A). However, in the primary cell culture model uptake of the siRNA was achieved in all 3 cell lines (AM, PAEC and T2P) within 6 hours of treatment (Figure 1A). As expected given the differential uptake of siRNA in the in vivo model, TLR4 protein expression following TLR4 siRNA sequence-2 treatment in vivo was reduced only in AM, not PAEC or T2P. Non-coding siRNA treated animals were used as controls and protein expression was assessed by relative optical densitometry (Figure 1B).

Figure 1.

Figure 1

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A. Localization and Uptake of TLR4 siRNA. Immunohistochemistry on lung sections following in vivo biotin labeled TLR4 siRNA administration demonstrated siRNA localization only to the AM 2 hours after treatment (n=4). Immunohistochemical analysis of alveolar macrophages, pulmonary artery endothelial cells and type 2 pneumocytes treated with biotin labeled TLR4 siRNA demonstrated uptake of the biotin labeled TLR4 siRNA by all three cell types within 6 hours of treatment (n=12 wells). AM = alveolar macrophage, PAEC = pulmonary artery endothelial cell, siRNA = short interference Ribonucleic Acid, T2P = type 2 pneumocyte, TLR = Toll-Like Receptor. Figure 1B. Reduction in TLR4 Expression with TLR4 siRNA. TLR4 expression following TLR4 siRNA sequence-2 treatment in vivo was reduced only in alveolar macrophages (by 92%, p <0.001), not pulmonary artery endothelial cells (19% reduction, p>0.05) or type 2 pneumocytes (6% increase, p>0.05) compared to non-coding siRNA controls as assessed by relative optical densitometry (n=4). However, there was 80% (p<0.01), 85% (p<0.001) and 82% (p<0.01) knockdown of TLR4 expression with TLR4 siRNA sequence-2 treatment of cultured alveolar macrophages, pulmonary artery endothelial cells and type 2 pneumocytes, respectively compared to non-coding controls siRNA (n=12 wells per group). AM = alveolar macrophage, PAEC = pulmonary artery endothelial cell, siRNA = short interference Ribonucleic Acid, T2P = type 2 pneumocyte, TLR = Toll-Like Receptor.

In the cultured cell lines undergoing in vitro hypoxia and re-oxygenation, there was consistent 80%, 85% and 82% knockdown of TLR4 expression with TLR4 siRNA treatment of cultured AM, PAEC and T2P, respectively compared to non-coding siRNA controls (Figure 1B). Sequence 2 of the TLR-4 siRNA was used in the experiments since it had the highest affinity and level of knockdown. Rats pretreated with different TLR4 siRNA sequences (sequences 1, 2, and 3) prior to undergoing IR demonstrated a 70%-92% reduction in TLR4 expression compared to non-coding siRNA controls as assessed by relative optical densitometry (Figure 2). Primary AM cultures treated with MyD88 or TLR2 siRNA sequences 1, 2, and 3 prior to undergoing HR demonstrated 63-83% and 78-87% reductions, respectively, in MyD88 and TLR2 expression, respectively, compared to non-coding siRNA controls (Figure 2). TLR4 sequence-2, TLR2 sequence-1 and MyD88 sequence-3 were deemed most efficacious at achieving knockdown and used for subsequent experiments. Ten nanomolar TLR4 siRNA sequence-2 treatment was the lowest dose with a strong knockdown efficacy (80%) and was used for all subsequent in vivo studies (Figure 3). In vitro, we performed dose response experiments with TLR4 concentrations of 10, 50, 100, 500 and 1000pM and observed similar knockdown efficacy at TLR4 siRNA concentrations of 100pM (data not shown).

Figure 2. Sequence Specific Knockdown Efficacy.

Figure 2

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In rats pretreated with TLR4 siRNA, sequences 1, 2, and 3, prior to undergoing ischemia and reperfusion, there was a 70% (p<0.01), 92% (p<0.001), and 89% (p<0.001) reduction, respectively, in TLR4 protein expression compared to non-coding siRNA controls as assessed by relative optical densitometry (n=3). Primary alveolar macrophage cultures treated with MyD88 siRNA sequences 1, 2, and 3 prior to undergoing hypoxia and reoxygenation demonstrated 76% (p<0.001), 63% (p<0.01), and 83% (p<0.001) reductions, respectively, in MyD88 expression compared to non-coding siRNA controls (n=12 wells). Primary alveolar macrophage cultures treated with TLR2 siRNA sequences 1, 2, and 3 prior to undergoing hypoxia and reoxygenation demonstrated 87% (p<0.001), 78% (p<0.001), and 84% (p<0.001) reductions, respectively, in TLR2 expression compared to non-coding siRNA controls (n=12 wells). MyD88 = Myeloid Differentiation Factor 88, siRNA = short interference Ribonucleic Acid, TLR = Toll-Like Receptor.

All three markers of lung injury were significantly increased in non-coding siRNA controls after undergoing IR compared to negative controls (Figure 4). However, with TLR4 siRNA treatment prior to undergoing IR, all three markers of lung injury were significantly reduced, compared to non-coding siRNA controls (Figure 4). CINC production was significantly increased in primary cell cultures treated with non-coding siRNA controls prior to undergoing HR production compared to negative controls (Figure 5). However, CINC production was only reduced in AM, not PAEC or T2P, treated with TLR4 siRNA prior to undergoing HR compared to non-coding siRNA controls (Figure 5). TLR2 expression was present and unaffected in AM, PAEC and T2P eluted from rats treated TLR4 siRNA compared to non-coding siRNA controls as assessed by relative optical densitometry (Figure 6). There was no significant difference in IFNγ or IFNβ production among rats or AM treated with TLR4, MyD88 or TLR2 siRNA in cationic lipid vector, lipid vector alone, or normal saline alone at 1, 6, or 24 hours after treatment (Figure 7, 6 hour data shown).

Figure 4. Reduced Lung Injury with TLR4 Knockdown.

Figure 4

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Vascular permeability, myeloperoxidase content and BAL leaukocyte count were increased by 11-fold, 42-fold, and 28-fold in non-coding siRNA controls after undergoing ischemia and reperfusion compared to negative controls which did not undergo ischemia and reperfusion (n=3). However, with TLR4 siRNA treatment prior to undergoing ischemia reperfusion, vascular permeability, myeloperoxidase content and BAL leaukocyte count were all reduced by 82% (p<0.001), 73% (p<0.01) and 96% (p<0.001), respectively, compared to non-coding siRNA controls. BAL = bronchoalveolar lavage, MPO = myeloperoxidase, siRNA = short interference Ribonucleic Acid, TLR = Toll-Like Receptor.

Figure 5. CINC Production Following TLR4 Knockdown in Cultured AM, PAEC and T2P.

Figure 5

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In cultured alveolar macrophages, pulmonary artery endothelial cells, and type 2 pneumocytes treated with non-coding siRNA controls prior to undergoing hypoxia and reperfusion, there was a 7-fold, 12-fold, and 9-fold increase in CINC production compared to negative controls which did not undergo hypoxia and reoxygenation. CINC production was reduced by 98% in alveolar macrophages treated with TLR4 siRNA prior to undergoing hypoxia and reperfusion compared to non-coding siRNA controls (n=12 wells per group). However, CINC production was not significantly changed in pulmonary artery endothelial cells (4% increase, p>0.05) or type 2 pneumocytes (9% reduction, p>0.05) treated with TLR4 siRNA prior to undergoing hypoxia and reperfusion compared to non-coding siRNA controls (n=12 wells per group). AM = alveolar macrophage, PAEC = pulmonary artery endothelial cell, siRNA = short interference Ribonucleic Acid, T2P = type 2 pneumocyte, TLR = Toll-Like Receptor.

Figure 6. Specificity of siRNA Knockdown.

Figure 6

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In alveolar macrophages, pulmonary artery endothelial cells, and type 2 pneumocytes eluted from rats treated with TLR4 siRNA, TLR2 expression was present and unaffected (7% reduction, p>0.05; 4% increase, p>0.05; and 7% increase, p>0.05; respectively), 48 hours after the initial siRNA dose, compared to non-coding siRNA controls as assessed by relative optical densitometry (n=3). AM = alveolar macrophage, PAEC = pulmonary artery endothelial cell, siRNA = short interference Ribonucleic Acid, T2P = type 2 pneumocyte, TLR = Toll-Like Receptor.

Figure 7. Interferon Production after siRNA Administration.

Figure 7

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There was no significant difference (p>0.05) in interferon-γ or interferon-β production among rats treated with TLR4, MyD88 or TLR2 siRNA in cationic lipid vector, lipid vector alone, or normal saline alone at 1, 6, or 24 hours after treatment (n=3, 6 hour data shown). Additionally, there was no significant difference (p>0.05) in interferon-γ or interferon-β production by alveolar macrophages treated with TLR4, MyD88 or TLR2 siRNA in cationic lipid vector, lipid vector alone, or normal saline alone at 1, 6, or 24 hours after treatment (n=12 wells per group, 6 hour data shown). IFN = interferon, MyD88 = Myeloid Differentiation Factor 88, siRNA = short interference Ribonucleic Acid, TLR = Toll-Like Receptor.

DISCUSSION

This study establishes the cell specific uptake of intravenously administered siRNA in the AM in the lung as well as our ability to transfect primary cell cultures of AM, PAEC, and T2P and validates this novel approach for target gene knockdown in our model of LIRI. Additionally, we have demonstrated reduced expression of the target gene using multiple unique siRNA sequences both in vivo and in vitro, as well as the specificity of the siRNA to reduce expression of only the target gene. Furthermore, we demonstrated that knockdown of TLR4 was associated with reduction in multiple markers of lung injury in vivo as well as decreased CINC production by AM. Dose response experiments identified the lowest most effective dose of siRNA in order to minimize OTE. Finally, we demonstrated that siRNA administration was not associated with IFN production in vivo or in vitro. Taken together, these results significantly increase the confidence with which the observed phenotype can be ascribed to knockdown of the target protein, and provide a powerful tool for studying the central role of the AM in the development of LIRI.

The validation of siRNA use in our model includes not only our results, but also experimental design and methodological considerations. While the exact chemical modifications to Stealth siRNA (Invitrogen) are proprietary, the specific siRNA molecules chosen avoid any known stretches of RNA that can induce an IFN response23, contain no sequence homology to each other when multiple siRNAs were used against the same target, and include chemical modifications ensuring that only the antisense strand of the siRNA duplex is processed by the endogenous RNAi machinery. Not only did we demonstrate that the target protein was reduced in vivo and in vitro, a critical component of siRNA validation, but we did so with multiple non-overlapping sequences and also demonstrated that TLR2 was not affected by TLR4 siRNA, thereby confirming specificity and addressing a concern that siRNA can affect expression of non-target TLR.26 Determining the lowest effective dose of siRNA was a significant component of these experiments, considering siRNA concentrations in excess of 100nM frequently produce non-specific OTE, whereas concentrations <20nM, and especially <10nM, rarely produce OTE.26,27 Induction of the IFN response can result from siRNA itself or even its vector, therefore regardless of any dose, design or manufacturer modifications, measurement of in vivo and in vitro IFN production is essential, and notably was not increased in our experiments. These steps, the appropriate design of siRNA, experimental design considerations and the confirmation of specificity, localization and observed phenotype, all contribute to the overall validation of siRNA use in our model of LIRL.

Intravenous siRNA administration has been repeatedly shown to transfect resident tissue inflammatory cells, particularly mononuclear cells, including the alveolar macrophage7,10-12, whereas studies employing intratracheal or intranasal siRNA administration have demonstrated inconsistent, erratic or non-specific cellular uptake of siRNA.28,29 Having previously identified the AM as the key coordinating cell type early in LIRI9, the intravenous approach for siRNA administration provided a likely, and subsequently correct, route for targeted, cell specific knockdown in the AM in vivo. Emerging methods for targeted intratracheal siRNA administration to the AM are under development30, yet should these potential approaches be successful, the validation strategy created for our study would be applicable, relevant and translatable.

Investigating LIRI requires not only pertinent and appropriate animal models, but cell culture models as well in order to discretely map critical cell signaling events that would otherwise be cumbersome or impossible to do in vivo. Acknowledging that in vitro studies fall short of reconstituting complex in vivo interactions, they serve as an important complement to the animal studies in order to characterize critical signaling events. Our warm hilar occlusion model is specific and reliable for the study of mechanisms of injury related to warm atelectatic ischemia and reperfusion. Our model is not only relevant, but addresses an important clinical problem that has a very predictable time of onset. This is in strong contrast to most types of acute inflammatory lung injury and allows for practical pretreatment and intervention. Difficulty with target cell transfection has been one of the primary deterrents to the broader in vivo application of siRNA to other model systems, including myocardial and cerebral systems.6-8 The overall validation strategy employed in this study nonetheless confirms siRNA technology to be particularly well suited for application in LIRI.

The immune compromised lung transplant recipient faces numerous challenges following implantation, LIRI being one of the earliest and most significant, with the potential to affect both acute and chronic outcomes. LIRI has a predictable onset, and increasingly, an identifiable set of molecular signaling events that have recognized activation sequences. The transient nature of siRNA mediated target mRNA degradation is ideally positioned for application in the setting of LIRI, with the ability to knockdown the target gene of interest during the most injurious phase of LIRI, only to have that protein expression reconstitute to participate in normal innate immune resistance to infection and rejection. The simplicity of siRNA belies the complicated validation tactics developed and employed in our model, necessary when using this technology, but the advantages and potential translatability of siRNA in the setting of LIRI justifies its ongoing examination and advancement.

Acknowledgments

Grant Support: The authors are grateful for the research funding from the National Institutes of Health, Bethesda, MD grant R01HL093097. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health nor its subsidiary Institutes and Centers.

Sources of Funding: NIH RO1-Role of TLR-4 in Lung Reperfusion Injury

Abbreviations and Acronyms

T2P

type 2 pneumocytes

PAEC

pulmonary artery endothelial cells

siRNA

short interference RNA

TLR2

toll-like receptor 2

TL4

toll-like receptor 4

MyD88

myeloid differentiation factor-88

OTE

off-target effects

LIRI

lung ischemia reperfusion injury

HR

hypoxia and reoxygenation

AM

alveolar macrophages

Footnotes

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AUTHORSHIP:
  • Patrick Phelan drafted and revised the manuscript, co-supervised the study, analyzed and interpreted the data. No conflicts of interest.
  • Heather E Merry revised the manuscript, executed the studies, analyzed and interpreted the data. No conflicts of interest.
  • Billanna Hwang revised the manuscript. No conflicts of interest.
  • Michael S Mulligan designed and supervised the study, and revised the manuscript. No conflicts of interest.

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