BLOOD COMPONENTS Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro Jennifer A. Muszynski,1,2,4 Justin Bale,2 Jyotsna Nateri,2 Kathleen Nicol,3 Yijie Wang,4 Valerie Wright,4 Clay B. Marsh,4 Mikhail A. Gavrilin,4 Anasuya Sarkar,4 Mark D. Wewers,4* and Mark W. Hall1,2,4*
BACKGROUND: We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. STUDY DESIGN AND METHODS: To determine the role of stored RBC unit–derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). RESULTS: Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPSinduced tumor necrosis factor-a production, 17,611 6 3,426 vs. 37,486 6 5,598 pg/mL; p 5 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant– induced monocyte suppression. CONCLUSION: Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.
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ed blood cell (RBC) transfusion is independently associated with risk of new infection in critically ill patients.1-10 Our prior ex vivo and in vitro work strongly suggests that stored RBCs progressively suppress monocyte function with increasing storage duration.11-13 However, mechanisms of RBC-induced monocyte suppression are poorly understood. Understanding these mechanisms is important because we and others have shown that monocyte suppression in critically ill patients is strongly associated with increased risks for the development of new nosocomial infection and death.13-19 An important measure of monocyte immune function is the ability of monocytes to produce the proinflammatory cytokine tumor necrosis factor (TNF)-a in response to stimulation with lipopolysaccharide (LPS). Clinically, low
ABBREVIATIONS: LMV(s) 5 large (larger) microvesicle(s); LPS 5 lipopolysaccharide; miRNA 5 microRNA; MV(s) 5 microvesicle(s); RCN 5 relative copy number; SMV(s) 5 small (smaller) microvesicle(s). From the 1Division of Critical Care Medicine; 2The Research Institute; 3Department of Pathology, Nationwide Children’s Hospital; and the 4Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio Address reprint requests to: Jennifer A. Muszynski, MD, Critical Care Medicine, Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205; e-mail: [email protected]. This work was supported by K12HD043372 from the National Institute of Child Health and Development (JAM) and The Research Institute at Nationwide Children’s Hospital. *MDW and MWH contributed equally. Received for publication December 16, 2014; revision received February 12, 2015; and accepted February 13, 2015. doi:10.1111/trf.13084 C 2015 AABB V
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LPS-induced TNF-a production capacity is associated with increased risks for nosocomial infection and mortality in critically ill patients across diverse diagnoses of trauma, sepsis, critical viral infections, multiple organ dysfunction syndrome, and status post–cardiopulmonary bypass.13-17,20 In our previous in vitro work, aliquots from prestorage leukoreduced RBC units directly suppress monocyte LPSinduced TNF-a production capacity as a function of RBC storage duration.11 This suppression occurs via a soluble mediator(s), with effects occurring at the mRNA level. The identity of the soluble mediator(s) remains unknown, although previous reports have implicated RBC-derived microvesicles (MVs). MVs are small phospholipid membrane-bound vesicles (40-1000 nm) shed from a variety of cell types that may play important roles in cell-tocell communication.21 MVs accumulate over time during RBC storage,22-24 but their effects on monocyte function are unclear, with some investigators reporting monocyte activation while others report monocyte suppression.25,26 We therefore designed a series of in vitro experiments to test the hypothesis that older stored RBC products induce monocyte suppression via RBC-derived MVs.
MATERIALS AND METHODS Study subjects This study was approved by the institutional review board at Nationwide Children’s Hospital. Ten healthy adult volunteers were enrolled after written informed consent. Subjects were excluded if they had a history of cancer or posttransplant status or were receiving immunosuppressive or anti-inflammatory medication (including the use of aspirin or nonsteroidal anti-inflammatory medication within the past 48 hr). Up to 120 mL of blood was drawn in EDTA tubes (Becton Dickinson, Franklin Lakes, NJ) for monocyte isolation. Monocytes were isolated within 30 minutes after blood draws and were used immediately in coculture models. Monocytes were isolated as previously described.11 Briefly, whole blood was diluted 1:1 with phosphate-buffered saline (PBS), and peripheral blood mononuclear cells (PBMNCs) were then collected by density gradient centrifugation using lymphocyte separation medium (Mediatech, Manassas, VA). Monocytes were further purified by positive selection using CD14 magnetic beads (Miltenyi Biotec, Auburn, CA) and were resuspended in complete medium (RPMI 1 10% fetal bovine serum 1 1% penicillin/streptomycin). Percent purity using this method is at least 98% as previously reported.27
RBC products and MV isolation Eleven RBC units were obtained from the blood bank at Nationwide Children’s Hospital. All RBC components were prestorage leukoreduced using RBC filters (Flex Excel, 1938 TRANSFUSION Volume 55, August 2015
Fenwal, Lake Zurich, IL) and stored in Adsol-containing additive solution (AS-1, n 5 10; or AS-3, n 5 1). Units were stored at 4 C according to standard blood banking practice. Units were used in coculture experiments when they had been stored for 7 6 1 days or when they had been stored for 30 6 3 days. These storage durations were chosen to reflect the usual range of storage durations for transfused RBCs at our institution. For each experiment, aliquots from RBC units were obtained using a sterile connection device and blood administration syringe sets (CODAN, Santa Ana, CA). To obtain raw RBC supernatant, aliquots were centrifuged at 800 3 g for 10 minutes at 4 C. The supernatant was taken and centrifuged again at 800 3 g for 10 minutes to remove any remaining cells. This centrifugation speed was determined by preliminary experiments to yield the highest number of MVs with the fewest remaining contaminating cells (including platelets [PLTs]). MVs were then isolated from the raw supernatants by one of two different methods to yield either larger MVs (LMVs) or smaller MVs (SMVs). LMVs were isolated from 1 mL of raw RBC supernatant by centrifugation at 16,000 3 g at 4 C for 20 minutes and were resuspended in 200 mL of complete medium (for coculture experiments), PBS (for electron microscopy), or annexin V binding buffer (for flow cytometry). Supernatants remaining after centrifugation were retained and labeled LMV–depleted supernatants. SMVs were isolated from separate 1-mL aliquots of raw RBC supernatant by ultracentrifugation at 100,000 3 g at 4 C for 60 minutes and were resuspended in 200 mL of complete medium, PBS, or annexin V binding buffer, as described. Supernatants remaining after centrifugation were retained and labeled SMV-depleted supernatants. Isolated MVs were visualized by transmission electron microscopy as follows. After isolation by centrifugation, the LMV or SMV pellet was resuspended in PBS and loaded onto Formvar-carbon–coated copper grids (Electron Microscopy Sciences, Hattfield, PA). Samples were stained with 2% uranyl acetate for 10 minutes, washed, and then dried overnight at room temperature. Images were obtained using a transmission electron microscope (Model H-7650, Hitachi, Harrodsburg, KY). Flow cytometry was used to characterize MV cells of origin. Immediately after isolation, LMV and SMV pellets were suspended in 200 mL of annexin V binding buffer (eBioscience, San Diego, CA). MVs were identified by side scatter properties and surface expression of phosphatidylserine using PercP-eFluor 710 annexin V (eBioscience). To determine cell(s) of origin, MV samples were stained in two panels using the following antibodies: Panel 1 (fluorescein isothiocyanate [FITC]–anti-CD235a [RBCs; Becton Dickinson], eFluor 450 anti-CD41 [PLTs; eBioscience], allophycocyanin [APC]–anti-CD19 [B cells; Becton Dickinson]) and Panel 2 (FITC–anti-CD14 [monocytes], V450– anti-CD3 [T cells], APC–anti-CD142 [endothelial cells;
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Becton Dickinson]). COMPtrol antibody capture beads (Spherotech, Lake Forest, IL) or single-stained samples were used to set compensation where appropriate. Positive and negative gates were set using a full-minus-one strategy with appropriate isotype controls. To quantify MV content in MV fractions versus MV-depleted supernatants, absolute quantity of MVs was determined using absolute counting beads (CountBright, Life Technologies, Grand Island, NY). Absolute counting beads were added to samples just before acquisition. Because of poor resolution for smaller particles with conventional flow cytometry, MV quantification was performed for LMV fractions versus LMV-depleted supernatants.28 Flow cytometry data were acquired using a cytometer (LSRII; Becton Dickinson) and were analyzed in computer software (FlowJo, TreeStar, Ashland, OR).
Coculture experiments We used our previously published monocyte coculture model to investigate the role of RBC unit–derived MVs in modulating monocyte function.11 Briefly, this model involves the coculture of healthy adult monocytes in 12well plates using a ratio of culture medium-to-blood product that approximates the volume ratio associated with a 20 mL/kg RBC transfusion. Healthy monocytes are cocultured with the blood product of interest followed by stimulation with LPS, with high levels of LPS-induced TNF-a production being typical for healthy cells and reduced levels being consistent with immunosuppression. This model has been adapted for our MV experiments as follows. For MV experiments, 1 3 106 monocytes in 800 mL of complete medium were treated with 200 mL of raw RBC supernatant, 200 mL of LMVs in complete medium, 200 mL of SMVs in complete medium, 200 mL of LMV-depleted supernatant, 200 mL of SMV-depleted supernatant, or 200 mL of complete medium (control) at 37 C for 4 hours in 12-well plates. After 4 hours, monocytes were stimulated with 1 ng/mL LPS (phenol-extracted from Salmonella abortus equii [Sigma, St Louis, MO]) and incubated for an additional 4 hours at 37 C. Unstimulated control wells were incubated under the same conditions for the same durations, but without the addition of LPS. Supernatants from LPS-stimulated and unstimulated monocytes were collected by centrifugation and stored at 280 C for batch analysis of TNF-a and interleukin (IL)-10 protein production. MV coculture experiments were performed in four replicates with a different monocyte donor and different RBC units for each replicate. For each experiment, RBC supernatant and MV fractions were obtained from RBC units that had been stored for 7 6 1 or 30 6 3 days. For heat inactivation experiments, RBC supernatants were heated to 100 C for 5 minutes and then allowed to cool to room temperature before use in coculture. For RNase-treated supernatants, 0.5 mg of RNase A (Life
Technologies) was added to RBC supernatant or to heatinactivated RBC supernatant just before coculture experiments. Heat inactivation experiments were performed six times with a different monocyte donor and RBC units per replicate. Cytokine production from coculture experiments (TNF-a, IL-10) as well as cytokine quantities in stored RBC products (TNF-a, IL-1b, IL-8, and IL-10) were quantified by chemiluminescence using an automated chemiluminometer (Immulite 1000, Siemens Healthcare Diagnostics, Deerfield, IL), with a lower limit of detection of 5 pg/mL for all cytokines. Manufacturer-provided standards and controls were used to ensure proper instrument calibration before each run.
RNA isolation and microRNA quantification To evaluate microRNA (miRNA) as a potential mediator of monocyte suppression in RBC units, miRNAs let-7e, mir16, and mir-195 were quantified in raw and treated RBC supernatants by real-time polymerase chain reaction (PCR) as follows. These miRNA were chosen from an exploratory analysis in which we profiled miRNA in fresher versus older RBC supernatants by real-time PCR and found increased relative expression among these immunomodulatory miRNA in older versus fresher RBC supernatants (data not shown). Aliquots of raw RBC supernatants used in coculture experiments were taken and kept frozen at 280 C before subsequent analysis. Just before RNA isolation, RBC supernatants were treated with heat, RNase, or heat plus RNase as described. RNA was isolated using the miRNeasy kit for serum and plasma (Qiagen, Valencia, CA) per the manufacturer’s instructions. Briefly, 1 mL of lysis reagent (QIAzol, Qiagen) was added to 200 mL of samples and incubated at room temperature for 5 minutes. Twenty-five fmol of synthetic Caenorhabditis elegans miRNA mixture (Syn-cel-miR-39, Syn-cel-miR-54; Qiagen) was added to each sample to serve as exogenous controls. Then 200 mL of chloroform was added to each sample and the aqueous phases containing RNA were purified. RNA (10 ng) was converted to cDNA using reverse transcription master mix with individual PCR primers (TaqMan, Life Technologies). Quantitative real-time PCR was performed in duplicate using TaqMan master mix (Life Technologies) using a real-time PCR system (Applied Biosystems Step One Plus, Life Technologies). The average cycle threshold (Ct) value for exogenous controls was used for normalization. Target miRNA expression is expressed as relative copy number (RCN) using the value 2(2DCt).
Statistical analysis Data presented are mean 6 standard error (SE). Differences between groups were analyzed using t test or paired t test, where appropriate. Analyses were performed using computer software (Prism6, GraphPad, Inc., La Jolla, CA). Volume 55, August 2015 TRANSFUSION 1939
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Fig. 1. Representative electron micrographs of isolated MVs and MV cells of origin by flow cytometry. Isolated MV fractions contained MVs of varying sizes for both LMVs (A; 125-1464 nm) and SMVs (B; 45-337 nm). Images are representative of two independent experiments. Images were obtained using a transmission electron microscope (Model H-7650, Hitachi) at a 15,0003 magnification. By flow cytometry, RBCs and PLTs were the most frequent MV cell(s) of origin for both SMVs (C) and LMVs (D) with very little expression of evaluated white blood cell or endothelial cell markers. Overall, there were no significant differences in MV cells of origin between LMVs and SMVs or among MV fractions from RBC units of varying storage durations ( , 7 days; w, 30 days). Data represent percent positive among isolated MVs and are expressed as mean (SE) from three individual experiments.
A p value of less than 0.05 was considered to be significant throughout.
RESULTS Isolated MVs from stored RBC units were visualized by transmission electron microscopy. LMVs isolated by 16,000 3 g centrifugation were variably sized ranging between 125 and 1464 nm, with a mean size of 479 6 101 nm (Fig. 1A). SMVs isolated by 100,000 3 g centrifugation were likewise variably sized ranging between 45 and 337 nm, with a mean size of 125 6 20 nm (Fig. 1B). For both LMVs and SMVs, the most frequent MV cells of origin were RBCs (Figs. 1C and 1D). Overall, there were no significant differences in MV cells of origin between LMV and SMV or among MV fractions over time, except for a trend toward increases in percent of PLT-derived MVs 1940 TRANSFUSION Volume 55, August 2015
from older RBC units. As expected, MV-depleted supernatants contained significantly fewer MVs compared to isolated MV fractions (17 6 6.5 MVs/mL vs. 10,325 6 1,548 MV/mL; n 5 3, p 5 0.02). Consistent with our previous study, raw supernatants from prestorage leukoreduced RBC units stored for 30 6 2 days suppressed monocyte function as evidenced by reduced LPS-induced TNF-a production capacity. Unexpectedly, MV fractions isolated from both fresher and older RBC units failed to induce monocyte suppression (Fig. 2). This was true for both LMVs and SMVs. Importantly, older RBC supernatants that had been depleted of MVs remained immunosuppressive to LPS-stimulated monocytes, suggesting a soluble mediator(s) outside of isolated MV fractions (Figs. 2A and 2B). The ability of monocytes to produce the anti-inflammatory cytokine IL10 in response to LPS stimulation was preserved in all
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Fig. 2. Supernatants from older stored RBCs suppressed monocyte function whereas MVs isolated from RBC supernatants did not. Healthy adult monocytes were cocultured with supernatants from prestorage leukoreduced RBC units that had been stored for 7 6 1 or 30 6 3 days. Monocyte cytokine production was measured after LPS stimulation (A-D) or without LPS (E, F). LPS-induced TNF-a production was impaired after exposure to raw supernatants from older RBC units (A, B). MVs isolated by 16,000 3 g centrifugation (LMV) did not induce monocyte suppression, but RBC supernatant that had been depleted of MV did (A). MVs isolated from 100,000 3 g centrifugation (SMVs) also did not induce monocyte suppression while SMV-depleted supernatants remained immunosuppressive (B). LPS-induced IL-10 production was preserved in all groups without significant differences compared to control monocytes (C, D). In the absence of LPS stimulation, TNF-a production was low in all groups (E, F; note the difference in y-axis scale). Data are mean (SE). n 5 4 experiments. *p < 0.05 compared to control. (A-F) w, raw RBC supernatant;
, LMV-depleted RBC supernatant;
LMV (A, C, E); w, raw RBC supernatant; (B, D, F)
, SMV (B, D, F).
, SMV-depleted RBC supernatant;
experimental groups, with no differences seen compared to control monocytes (Figs. 2C and 2D). In the absence of LPS stimulation, TNF-a levels in the coculture experiments were low in all groups, with no differences seen between experimental groups and controls (Figs. 2E and 2F), suggesting the absence of contaminating stimulants.
,
Levels of the cytokines IL-1b, IL-8, and IL-10 were not detectable in the supernatants of older or fresher RBC units. Supernatants from older RBC units also did not demonstrate elevated TNF-a concentrations, but rather had decreased amounts of TNF-a relative to fresher units (15 6 4 pg/mL vs. 113 6 43 pg/mL, respectively; p 5 0.01). Volume 55, August 2015 TRANSFUSION 1941
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As MVs did not appear to be the soluble mediators of the observed reduction in LPS-induced TNF-a production, we next performed a series of experiments designed to determine whether the responsible mediator(s) may be soluble RNA alone, protein, or protein-bound RNA (Fig. 3A). When monocytes were cocultured with older RBC supernatants, treatment of those supernatants with RNase alone to remove soluble RNAs did not remove their immunosuppressive effects. Likewise, heat inactivation (to degrade protein) only partially prevented the immunosuppressive effects of older RBC supernatants. However, treating RBC supernatants with heat inactivation followed by RNase (to degrade protein-bound RNA) prevented monocyte suppression. We hypothesize that the responsible protein-bound RNA may be miRNA. To explore whether heat and RNase treatment of RBC supernatants results in decreased miRNA levels, we quantified select miRNA (let 7-e, miR16, miR-195) in treated compared to untreated RBC supernatants by real-time PCR. As expected, the heat and RNase–treated supernatants had significantly decreased levels of let-7e (RCN, 2.6 vs, 15.6; p 5 0.04) and miR-195 (RCN, 0.46 vs. 14.1; p 5 0.04) with a trend toward decrease in miR-16 (RCN, 47 vs. 780; p 5 0.07; Fig. 3B).
DISCUSSION With a marker of innate immune function that has been repeatedly observed to be clinically relevant in critically ill patients, supernatants from older units of prestorage leukoreduced RBC suppressed monocyte function in vitro while MV fractions isolated from the same units did not. Protein-bound RNA are implicated as potential soluble mediators of monocyte suppression. These may represent miRNA. Transfusion-related immunomodulation has long been recognized but remains poorly understood. Some of the well-described but relatively rare complications of RBC transfusion (such as transfusion-related acute lung injury and nonhemolytic transfusion reactions) likely arise from proinflammatory mediators. However, a growing body of literature strongly supports the notion that on balance, RBC transfusion may be immunosuppressive. As early as the 1970s investigators noted that in transplant recipients, allogeneic blood transfusion was associated with lower rates of organ rejection—suggesting that RBC transfusion may be immunosuppressive.29,30 Since then, a number of studies have demonstrated independent associations between RBC transfusion and increased risks of nosocomial infection and cancer recurrence, consistent with overall immunosuppressive effects of RBC transfusion.6,8-10,31-34 Further, infection risks may be highest for patients who receive RBCs with the longest storage durations, suggesting that RBCs with longer storage durations may be particularly immunosuppressive.35-39 1942 TRANSFUSION Volume 55, August 2015
Monocytes are an important cell type in the innate immune response. In clinical studies by our group and others, monocyte suppression, characterized by low ex vivo LPS-induced TNF-a production capacity, is significantly associated with increased risks of nosocomial infection and mortality across multiple critically ill patient populations.13,15-17 Further, we have previously demonstrated in two separate cohorts of transfused critically ill children associations between longer RBC storage durations and suppressed LPS-induced TNF-a production capacity after transfusion relative to fresher RBCs.12,13 In in vitro studies by our group and others, stored RBCs directly suppress monocyte TNF-a production capacity as a function of RBC storage duration by unknown mechanisms.11,40,41 In our prior work, monocyte suppression occurred in the absence of cell-to-cell contact, suggesting a soluble mediator(s). This study uses our monocyte coculture model to evaluate the effects of RBC unit–derived MVs on monocyte function. Using this approach, MVs did not induce monocyte suppression while RBC supernatants that had been depleted of MVs did, suggesting soluble mediator(s) outside of isolated MV fractions. MVs are small, phospholipid membrane vesicles that are shed by a variety of cell types, including RBCs. Previous studies have demonstrated that MVs accumulate over time during RBC storage and may have immunomodulatory function.25,26 Sadallah and colleagues26 exposed human monocytederived macrophages to MVs that had been isolated from RBCs that had been stored for 25 days in saline-adenineglucose-mannitol. In contrast to this study, macrophages exposed to RBC-derived MVs demonstrated modest decreases in LPS-induced TNF-a production. Differences between the two studies may be related to differences in response to MVs by monocytes compared to macrophages.42 We chose to focus our studies on monocytes because circulating monocytes would be more likely to come into contact with transfusion-delivered MVs than would tissue macrophages. Similarly, a growing body of literature suggests that microparticles and other bioactive lipids found within stored RBC products may induce proinflammatory effects on neutrophils, another important cell type of the innate immune response.43,44 Our data suggest that effects on monocytes may be very different and may occur due to different mechanisms. Thus, immunomodulatory effects of RBC supernatants and RBC unit–derived MVs among different immunologic cell types remain important areas for future research. Regarding monocyte exposure to RBC unit–derived microparticles, Danesh and colleagues25 found increases in unstimulated monocyte cytokine production along with T-cell proliferation among PBMNCs after exposure to stored RBC-derived exosomes (SMV). Importantly, monocyte cytokine responses were measured after exposure to vesicles alone without the addition of LPS. Our study
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Fig. 3. Heat inactivation followed by RNase treatment removes immunosuppressive effects of older stored RBC supernatants. Monocyte LPS-induced TNF-a production was intact and did not differ significantly from controls after exposure to supernatants from 30-day-old RBC units that had been treated with heat inactivation followed by RNase (to degrade protein-bound RNA). Neither RNase treatment alone nor heat inactivation alone was sufficient to remove immunosuppressive effects (A). Heat plus RNase treatment was associated with reduced quantities of select miRNA compared to raw RBC supernatants (B). Data are mean (SE). n 5 6 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control monocytes (A) or raw RBC supernatant (B). ( 7e; ( ) miR-195; (w) miR-16.
likewise showed a modest although not significant increase in TNF-a after exposure to SMV in the absence of LPS. This response, however, was not associated with significant changes in LPS-induced TNF-a production, suggesting that it was not sufficient to induce either significant initial monocyte activation or subsequent suppression. Potential soluble mediator(s) of RBC storage-related immunomodulation may include, among others, bioactive lipids, iron, or miRNA.44,45 In our previous work, impaired LPS-induced TNF-a response occurred at both protein and mRNA levels, while IL-10 protein and mRNA production were preserved.11 This apparent specificity of response was recapitulated in our current experiments. These data raise the possibility that miRNA could be responsible for these effects. miRNA are small noncoding RNA that function to regulate specific genes or groups of genes. While circulating miRNA can be found associated with MV populations, recent reports suggest that the majority of circulating plasma miRNAs may exist outside of MVs.46 These MV-free miRNAs are likely to be resistant to RNase treatment (and thus able to remain intact in the setting of normal plasma RNases) by virtue of association with chaperone proteins such as Argonate 2. Despite the fact that erythrocytes lack a nucleus, stored RBC units do contain miRNA and miRNA levels have been shown to change over time during RBC storage through unknown mechanisms.47 Thus, it is plausible that immunomodulatory miRNA may accumulate in RBC supernatants and induce immune cell suppression. Whether miRNA are
) let-
the responsible mediators of immunosuppressive effects of stored RBCs cannot be determined from our study, although prevention of RBC supernatant–induced monocyte suppression by heat inactivation followed by RNase treatment implicates protein-bound RNA and provides intriguing data to support this hypothesis. Future studies will aim to comprehensively determine miRNA content of stored RBC supernatants across a spectrum of storage durations, to determine mechanisms of miRNA accumulation in the supernatant fraction of stored RBC units and to provide causal evidence to support the potential role of miRNA in stored RBC– induced monocyte suppression. Our study has important limitations. It is possible that additional soluble mediators may have been spun down with the MVs that counteracted the effects of the vesicles themselves. However, given that the MV fractions did not induce significant monocyte activation and that the magnitude of monocyte suppression seen with supernatants remaining after MV isolation was very similar to that seen with raw RBC supernatants, this possibility appears unlikely. Likewise, we are unable to say that MVdepleted supernatants were completely devoid of MVs. However, the differences in immunosuppressive effects between isolated MV fractions and MV-depleted supernatants were striking. Additionally, we chose to focus our studies on stimulated monocyte cytokine production capacities because of the clinical relevance of this particular measure of monocyte function in critically ill patients. Thus we are unable to comment on immunomodulatory effects of stored RBC supernatants or RBC-derived MVs Volume 55, August 2015 TRANSFUSION 1943
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on other immune cell types or on other measures of monocyte function. Along similar lines, these studies were performed using healthy donor monocytes and it is possible that results may have been more striking if a two-hit model employing monocytes from critically ill patients had been used. We view these areas to be highly important topics for ongoing and future study. Finally, the magnitude of immunosuppressive effects on monocytes may have been greater if we had compared Day 0 or Day 1 RBC units to Day 42 units. We chose instead to focus our studies on Day 7 versus Day 30 RBC units to mirror the typical range of storage durations of transfused RBCs in our institution. Even with this more pragmatic approach, differences between fresher and older RBC units were apparent. In conclusion, we found that while supernatants from prestorage leukoreduced RBC units stored for 30 6 3 days suppressed monocyte function in vitro, MVs isolated from RBC units did not appear to be responsible for monocyte suppression. Protein-bound RNA are implicated in this process, which may represent miRNA. Further study is warranted to determine the role of miRNA and other potential soluble mediators in RBC supernatant-induced monocyte suppression and to translate mechanisms found in vitro to the bedside.
6. Sadjadi J, Cureton EL, Twomey P, et al. Transfusion, not just injury severity, leads to posttrauma infection: a matched cohort study. Am Surg 2009;75:307-12. 7. Shorr AF, Jackson WL. Transfusion practice and nosocomial infection: assessing the evidence. Curr Opin Crit Care 2005; 11:468-72. 8. Taylor RW, Manganaro L, O’Brien J, et al. Impact of allogenic packed red blood cell transfusion on nosocomial infection rates in the critically ill patient. Crit Care Med 2002;30: 2249-54. 9. Taylor RW, O’Brien J, Trottier SJ, et al. Red blood cell transfusions and nosocomial infections in critically ill patients. Crit Care Med 2006;34:2302-8. 10. White M, Barron J, Gornbein J, et al. Are red blood cell transfusions associated with nosocomial infections in pediatric intensive care units? Pediatr Crit Care Med 2010;11: 464-8. 11. Muszynski J, Nateri J, Nicol K, et al. Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution. Transfusion 2012;52: 794-802. 12. Muszynski JA, Frazier E, Nofziger R, et al. Red blood cell transfusion and immune function in critically ill children: a prospective observational study. Transfusion 2015;55: 766-74. 13. Muszynski JA, Nofziger R, Greathouse K, et al. Innate
ACKNOWLEDGMENT
immune function predicts the development of nosocomial infection in critically injured children. Shock 2014;42:313-21.
The authors thank Cindy McAllister in the morphology core at The Research Institute at Nationwide Children’s Hospital for her assistance with electron microscopy.
14. Allen ML, Hoschtitzky JA, Peters MJ, et al. Interleukin-10 and its role in clinical immunoparalysis following pediatric cardiac surgery. Crit Care Med 2006;34:2658-65. 15. Cornell TT, Sun L, Hall MW, et al. Clinical implications and molecular mechanisms of immunoparalysis after cardiopul-
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
monary bypass. J Thorac Cardiovasc Surg 2012;143:11606.e1. 16. Hall MW, Geyer SM, Guo CY, et al. Innate immune function and mortality in critically ill children with influenza: a multi-
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position, oxidation, and signaling components. Transfusion 2008;48:1943-53.
risk pediatric cardiac surgery. Ann Thorac Surg 2012;93: 1563-9.
23. Rubin O, Crettaz D, Canellini G, et al. Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools. Vox Sang 2008;95:288-97. 24. Salzer U, Zhu R, Luten M, et al. Vesicles generated during storage of red cells are rich in the lipid raft marker stomatin. Transfusion 2008;48:451-62. 25. Danesh A, Inglis HC, Jackman RP, et al. Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro. Blood 2014;123:687-96. 26. Sadallah S, Eken C, Schifferli JA. Erythrocyte-derived ectosomes have immunosuppressive properties. J Leukoc Biol 2008;84:1316-25. 27. Gavrilin MA, Bouakl IJ, Knatz NL, et al. Internalization and phagosome escape required for Francisella to induce human monocyte IL-1beta processing and release. Proc Natl Acad Sci U S A 2006;103:141-6. € gger U, Rudy CK, E Etter M, et al. Imaging flow 28. Erdbru cytometry elucidates limitations of microparticle analysis by conventional flow cytometry. Cytometry A 2014;85:756-70. 29. Opelz G, Sengar DP, Mickey MR, et al. Effect of blood transfusions on subsequent kidney transplants. Transplant Proc 1973;5:253-9. 30. van Es AA, Balner H. Effect of pretransplant transfusions on kidney allograft survival. Transplant Proc 1979;11:127-37. 31. Rachoin JS, Daher R, Schorr C, et al. Microbiology, time course and clinical characteristics of infection in critically ill patients receiving packed red blood cell transfusion. Vox Sang 2009;97:294-302. 32. Shorr AF, Jackson WL, Kelly KM, et al. Transfusion practice and blood stream infections in critically ill patients. Chest 2005;127:1722-8. 33. Cata JP, Wang H, Gottumukkala V, et al. Inflammatory
37. Offner PJ, Moore EE, Biffl WL, et al. Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg 2002;137:711-16; discussion 716-7. 38. Ranucci M, Carlucci C, Isgro G, et al. Duration of red blood cell storage and outcomes in pediatric cardiac surgery: an association found for pump prime blood. Crit Care 2009;13: R207. 39. Weinberg JA, McGwin G Jr, Vandromme MJ, et al. Duration of red cell storage influences mortality after trauma. J Trauma 2010;69:1427-31. 40. Biedler AE, Schneider SO, Seyfert U, et al. Impact of alloantigens and storage-associated factors on stimulated cytokine response in an in vitro model of blood transfusion. Anesthesiology 2002;97:1102-9. 41. Mynster T. Effects of red cell storage and lysis on in vitro cytokine release. Transfus Apher Sci 2001;25:17-23. 42. Mikolajczyk TP, Skrzeczynska-Moncznik JE, Zarebski MA, et al. Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells. Immunology 2009;128: 103-13. 43. Belizaire RM, Prakash PS, Richter JR, et al. Microparticles from stored red blood cells activate neutrophils and cause lung injury after hemorrhage and resuscitation. J Am Coll Surg 2012;214:648-57. 44. Kent MW, Kelher MR, West FB, et al. The pro-inflammatory potential of microparticles in red blood cell units. Transfus Med 2014;24:176-81. 45. Wang D, Cortes-Punch I, Sun J, et al. Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia. Transfusion 2014; 54:1712-24. 46. Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 com-
response, immunosuppression, and cancer recurrence after
plexes carry a population of circulating microRNAs inde-
perioperative blood transfusions. Br J Anaesth 2013;110: 690-701.
pendent of vesicles in human plasma. Proc Natl Acad Sci U S A 2011;108:5003-8.
34. Luan H, Ye F, Wu L, et al. Perioperative blood transfusion adversely affects prognosis after resection of lung cancer: a systematic review and a meta-analysis. BMC Surg 2014;14:34.
47. Kannan M, Atreya C. Differential profiling of human red blood cells during storage for 52 selected microRNAs. Transfusion 2010;50:1581-8.
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BACKGROUND: We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. STUDY DESIGN AND METHODS: To determine the role of stored RBC unit–derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). RESULTS: Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPSinduced tumor necrosis factor-a production, 17,611 6 3,426 vs. 37,486 6 5,598 pg/mL; p 5 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant– induced monocyte suppression. CONCLUSION: Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.
R
ed blood cell (RBC) transfusion is independently associated with risk of new infection in critically ill patients.1-10 Our prior ex vivo and in vitro work strongly suggests that stored RBCs progressively suppress monocyte function with increasing storage duration.11-13 However, mechanisms of RBC-induced monocyte suppression are poorly understood. Understanding these mechanisms is important because we and others have shown that monocyte suppression in critically ill patients is strongly associated with increased risks for the development of new nosocomial infection and death.13-19 An important measure of monocyte immune function is the ability of monocytes to produce the proinflammatory cytokine tumor necrosis factor (TNF)-a in response to stimulation with lipopolysaccharide (LPS). Clinically, low
ABBREVIATIONS: LMV(s) 5 large (larger) microvesicle(s); LPS 5 lipopolysaccharide; miRNA 5 microRNA; MV(s) 5 microvesicle(s); RCN 5 relative copy number; SMV(s) 5 small (smaller) microvesicle(s). From the 1Division of Critical Care Medicine; 2The Research Institute; 3Department of Pathology, Nationwide Children’s Hospital; and the 4Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio Address reprint requests to: Jennifer A. Muszynski, MD, Critical Care Medicine, Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205; e-mail: [email protected]. This work was supported by K12HD043372 from the National Institute of Child Health and Development (JAM) and The Research Institute at Nationwide Children’s Hospital. *MDW and MWH contributed equally. Received for publication December 16, 2014; revision received February 12, 2015; and accepted February 13, 2015. doi:10.1111/trf.13084 C 2015 AABB V
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LPS-induced TNF-a production capacity is associated with increased risks for nosocomial infection and mortality in critically ill patients across diverse diagnoses of trauma, sepsis, critical viral infections, multiple organ dysfunction syndrome, and status post–cardiopulmonary bypass.13-17,20 In our previous in vitro work, aliquots from prestorage leukoreduced RBC units directly suppress monocyte LPSinduced TNF-a production capacity as a function of RBC storage duration.11 This suppression occurs via a soluble mediator(s), with effects occurring at the mRNA level. The identity of the soluble mediator(s) remains unknown, although previous reports have implicated RBC-derived microvesicles (MVs). MVs are small phospholipid membrane-bound vesicles (40-1000 nm) shed from a variety of cell types that may play important roles in cell-tocell communication.21 MVs accumulate over time during RBC storage,22-24 but their effects on monocyte function are unclear, with some investigators reporting monocyte activation while others report monocyte suppression.25,26 We therefore designed a series of in vitro experiments to test the hypothesis that older stored RBC products induce monocyte suppression via RBC-derived MVs.
MATERIALS AND METHODS Study subjects This study was approved by the institutional review board at Nationwide Children’s Hospital. Ten healthy adult volunteers were enrolled after written informed consent. Subjects were excluded if they had a history of cancer or posttransplant status or were receiving immunosuppressive or anti-inflammatory medication (including the use of aspirin or nonsteroidal anti-inflammatory medication within the past 48 hr). Up to 120 mL of blood was drawn in EDTA tubes (Becton Dickinson, Franklin Lakes, NJ) for monocyte isolation. Monocytes were isolated within 30 minutes after blood draws and were used immediately in coculture models. Monocytes were isolated as previously described.11 Briefly, whole blood was diluted 1:1 with phosphate-buffered saline (PBS), and peripheral blood mononuclear cells (PBMNCs) were then collected by density gradient centrifugation using lymphocyte separation medium (Mediatech, Manassas, VA). Monocytes were further purified by positive selection using CD14 magnetic beads (Miltenyi Biotec, Auburn, CA) and were resuspended in complete medium (RPMI 1 10% fetal bovine serum 1 1% penicillin/streptomycin). Percent purity using this method is at least 98% as previously reported.27
RBC products and MV isolation Eleven RBC units were obtained from the blood bank at Nationwide Children’s Hospital. All RBC components were prestorage leukoreduced using RBC filters (Flex Excel, 1938 TRANSFUSION Volume 55, August 2015
Fenwal, Lake Zurich, IL) and stored in Adsol-containing additive solution (AS-1, n 5 10; or AS-3, n 5 1). Units were stored at 4 C according to standard blood banking practice. Units were used in coculture experiments when they had been stored for 7 6 1 days or when they had been stored for 30 6 3 days. These storage durations were chosen to reflect the usual range of storage durations for transfused RBCs at our institution. For each experiment, aliquots from RBC units were obtained using a sterile connection device and blood administration syringe sets (CODAN, Santa Ana, CA). To obtain raw RBC supernatant, aliquots were centrifuged at 800 3 g for 10 minutes at 4 C. The supernatant was taken and centrifuged again at 800 3 g for 10 minutes to remove any remaining cells. This centrifugation speed was determined by preliminary experiments to yield the highest number of MVs with the fewest remaining contaminating cells (including platelets [PLTs]). MVs were then isolated from the raw supernatants by one of two different methods to yield either larger MVs (LMVs) or smaller MVs (SMVs). LMVs were isolated from 1 mL of raw RBC supernatant by centrifugation at 16,000 3 g at 4 C for 20 minutes and were resuspended in 200 mL of complete medium (for coculture experiments), PBS (for electron microscopy), or annexin V binding buffer (for flow cytometry). Supernatants remaining after centrifugation were retained and labeled LMV–depleted supernatants. SMVs were isolated from separate 1-mL aliquots of raw RBC supernatant by ultracentrifugation at 100,000 3 g at 4 C for 60 minutes and were resuspended in 200 mL of complete medium, PBS, or annexin V binding buffer, as described. Supernatants remaining after centrifugation were retained and labeled SMV-depleted supernatants. Isolated MVs were visualized by transmission electron microscopy as follows. After isolation by centrifugation, the LMV or SMV pellet was resuspended in PBS and loaded onto Formvar-carbon–coated copper grids (Electron Microscopy Sciences, Hattfield, PA). Samples were stained with 2% uranyl acetate for 10 minutes, washed, and then dried overnight at room temperature. Images were obtained using a transmission electron microscope (Model H-7650, Hitachi, Harrodsburg, KY). Flow cytometry was used to characterize MV cells of origin. Immediately after isolation, LMV and SMV pellets were suspended in 200 mL of annexin V binding buffer (eBioscience, San Diego, CA). MVs were identified by side scatter properties and surface expression of phosphatidylserine using PercP-eFluor 710 annexin V (eBioscience). To determine cell(s) of origin, MV samples were stained in two panels using the following antibodies: Panel 1 (fluorescein isothiocyanate [FITC]–anti-CD235a [RBCs; Becton Dickinson], eFluor 450 anti-CD41 [PLTs; eBioscience], allophycocyanin [APC]–anti-CD19 [B cells; Becton Dickinson]) and Panel 2 (FITC–anti-CD14 [monocytes], V450– anti-CD3 [T cells], APC–anti-CD142 [endothelial cells;
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Becton Dickinson]). COMPtrol antibody capture beads (Spherotech, Lake Forest, IL) or single-stained samples were used to set compensation where appropriate. Positive and negative gates were set using a full-minus-one strategy with appropriate isotype controls. To quantify MV content in MV fractions versus MV-depleted supernatants, absolute quantity of MVs was determined using absolute counting beads (CountBright, Life Technologies, Grand Island, NY). Absolute counting beads were added to samples just before acquisition. Because of poor resolution for smaller particles with conventional flow cytometry, MV quantification was performed for LMV fractions versus LMV-depleted supernatants.28 Flow cytometry data were acquired using a cytometer (LSRII; Becton Dickinson) and were analyzed in computer software (FlowJo, TreeStar, Ashland, OR).
Coculture experiments We used our previously published monocyte coculture model to investigate the role of RBC unit–derived MVs in modulating monocyte function.11 Briefly, this model involves the coculture of healthy adult monocytes in 12well plates using a ratio of culture medium-to-blood product that approximates the volume ratio associated with a 20 mL/kg RBC transfusion. Healthy monocytes are cocultured with the blood product of interest followed by stimulation with LPS, with high levels of LPS-induced TNF-a production being typical for healthy cells and reduced levels being consistent with immunosuppression. This model has been adapted for our MV experiments as follows. For MV experiments, 1 3 106 monocytes in 800 mL of complete medium were treated with 200 mL of raw RBC supernatant, 200 mL of LMVs in complete medium, 200 mL of SMVs in complete medium, 200 mL of LMV-depleted supernatant, 200 mL of SMV-depleted supernatant, or 200 mL of complete medium (control) at 37 C for 4 hours in 12-well plates. After 4 hours, monocytes were stimulated with 1 ng/mL LPS (phenol-extracted from Salmonella abortus equii [Sigma, St Louis, MO]) and incubated for an additional 4 hours at 37 C. Unstimulated control wells were incubated under the same conditions for the same durations, but without the addition of LPS. Supernatants from LPS-stimulated and unstimulated monocytes were collected by centrifugation and stored at 280 C for batch analysis of TNF-a and interleukin (IL)-10 protein production. MV coculture experiments were performed in four replicates with a different monocyte donor and different RBC units for each replicate. For each experiment, RBC supernatant and MV fractions were obtained from RBC units that had been stored for 7 6 1 or 30 6 3 days. For heat inactivation experiments, RBC supernatants were heated to 100 C for 5 minutes and then allowed to cool to room temperature before use in coculture. For RNase-treated supernatants, 0.5 mg of RNase A (Life
Technologies) was added to RBC supernatant or to heatinactivated RBC supernatant just before coculture experiments. Heat inactivation experiments were performed six times with a different monocyte donor and RBC units per replicate. Cytokine production from coculture experiments (TNF-a, IL-10) as well as cytokine quantities in stored RBC products (TNF-a, IL-1b, IL-8, and IL-10) were quantified by chemiluminescence using an automated chemiluminometer (Immulite 1000, Siemens Healthcare Diagnostics, Deerfield, IL), with a lower limit of detection of 5 pg/mL for all cytokines. Manufacturer-provided standards and controls were used to ensure proper instrument calibration before each run.
RNA isolation and microRNA quantification To evaluate microRNA (miRNA) as a potential mediator of monocyte suppression in RBC units, miRNAs let-7e, mir16, and mir-195 were quantified in raw and treated RBC supernatants by real-time polymerase chain reaction (PCR) as follows. These miRNA were chosen from an exploratory analysis in which we profiled miRNA in fresher versus older RBC supernatants by real-time PCR and found increased relative expression among these immunomodulatory miRNA in older versus fresher RBC supernatants (data not shown). Aliquots of raw RBC supernatants used in coculture experiments were taken and kept frozen at 280 C before subsequent analysis. Just before RNA isolation, RBC supernatants were treated with heat, RNase, or heat plus RNase as described. RNA was isolated using the miRNeasy kit for serum and plasma (Qiagen, Valencia, CA) per the manufacturer’s instructions. Briefly, 1 mL of lysis reagent (QIAzol, Qiagen) was added to 200 mL of samples and incubated at room temperature for 5 minutes. Twenty-five fmol of synthetic Caenorhabditis elegans miRNA mixture (Syn-cel-miR-39, Syn-cel-miR-54; Qiagen) was added to each sample to serve as exogenous controls. Then 200 mL of chloroform was added to each sample and the aqueous phases containing RNA were purified. RNA (10 ng) was converted to cDNA using reverse transcription master mix with individual PCR primers (TaqMan, Life Technologies). Quantitative real-time PCR was performed in duplicate using TaqMan master mix (Life Technologies) using a real-time PCR system (Applied Biosystems Step One Plus, Life Technologies). The average cycle threshold (Ct) value for exogenous controls was used for normalization. Target miRNA expression is expressed as relative copy number (RCN) using the value 2(2DCt).
Statistical analysis Data presented are mean 6 standard error (SE). Differences between groups were analyzed using t test or paired t test, where appropriate. Analyses were performed using computer software (Prism6, GraphPad, Inc., La Jolla, CA). Volume 55, August 2015 TRANSFUSION 1939
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Fig. 1. Representative electron micrographs of isolated MVs and MV cells of origin by flow cytometry. Isolated MV fractions contained MVs of varying sizes for both LMVs (A; 125-1464 nm) and SMVs (B; 45-337 nm). Images are representative of two independent experiments. Images were obtained using a transmission electron microscope (Model H-7650, Hitachi) at a 15,0003 magnification. By flow cytometry, RBCs and PLTs were the most frequent MV cell(s) of origin for both SMVs (C) and LMVs (D) with very little expression of evaluated white blood cell or endothelial cell markers. Overall, there were no significant differences in MV cells of origin between LMVs and SMVs or among MV fractions from RBC units of varying storage durations ( , 7 days; w, 30 days). Data represent percent positive among isolated MVs and are expressed as mean (SE) from three individual experiments.
A p value of less than 0.05 was considered to be significant throughout.
RESULTS Isolated MVs from stored RBC units were visualized by transmission electron microscopy. LMVs isolated by 16,000 3 g centrifugation were variably sized ranging between 125 and 1464 nm, with a mean size of 479 6 101 nm (Fig. 1A). SMVs isolated by 100,000 3 g centrifugation were likewise variably sized ranging between 45 and 337 nm, with a mean size of 125 6 20 nm (Fig. 1B). For both LMVs and SMVs, the most frequent MV cells of origin were RBCs (Figs. 1C and 1D). Overall, there were no significant differences in MV cells of origin between LMV and SMV or among MV fractions over time, except for a trend toward increases in percent of PLT-derived MVs 1940 TRANSFUSION Volume 55, August 2015
from older RBC units. As expected, MV-depleted supernatants contained significantly fewer MVs compared to isolated MV fractions (17 6 6.5 MVs/mL vs. 10,325 6 1,548 MV/mL; n 5 3, p 5 0.02). Consistent with our previous study, raw supernatants from prestorage leukoreduced RBC units stored for 30 6 2 days suppressed monocyte function as evidenced by reduced LPS-induced TNF-a production capacity. Unexpectedly, MV fractions isolated from both fresher and older RBC units failed to induce monocyte suppression (Fig. 2). This was true for both LMVs and SMVs. Importantly, older RBC supernatants that had been depleted of MVs remained immunosuppressive to LPS-stimulated monocytes, suggesting a soluble mediator(s) outside of isolated MV fractions (Figs. 2A and 2B). The ability of monocytes to produce the anti-inflammatory cytokine IL10 in response to LPS stimulation was preserved in all
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Fig. 2. Supernatants from older stored RBCs suppressed monocyte function whereas MVs isolated from RBC supernatants did not. Healthy adult monocytes were cocultured with supernatants from prestorage leukoreduced RBC units that had been stored for 7 6 1 or 30 6 3 days. Monocyte cytokine production was measured after LPS stimulation (A-D) or without LPS (E, F). LPS-induced TNF-a production was impaired after exposure to raw supernatants from older RBC units (A, B). MVs isolated by 16,000 3 g centrifugation (LMV) did not induce monocyte suppression, but RBC supernatant that had been depleted of MV did (A). MVs isolated from 100,000 3 g centrifugation (SMVs) also did not induce monocyte suppression while SMV-depleted supernatants remained immunosuppressive (B). LPS-induced IL-10 production was preserved in all groups without significant differences compared to control monocytes (C, D). In the absence of LPS stimulation, TNF-a production was low in all groups (E, F; note the difference in y-axis scale). Data are mean (SE). n 5 4 experiments. *p < 0.05 compared to control. (A-F) w, raw RBC supernatant;
, LMV-depleted RBC supernatant;
LMV (A, C, E); w, raw RBC supernatant; (B, D, F)
, SMV (B, D, F).
, SMV-depleted RBC supernatant;
experimental groups, with no differences seen compared to control monocytes (Figs. 2C and 2D). In the absence of LPS stimulation, TNF-a levels in the coculture experiments were low in all groups, with no differences seen between experimental groups and controls (Figs. 2E and 2F), suggesting the absence of contaminating stimulants.
,
Levels of the cytokines IL-1b, IL-8, and IL-10 were not detectable in the supernatants of older or fresher RBC units. Supernatants from older RBC units also did not demonstrate elevated TNF-a concentrations, but rather had decreased amounts of TNF-a relative to fresher units (15 6 4 pg/mL vs. 113 6 43 pg/mL, respectively; p 5 0.01). Volume 55, August 2015 TRANSFUSION 1941
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As MVs did not appear to be the soluble mediators of the observed reduction in LPS-induced TNF-a production, we next performed a series of experiments designed to determine whether the responsible mediator(s) may be soluble RNA alone, protein, or protein-bound RNA (Fig. 3A). When monocytes were cocultured with older RBC supernatants, treatment of those supernatants with RNase alone to remove soluble RNAs did not remove their immunosuppressive effects. Likewise, heat inactivation (to degrade protein) only partially prevented the immunosuppressive effects of older RBC supernatants. However, treating RBC supernatants with heat inactivation followed by RNase (to degrade protein-bound RNA) prevented monocyte suppression. We hypothesize that the responsible protein-bound RNA may be miRNA. To explore whether heat and RNase treatment of RBC supernatants results in decreased miRNA levels, we quantified select miRNA (let 7-e, miR16, miR-195) in treated compared to untreated RBC supernatants by real-time PCR. As expected, the heat and RNase–treated supernatants had significantly decreased levels of let-7e (RCN, 2.6 vs, 15.6; p 5 0.04) and miR-195 (RCN, 0.46 vs. 14.1; p 5 0.04) with a trend toward decrease in miR-16 (RCN, 47 vs. 780; p 5 0.07; Fig. 3B).
DISCUSSION With a marker of innate immune function that has been repeatedly observed to be clinically relevant in critically ill patients, supernatants from older units of prestorage leukoreduced RBC suppressed monocyte function in vitro while MV fractions isolated from the same units did not. Protein-bound RNA are implicated as potential soluble mediators of monocyte suppression. These may represent miRNA. Transfusion-related immunomodulation has long been recognized but remains poorly understood. Some of the well-described but relatively rare complications of RBC transfusion (such as transfusion-related acute lung injury and nonhemolytic transfusion reactions) likely arise from proinflammatory mediators. However, a growing body of literature strongly supports the notion that on balance, RBC transfusion may be immunosuppressive. As early as the 1970s investigators noted that in transplant recipients, allogeneic blood transfusion was associated with lower rates of organ rejection—suggesting that RBC transfusion may be immunosuppressive.29,30 Since then, a number of studies have demonstrated independent associations between RBC transfusion and increased risks of nosocomial infection and cancer recurrence, consistent with overall immunosuppressive effects of RBC transfusion.6,8-10,31-34 Further, infection risks may be highest for patients who receive RBCs with the longest storage durations, suggesting that RBCs with longer storage durations may be particularly immunosuppressive.35-39 1942 TRANSFUSION Volume 55, August 2015
Monocytes are an important cell type in the innate immune response. In clinical studies by our group and others, monocyte suppression, characterized by low ex vivo LPS-induced TNF-a production capacity, is significantly associated with increased risks of nosocomial infection and mortality across multiple critically ill patient populations.13,15-17 Further, we have previously demonstrated in two separate cohorts of transfused critically ill children associations between longer RBC storage durations and suppressed LPS-induced TNF-a production capacity after transfusion relative to fresher RBCs.12,13 In in vitro studies by our group and others, stored RBCs directly suppress monocyte TNF-a production capacity as a function of RBC storage duration by unknown mechanisms.11,40,41 In our prior work, monocyte suppression occurred in the absence of cell-to-cell contact, suggesting a soluble mediator(s). This study uses our monocyte coculture model to evaluate the effects of RBC unit–derived MVs on monocyte function. Using this approach, MVs did not induce monocyte suppression while RBC supernatants that had been depleted of MVs did, suggesting soluble mediator(s) outside of isolated MV fractions. MVs are small, phospholipid membrane vesicles that are shed by a variety of cell types, including RBCs. Previous studies have demonstrated that MVs accumulate over time during RBC storage and may have immunomodulatory function.25,26 Sadallah and colleagues26 exposed human monocytederived macrophages to MVs that had been isolated from RBCs that had been stored for 25 days in saline-adenineglucose-mannitol. In contrast to this study, macrophages exposed to RBC-derived MVs demonstrated modest decreases in LPS-induced TNF-a production. Differences between the two studies may be related to differences in response to MVs by monocytes compared to macrophages.42 We chose to focus our studies on monocytes because circulating monocytes would be more likely to come into contact with transfusion-delivered MVs than would tissue macrophages. Similarly, a growing body of literature suggests that microparticles and other bioactive lipids found within stored RBC products may induce proinflammatory effects on neutrophils, another important cell type of the innate immune response.43,44 Our data suggest that effects on monocytes may be very different and may occur due to different mechanisms. Thus, immunomodulatory effects of RBC supernatants and RBC unit–derived MVs among different immunologic cell types remain important areas for future research. Regarding monocyte exposure to RBC unit–derived microparticles, Danesh and colleagues25 found increases in unstimulated monocyte cytokine production along with T-cell proliferation among PBMNCs after exposure to stored RBC-derived exosomes (SMV). Importantly, monocyte cytokine responses were measured after exposure to vesicles alone without the addition of LPS. Our study
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Fig. 3. Heat inactivation followed by RNase treatment removes immunosuppressive effects of older stored RBC supernatants. Monocyte LPS-induced TNF-a production was intact and did not differ significantly from controls after exposure to supernatants from 30-day-old RBC units that had been treated with heat inactivation followed by RNase (to degrade protein-bound RNA). Neither RNase treatment alone nor heat inactivation alone was sufficient to remove immunosuppressive effects (A). Heat plus RNase treatment was associated with reduced quantities of select miRNA compared to raw RBC supernatants (B). Data are mean (SE). n 5 6 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control monocytes (A) or raw RBC supernatant (B). ( 7e; ( ) miR-195; (w) miR-16.
likewise showed a modest although not significant increase in TNF-a after exposure to SMV in the absence of LPS. This response, however, was not associated with significant changes in LPS-induced TNF-a production, suggesting that it was not sufficient to induce either significant initial monocyte activation or subsequent suppression. Potential soluble mediator(s) of RBC storage-related immunomodulation may include, among others, bioactive lipids, iron, or miRNA.44,45 In our previous work, impaired LPS-induced TNF-a response occurred at both protein and mRNA levels, while IL-10 protein and mRNA production were preserved.11 This apparent specificity of response was recapitulated in our current experiments. These data raise the possibility that miRNA could be responsible for these effects. miRNA are small noncoding RNA that function to regulate specific genes or groups of genes. While circulating miRNA can be found associated with MV populations, recent reports suggest that the majority of circulating plasma miRNAs may exist outside of MVs.46 These MV-free miRNAs are likely to be resistant to RNase treatment (and thus able to remain intact in the setting of normal plasma RNases) by virtue of association with chaperone proteins such as Argonate 2. Despite the fact that erythrocytes lack a nucleus, stored RBC units do contain miRNA and miRNA levels have been shown to change over time during RBC storage through unknown mechanisms.47 Thus, it is plausible that immunomodulatory miRNA may accumulate in RBC supernatants and induce immune cell suppression. Whether miRNA are
) let-
the responsible mediators of immunosuppressive effects of stored RBCs cannot be determined from our study, although prevention of RBC supernatant–induced monocyte suppression by heat inactivation followed by RNase treatment implicates protein-bound RNA and provides intriguing data to support this hypothesis. Future studies will aim to comprehensively determine miRNA content of stored RBC supernatants across a spectrum of storage durations, to determine mechanisms of miRNA accumulation in the supernatant fraction of stored RBC units and to provide causal evidence to support the potential role of miRNA in stored RBC– induced monocyte suppression. Our study has important limitations. It is possible that additional soluble mediators may have been spun down with the MVs that counteracted the effects of the vesicles themselves. However, given that the MV fractions did not induce significant monocyte activation and that the magnitude of monocyte suppression seen with supernatants remaining after MV isolation was very similar to that seen with raw RBC supernatants, this possibility appears unlikely. Likewise, we are unable to say that MVdepleted supernatants were completely devoid of MVs. However, the differences in immunosuppressive effects between isolated MV fractions and MV-depleted supernatants were striking. Additionally, we chose to focus our studies on stimulated monocyte cytokine production capacities because of the clinical relevance of this particular measure of monocyte function in critically ill patients. Thus we are unable to comment on immunomodulatory effects of stored RBC supernatants or RBC-derived MVs Volume 55, August 2015 TRANSFUSION 1943
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on other immune cell types or on other measures of monocyte function. Along similar lines, these studies were performed using healthy donor monocytes and it is possible that results may have been more striking if a two-hit model employing monocytes from critically ill patients had been used. We view these areas to be highly important topics for ongoing and future study. Finally, the magnitude of immunosuppressive effects on monocytes may have been greater if we had compared Day 0 or Day 1 RBC units to Day 42 units. We chose instead to focus our studies on Day 7 versus Day 30 RBC units to mirror the typical range of storage durations of transfused RBCs in our institution. Even with this more pragmatic approach, differences between fresher and older RBC units were apparent. In conclusion, we found that while supernatants from prestorage leukoreduced RBC units stored for 30 6 3 days suppressed monocyte function in vitro, MVs isolated from RBC units did not appear to be responsible for monocyte suppression. Protein-bound RNA are implicated in this process, which may represent miRNA. Further study is warranted to determine the role of miRNA and other potential soluble mediators in RBC supernatant-induced monocyte suppression and to translate mechanisms found in vitro to the bedside.
6. Sadjadi J, Cureton EL, Twomey P, et al. Transfusion, not just injury severity, leads to posttrauma infection: a matched cohort study. Am Surg 2009;75:307-12. 7. Shorr AF, Jackson WL. Transfusion practice and nosocomial infection: assessing the evidence. Curr Opin Crit Care 2005; 11:468-72. 8. Taylor RW, Manganaro L, O’Brien J, et al. Impact of allogenic packed red blood cell transfusion on nosocomial infection rates in the critically ill patient. Crit Care Med 2002;30: 2249-54. 9. Taylor RW, O’Brien J, Trottier SJ, et al. Red blood cell transfusions and nosocomial infections in critically ill patients. Crit Care Med 2006;34:2302-8. 10. White M, Barron J, Gornbein J, et al. Are red blood cell transfusions associated with nosocomial infections in pediatric intensive care units? Pediatr Crit Care Med 2010;11: 464-8. 11. Muszynski J, Nateri J, Nicol K, et al. Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution. Transfusion 2012;52: 794-802. 12. Muszynski JA, Frazier E, Nofziger R, et al. Red blood cell transfusion and immune function in critically ill children: a prospective observational study. Transfusion 2015;55: 766-74. 13. Muszynski JA, Nofziger R, Greathouse K, et al. Innate
ACKNOWLEDGMENT
immune function predicts the development of nosocomial infection in critically injured children. Shock 2014;42:313-21.
The authors thank Cindy McAllister in the morphology core at The Research Institute at Nationwide Children’s Hospital for her assistance with electron microscopy.
14. Allen ML, Hoschtitzky JA, Peters MJ, et al. Interleukin-10 and its role in clinical immunoparalysis following pediatric cardiac surgery. Crit Care Med 2006;34:2658-65. 15. Cornell TT, Sun L, Hall MW, et al. Clinical implications and molecular mechanisms of immunoparalysis after cardiopul-
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
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